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CD1 MR1 2017 Delegate Booklet

TABLE OF CONTENTS

CONFERENCE SPONSORS .................................................................. Inside Cover

WELCOME FROM THE CONFERENCE ORGANISERS ............................................. 2

ORGANISING COMMITTEE 2017 ......................................................................... 3

INVITED SPEAKERS .............................................................................................. 4

DELEGATE INFORMATION................................................................................. 19

VENUE MAP ...................................................................................................... 22

CONFERENCE PROGRAM .................................................................................. 24

FRIDAY 3RD NOVEMBER 2017 24

SATURDAY 4TH NOVEMBER 2017 25

SUNDAY 5TH NOVEMBER 2017 28

MONDAY 6TH NOVEMBER 2017 32

TUESDAY 7TH NOVEMBER 2017 35

ORAL ABSTRACTS .............................................................................................. 36

RAPID FIRE ABSTRACTS ..................................................................................... 67

POSTER SESSION 1 LISTING - ODD NUMBERS ................................................... 72

POSTER SESSION 2 LISTING - EVEN NUMBERS .................................................. 78

POSTER ABSTRACTS .......................................................................................... 85

SPONSORS AND EXHIBITORS .......................................................................... 132

ATTENDEES ..................................................................................................... 136

NOTES ............................................................................................................. 140


WELCOME FROM THE CONFERENCE ORGANISERS Dear Delegates, Thank you for joining us in California, a state that is almost a country unto itself. (Our sincere apologies for having an orange president; we would highly recommend for us all to stay away from rude political jokes as they have become too easy and a sad reminder of the situation we are in). Instead, let’s be thankful the fires are under control, focus on our outstanding meeting, and enjoy our great privilege to be scientists and having the best job in the world. We have tried, like in our previous gatherings, to mix old and new (speakers), MAITs, NKTs, and group I CD1 reactive cells, traditional with unusual, expected with surprising. Challenge the speakers during the sessions, press the poster presenters with your astute questions, freely share ideas, expand your horizons by talking to your neighbor at meals, and smile to communicate your ecstasy. The sessions have been designed so that nearly all of the talks will be short (15 minutes), and will present a highly focused story. However, we have sandwiched the meeting with three special lectures, two bookends and a middle, to ensure that your minds and your attention spans will remain flexible. The best of the younger generation will also be given the chance to compete for $$, the currency of the land, and we expect nothing less from them than the best. Socially, we will offer outings to explore the surrounding areas, and will not require you to take part in brutal local sports activities like football or baseball. Everybody is welcome to call everybody else “Dude” and as a primer, may want to watch the Big Lebowski to understand the full and profound meaning of the word. It is not as simple as the Australian “Mate” which refers only to “we came on the same boat and weren’t in first class cabin”, instead, it is complex, intricate, profoundly absurd and therefore existentialist. We are looking to make your stay pleasurable and memorable. Jenny Gumperz, Mitchell Kronenberg & Luc Teyton


ORGANISING COMMITTEE 2017

Associate Professor Jenny Gumperz University of Wisconsin Dr. Gumperz is Associate Professor of Medical Microbiology and Immunology at the University of Wisconsin-Madison. She completed her B.A. at the University of California at Santa Cruz, then worked for several years as a chemist at Molecular Devices Corp. She completed her Ph.D. at Stanford University under the mentorship of Dr. Peter Parham, where she collaborated with Dr. Lewis Lanier's group to investigate human Natural Killer cell recognition of HLA molecules. During her postdoctoral studies at Harvard University in the laboratory of Dr. Michael Brenner, she focused on the functions and self-antigen specificities of invariant Natural Killer T cells. Dr. Gumperz joined the faculty of the University of Wisconsin in 2003,

where her current research focuses on using humanized mouse models to dissect the activation and effector pathways of human innate lymphocytes in vivo, with the ultimate goal of furthering the development of clinical approaches using these cells to treat cancer, inflammation, and infectious diseases.

Dr Mitchell Kronenberg La Jolla Institute Mitchell Kronenberg received B.A. in from Columbia University and a Ph.D. in Biochemistry from the California Institute of Technology (Caltech), where he continued as a postdoctoral fellow in the laboratory of Dr. Leroy Hood. Mitchell served on the faculty of UCLA from 1986-1997. He moved to the La Jolla Institute for Allergy & Immunology (LJI) in 1997, and was appointed President in 2003. He is responsible for the leadership and overall administration of the Institute, which has more than 375 employees. Mitchell’s research interests include T lymphocyte biology, mucosal immunology, and inflammatory and autoimmune disease models. His laboratory has carried out research on diverse topics related to invariant natural

killer T (iNKT) cells, including antigen processing and presentation of lipid antigens by CD1d, the identification of microbial antigens, the role of iNKT cells in the response to infections, and the developmental programs driving iNKT cell differentiation. Mitchell started this meeting series, with the first meeting in San Diego in 1999, and is pleased to participate with Jenny and Luc in organizing the Napa meeting.

Professor Luc Teyton The Scripps Research Institute M.D., Ph.D. from Paris University. Arrived in the USA in 1987 for a post-doctoral fellowship at the Scripps Research Institute. TSRI faculty member since 1991. Initially, his laboratory was focused on antigen presentation and discovered the double role of the invariant chain, and expressed the first recombinant I-A MHC class II molecules. Interest in structural biology led to the determination of the first structures of I-A and diabetogenic MHC molecules, as well as the first structure of a T cell receptor alone and in complex with pMHC. His laboratory focused its attention on lipid transport, loading onto CD1 molecules and recognition by immune cells around the turn of the new century. This work produced the first CD1d tetramers, and has

allowed the discovery of the role of saposins in lipid antigen presentation, the elucidation of the role of NKT cells in antigen priming, the first two structures of CD1-lipid complexes, the role of serum transport for antigen presentation, and more recently the discovery of the endogenous ligand of NKT cells. A strong emphasis of his laboratory is now to translate basic knowledge to human immunology.


INVITED SPEAKERS

KEYNOTE SPEAKERS

Dr Yasmine Belkaid National Institute of Allergy and Infectious Disease Dr. Yasmine Belkaid obtained her Ph.D. in 1996 from the Pasteur Institute in France exploring innate immune responses to parasitic infections. Her work explores mechanisms that regulate host immune responses to microbes at barrier sites and revealed key roles for the microbiota and dietary factors in the maintenance of tissue immunity and homeostasis. Following a postdoctoral fellowship at the National Institute of Allergy and Infectious Diseases (NIAID) in the US, she joined the Children’s Hospital Research Foundation in Cincinnati as an assistant

professor. In 2005, she joined NIAID and was appointed Senior Scientist in 2008. Dr Belkaid is also an Adjunct professor in the department of Pathology at the University of Pennsylvania. She is currently the chief of the Mucosal Immunology section and Director of the NIAID Microbiome initiative.

Paolo Dellabona Ospedale San Raffaele Paolo Dellabona graduated in Medicine and obtained a Ph.D. in Medical Genetics from the University of Torino. He is currently joint-head with Giulia Casorati of the Experimental Immunology Unit and head of the Division of Immunology, Transplantation and Infectious Diseases at the San Raffaele Scientific Institute in Milano, Italy. The main scientific interest of the Casorati/Dellabona lab concerns the understanding of the development and anti-tumor functions of CD1-restricted T and NKT lymphocytes.

Professor Mark Exley Brigham and Women’s Hospital, USA Mark Exley gained his BS, MS, and PhD. from London University and post-doc at the Dana Farber Cancer Institute, Harvard Medical School (HMS). Mark worked at Immulogic Inc on novel stimulating as well as tolerising vaccines, before returning to HMS as Faculty in 1996. Mark has worked on then newly-emerging human as well as murine CD1d-reactive ‘NKT’ cell populations for over 20 years. Mark was Professor at the University of Manchester, UK and Visiting Professor at HMS 2013-2015, before returning to the US as VP Cellular Immunology at Agenus Inc., retaining honorary appointments at Manchester and HMS. His research continues to focus on regulation of immunity by NKT and other cells and how this knowledge

can be exploited clinically in cancer, hepatitis, and other diseases, such as obesity. He has been involved in clinical trials of NKT antibody 6B11 that he co-developed in cell therapy and humanized derivative in vivo, as well as other immuno-therapeutic approaches.


Dr Dale Godfrey University of Melbourne Godfrey has worked in the field of T cell biology for over 25 years. His initial studies were focused on T cell development in the thymus, where he mapped the DN1-DN4 thymocyte developmental sequence. Godfrey then shifted his focus to unconventional T cells, initially in the field of CD1d-restricted lipid antigen reactive NKT cell biology. These studies included several breakthroughs in our understanding of NKT cell development, their role in disease, and the role of diverse NKT cell TCRs in differential lipid antigen recognition. More recently, Godfrey has expanded his interest to encompass Type 2 CD1d-restricted NKT cells as well as CD1a,b,c

restricted T cells, MR1-restricted MAIT cells and gd T cells. Godfrey is an NHMRC Senior Principal Research Fellow, Immunology Theme Leader at the Doherty Institute at University of Melbourne, and a Past President of the Australasian Society for Immunology (ASI).

Professor James McCluskey University of Melbourne James McCluskey is Deputy Vice Chancellor Research at The University of Melbourne. After completing his physician training, he worked at the National Institutes of Health (USA) from 1983-87, then worked at Monash University training in pathology. From 1997-2010 he was Chair in Microbiology and Immunology at The University of Melbourne. He has published more than 300 scientific articles on how genes control immunity, mechanisms of autoimmune disease, immune recognition and the basis of transplantation matching. He was awarded the Parr Prize from the Australian Rheumatism Association, the Priscilla Kincaid Smith Oration and

medal of the Royal Australian College of Physicians, the Rose Payne Award from the American Society for Histocompatibility and Immunogenetics (ASHI), The Ceppellini award from the European Federation for Immunogenetics, the International Roche Organ Transplantation Fund Recognition Prize for Excellence in Organ Transplantation Research jointly with Jamie Rossjohn, an Australian Museum Eureka award for scientific Research (McCluskey, Kjer-Nielsen and Rossjohn) and NHMRC Research Excellence Award for top NHMRC Program Grant in 2012. He received GSK Research Excellence Award and the 2016 Victoria Prize for Life Sciences (both jointly with Jamie Rossjohn). In 2010 he was elected to the Faculty of Science, Royal College of Pathologists Australasia. He is a Fellow Australian Academy of Science, Fellow of the Australian Academy of Health and Medical Sciences, Fellow of the Royal Australian College of Physicians and Fellow of the Royal College of Pathologists Australasia. He led the development, funding and establishment of the Peter Doherty Institute for Infection and Immunity ($207M), bringing together researchers, infectious disease physicians and public health laboratories jointly with Melbourne Health (resembling the US CDC).

Professor Charles Serhan Brigham and Women’s Hospital, Harvard Medical School Charles is the Simon Gelman Professor of Anaesthesia (Biochemistry and Molecular Pharmacology) at Harvard Medical School, Professor of Oral Medicine, Infection and Immunity at HSDM, and Director of the Center for Experimental Therapeutics and Reperfusion Injury at Brigham and Women’s Hospital. He received a BS in biochemistry from Stony Brook University followed by a doctorate in experimental pathology and medical sciences from New York University School of Medicine. Visiting scientist and post-doctoral fellow at the Karolinska Institutet, Stockholm with Professor Samuelsson 1982 Nobel Laureate. 1987 joined the faculty at Harvard Medical School and received an honorary degree from Harvard University (1996). He

received several awards including an NIH MERIT award and has delivered over 50 keynote and plenary lectures. In 2016, he received the IUBMB Lecture Metal and Ross Prize in Molecular Medicine.


SESSION CHAIRS - INTERNATIONAL ADVISORY COMMITTEE

Professon Erin Adams University of Chicago The Adams lab is interested in nonclassical and MHC-like molecules and the cell populations that respond to them. We have investigated CD1, MR1 and γδ T cell reactivity and the interplay between nonclassical MHC and other systems that regulate defense, homeostasis and reproduction. We use a combination of structural, biochemical and functional approaches to investigate these systems, with the goal of understanding the molecular mechanisms that regulate them in human health, reproduction and disease.

Dr Patricia Barral King’s College London Dr Patricia Barral obtained her PhD from the Universidad Complutense de Madrid (Spain) and trained as a postdoctoral fellow at the London Research Institute (UK). She is currently a lecturer at King’s College London and group leader at the Francis Crick Institute (London, UK). Her group’s interest is to understand the cellular mechanisms that initiate and shape immune responses, with a particular emphasis on studying the biology and function of NKT cells in health and disease.

Dr Albert Bendelac The University of Chicago Dr Bendelac obtained his MD PhD in Paris, France and joined the Ronald Schwartz’s laboratory for postdoctoral research during which he first identified NKT cells. He established his independent laboratory at Princeton University in 1994 and moved to the University of Chicago in 2002 to become Chair of the Committee on Immunology.Dr Bendelac has a longstanding interest in the development and function of innate and innate-like lymphocytes. His early studies identified NKT cells, demonstrated their recognition of CD1d-lipid complexes by semi-invariant TCRs, and characterized the cell biology of lipid antigen capture, processing

and loading. The Bendelac group used CD1d tetramers to characterize the different stages and the molecular mechanisms of NKT cell development. These studies led in the identification of their master transcription factor PLZF. More recently, the critical role of PLZF was extended to innate lymphoid cells (ILC) and PLZF expression was used to define the common ILC precursor in the bone marrow.

Professor Richard Blumberg Brigham and Women’s Hospital, USA Dr. Blumberg is Professor of Medicine, Harvard Medical School, Chief of Gastroenterology, Brigham and Women’s Hospital, co-Director of the Harvard Digestive Diseases Center and past-Director of the Brigham Research Institute. He has directed a National Institutes of Health funded laboratory since 1989 which has a particular emphasis on the immunologic functions associated with the cross-talk between microbes, the epithelium and immune system and focused on non-classical MHC molecules such CD1 and the neonatal crystallizable fragment receptor (FcRn). He also has major interests in the function of the unfolded protein

response in determining intestinal inflammation and how carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) function in regulating T cell biology. Dr. Blumberg has been the recipient of the an NIH Method to Extend Research in Time (M.E.R.I.T) Award (2005), the William Beaumont Prize (2009), the CCFA Scientific Achievement Award in Inflammatory Bowel Disease Basic Research (2012), a Lifetime Scientific Achievement Award from the Society for Mucosal Immunology (2015), the Lloyd Mayer Award in Mucosal Immunology and is an elected member of the Norwegian Academy of Sciences.


Professor Michael Brenner Brigham and Women’s Hospital, USA Michael B Brenner MD is Theodore Bevier Bayles Professor of Medicine, at Harvard Medical School, and Chief of the Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, USA. My lab research examines basic and translational studies in T cell biology, antigen presentation, microbial and autoimmunity. We identified and characterised gd T cells and identified the pathway of lipid antigen presentation by CD1. More recently we defined the mechanisms that activate iNKT cells including identifying the lipid antigens and cytokines that drive their activation and the cross-talk between iNKT cells and DCs. Besides studies in microbial immunity and host defense, we are defining the regulatory roles of iNKT

cells and gd in maintaining a healthy Th2 like state in adipose tissue – and a surprizing role in controlling body temperature. In other recent studies in autoimmune diseases, we also have defined specific fibroblast subpopulations that are pathologically expanded in disease, and a new T helper cell population, T peripheral helper (Tph) cells that drive autoantibody production in autoimmune diseases. I am now leading the Human Immunology Center at Brigham and Women’s Hospital that designs and implements high dimensional immunophenotyping, single cell transcriptional analyses and functional studies to deconstruct human autoimmune disorders.

Professor Vincenzo Cerundolo The University of Oxford Vincenzo Cerundolo is Professor of Immunology and Director of the MRC Human Immunology Unit of the University of Oxford. He graduated in Medicine at the University of Padua, Italy where he completed his PhD in Immunology. In 1990 he described the first human antigen processing deficient cells, which led to the cloning and characterisation of TAP1 and TAP2 genes. Vincenzo was appointed Professor of Immunology at the University of Oxford in 2000 and became Director of the MRC Human Immunology Unit in 2010. In 2012 he was appointed Head of the Investigative Medicine Division of the Radcliffe Department of Medicine of the University of Oxford. He is a Fellow of Merton College, of the Royal College of Pathologists,

Batsheva Fellow of the Israeli Academy of Medical Sciences, and a Fellow of the Academy of Medical Sciences, UK. Research carried out in Professor Cerundolo’s laboratory is mainly focussed on gaining a better understanding of the mechanisms that control the cell-cell interplay required for optimal expansion and activation of tumour-specific T cell populations and to apply this knowledge to the development of better treatment strategies in cancer patients.

Assistant Professor Annemieke de Jong Columbia University Annemieke de Jong is an Assistant Professor at Columbia University, New York, in the department of Dermatology. Her research focuses on T cell functions in human skin, in particular the role of lipid antigen presentation, and mechanisms by which the activation of CD1-restricted T cells is regulated, both in response to infection and in inflammatory skin disease. She obtained her PhD in Immunology from Leiden University, The Netherlands, and developed an interest in the functions of CD1-restricted T cells during her postdoctoral fellowship in Branch Moody's lab, Harvard Medical School. Here, she worked on the

characterization of CD1-restricted T cells subsets in humans, including CD1a-autoreactive T cells, and the identification of skin lipid antigens recognized by these T cells.


Professor Dirk Elewaut Ghent University Hospital Dirk Elewaut is a full professor of rheumatology and immunology and Chair of the Department of Rheumatology at Ghent University Hospital, a EULAR and FOCIS center of excellence. He obtained his MD at Ghent University in 1991 and his PhD in 1997 at the same institution. Following postdoctoral research at the University of California San Diego and the La Jolla Institute for Allergy and Immunology he joined the faculty of the Department of Rheumatology at Ghent University Hospital in 2001. He has published more than 230 scientific publications, and is heading a team of 20 researchers of the Laboratory of Molecular Immunology and Inflammation at the same department. He joined the Inflammation Research

Center (IRC) of the Flanders Research Institute for Biotechnology (VIB) in 2015 as principal investigator. His research interests are centered around translational aspects of immune regulation to combat inflammatory arthritis and associated joint damage, with special focus on iNKT cells and related innate like T cells.

Professor Martin Flajnik University of Maryland Martin Flajnik graduated from Penn State in 1978, and earned an MS (1979) and a PhD (1983) from the University of Rochester (NY) in Microbiology and Immunology. He was a postdoc at the Basel Institute for Immunology under Louis Du Pasquier (1983-88), and rose to professor in his first independent academic position at the University of Miami (FL) (1988-97). He has been a professor at the University of Maryland Baltimore SOM since 1998. His work is centered on the evolution of the adaptive immune system, with the major goal being to probe the origins of adaptive immunity. He was involved in the early biochemical and molecular characterization of the MHC in lower vertebrates, and he has identified several

immunoglobulin and T cell receptor isotypes in ectothermic vertebrates, and he has contributed to the understanding of single-domain antibodies. His lab recently has become interested in the evolution of lymphoid tissues.

Professor Laurent Gapin University of Colorado Laurent Gapin is a full professor of Immunology at the University of Colorado Denver. He obtained is PhD in 1997 at the University Paris 7. Following post-doctoral research at the La Jolla Institute for Allergy and Immunology, he joined the faculty of the Department of Immunology and Microbiology at the University of Colorado Denver and National Jewish Health. The Gapin’s lab has been studying the development, function and specificity of Natural Killer T cells for the past 20 years.

Professor Kristin Hogquist University of Minnesota Dr. Hogquist obtained her doctoral degree in Immunology from Washington University (St. Louis, MO) and did post-doctoral work at the University of Washington (Seattle, WA), where she first became interested in T cell development. She started as an Assistant Professor at the University of Minnesota in 1995, and currently holds the David M. Brown Endowed Professorship in the Department of Laboratory Medicine and Pathology there, and is Associate Director of the Center for Immunology. Her research program is focused on T cell development

in the thymus, particularly positive and negative selection, tolerance, and the human immune response to chronic infection.


Professor Paul Klenerman University of Oxford Professor Paul Klenerman trained as a clinician in infectious diseases, and completed his PhD on HIV at Oxford, and then his postdoctoral on LCMV with Rolf Zinkernagel and Hans Hengartner in Zurich. He now holds a chair at the Translational Gastroenterology Unit at Oxford. The lab is focused on host defence and inflammation and they work on hepatitis C and other persistent infections. More recently they have become interested in the role of CD161+ T cells in general and MAIT cells in particular in the liver and gut, given the dominance of these

cell types. They are trying to understand if and how such cell types play a role in viral infections, as well as inflammatory diseases of the liver and gut.

Professor Paul Kubes University of Calgary Dr. Paul Kubes is a Professor at the University of Calgary Faculty of Medicine and Founding Director of the Snyder Institute for Chronic Diseases. He also holds a Canada Research Chair in Leukocyte Recruitment in inflammatory disease. Dr. Kubes has received numerous awards including the CIHR Investigator of the Year in 2011 for his basic science work on how the brain affects immunity. He has also received the Alberta Science and Technology Award and the Henry Friesen Award. Dr. Kubes has published basic science work in Cell, Science and the Nature journals and also has publications in both clinical journals including Lancet and more translational journals (JCI). His latest work has uncovered a key role for peritoneal cavity

macrophage in healing visceral organs. Dr. Kubes has extensive review experience with CIHR having been part of numerous committees including the Immunology panel, Cardiovascular A and B panel, the CIHR scholar panel and the Banting postdoctoral panel. He also served as a member of CIHR Governing Council and is chair of the college chairs. In addition, he has reviewed for NIH and he co-chairs the Gairdner Research Committee.

Associate Professor Elizabeth Leadbetter University of Texas Health Science Center Elizabeth Leadbetter received her Ph.D. in 2002 from Boston University School of Medicine. Her graduate work with Dr. Ann Marshak-Rothstein provided the initial description of activation of B cells via immune complex mediated co-ligation of the B cell receptor and Toll-like receptor 9. This was one of the first examples of cross-talk between an ancient, germline-encoded innate immune receptor and the flexible, gene-rearrangement-based adaptive immune system. She next applied this concept to a new innate player, invariant NKT cells, during her postdoctoral fellowship at Brigham & Women’s Hospital; Harvard Medical School, Boston, MA in the lab of Dr. Michael Brenner. In 2009, she joined the faculty at the Trudeau

Institute in Saranac Lake, NY as an Assistant Member. In 2015 she became an Associate Professor at the University of Texas Health Science Center at San Antonio where she is currently investigating the cooperation and cross-regulation of iNKT and B cells using human clinical samples and murine models of vaccination, infection, autoimmunity, and metabolic disease.

Dr Agnes Lehuen University Paris Descartes Agnes Lehuen is director of the laboratory “Immunology of diabetes” at Institut Cochin, INSERM-CNRS, University Paris Descartes, France and Co-coordinator of the Laboratory of Excellence INFLAMEX. Dr Lehuen has made important contributions in the field of autoimmunity and more particularly in type 1 diabetes. Her research group discovered that iNKT cells, prevents the development of several autoimmune diseases such as type 1 diabetes and multiple sclerosis using mouse models. Her group also showed that in the context of infection-induced type 1 diabetes, iNKT cells cooperate with other innate immune cells to

develop efficient immune response against viruses while inhibiting potential damage of the pancreas. Her studies


unveiled the role of neutrophils and plasmacytoid DC in the initiation of the autoimmune response against pancreatic beta cells. More recently, her laboratory showed that MAIT cells are altered in type 2 diabetic and obese patients.

Dr David Lewinson Oregon Health and Science University David M. Lewinsohn is a Professor of Pulmonary and Critical Care Medicine at Oregon Health & Sciences University and holds adjunct appointments in Molecular Microbiology & Immunology as well as the Vaccine and Gene Therapy Institute. Dr. Lewinsohn received his undergraduate degree from Haverford College, his MD and PhD from Stanford University School of Medicine, internal medicine training at the University of California, San Francisco, and fellowship training in Pulmonary and Critical Care Medicine at the University of Washington. Dr. Lewinsohn’s research interest is in Tuberculosis Immunology. The core research question has been that of how the immune system can find those cells harboring the intracellular bacterium. As a result, he has been particularly interested in the role of

CD8+ T cells. The human CD8+ T cell response to Mtb is comprised of both classically and non-classically restricted T cells, with those restricted by MR1 found at high frequencies in both the peripheral blood and lung. Oligoclonal expansions of these cells in the lung in the setting of infection with Mtb would suggest a relationship between the MR1T cell response and selective microbial ligands. In this presentation, the characterization and synthesis of some of these ligands will be discussed.

Professor Joel Linden LaJolla Institute for Allergy and Immunology After receiving a PhD in Pharmacology from the University of Virginia my laboratory developed an interested in adenosine receptors. We have focused on investigating adenosine’s cardiovascular and immunomodulatory effects. The activation of adenosine A2A receptors (A2AR) produces anti-inflammatory responses in most immune cells including: neutrophils, macrophages, DCs, mast cells, NK cells, T effector cells and iNKT cells. T regulatory cells act in part by producing adenosine. The activation of A2ARs at the time of

reperfusion following ischemia to heart, liver, kidney, spinal cord or skin was found to reduce tissue inflammation and reperfusion injury. Based on targeted deletion of A2ARs from immune cells, the primary cell type by which adenosine protects tissues was identified as the iNKT cell. Deletion of iNKT cells, or blockade of CD1d-restricted activation was found to produce generalized tissue protection from ischemia reperfusion-injury. Currently we are investigating how activation-induced induction of multiple purinergic signaling molecules limits the extent and duration of iNKT cell activation.

Assistanct Professor Lydia Lynch Brigham and Women’s Hospital, USA The Lynch Lab studies immunometabolism- how the immune and metabolic systems interact. Our research focuses on the role of innate immune cells in regulating systemic metabolism, and coming full circle to the effects of altered systemic metabolism on immune cell function. Thus, we study immunometabolism at both the organismal and cell-intrinsic level. In particular, we study the role of innate immune cells including iNKT cells and gd T cells in adipose tissue in metabolic regulation. We have found that human and murine adipose tissue is enriched for iNKT cells which are a unique subset of regulatory iNKT cells that play an unexpected role in regulating metabolism and weight (Eur J.

Immunol, 2009, Immunity, 2012 Nature Immunology, 2015, Cell Metabolism, 2016). The goals of the lab are to understand why iNKT cells and CD1d molecules are conserved in adipose tissue, and to target adipose iNKT cells, or other innate immune populations, for protection against metabolic disorder.


Associate Professor Thierry Mallevaey University of Toronto The Mallevaey lab studies how innate lymphocytes develop and acquire their effector properties, and how they interact with commensal and pathogenic microorganisms to regulate mucosal immune responses. Dr. Mallevaey obtained his PhD in 2006 at the University of Lille II and Pasteur Institute of Lille, and trained in the Gapin lab at the University of Colorado Denver and National Jewish Health. Dr. Mallevaey is an Associate Professor at the University of Toronto and holds a Canada Research Chair in NKT cell Immunobiology. Dr Leonid Metelitsa Texas Children’s Cancer Center Dr. Leonid Metelitsa is a Professor of Pediatrics and Immunology at Baylor College of Medicine. His research is focused on understanding the role of Vα24-invariant Natural Killer T (NKT) cells in tumor immunity and developing NKT cell-based cancer immunotherapy. He originally demonstrated that NKT cells localize to primary tumors in humans and their presence at the tumor site is associated with good outcomes (Metelitsa et al., JEM, 2004). Metelitsa and his team then revealed the underlying mechanistic basis of NKT cell tumor localization and function in the tumor microenvironment (Song et al., JCI, 2007; Song et al., JCI, 2009, Liu et al., JCI, 2012). They also developed original technologies for NKT cell isolation,

genetic modification with chimeric antigen receptors (CAR), and expansion to clinical scale (Heczey et al., Blood, 2014; Tian et al., JCI, 2016). These technologies have been licensed to industry for clinical testing and commercialization.

Professor Branch Moody Harvard Medical School The central focus of the Moody laboratory is to understand how dendritic cells, CD1 proteins and T cell receptors mediate immune response to M. tuberculosis infection and in autoimmune disease. Focusing on the human CD1 system for more than two decades, we identified glucose monomycolate and have used this antigen to dissect the cellular and molecular mechanisms lipid antigen presentation as well as patterns of host response. We have discovered many lipid antigens for T cells, including dideoxymycobactins, phosphomycoketides and skin oils. Moving from in vitro molecular studies to translational

immunology, we worked with John Altman to develop human CD1a, CD1b and CD1c tetramers. Most recently we have created a mass spectrometry platform for nearly simultaneous detection of thousands of lipids present in human cells or mycobacteria and used this system to identify ligands for CD1 proteins and antigens for T cells.

Professor Shinichiro Motohashi Chiba University Hospital Center for Advanced Medicine Shinichiro Motohashi is Professor of Medical Immunology at Chiba University, Japan. He received his M.D. and Ph.D. from Chiba University, where he continued as a postdoctoral fellow in the laboratory of Dr. Masaru Taniguchi and joined the faculty member in 2005. Currently, Motohashi’s lab focuses on the development of NKT cell-mediated immunotherapy for non-small cell lung cancer and head and neck cancer, the novel therapeutics supported by Chiba University for its research and development. They conduct translational research to

advance their basic studies of a-galactosylceramide-pulsed dendritic cells to clinical studies, and are now working within the framework of the clinical research, regulated by the Japanese Ministry of Health, Labour and Welfare. They are engaged in a joint project with RIKEN to develop adoptive immunotherapy with induced pluripotent stem (iPS) derived NKT cells. They also develop the biomarkers which can predict the response of immunotherapy.


Professor Derek Sant’Angelo Rutgers Robert WoodJohnson Medical School Derek Sant’Angelo received his B.S. from the University of Michigan, and his Ph.D. from Rutgers University. Derek was an HHMI Postdoctoral Fellow at the Yale under the mentorship of Dr. Charles A. Janeway, Jr. As a faculty member at Memorial Sloan-Kettering, Derek initiated studies that led to the discovery that the BTB-ZF transcription factor, PLZF, is essential for NKT cell effector functions. In 2011, Derek moved to Rutgers Robert Wood Johnson Medical School. In 2015 he was appointed Associate Director for Basic Science of the Child Health Institute. Derek has continued to work on identifying key transcription factors

that define and maintain the functional identity of lymphocytes. Current interests include TCR mediated control of adipose resident iNKT cells and how alteration of PLZF expression impacts the onset of obesity and related metabolic disorders.

Dr Chun-Ru Wang Northwestern University Dr. Wang and the researchers in her lab are interested in understanding how group 1 and group 2 CD1-restricted T cells are selected and regulated during development and how they contribute to the control of autoimmunity, anti-tumor immunity and infectious diseases. Her group also studies the immune function of other MHC Ib molecules, such as H2-M3, Qa-1, Qa-2 and MR1. Dr Florian Winau Boston Children’s Hospital Dr. Winau is a Principal Investigator in the Program in Cellular and Molecular Medicine (PCMM) at Boston Children’s Hospital, and a faculty member in the Department of Microbiology and Immunobiology at Harvard Medical School. Dr. Winau received his M.D. from the Charité Humboldt University in Berlin, Germany. He did his post-doctoral work at the Max-Planck-Institute for Infection Biology in Berlin, before establishing his laboratory at Harvard Medical School. The Winau Lab studies diverse aspects of antigen presentation and T cell biology. He is especially interested in the role of lipids in Immunology, acting as antigens

or regulators of immune responses. Dr. Winau’s recent research endeavors in the CD1 field include the study of the in vivo role of CD1a on Langerhans cells in inflammatory skin disease, the epigenetic regulation of NKT cell development in the thymus, and the elucidation of the enzymatic pathways that generate endogenous α-glycosylceramides for NKT cell selection and activation.

Dr Gerhard Wingender Izmir Biomedicine and Genome Centre Since 2015 Dr. Gerhard Wingender is an Assistant Professor at the Izmir Biomedicine and Genome Center (IBG, Izmir, Turkey), and the manager of IBG’s Flow Cytometry and Cell Sorting Facility. He received his Ph.D. from the University of Heidelberg (Germany) before joining the laboratory of Dr. Mitchell Kronenberg as a postdoctoral fellow at the La Jolla Institute for Allergy and Immunology (LJI, La Jolla, USA). At LJI he worked on the impact of environmental antigens in the airways and intestine on iNKT cell development and function. Furthermore, together with his colleagues he described regulatory NKT10 cells. His current research interest is the role of NKT10 cells in autoimmune diseases, in particular in multiple

sclerosis. Additionally, he and his group investigate the function of iNKT and MAIT cells in airway hypersensitivity and multiple myeloma.


INVITED SPEAKERS Dr Jay Berzofsky National Cancer Institute, National Institutes of Health Dr. Jay Berzofsky was appointed Chief of the new Vaccine Branch, Center for Cancer Research, National Cancer Institute, in 2003, after being Chief of the Molecular Immunogenetics and Vaccine Research Section, Metabolism Branch, National Cancer Institute, NIH, since 1987. He graduated Summa cum Laude from Harvard (1967), and received a Ph.D. and M.D. from Albert Einstein College of Medicine. After interning at Massachusetts General Hospital, he joined NIH in 1974. Dr. Berzofsky's research has focused on antigen processing and presentation by MHC molecules, the

structure of antigenic determinants, cytokine and regulatory cell control of T cell function and avidity, and translation to the design of vaccines for AIDS, cancer, and viruses causing cancer. He has 497 scientific publications and a number of awards. He was elected as President of the American Society for Clinical Investigation (1993-94), a member of the Association of American Physicians, and a Fellow of the American Association for the Advancement of Science, and was elected Distinguished Alumnus of the Year for 2007 by the Albert Einstein College of Medicine. He was also elected Chair of the Medical Sciences Section of the American Association for the Advancement of Science ( S) for 2007-2008. In 2008, he received the NIH Director’s Award and the NCI Merit Award, and in 2011 he received another NCI Director’s Merit Award.

Professor Susana Cardell University of Gothenborg Susanna Cardell obtained her PhD on studies of Th cell subsets at Stockholm University. She was a postdoctoral fellow with the Mathis/Benoist laboratory at the IGBMC in Strasbourg, France, where she discovered the CD1d-restricted T cells with diverse TCR, now called type 2 NKT cells. She set up her independent laboratory at Lund University in 1995, and in 2005 she moved to the University of Gothenburg

where she is professor of immune regulation and head of the Department of Microbiology and Immunology. Her laboratory has studied the development and function of type 2 NKT cells, and identified glycolipid ligands for these cells, including isoforms of sulfatide. More recently, they have demonstrated that TCR Vdelta1 T cells, that were previously shown to include sulfatide-CD1d reactive T cells, show increased activation and association with disease severity in newly diagnosed multiple sclerosis. They have also demonstrated that type 1 NKT cells promote intestinal tumors, by supporting an immunosuppressive microenvironment.

Dr Siobhan Cowley Food and Drug Administration Siobhan Cowley completed her Ph.D. at the University of Victoria in the Department of Biochemistry and Microbiology in Victoria, British Columbia, Canada. She did her post-doctoral work at the University of British Columbia. She is currently a Principal Investigator in the Laboratory of Mucosal Pathogens and Cellular Immunology at the FDA. Her laboratory is primarily interested in understanding mucosal immune responses to pathogens, with an eye towards aiding the development of mucosal vaccines and adjuvants. In particular, a major recent focus of her lab has been the use of Francisella tularensis as a model intracellular pathogen to define the in vivo

role of MAIT cells in pulmonary immune responses.


Assistant Professor Louise D’Cruz University of Pittsburg Louise D'Cruz is an Assistant Professor of Immunology at the University of Pittsburgh School of Medicine. Dr. D'Cruz studied Genetics at Trinity College Dublin, where she graduated in 2003. In 2006, she completed her Ph.D. at the University of Vienna, under the mentorship of Dr. Ludger Klein, where she studied the factors that influence regulatory T cell development, essential cells that provide protection against autoimmunity. In 2007, Dr. D'Cruz moved to UC San Diego, where she completed her post doc with Dr. Ananda Goldrath, investigating the transcription

factors and proteins responsible for the development and function of CD8+ effector and memory T cells as well as invariant Natural Killer T (iNKT) cells. In 2014, she moved to the University of Pittsburgh to begin her independent research program. Dr. D'Cruz's lab focuses on the transcriptional regulation of iNKT cell development, differentiation and activation.

Stephan Gadola Roche Stephan’s interest in academic research was sparked after he had characterized a human primary immune deficiency in the HLA class I pathway. In Enzo Cerundolo’s lab he built up CD1 research in Oxford. Early contributions to the CD1 field include refolded CD1d-tetramers, the first CD1/ligand (CD1b) and first human CD1d structure, and studies on CD1d-specific T cells, including the identification of human Va24-negative CD1d/a-GalCer specific T cells. After returning to Switzerland he focused on TCR-CD1d interactions, resulting in the first iNKT TCR structures and the

demonstration that the human iNKT repertoire contains clones of highly different iNKT TCR affinities based on differences within CDR3β. After returning to the UK/Southampton, his group analyzed the clonal iNKT repertoire in early rheumatoid arthrtitis and type 1 diabetes. Recently, the group published a novel ligand-saturated human CD1c structure, revealing major conformational changes of CD1c upon ligand binding that determine TCR recognition, and identifying cholesteryl esters as a new class of ligands for CD1c. In 2016, Stephan has joined the Immunology & Inflammation group at Roche/Basel as a translational project leader, while still pursuing his academic research and regularly seeing patients in his Rheumatology practice.

Dr Kazuya Iwabuchi Kitasato University Dr Iwabuchi completed his PhD at Hokkaido University, Sapporo Japan in 1986. He was a research associate at Dr Loh's Lab between 1989 and 1991. Dr Iwabuchi attended the 3rd NKT and CD1 International Workshop held at Herron Island, Australia in 2004, and has since been facinated by studies of NKT and CD1 molecules. His interest now focuses on the mechanisms how NKT cells modulate the development of obesity and why NKT cells affect the development in opposite

direction in some cases with his colleague Masashi Satoh..


Professor Mikael Karlsson Karolinska Institutet Mikael Karlsson graduated from Uppsala University 1997 and received his Ph.D. in 2000 from the same University where he studied under Prof. Birgitta Heyman. In 2005 he completed his postdoctoral research at the Rockefeller University in New York in the lab of prof. Jeffrey Ravetch. After this he joined the faculty at Dept. of Medicine at Karolinska Institutet, Stockholm receiving an Assistant prof. position appointed by the Swedish Research Council. In 2009 he became Associate professor and in 2010 he received a senior research position appointed by the Swedish Research Council. Since 2016 he is a Professor of Immunology at the department

of Microbiology, Tumor and Cell biology. Dr Karlsson is an elected member of the Henry Kunkel Society of Immunology as well as a co-chair of the Swedish Society for Immunology and head of the KI inflammation and immunology network (KiiM).

Professor Jim Kaufman University of Cambridge As a PhD student at Harvard in the late 1970s, Jim Kaufman identified and characterized class II molecules encoded in the human major histocompatibility complex (MHC). A model based on a chance observation led to his life-long interest in the evolution of immunity. At the Basel Institute for Immunology, he looked for MHC molecules and genes in many organisms. Eventually, he focused on the chicken, the only non-mammalian vertebrate for which there was extensive

information on pathogens, vaccines, immune responses, genetics and development. The work continued at the Institute for Animal Health in Compton UK, where he showed the importance of genomic organisation and gene co-evolution in the evolution of the MHC. For the last ten years, he has been the Professor of Comparative Immunogenetics at the University of Cambridge, developing scenarios for the origin and evolution of the adaptive immune system of jawed vertebrates, refining our understanding of how chickens respond to infectious pathogens, and using the simpler chicken MHC to discover phenomena difficult to discern in mammals.

Dr Olivier Lantz Institut Curle France Olivier Lantz heads the clinical immunology laboratory at the Institut Curie Hospital in Paris. He also heads a basic immunology research group in the research center. He received his MD and Ph.D. degrees from Orsay University in 1986 and 1990, respectively. Before joining Institut Curie in 2000, he was associate professor at the Medical University of Paris South. He is author or coauthor of over 130 peer-reviewed articles. He is strongly involved in translational immuno-oncology

(immunomonitoring, biomarkers, preclinical assessment of anti tumor immunotherapies). His research interests are focused on basic immunology (characterization of NKT and MAIT cells, biology of CD4 T cells, interactions between the immune system and tumors). He discovered MAIT cells and performed their characterization in humans and mice. He demonstrated their anti-microbial reactivity restricted by MR1. He currently focus on the development of MAIT cells and the analysis of their function in mouse models.


Professor Claudia Mauri University College London Claudia Mauri is Professor of Immunology and Vice-Dean International Faculty medical Science at University College in London. She received her Doctor of Biology with magna cum laude in 1989 and PhD equivalent in 1996 from the University La Sapienza in Rome, Italy. She performed postdoctoral work in London at The Kennedy Institute of Rheumatology, Imperial College, UK. She moved to University College London in 2002 where she established her group. Her main research interest lies in

understanding the mechanisms driving autoimmunity with a particular interest in understanding the function of regulatory B cells in experimental models of rheumatic disease and in patients with Systemic Lupus Erythematosus and Rheumatoid Arthritis.

Professor Gavin Painter The Ferrier Research Institute, Victoria University of Wellington, New Zealand Gavin received his PhD in chemistry from the University of Otago in 1995. After a postdoc at Cambridge University (UK) he joined The Ferrier Research Institute of Victoria University of Wellington in New Zealand where he holds a professorial position and leads the Chemical Immunology Team. His research includes the synthesis and activity of phosphatidylinositol phosphates (PIPs), phosphatidylinositol mannosides (PIMs) and glycolipids. His current research activities include developing synthetic vaccines for cancer and infectious disease that utilize the activity of semi-

activated T cells known as innate-like T cells. This approach is complementary to most other vaccine approaches, which rely on triggering pattern recognition receptors located on antigen-presenting cells. He is also a science co-founder of Biotech Avalia Immunotherapies where he holds the positon of chief technology officer (CTO). Avalia Immunotherapies was founded in March 2015 to commercialize IP assets generated from his collaborative research.

Professor Jamie Rossjohn Monash University He is currently an ARC Australian Laureate Fellow (2017-2021) and previously a NHMRC Australia Fellow (2011-2016) and ARC Federation Fellow (2007-11). He is the Head of the Infection and Immunity Program of the Biomedicine Discovery Institute at Monash University and Professor of Structural Immunology at the School of Medicine, Cardiff University. Rossjohn is known for his contributions to the understanding the molecular basis underpinning immunity. He has used structural

biology to explain pre-T-cell receptor (TCR) self-association in T-cell development, and how the TCR specifically recognises polymorphic Human Leukocyte Antigen (HLA) molecules in the context of viral immunity and aberrant T-cell reactivity. He has unearthed structural mechanisms of HLA polymorphism impacting on drug and food hypersensitivities, as well as Natural Killer cell receptor recognition. He has pioneered our molecular understanding of lipid-based immunity by T cells, revealing that it can differ fundamentally from peptide-mediated adaptive immunity. Recently he has provided a structural basis of how vitamin B metabolites can be presented and recognised by the immune system, revealing a new class of antigen.


Dr Mariolina Salio University of Oxford Mariolina Salio obtained her degree in Medicine from the University of Torino, Italy and worked at the Basel Institute for Immunology before joining the MRC Human Immunology Unit in Oxford, where she is a junior PI. She has a long-standing interest in the interface between innate and adaptive immunity, and her research has focussed on the mechanisms of DC activation by NKT cells and more recently by MAIT cells. Ultimately, her interest lies in designing strategies to harness innate-like T cells

to improve adaptive immunity in cancer.

Professor Masaru Taniguchi Riken Centre for Integrative Medical Sciences, Japan Masaru Taniguchi received his M.D. and Ph.D. degrees at Chiba University School of Medicine (1974). He was appointed a Professor of Immunology at Chiba University School of Medicine (1980-2004), served as the Dean there (1996-2000) and President of the Japanese Society for Immunology (1997-1998). He was then appointed the founding Director of the RIKEN RCAI (2001-2013) and the Senior Advisor at RIKEN IMS in 2013. He identified an NKT cell and its unique invariant

antigen receptor expression in 1986-1990, which was selected by the American Association of Immunologists in 2014 as one of the “Pillars of Immunology”, and also discovered an NKT cell ligand, a-galactosylceramide in 1997 and developed NKT cell-deficient mice in 1997. He gave a Special Lecture for the Emperor on Allergy and Immunology (2000). He was awarded the Medal with Purple Ribbon in 2004 and the Order of the Sacred Treasure in 2016 by the Emperor of Japan.

Dr Ajitha Thanabalasuriar University of Calgary Ajitha Thanabalasuriar completed her Ph.D. at McGill University in the Department of Microbiology and Immunology in 2013. During her Ph.D. she studied host-pathogen interactions between a bacterial virulence protein, NleA found in pathogenic E. coli, and mammalian secretary complex, the COPII complex. After completing her Ph.D. Ajitha pursued a postdoctoral fellowship with Dr. Paul Kubes at the University of Calgary. Here she is studying the mechanism of activation of

pulmonary iNKT cells. Ajitha has developed in vivo imaging approaches to understand pulmonary iNKT cell behavior, function, and subsets. Dr. Thanabalasuriar is currently a Banting postdoctoral fellow, the highest ranked fellowship for postdocs in Canada, and she has published her work in the journals Cell Reports and Journal of Clinical Investigations in the past year.

Assistant Professor Ildiko Van Rhijn Brigham and Women’s Hospital, Harvard Medical School Ildiko Van Rhijn obtained her PhD in immunology at the University of Utrecht in the Netherlands and worked at its School of Veterinary Medicine before joining the Division of Rheumatology, Immunology, and Allergy of the Brigham and Women’s Hospital and Harvard Medical School, where she is an Assistant Professor. She has a long standing interest in human CD1-resticted T cells, the antigens they recognize, and their T cell receptor repertoire. Using human biospecimens, she aims to understand the mechanisms and function of self recognition within the CD1 system and the contribution of CD1-restricted T cells to immunity against bacterial

pathogens.


Associate Professor Tonya Webb University of Maryland Dr. Webb earned a PhD degree from Indiana University in the laboratory of Dr. Randy Brutkiewicz. She has conducted research as a Leadership Alliance/ Howard Hughes fellow at Dartmouth College. She worked on mechanisms regulating immune tolerance, as a postdoc in Dr. David Wilkes’ lab. Her second postdoctoral fellowship focused on cancer immunology at Johns Hopkins. She joined the University of Maryland School of Medicine faculty in 2009. Her current research focuses on the role of NKT cells in cancer. She is working to develop NKT cell-based

cancer immunotherapy using natural and artificial platforms. However, many cancer patients have a reduction in both NKT cell number and function, and these deficits currently limit their potential clinical application. She is trying to understand why cancer patients have fewer NKT cells, and design novel therapeutic strategies to restore NKT cell number and function in these patients.

Assistant Professor Lili Yang University of California Los Angeles Lili received her B.S. degree in Biology from the University of Science & Technology of China (USTC) in 1997, her M.S. degree in Biomedical Sciences from the University of California, Riverside (UCR) in 1999, and her Ph.D. degree in Biology from the California Institute of Technology (Caltech) in 2004. She obtained her Ph.D. training at the Laboratory of David Baltimore. Post-graduation, she stayed at Caltech and led a multi-institutional Engineering Immunity Program from 2004 to 2012, developing gene- and cell-based immunotherapies for cancer and HIV/AIDS. Her work resulted in over 24 peer-reviewed publications, 8 patents and

2 clinical trials. She joined the University of California, Los Angeles (UCLA) as an Assistant Professor in January of 2013. Her laboratory at UCLA studies tumor immunology and cancer immunotherapy, with a special focus on stem cell-engineered immunotherapy for cancer. Based on her research achievements, Lili has received multiple awards including an ASGCT Outstanding New Investigator Award, an AAI Young Investigator Award, a NIH Director’s New Innovator Award, and a CIRM Partnering Opportunity for Translational Research Award, a STOP CANCER Research Career Development Award, a Forbeck Scholar Award, and a TR35 Award.


DELEGATE INFORMATION

VENUE DETAILS Silverado Resort and Spa, California 1600 Atlas Peak Road Napa Valley, CA 94558 www.silveradoresort.com

• All scientific sessions will be in the Silverado Ballroom • All catered breaks will be served among the exhibition display in the Silverado Ballroom and on the

Fairway Deck. • Poster Sessions will run on the Fairway Deck • Breakfast may be included with your accommodation booking at Silverado. Refer to your booking

reference for further details.

ORGANISER’S OFFICE AND REGISTRATION DESK

The Organisers registration desk is located within the Silverado Ballroom Foyer, right in front of the Fairway Deck doors. Any enquiries about registration or the conference can be directed to ASN staff there. The registration desk hours are:

Friday: 3:00PM – 7:00PM Saturday:7:30AM – 6:15PM Sunday: 7:30AM – 6:15PM Monday: 7:30AM – 7:15PM Tuesday: 7:30AM – 12:00PM

REGISTRATION

Conference delegates receive the following services as part of their registration: • Access to the sessions of your choice

• Conference satchel complete with conference book

• Conference App

• Morning and/or afternoon tea for the days of nominated attendance

• Lunches on the days of nominated attendance

• Welcome Reception (Friday)

• Gala Dinner (Monday)

NAME TAGS Delegates are required to wear their name tags to all scientific and catered sessions. SPEAKERS PRESENTATIONS Oral presentations can be brought on USB and loaded directly onto the presentation computers within the session rooms. Speakers are asked to bring their presentation to the registration desk half an hour before their session commences as ASN staff will load the talks to the computer for them. Alternatively, speakers can bring their own laptops which can be connected to the projectors within the presentation rooms.


POSTER PRESENTATIONS There are two poster sessions running at CD1-MR1 in 2017. If you have an odd Poster number you can display your presentation from 3.00pm on Friday until the end of Poster Session 1 on Sunday (when you should remove your poster). Those with an Even number can display their poster from first thing Monday Morning and they can remain up until the conference concludes on Tuesday. The approved way of attaching your poster is with Velcro (or pins), which will be provided on your poster board.

MOBILE PHONES Please ensure your mobile phone is switched to silent mode during sessions.

EMERGENCY CONTACTS Hospital Napa State Hospital 2100 Napa Vallejo Hwy, Napa, CA 94558, USA + 1 707-253-5000 GP Queen of the Valley Medical Center 1000 Trancas St, Napa, CA 94558, USA +1 707-252-4411 Police Napa City Police Department Police Department, Napa, CA, USA + 1 707-257-9223

INSURANCE The registration fee does not include insurance of any kind and the Meeting Secretariat cannot take any responsibility for any participant failing to arrange their own insurance. Delegates are encouraged to make their own insurance arrangements to cover any loss caused by unforeseen delay, circumstance or cancellation. RETURN BUS TRANSFERS

Bus will depart at 3:00PM from San Francisco International Airport to Silverado Resort (via the Golden Gate Bridge) on Friday the 3rd of November 2017 arriving at 4:30PM or 5:00PM and return to San Francisco International Airport at 12:30PM from Silverado Resort. If you have not signed up to the bus transfer, please see the registration desk for availability.

SOCIAL FUNCTIONS AND ACTIVITIES

Welcome Reception The Welcome Function is a fantastic opportunity for delegates to catch up with old friends from past conferences. This is also a great networking opportunity for students and the trade. Tickets are included in

your registration, additional/partner tickets can be added for AUD $90 each.

Date: Friday 3 November

Time: 5:00pm - 7:00pm

Venue: Fairway Deck, Silverado Resort

Gala Dinner The Gala Dinner is without a doubt the social highlight of the conference. Enjoy a 2-course meal of local Napa Valley wine and produce, while catching up with friends old and new. This is an event not to miss! Tickets are $80 for registrants and $120 for partners/additional persons.

Date: Monday 6 November

Time: 7:30pm - 9:30pm

Venue: Garden Pavilion, Silverado Resort


Saturday Afternoon Activities

Activity 1 - Oxbow Public Market & Madonna Estate Winery

Take the bus to Oxbow Public Market and visit one of the 22 stalls situated in the 40,000 square foot marketplace.

Next you will be treated to a tasting of 5 different wines at Madonna Estate Winery in Napa Valley. You will then

return to Silverado Resort at 4:30pm for the next session.

Date: Saturday 4 November

Time: 12:00pm - 4:30pm

Cost: $25, includes lunchbox

To purchase a ticket please see the registration desk.

Activity 2 - Bothe-Napa Valley State Park & Old Faithful Geyser of California

Get acquainted with the natural geography of the Napa Valley! This tour will include an opportunity to eat a picnic

lunch and take a walk in the woods at the Petrified Forest and/or the Bothe-Napa Valley State park, as well as paying

a visit to the Old Faithful Geyser, a product of the region's underground geothermal activity. The bus will then return

you to Silverado Resort for the next session commencing at 4:30pm.

Date: Saturday 4 November

Time: 12:00pm - 4:30pm

Cost: $25, includes lunchbox

To purchase a ticket please see the registration desk.

WIRELESS INTERNET There is complimentary wireless internet within Silverado Ballroom. Simply select the Silverado network and accepts the terms and conditions on the website to connect. CONFERENCE APP The App is displayed in a simple and easy to read format on your phone, iPad, or even your computer. To get the ‘App’, please open the below link in your internet browser on your phone, iPad or laptop.

http://cd1-mr1-2017.m.asnevents.com.au

You will be prompted to add an icon onto your device home screen. The Smartphone/Mobile Device ‘App’ will allow you to: • View the full conference program • View all abstracts for the conference • Save your favourite sessions and plan your day • Take notes which will then be saved and downloaded from your registration profile.

To use most of these functions, you will be prompted to ‘log in” each day. Simply enter the same email & password which you used to register. HOTEL CHECK IN - OUTS Delegates can check into their rooms any time after 4.00pm. Check out is from 11.00am. DISCLAIMER The hosts, organisers and participating societies are not responsible for, or represented by, the opinions expressed by participants in either the sessions or their written abstracts. SMOKING Smoking is not permitted in the venue.


VENUE MAP


CONFERENCE PROGRAM

FRIDAY 3RD NOVEMBER 2017

Registration Open

3:00PM - 7:00PM Fairway Deck

Welcome Reception


Opening Keynote Lecture

7:00PM - 8:00PM Silverado Ballroom

Chair: Mitch Kronenberg

7:00 PM Mitch Kronenberg Opening remarks

7:10 PM Dale Godfrey From αβTCR+ DN T cells to NKT cells to CD1a, b and c restricted T cells and MR1-restricted MAIT cells: An unexpected journey. abs# 1

Translational Directions


Chairs: Jenny Gumperz

8:00 PM Paolo Dellabona Clinical harnessing of lipid-specific T cell responses abs# 2

8:20 PM Mark Exley Translational Directions: CD1d-restricted NKT cell populations abs# 3

8:40 PM Jim McCluskey Specific identification, tracking and harnessing of Mucosal Associated Invariant T (MAIT) cells in health and disease abs# 4


SATURDAY 4TH NOVEMBER 2017

Registration Open

7:30AM - 6:15PM Fairway Deck

Session 1: Evolutionary perspectives on innate T cells, CD1-MR1 molecules

8:00AM - 9:20AM Silverado Ballroom

Chairs: Albert Bendelac & Martin Flajnik

8:00 AM Albert Bendelac The transcription factor PLZF directs the acquisition of effector programs during innate and innate-like lymphocyte development abs# 5

8:20 AM Jacques Robert E. Edholm MHC class I-like XNC4 tetramers identify multiple invariant TCR rearrangements during mycobacteria infection in the amphibian Xenopus abs# 6

8:40 AM Jim Kaufman Evolution of MHC class I, CD1 and class II genes: how did it happen? abs# 7

9:00 AM Martin Flajnik Y. Ohta, M. Kasahara Evolutionary origins of CD1 and MHC class I abs# 8

Morning Tea

9:20AM - 9:50AM Fairway Deck

Session 2: Antigen discovery, recognition, structure


Chairs: Erin Adams & Branch Moody

9:50 AM Branch Moody Size Motifs and Misfit Lipids in Human CD1b Proteins abs# 9

10:10 AM Jamie Rossjohn Living Under the A’ Roof abs# 10

10:30 AM Stephan Gadola The hidden virtues of CD1b and CD1c antigen presentation abs#11


10:50 AM Adam E Shahine I. Van Rhijn, T. Cheng, S. Iwany, S. Gras, D. Moody, J. Rossjohn A molecular basis of human T cell receptor autoreactivity towards CD1b and self-phospholipids abs# 12

11:10 AM David Lewinsohn Exploring MR1 Mycobacterial Ligands and Antigens abs# 13

11:30 AM Erin Adams Identifying Antigens that are relevant for T cell Reactivity in Humans abs# 14

Lunch


Take Away Lunch Boxes Provided Lunch sponsored by

Afternoon Activities

12:00PM - 4:30PM

Rapid Fire Session 1


Chairs: Annemieke de Jong & Laurent Gapin

4:45 PM Annemieke de Jong G.C. Monnot, C. Tejeda, A. Chang, H.F. Jiang, W. Zeng, C. Rhode, S. Cremers Regulation of CD1a-dependent T cell activation in human skin abs# 201

5:05 PM Edy Kim H. Ner-gaon, J. Choi, J. Guo, C. Benoist, T. Shay, M.B. Brenner Natural Killer T cells are master regulators of post-sepsis immunosuppression abs# 203

5:15 PM Vipin Kumar I. Marrero, I. Maricic, A. Eguchi, S. Dasgupta, A. Feldstein, C. Hernandez, R. Loomba A dominant immune mechanism mediated by type I NKT cell subsets in different phases of non-alcoholic fatty liver disease abs# 205

5:25 PM Ageliki Tsagaratou S. Rautio , E. Gonzalez Avalos, J. Scott-Browne , S. Togher , L. Chavez, E.V. Rothenberg , H. Lähdesmäki, A. Rao TET proteins control iNKT cell lineage specification and TCR mediated expansion abs# 207


5:35 PM James Ussher T. Williams, R. Lamichhane, S. de la Harpe, J. Tyndall, A. Vernall Bacteria license antigen presenting cells for MR1-mediated MAIT cell activation abs# 209

5:45 PM Peter van den Elzen J. Tuengel, D. Zheng, W. Panenka, A. Stax Cerebrospinal fluid and serum lipoprotein distribution affects the antigenicity of sulfatide for human iNKT cells abs# 211

5:55 PM Laurent Gapin S. Sundararaj, J. Zhang, S. Krovi, R. Bedel, K. Tuttle, N. Veerapen, G.S. Besra, J. Le Nours, J. Matsuda, J. Rossjohn The differing roles of CD1d2 and CD1d1 proteins in type I Natural Killer T cell development and function abs# 213

Dinner Break (delegates own arrangements)

6:15PM - 7:30PM

Session 3: Anti-microbial defense


Chairs: David Lewinsohn & Chyung-Ru Wang

7:30 PM Gavin Painter Augmenting influenza-specific T cell memory generation with a NKT cell-dependent glycolipid-peptide vaccine abs# 22

7:50 PM Catherine M Crosby Z. Mikulski, M. Kronenberg NKT cell subsets involved in host defense from pulmonary Streptococcus pneumoniae infection abs# 23

8:10 PM Melanie Harriff C. McMurtrey, N. Hartmann, M. Sorensen, J. Mizgerd, W. Hildebrand, M. Kronenberg, D. Lewinsohn Riboflavin metabolism variation among clinical isolates of Streptococcus pneumoniae results in differential activation of MAIT cells abs# 24

8:30 PM Ildiko Van Rhijn CD1a, CD1b, and CD1c tetramers detect antigen-specific T cells in a tuberculosis cohort abs# 25

8:50 PM Chyung-Ru Wang S. Shang, Y. Bian, S. Siddiqui, J. Zhao, Y. He MHC class Ib molecules in host defense against Mycobacterium tuberculosis infection abs# 26


SUNDAY 5TH NOVEMBER 2017

Registration Open


Session 4: Antigen presentation and APC interactions


Chairs: Mitchell Kronenberg & Elizabeth Leadbetter

8:00 AM Melissa Bedard M. Salio, D. Priestman, D. Shrestha, C. Eggeling, F. Platt, R. Blumberg, V. Cerundolo A novel mechanism of iNKT cell activation mediated by ER-stressed antigen presenting cells abs# 27

8:20 AM Nadine Hartmann M. Kronenberg Immune evasion mechanism of Streptococcus pneumoniae - gain fat to lose sugar abs# 28

8:40 AM Mariolina Salio O. Gasser, C. Gonzalez-Lopez, N. Veerapen, U. Gileadi, G. Napolitani, R. Anderson, G. Painter, G.S. Besra, I.F. Hermans, V. Cerundolo Activation of human mucosal-associated invariant T cells induces CD40L-dependent maturation of monocyte-derived and primary dendritic cells abs# 29

9:00 AM Jose Villadangos H.E. McWilliam, M. Zorkau, S. Wormald, J.D. Mintern, J. McCluskey, J. Rossjohn Towards characterization of the MR1 antigen presentation machinery abs# 30

9:20 AM Elizabeth Leadbetter iNKT cells support cytokine-producing B cells in adipose tissue and are required to limit symptoms of metabolic disease abs# 31

Morning Tea



Session 5: Development, Selection, Subsets


Chairs: Kristin Hogquist & Derek Sant'Angelo

10:15 AM Kristin Hogquist H. Wang, C.N. Miller, E.R. Breed, Y. Lee, M.S. Anderson iNKT cells require TCR and cytokine signals from distinct APC to support IL-4 production and shape thymic development abs# 32

10:35 AM Louise DCruz H.M. Buechel, A.B. Frias, L. Beppu How E and Id proteins regulate iNKT cell development, subset differentiation and function abs# 33

10:55 AM Hui-Fern Koay S. Su, D. Zalcenstein, C. Seillet, M. Ritchie, S. Naik, A. Uldrich, J. McCluskey, G. Belz, D. Pellicci, D. Godfrey Development of functional MAIT cells. abs# 34

11:15 AM Masaru Taniguchi

A novel alternative pathway of V14+ NKT cell development directly from CD4-

CD8- double-negative thymocytes, bypassing the CD4+CD8+ double-positive stage abs# 35

11:35 AM Derek Sant'Angelo Are fat NKT cells born that way? abs# 36

Lunch


Lunch sponsored by

Poster Session 1 - Odd Numbers + Rapid Fire 1 Posters


Social Break

3:00PM - 3:45PM


Rapid Fire Session 2


Chairs: Patricia Barral & Florian Winau

3:45 PM Patricia Barral iNKT cells in intestinal immunity abs# 200

4:05 PM Alexandra J Corbett C. D'Souza, T. Pediongco, H. Wang, J.Y. Scheerlinck, L. Kostenko, R. Esterbauer, A. Stent, S.B. Eckle, B.S. Meehan, R.A. Strugnell, H. Cao, L. Liu, J.Y. Mak, G. Lovrecz, L. Lu, D.P. Fairlie, J. Rossjohn, J. McCluskey, A.L. Every, Z. Chen Mucosal-associated invariant T cells augment immunopathology in chronic Helicobacter pylori infection abs# 202

4:15 PM Sai Harsha Krovi K.D. Tuttle, J. Zhang, R. Bedel, L.K. Peterson, L.L. Dragone, K. Riemondy, J. Hesselberth, J. Scott-Browne, L. Gapin The TCR signal strength influences the specification of iNKT subsets abs# 204

4:25 PM Marion Salou A. Goubet, V. Premel, A. Darbois, R. Alonso, F. Legoux, O. Lantz MAIT and iNKT cell subsets share similar tissue residency programs abs# 206

4:35 PM Susannah C Shissler T.J. Webb Defective NKT cell development in BRCA-1 mutant mice abs# 208

4:45 PM Takashi Yamamura Oral administration of iNKT cell ligand OCH induces anti-inflammatory immune

responses in healthy human subjects and multiple sclerosis：Results of

investigator-initiated, first-in-human phase 1 study abs# 210

4:55 PM Florian Winau Epigenetic regulation of NKT cell development abs# 212

Special Seminar


Chair: Luc Teyton

5:15 PM Charles Serhan Resolvins & pro-resolving mediator signals in resolution of inflammation abs# 44

Dinner Break (delegates own arrangements)

6:15PM - 7:30PM


Session 6: Insights into the biology of innate T cells from imaging and transcriptomics


Chairs: Michael Brenner & Paul Kubes

7:30 PM Patrick J. Brennan M. Gutierrez-Arcelus, A.R. Mola, S.K. Hannes, N.C. Teslovich, W.F. Watts, M.B. Brenner, S. Raychaudhuri Defining a human T cell innateness gradient abs# 45

7:50 PM Michael Brenner C. Donado, P. Brennan iNKT cell – DC cross-talk after microbial PAMP encounter abs# 46

8:10 PM Luc Teyton L. Kain, M. Holt, L. Gioia, S. Head, P. Savage, F. Winau, A. Su Using single cell analysis to understand the thymic selection of NKT cells abs# 47

8:30 PM Lili Yang Stem Cell-Engineered iNKT Cells for Cancer Immunotherapy abs# 48

8:50 PM Ajitha Thanabalasuriar A.S. Neupane, J. Wang, M.F. Krummel, P. Kubes iNKT cell emigration out of the lung vasculature during inflammation requires neutrophils and monocyte-derived dendritic cells abs# 49


MONDAY 6TH NOVEMBER 2017

Registration Open


Session 7: Metabolism and Inflammation


Chairs: Joel Linden & Lydia Lynch

8:00 AM Joel Linden J.C. Yu, G. Lin Induction of anti-inflammatory purinergic signaling in activated human iNKT cells abs# 50

8:20 AM Mikael Karlsson Exogenous glycolipids override a regulatory role of iNKT cells during auto-inflammation abs# 51

8:40 AM Koen Venken M. Favreau, E. Menu, D. Gaublomme, K. Vanderkerken, S. Faict, K. Maes, E. De Bruyne, S. Govindarajan, M. Drennan, X. Leleu, K. Jochmans, L. Zabeau, J. Tavernier, D. Elewaut Leptin receptor antagonism of iNKT cell function: a novel strategy to combat multiple myeloma abs# 52

9:00 AM Kazuya Iwabuchi K. Fujita, M. Iizuka, L. Van Kaer, M. Satoh NKT cell-adipocyte interactions play an important role in the development of obesity abs# 53

9:20 AM Lydia Lynch A.C. Kohlgruber, M.B. Brenner Adipose tissue resident innate PLZF+ gd T cells are key players in immunometabolic homeostasis abs# 54

Morning Tea



Session 8: Autoimmunity and Tolerance


Chairs: Dirk Elewaut & Agnes Lehuen

10:15 AM Dirk Elewaut RORγt inhibition selectively targets pathogenic human iNKT and γδ-T cells enriched in Spondyloarthritis while preserving IL-22 responses abs# 55

10:35 AM Gennaro De Libero M. Lepore, L. Mori A functionally diverse population of human T cells recognizes non-microbial antigens presented by MR1 abs# 56

10:55 AM Claudia Mauri K. Oleinika, E. Rosser Regulatory B cells control the differentiation of suppressive iNKTcells via CD1d. abs# 57

11:15 AM Siddhartha Sharma B. Abe, A. Costanzo, M. Holt, G. Konijeti, L. Teyton Towards the definition of the role of MAIT cells in ulcerative colitis by single cell studies abs# 58

11:35 AM Agnes Lehuen Dual role of MAIT cells in diabetes abs# 59

Lunch


Lunch sponsored by

Session 9: Cancer and immunotherapy


Chairs: Leonid Metelitsa & Shinichiro Motohashi

1:30 PM Shinichiro Motohashi NKT cell-targeted immunotherapy for non-small cell lung cancer abs# 60

1:50 PM Jay Berzofsky J.J. O'Konek, E. Ambrosino, A.C. Bloom, Z. Xia, M. Terabe Immunity to the same tumor in lungs vs skin is differentially regulated by NKT cells or Treg cells and is asymmetrically cross-protective abs# 61


2:10 PM Michela Consonni C. Garavaglia, C. de Lalla, A. Bigi, M. Casucci, A. Bondanza, M. Lepore, L. Mori, G. De Libero, F. Ciceri, P. Dellabona, G. Casorati Retargeting T cells against leukemia by lipid-specific TCR transfer abs# 62

2:30 PM Jenny E Gumperz N.A. Zumwalde, A. Sharma, X. Xu, S.C. Kenney Investigating anti-lymphoma effects of human CD4+ iNKT cells in vivo: effectors or adjuvants? abs# 63

2:50 PM Tonya Webb Profiling NKT cell responses in cancer patients to identify immune biomarkers abs# 64

3:10 PM Leonid Metelitsa Harnessing natural and engineered properties of NKT cells for adoptive cancer immunotherapy abs# 65

Afternoon Tea


Session 10: Regional immunity


Chairs: Richard Blumberg & Paul Klenerman

4:00 PM Richard Blumberg AhR regulation of CD1d function in the intestinal epithelium abs# 66

4:20 PM Susanna Cardell iNKT cell suppression of tumor immunity in intestinal polyposis abs# 67

4:40 PM Ji Hyung Kim Y. Hu, T. Yongqing, J. Kim, V.A. Hughes, J. . Nours, E.A. Marquez, A.W. Purcell, Q. Wan, M. Sugita, J. Rossjohn, F. Winau CD1a: a key player in inflammatory skin diseases abs# 68

5:00 PM Paul Klenerman Viruses and MAIT cells abs# 69

Poster Session 2 - Even Numbers + Rapid Fire 2 Posters


Gala Dinner

7:30PM - 9:30PM Garden Pavilion


TUESDAY 7TH NOVEMBER 2017

Registration Open


Session 11: Environmental antigens and Microbiota


Chairs: Thierry Mallevaey & Gerhard Wingender

8:30 AM Thomas Gensollen Colonic iNKT cell establishment is specifically regulated by macrophages during early life. abs# 70

8:50 AM Shouxiong Huang M. Sharma, X. Zhang, S. Zhang, L. Niu, S. Ho, A. Chen Inhibition of endocytic lipid antigen presentation by common lipophilic environmental pollutants abs# 71

9:10 AM Olivier Lantz G.B. Youssef, M. Tourret, M. Salou, L. Ghazarian, S. Caillat-Zucman Ontogeny of human mucosal-associated invariant T cells and related T cell subsets abs# 72

9:30 AM Gerhard Wingender Possible protective roles of MAIT cells and environmental iNKT cell antigens in asthma in inner-city children. abs# 73

Morning Tea


Closing Keynote Lecture


Chair: Luc Teyton

10:20 AM Yasmine Belkaid Homeostatic immunity and the microbiota abs# 74

Awards & Closing Remarks

11:20AM - 12:00PM Silverado Ballroom

Awards sponsored by


ORAL ABSTRACTS

1

From αβTCR+ DN T cells to NKT cells to CD1a, b and c restricted T cells and MR1-restricted MAIT cells: An unexpected journey.

Dale Godfrey1, 2 1. Department of Microbiology and Immunology, Peter Doherty Institute, University of Melbourne, Melbourne, VIC, Australia

2. ARC Centre of Excellence for Advanced Molecular Imaging at the University of Melbourne, Melbourne

Thirty years ago, everyone knew that αβTCR+ T cells were either CD4+ MHC class II restricted (helper) cells or CD8+ MHC class I restricted (cytotoxic) T cells. Subsequently, many (but unfortunately not all) immunologists have come to realise that there are other types of αβTCR+ T cells, that in some cases are highly abundant and functionally unique components of the immune system. Early studies with αβTCR+ CD4-CD8- (αβDN) T cells and NK1.1+ T cells evolved into the extensive collection of CD1d restricted NKT cells (Types I and II), CD1a, CD1b, CD1c restricted T cells; and MR1 restricted MAIT cells that brings the delegates of this conference together every 2 years. I will present an overview of my early forays into this field (alongside many valued team members and collaborators) working with αβDN T cells, through to developmental and functional studies of NKT cells and subsets thereof. Our more recent studies have led to the identification of atypical populations of CD1d-restricted T cells and the molecular and structural basis of their antigen reactivity. We are also making great headway in the identification, functional and molecular analysis of human CD1a, CD1b and CD1c restricted T cells and their antigen reactivity. Most recently, our studies have encompassed MR1-restricted MAIT cells where we have mapped a developmental pathway and identified several key factors that regulate the development of these cells and lastly, I will discuss our recent studies of atypical MR1-restricted αβ and γδ T cells.

2

Clinical harnessing of lipid-specific T cell responses

Paolo Dellabona1 1. San Raffaele Scientific Institute, Milano, Italy

CD1 antigen presenting molecules are poorly polymorphic; this makes extremely attractive harnessing CD1-restricted T cells for clinical use, because their reactivity would be donor-unrestricted, hence universal. CD1d-restricted iNKT cells have been implicated in a number of pathological situations and are already exploited for clinical use, particularly in cancer immunotherapy. Clinical implications and exploitation of group 1 CD1 T cell responses are less explored, even though basic and pre-clinical studies are accumulating, suggesting intriguing pathophysiological involvements for these cells. Xenoreactive group 1 CD1 restricted T cells specific for bacterial lipid antigens have long been implicated in the immune control of infectious pathogens, particularly Mycobacterial ones, suggesting the generation of group 1 CD1-restricted protective lipid vaccines. New data are revealing the potential clinical relevance also for autoreactive group 1 CD1 restricted T cell responses directed against stress-related self-lipids, which may be involved in tumor immunesurveillance or autoimmunity, two conditions in which self-antigens are the targets of the immune response. Targeting or blocking recognition of self-lipid antigens may become viable therapeutic options in cancer and autoimmunity, respectively. Continuous investigation on fundamental aspects of group 1 CD1 restricted T cell response will fuel the promise of appropriate clinical translation.


3

Translational Directions: CD1d-restricted NKT cell populations

Mark Exley1 1. University of Manchester, Manchester, ACT, United Kingdom

Since various ‘NKT’ cell populations were described over 20 years ago in man and model organisms, thoughts have turned to exploiting them in treatment of disease. Much encouraging model data and preclinical human progress supported the diagnostic and therapeutic potential of NKT cells in general and iNKT cells in particular. For example, NKT IFNg production stratifies for survival of cancer patients. Unlike other CD1d-restricted T cells, iNKT can be uniformly and highly specifically manipulated with certain lipids and antibodies ex vivo and in vivo. Safety studies showed iNKT-based approaches are well-tolerated. More recent clinical trials worldwide have shown clear anti-tumor potential for iNKT-based treatments. Such approaches maybe optimized by genetic modification &/or combination with complementary therapies. Other diseases where iNKT may have positive impact will also be summarized, along with some notes of caution.

4

Specific identification, tracking and harnessing of Mucosal Associated Invariant T (MAIT) cells in health and disease

Jim McCluskey1 1. Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria 3000, Australia.

Mucosal associated invariant T (MAIT) cells recognize conserved microbial metabolites from riboflavin synthesis. Their striking evolutionary conservation and relative abundance, implicate them in antibacterial host defense. Yet their role in protection against clinically significant pathogens, and as potential vaccine targets, remain unknown. The development of Ag-specific MR1-tetramers has allowed more specific tracking and phenotyping of MAIT cells, facilitating their biological characterisation and exploration of translational opportunities.

We have investigated MAIT cell function in two infectious disease models. Firstly, we noted that following lung infection with Salmonella typhimurium, MAIT cells were rapidly enriched in the lungs of C57BL/6 mice and displayed an activated/memory phenotype. The majority of cells produced interleukin-17 while smaller subsets produced interferon-g or tumor necrosis factor, detected directly ex vivo. However, MAIT cells did not offer any protection measured by bacterial clearance.

Secondly, murine Legionella infection also induced MR1-dependent MAIT cell activation and rapid pulmonary accumulation of MAIT cells, but in contrast, this response was associated with immune protection evident in normal, immunocompetent mice. Peeling away layers of adaptive immunity was revealing. MAIT cell protection was even more apparent in mice lacking CD4+ cells, whilst profoundly immunodeficient RAG2-/-gC-/- mice could be substantially rescued from uniformly lethal Legionella infection by adoptively-transferred MAIT cells. This protection was dependent on MR1, IFN-g and GM-CSF, but not IL-17, TNF-a or perforin.

Protection in the Legionella model was enhanced in mice that were first primed intransally with 5-OP-RU and a costimulus to boost MAIT cells before infectious challenge. Accordingly, MAIT cells not only protect against the major human lung pathogen Legionella, but can potentially be harnessed as vaccine targets to enhance protection through simple intranasal delivery of purified antigen and a defined adjuvant.


5

The transcription factor PLZF directs the acquisition of effector programs during innate and innate-like lymphocyte development

Albert Bendelac1 1. The University of Chicago, Chicago, IL, United States

Zbtb16-encoded PLZF is a signature transcription factor (TF) that directs the acquisition of T helper effector programs during the development of multiple innate lymphocyte lineages, including NKT, ILC, MAIT and gammadelta ineages. It is also essential in osteoblast and spermatogonial development. How Zbtb16 itself is regulated in different lineages is incompletely understood. By systematic Crispr/Cas9-assisted deletions of chromatin accessible regions within the Zbtb16 locus in mouse, we identified a dominant critical enhancer controlling PLZF expression exclusively in innate lymphoid lineages, including ILC, and NKT, MAIT and gammadelta lineages precursors. Multiple sites within this enhancer expressed canonical motifs for the TF Runx1, which was essential for the development of both ILC and NKT lineages. Notably, some of these regulatory sites exerted an impact on NKT cell development by controlling the kinetic rather than the overall level of PLZF expression. Thus, a comprehensive unbiased analysis of regulatory elements in vivo revealed critical new mechanisms of PLZF regulation shared between innate and innate-like lymphoid lineages.

6

MHC class I-like XNC4 tetramers identify multiple invariant TCR rearrangements during mycobacteria infection in the amphibian Xenopus

Jacques Robert1, Eva-Stina Edholm1 1. University of Rochester, Rochester, NEW YORK, United States

A predominant system of multiple MHC class I-like restricted innate-like T cells have been identified in Xenopus laevis tadpoles. Using a combination of different reverse genetic approaches, we have identified two, functionally distinct iT cell populations (iVα6 and Vα45) restricted by the Xenopus MHC class I-like genes XNC10 and XNC4, respectively. While iVα6 T cells are critical for anti-ranaviral immunity, iVα45 T cells are dispensable for ranaviral immunity but required for anti-mycobacterial immune defense. Using Xenopus XNC10 tetramers (XNC10-T) we have previously reported the isolation of two populations of iVα6 T cells reminiscent of type I and type II iNKT cells. Here, we successfully generated XNC4 tetramers (XNC4-T) that bind to a distinct but more heterogeneous population of T cells than XNC10-T. Notably, TCRα repertoire analysis of XNC4 tetramer positive (cells sorted from either the spleen or the liver during early (6 days) and late (36 days) stages following Mycobacterium marinum (Mm) infections exhibited different bias. As expected XNC4-T sorted cells from the early stage of Mm infection showed a predominant usage of Va45. In contrast, XNC4-T positive cells isolated from late stage Mm infection did not express Vα45 but did rather displayed a highly biased TCRα repertoire with different dominant Vα segments in infected spleen and liver. These data suggest that MHC class I-like XNC4 may interact with different innate-like or invariant T cell effectors.


7

Evolution of MHC class I, CD1 and class II genes: how did it happen?

Jim Kaufman1 1. University of Cambridge, Cambridge, United Kingdom

After some 40 years of research, it seems clear that the adaptive immune system of jawed vertebrates based on major histocompatibility complex (MHC) class I and class II, αβ and γδ T cell receptor, and immunoglobulin genes arose sometime before the lineage leading to cartilaginous fish. Observations in many organisms, including seminal discoveries in the chicken, suggest that all these genetic components along with natural killer (NK) receptor genes arose in a single genetic region, allowing duplication and divergence of genes to diversify the functions, and co-evolution between structurally-unrelated components to make molecular pathways. If this view is correct, then the “primordial MHC” must have fallen apart somehow to give rise to contemporary genomes.

The existence of two rounds of genome-wide duplication (2R) at the base of the vertebrate lineage suggests one mechanism to distribute the genes of the primordial MHC to various paralogous regions, giving potential for the evolution of new functions (“neofunctionalisation”). The presence of the non-classical class I genes CD1, MR1 and the Fc receptor of neonates (FcRn) genes in MHC paralogous regions of the human genome might mean that (the ancestors of) these genes were moved to paralogous regions during 2R, making them very ancient. Such an interpretation could fit with early suggestions that CD1 has properties of both class I and class II molecules.

However, CD1 genes might be relatively recent and translocated from the MHC into an MHC-paralogous region during chromosomal re-organisations in the lineage leading to placental mammals. This alternative view has been based on sequence comparisons, location of CD1 genes in the chicken MHC, and lack of discernible CD1 genes in amphibians and fish. Re-examination of the MHC paralogous regions, consideration of various sequence features of MHC molecules, and more complete genomic sequences may help to choose between the various interpretations.

8

Evolutionary origins of CD1 and MHC class I

Martin Flajnik1, Yuko Ohta1, Masanori Kasahara2 1. Department of Microbiology and Immunology, University of Maryland, Baltimore, Maryland, USA

2. Department of Pathology, Hokkaido University, Sapporo, Japan

Since Milstein’s discovery of CD1 in 1986, it was proposed that CD1 is an ancient class I molecule, perhaps predating the emergence of classical class I and class II. It was further proposed, by Kasahara and colleagues, that CD1’s presence on human chromosome 1, in a potential MHC paralogue, was a consequence of genome-wide duplications (2R hypothesis) at the dawn of vertebrate history. Our analysis of MHC paralogous regions in many vertebrates, and focusing on the amphibian Xenopus paralogous regions, shows that CD1’s presence on human chromosome 1 is not a consequence of 2R, rather a translocation from the bona fide MHC early in the evolution of mammals. A bevy of other immune genes, such as FcR and SLAM, were also part of this translocation. This new idea is consistent with CD1’s presence in the MHC-proper of some birds and reptiles. Although CD1’s location on human chromosome 1 is not a consequence of 2R, it is indeed ancient, probably having arisen after 1R (the first genome-wide duplication) in the proto MHC, based on CD1’s presence on a true paralogous region (chr 19) in reptiles. Furthermore, we have found other class I molecules encoded in this paralogous region (chr 19) in amphibians, reptiles, and mammals, also consistent with class I’s early emergence. In summary, CD1 arose early in evolution in the proto MHC, but seems to have been lost in fish and amphibians, its function perhaps assumed by other nonclassical class I molecules in these taxa. The data also suggest that both classical and nonclassical class I genes predated the emergence of class II in evolution.


9

Size Motifs and Misfit lipids in Human CD1b Proteins

Branch Moody1, Shouxiong Huang1, Tan-Yun Cheng1, Adam Shahine2, Jamie Rossjohn2, Stephanie Gras2 1. Brigham and Women's Hospital, Harvard Medical School, Boston, MA, United States

2. Biochemistry and Molecular Biology, Monash University, Melbourne, Australia

Information about CD1-lipid complex formation comes mainly from antigen presentation assays and crystal structures in which the experimentalist adds a specific lipid of interest. To measure the spectrum of endogenous self lipids captured by cellular CD1 proteins, we developed a high performance liquid chromatography-time of flight mass spectrometry-based lipidomics platform. This platform separated and enumerated lipids with low rates of false positive ligand detection. Human CD1b proteins bound hundreds of ligands that spanned a broad range of polarity, including hydrophobes, glycolipids, phospholipids, phosphoglycolipids and zwitterionic lipids. After solving the structures of lipid types that bind multiple CD1 proteins, we used these promiscuously binding lipids as tools to probe the functional size capacity of CD1 clefts and matched these to the volumes of the hydrophobic clefts observed in crystal structures. Unlike control proteins, CD1b ligands showed a mismatch in lipid anchor length as compared to the volume of the hydrophobic cleft. These functional and structural data support a two-chamber model for lipid capture by cellular CD1b proteins. Deeply buried scaffold lipids and protruding antigenic ligands simultaneously bind newly translated CD1b proteins. Based on exchange of one or two cleft blocking lipids, this mechanism provides a solution to the previously unexplained phenomenon in which small exogenous lipids readily load onto CD1b, whereas loading of large bacterial lipids require lysosomal recycling.

10

Living Under the A’ Roof

Jamie Rossjohn1 1. Monash University, Clayton, VIC, Australia

T cells can recognise lipid-based antigens when presented by the CD1 family, of which there are four members of antigen-presenting molecules, CD1a-d. Each CD1 member possesses unique structural features that suggest distinct functional roles in the context of protective immunity and aberrant T cell reactivity. Moreover, each CD1 isoform presents specific challenges for T cell receptor (TCR) recognition. I shall compare and contrast TCR-CD1-lipid recognition, revealing that it can fundamentally differ from that of TCR-MHC-peptide recognition. This suggests that lipid-mediated presentation and recognition should move out of the shadow of MHC biology.

11

The hidden virtues of CD1b and CD1c antigen presentation

Stephan Gadola1 1. F.Hoffmann-La Roche & University of Southampton/UK, Basel, BASEL, Switzerland

Functional differentiation between the four antigen-presenting human CD1 isoforms, CD1a-d, is achieved through their lipid-binding properties. Compared to structurally-related MHC class I molecules, a far greater proportion of the CD1/ligand complex volume is made up by the ligand or ligands buried within the antigen binding groove. This is most pronounced in CD1b and CD1c. In consequence, the degree of ligand saturation of the antigen binding grooves in these CD1 proteins can exert major impact on overall CD1 conformation and thus T cell recognition. In my talk, I will discuss our group’s recent insights into CD1b and CD1c ligand structures, their binding to human T cell receptors, and implications for human disease.


12

A molecular basis of human T cell receptor autoreactivity towards CD1b and self-phospholipids

Adam E Shahine2, 1, Ildiko Van Rhijn3, 4, Tan-Yun Cheng4, Sarah Iwany4, Stephanie Gras2, 1, D Branch Moody4, Jamie

Rossjohn2, 5, 1 1. Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia

2. Australian Research Council Center of Excellence in Advanced Molecular Imaging, Melbourne

3. Department of Infectious Diseases and Immunology, University Utrecht, Utrecht, Netherlands

4. Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA

5. Institute of Infection and Immunity, Cardiff University School of Medicine, Cardiff, Wales, UK

Central to both innate and adaptive immunity is the mediation of T-cell recognition of antigen presenting molecules presenting antigens by T-cell receptors (TCR). CD1 molecules are Major Histocompatibility Complex class I like molecules involved in presentation of foreign and self-lipid antigens. CD1b, a member of group 1 CD1 molecules along with CD1a and CD1c, exhibits the largest hydrophobic antigen binding cleft, capable of presenting foreign and self-lipids with long carbon tails. While the first molecular mechanisms regulating T-cell reactivity towards microbial lipids presented by CD1b has recently been portrayed in the context of Mycobacterium tuberculosis infection, the structural mechanisms surrounding T-cell autoreactivity towards self-lipid antigens are unknown. Following up from decades-long interest in phospholipid-specific antibodies, recent work shows that human T-cells, including the named clones PG90, PG10, HJ1, recognize CD1b presenting self-phospholipids, circulate in human blood and mediate autoimmune skin disease in a human TCR transgenic model. Our elution studies demonstrate that while CD1b presents common membrane phospholipids, alpha/beta TCR reactivity is specifically skewed towards the rare self-phospholipid, phosphatidylglycerol (PG). Cellular levels of PG are low in absolute terms, highly regulated, and are predominantly localised within mitochondria under normal physiological conditions. Biophysical analysis of these TCRs by surface plasmon resonance reveals a high sub-micromolar affinity interaction for CD1b-PG. Structural characterisation of the PG90 TCR-CD1b-PG complex provides the first insights into the molecular mechanisms for rare phospholipid discrimination. The crystal structure demonstrates a dramatic induced fit model over the PG polar headgroup, primarily driven by rearrangement of the TCR CDR3 loops. Moulding of the TCR CDR3 loops forms a small, electrostatically positive pocket, denoted as a “cationic cup”, which selects against ubiquitous phospholipids through electrostatic or stearic hindrance. This provides insight into mechanisms of TCR autoreactivity geared towards rare self-phospholipids, thus improving our understanding towards detrimental T-cell autoreactivity against self-lipid antigens.

13

Exploring MR1 Mycobacterial Ligands and Antigens

David Lewinsohn1 1. OHSU / PVAMC, Portland, OREGON, United States

MAIT cells have been termed innate because they are restricted by the highly conserved MR1 antigen presentation molecule, and based on their limited TCR usage. This concept is further reinforced by the discovery of riboflavin metabolites as key MR1 antigens for many microbes. However, analysis of TCR usage in the context of particular microbial stimulation reveals selective TCR usage. This observation supports the the hypothesis that MR1 presents a diverse array of ligands, and that these in turn are selectively recognized by MR1 restricted T cells (MR1Ts). This hypothesis is further supported by the observation that MR1Ts present in the BAL of patients with TB are uniquely enriched relative to the peripheral blood. To explore the ligandome of MR1 in the context of infection, the Adams laboratory enriched for microbial ligands by producing recombinant MR1 in the context of a microbial infection. The Hildebrand laboratory then used tandem mass-spectrometry and molecular network analyses to visualize the array of MR1-presented microbial ligands and to subsequently identify several of these ligands. Synthetic analogs of these newly identified MR1 microbial ligands were tested for MR1 binding and for their capacity to activate MR1Ts with divergent TCR. Our results unambiguously demonstrate the capacity of different TCRs to discriminate among MR1 ligands and support the hypothesis that microbial infection results in the selective expansion of MR1Ts.


14

Identifying Antigens that are relevant for T cell Reactivity in Humans

Erin Adams1 1. University of Chicago, Chicago, IL, United States

The antigens that specifically regulate human gamma delta T cell activity have largely remained undefined. Our characterization of CD1d-lipid reactive gamma delta T cells in human blood has provided an initial insight into the signals that Vd1 gamma delta T cells respond to and demonstrated that some gamma deltas rely on MHC antigen presentation. We have continued to study the antigen reactivity of these cells, exploring their lipid reactivity, MHC restriction and molecular interactions. We have also demonstrated that CD1d reactive gamma deltas are present in human mucosal tissues and tumors.

22

Augmenting influenza-specific T cell memory generation with a NKT cell-dependent glycolipid-peptide vaccine

Regan J Anderson1, Jasmine Li2, Lukasz Kedzierski2, Benjamin J Compton1, Taryn L Osmond3, Ching-wen Tang3, Hui-Fern Koay4, Lauren R Holz4, Astrid Authier-Hall3, Robert Weinkove3, Dale I Godfrey4, 5, Ian F Hermans3, 6, 7, Stephen J Turner, Gavin

F Painter1 1. The Ferrier Research Institute, Victoria University of Wellington, PO Box 33436, Lower Hutt 5046, New Zealand

2. Department of Microbiology, Biomedical Discovery Institute, Monash University, Clayton, Australia

3. Malaghan Institute of Medical Research, PO Box 7060, Wellington 6242, New Zealand

4. Department of Microbiology and Immunology, at the Doherty Institute for Infection and Immunity, The University of Melbourne, 792 Elizabeth St, Melbourne, Vic, Australia

5. Australian Research Council Centre of Excellence in Advanced Molecular Imaging, University of Melbourne, Parkville, Victoria 3010, Australia

6. Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, 3 Symonds St, Auckland Central, 1142, New Zealand

7. Avalia Immunotherapies Limited, Gracefield Innovation Quarter, 69 Gracefield Rd, Lower Hutt 5010, New Zealand

The development of a universal vaccine for influenza A virus (IAV) that does not require seasonal modification is a long-standing health goal, particularly in the context of the increasing threat of new global pandemics. Vaccines that specifically induce T cell responses are of considerable interest because they can target viral proteins that are more likely to be shared between different virus strains and subtypes and hence provide effective cross-reactive IAV immunity. From a practical perspective, such vaccines should induce T cell responses with long-lasting memory, while also being simple to manufacture and cost-effective. Here we describe the synthesis and evaluation of a vaccine platform based on solid phase peptide synthesis and bio-orthogonal conjugation methodologies. The chemical approach involves covalently attaching synthetic long peptides from a virus-associated protein to a powerful adjuvant molecule, α-galactosylceramide (α-GalCer). Strain-promoted azide-alkyne cycloaddition is used as a simple and efficient method for conjugation, and pseudo-proline methodology is used to increase the efficiency of the peptide synthesis. α-GalCer is a glycolipid that stimulates NKT cells, a population of lymphoid-resident immune cells that can provide potent stimulatory signals to antigen-presenting cells engaged in driving proliferation and differentiation of peptide-specific T cells. When used in mice, the vaccine induced T cell responses that provided effective prophylactic protection against IAV infection, with the speed of viral clearance greater than that seen from previous viral exposure. Strong antigen-specific responses were also observed in human blood. These findings are significant because the vaccines are highly defined, quick to synthesize, and easily characterized, and are therefore appropriate for large scale affordable manufacture.


23

NKT cell subsets involved in host defense from pulmonary Streptococcus pneumoniae infection

Catherine M Crosby1, Zbigniew Mikulski1, Mitchell Kronenberg1 1. La Jolla Institute for Allergy and Immunology, La Jolla, CA, United States

Invariant natural killer T cells (iNKT) are a population of innate-like T lymphocytes with crucial roles in protective responses to infectious agents, particularly Streptococcus pneumoniae. Despite their abundance in the lungs, there is limited understanding of how iNKT cells mediate protection there. Prior work from our group showed after pulmonary S. pneumoniae infection, iNKT cells are rapidly activated in a CD1d-dependent manner. Similar to CD4+ T cells, iNKT cells have been sub-classified into NKT1, 2, and 17 subsets. Here we investigated the roles of these subsets in protection from S. pneumoniae. In uninfected mouse lungs, NKT1 and NKT17 cells were the predominant iNKT subsets, with most NKT17 cells in the tissue and NKT1 cells mainly within the vasculature. We utilized fluorescent bacteria, flow cytometry, and intravital microscopy to analyze the lungs during infection. By 24 hours, the bacteria localized within the tissue, and were taken up by alveolar macrophages, neutrophils, and to a lesser extent DCs. As early as 14 hours, most of the lung NKT17 cells produced IL-17, while only a minor percentage of NKT1 cells produced IFN-γ. This correlated with data from Nur77GFP reporter mice that indicate TCR-mediated activation. At 24 hours, approximately 75% of NKT17 but only 30% of NKT1 cells were TCR-activated; suggesting NKT17 cells may play a dominant role early in infection. Yet, at the same time, NKT1 cells expanded to become the major subset in the lungs, suggesting they also could be relevant for protection. In summary, our data show not only differential localization of iNKT cell subsets in the lung, but also differences in the timing of their TCR-mediated activation, suggesting the NKT17 subset could be particularly important for protection. Experiments are underway to determine which cell type(s) presents antigen, whether iNKT cells produce other cytokines, and the consequences of removing individual subsets.

24

Riboflavin metabolism variation among clinical isolates of Streptococcus pneumoniae results in differential activation of MAIT cells

Curtis McMurtrey1, Nadine Hartmann2, Michelle Sorensen3, Joseph Mizgerd4, William Hildebrand1, Mitchell Kronenberg2,

David Lewinsohn5, 6, Melanie Harriff5, 6 1. University of Oklahoma Health Sciences University, Oklahoma City

2. La Jolla Institute for Allergy and Immunology, La Jolla

3. Oregon Health & Sciences University, Portland, OR, USA

4. Boston University School of Medicine, Boston

5. VA Portland Health Care System, Portland, OR, USA

6. Pulmonary and Critical Care Medicine, Oregon Health & Sciences University, Portland, OR, USA

Streptococcus pneumoniae is an important bacterial pathogen commonly causing a range of non-invasive and invasive diseases. The mechanisms underlying variability in the ability of distinct S. pneumococcus serotypes and genetic backgrounds to transition from nasopharyngeal colonization to disease-causing pathogen are not well-defined. Mucosal-associated invariant T (MAIT) cells are highly prevalent in mucosal tissues such as the airways and are thought to play an important role in the early response to infection with bacterial pathogens. The ability of MAIT cells to recognize and contain infection with S. pneumoniae is not known. In the present study, we tested the ability of human MAIT cells to respond to and control infection with a panel of clinical isolates of S. pneumoniae serotype 19A, a non-vaccine serotype linked to invasive pneumococcal disease. We found that while MAIT cells were capable of responding in vitro to human dendritic cells and airway epithelial cells infected with S. pneumoniae, the magnitude of response to cells infected with different serotype 19A isolates, determined by genetic differences in the expression of the riboflavin biosynthesis pathway. The differences in MAIT cell release of cytokines correlated to differences in the ability of MAIT cells to control S. pneumoniae in vitro and persisted in MAIT cells isolated from a mouse challenge model. Together, these results demonstrate that there are genetic differences in riboflavin metabolism among clinical isolates of the same serotype that determine MAIT cell function in response to infection with S. pneumoniae. These differences are critical in considering the role that MAIT cells play in early responses to pneumococcal infection and determining whether invasive disease will develop.


25

CD1a, CD1b, and CD1c tetramers detect antigen-specific T cells in a tuberculosis cohort

Ildiko Van Rhijn1 1. Brigham and Women's Hospital-Harvard Medical School, Boston, MA, United States

A key question in CD1 biology that is relevant to its translational potential is how CD1-specific T cells respond during natural infection in humans. Two extreme possibilities are: 1) CD1a, CD1b, and CD1c-specific T cells expand from a naive, low precursor frequency population and acquire memory function and phenotype during infection, just like MHC-restricted T cells, or 2) CD1a, CD1b, and CD1c-specific T cells behave like CD1d-specific NKT cells and have a constitutively activated phenotype that does not change during infection, as well as an unchanged frequency. To address this question, we use CD1 tetramers loaded with synthetic versions of lipid antigens that are present in Mycobacterium tuberculosis bacteria. We use seven different tetramers to study T cells in a patient cohort from Lima, Peru, consisting of 50 active tuberculosis cases, 50 uninfected household contacts, and 50 infected, but non-progressing household contacts. These experiments establish immunodominance among the used lipid antigens and frequency of tetramer-positive T cell populations. Another question in the CD1a, CD1b, and CD1c field is whether the T cell repertoire of the responding human T cells is predictable and interpretable. This question is addressed by high throughput sequencing of tetramer-positive T cell populations. Together, our datasets help answer the question whether CD1-presented lipid antigens can be of use in vaccination and diagnostics.

26

MHC class Ib molecules in host defense against Mycobacterium tuberculosis infection

Chyung-Ru Wang1, Shaobin Shang1, Yan Bian1, Sarah Siddiqui1, Jie Zhao1, Ying He1 1. Department of Microbiology and Immunology, Northwestern University, Chicago, Illinois, USA

MHC class Ib genes comprise the majority of the class I family and encode a number of molecules that are expressed at lower levels than class Ia and have limited polymorphism. While much is known about the contribution of MHC Ia at different stages of Mycobacterium tuberculosis (Mtb) infection, our understanding of the diverse group of MHC Ib molecules in host defense against Mtb is quite limited in comparison. We have previously shown that M3 presents several N-formylated Mtb peptides to CD8+ cytotoxic T cells during Mtb infection. Using mice that lack MHC Ia/M3 (Kb-/-Db-/-M3-/-), we demonstrated that non-M3 MHC Ib-restricted CD8+ T cells recognized several Mtb protein antigens, showed polyfunctional capacity, and provided protection against Mtb infection, with Qa-2-restricted CD8+ T cells constituting a large proportion of the MHC Ib-restricted CD8+ response. Interestingly, we also found that the number of MAIT cells increased significantly during Mtb infection and they comprised a substantial proportion of IL-17-producing T cells in Mtb-infected mice. In addition, we found that Qa-1 could present multiple HLA-E-binding Mtb peptides to CD8+ T cells during Mtb infection, and Qa-1-/- mice were more susceptible to high-dose Mtb infection compared to wild-type controls. The increased susceptibility of Qa-1-/- mice was associated with dysregulated T cells that were more activated, produced higher levels of pro-inflammatory cytokines, and were more prone to cell death. Thus, our studies demonstrate that MHC Ib molecules participate in the immune response to Mtb infection by presenting diverse Mtb antigens to T cells and modulating anti-Mtb immune responses.


27

A novel mechanism of iNKT cell activation mediated by ER-stressed antigen presenting cells

Melissa Bedard1, Mariolina Salio1, David Priestman2, Dilip Shrestha1, Christian Eggeling1, Frances Platt2, Richard Blumberg3,

Vincenzo Cerundolo1 1. MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, Oxfordshire, United Kingdom

2. Department of Pharmacology, University of Oxford, Oxford, Oxfordshire, United Kingdom

3. Department of Medicine, Division of Gastroenterology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America

iNKT cells have a prominent role in anti-tumor immune responses. Indeed, iNKT cell deficient mice have a higher incidence of methylcholanthrene-induced sarcoma, indicative of defective immune-surveillance.1 However, it is unclear how iNKT cells become activated in the context of cancer. Accumulating evidence implicates endoplasmic reticulum (ER) stress in the development and progression of many diseases, including cancer. ER-stress triggers the unfolded protein response (UPR). In turn, the UPR activates numerous signaling pathways, many of which overlap with lipid biosynthetic and immune response pathways. This intersection raises the possibility that the UPR modulates endogenous lipid antigen presentation on CD1d+ myeloid populations and CD1d+ tumor cells, resulting in enhanced iNKT cell activation in cancer. To investigate this hypothesis, iNKT cells were co-cultured with monocyte-derived dendritic cells or THP1 cells pre-treated with thapsigargin. Thapsigargin-treated cells activated iNKT cells in a CD1d-dependent manner. Given that CD1d expression levels do not change between untreated and thapsigargin-treated antigen presenting cells, we are identifying the activating self-lipid antigen(s) preferentially loaded onto CD1d molecules in ER-stressed antigen presenting cells and through molecular techniques are investigating which branch of the UPR is responsible for changes in the lipid antigen profile of ER-stressed cells.

1. 1. Crowe NY, Smyth MJ, Godfrey DI. A critical role for natural killer T cells in immunosurveillance of methylcholanthrene-induced sarcomas. The Journal of experimental medicine (2002) 196(1):119-27. PubMed PMID: 12093876; PubMed Central PMCID: PMCPMC2194015.

28

Immune evasion mechanism of Streptococcus pneumoniae - gain fat to lose sugar

Nadine Hartmann1, Mitchell Kronenberg1 1. La Jolla Institute For Allergy And Immunology, La Jolla, CALIFORNIA, United States

Streptococcus pneumoniae is a global cause of morbidity and mortality. During pulmonary S. pneumoniae infection, a unique and versatile subset of T lymphocytes called invariant natural killer T (iNKT) cells plays a critical role in host protection of mice. The microbial antigen in S. pneumoniae is an abundant, glucosylated diacylglycerol molecule containing vaccenic acid (18:1n7). Antigenic activity of this molecule is lost when vaccenic acid is substituted, even with closely related fatty acids such as oleic acid (18:1n9). In order to investigate the possible contribution of self-lipids as opposed to microbial lipids during Streptococcus infection, we generated an S. pneumoniae mutant with an altered fatty acid biosynthesis that is unable to produce the previously identified iNKT cell antigen. This mutant is highly dependent upon exogenous C18 fatty acids for its growth, and shows a strongly reduced ability to stimulate protective iNKT cell responses in vitro and in vivo. Mice infected with this mutant had an elevated bacterial burden and lower survival rate when compared to mice infected with the wild-type strain, demonstrating that foreign antigen recognition is the dominant pathway for iNKT cell stimulation in mice. Interestingly, wild-type bacteria rapidly turn off expression of genes involved in fatty acid biosynthesis in the presence of abundant host fatty acids suggesting a mechanism to save metabolic energy. By manipulating serum free fatty acid levels in the host, we show that the switch from synthesis of vaccenic acid to incorporation of exogenous fatty acids from the host also provides an important immune evasion strategy for S. pneumoniae to prevent its recognition by iNKT cells.


29

Activation of human mucosal-associated invariant T cells induces CD40L-dependent maturation of monocyte-derived and primary dendritic cells

Mariolina Salio1, Olivier Gasser2, Claudia Gonzalez-Lopez1, Natasha Veerapen3, Uzi Gileadi1, Giorgio Napolitani1, Regan

Anderson4, Gavin Painter4, Gurdyal S Besra3, Ian F Hermans2, 5, Vincenzo Cerundolo1 1. MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, Headington, Oxford, UK

2. Malaghan Institute of Medical Research, School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand

3. School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK

4. The Ferrier Research Institute, Victoria University of Wellington, Lower Hutt, New Zealand

5. Maurice Wilkins Centre for Molecular Biodiscovery, Auckland, New Zealand

Mucosal-associated invariant T (MAIT) cells are innate T cells recognising intermediates of the vitamin B2 biosynthetic pathway presented by the monomorphic MR1 molecule. To date it remains unclear whether in addition to their cytolytic activity important in antimicrobial defence, MAIT cells have also immune-modulatory functions, which could enhance DC maturation. Here, we investigated the molecular mechanisms dictating the interactions between human MAIT cells and dendritic cells (DC) and demonstrate that human MAIT cells mature monocyte-derived and primary DC in an MR1- and CD40L-dependent manner. Furthermore, we show that MAIT cell derived signals synergise with microbial stimuli to induce secretion of bioactive IL-12 by DC. Activation of human MAIT cells in whole blood leads to MR1- and cytokine-dependent NK cell transactivation. Our results underscore an important property of MAIT cells, which can be of translational relevance to rapidly orchestrate adaptive immunity through DC maturation.

30

Towards characterization of the MR1 antigen presentation machinery

Hamish EG McWilliam1, Matthew Zorkau1, Sam Wormald, Justine D Mintern1, James McCluskey1, Jamie Rossjohn2, Jose

Villadangos1 1. University of Melbourne, Parkville, VIC, Australia

2. Infection and Immunity Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia

MR1 is the only antigen presenting molecule that displays a pathogen metabolic signature, the Vitamin B-related antigens (VitBAg). This allows the MR1-restricted MAIT cells to detect infection even if the antigen presenting cell has not physically encountered the pathogen. Given the distinct nature of MR1 ligands and their role, the MR1 presentation pathway is expected to possess unique features absent in other pathways of antigen presentation. Indeed, we recently showed that MR1 is the only human antigen presenting molecule that does not constitutively present self-ligands. Instead the majority of MR1 accumulates in the endoplasmic reticulum (ER) as a pool of empty molecules until a VitBAg forms a covalent bond (Schiff base) with a Lys residue in the antigen binding site of MR1. This acts as a “molecular switch” that triggers complete folding and egress of MR1 molecules from the ER to the cell surface (1). We hypothesised that MR1 requires molecular machinery along this pathway, therefore we aimed to characterise these accessory proteins that enable MR1 presentation of VBAg. Using genome-wide CRISPR-Cas9 screens and proteomics approaches we identified chaperones required for MR1 stabilisation, folding, and trafficking to and from the cell surface. We found that tapasin and tapasin-related protein both bound to ER-resident MR1, and the deletion of these resulted in a loss of the majority of the ligand-receptive MR1 pool due to faster degradation of MR1. While nascent molecules could still load and present VitBAg without these chaperones, the loss of the MR1 pool resulted in a significantly reduced presentation of VitBAg. Therefore, these chaperones play a key role in stabilising empty MR1 in the ER for the rapid presentation of bacterial metabolites, and illustrates a role beyond facilitating MHC class I peptide loading.

(1) McWilliam et al, Nat Immunol 2016


31

iNKT cells support cytokine-producing B cells in adipose tissue and are required to limit symptoms of metabolic disease

Elizabeth Leadbetter1 1. Microbiology, Immunology, and Molecular Genetics, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA

Obesity is an inflammatory disease that leads to chronic, low-grade inflammation in adipose tissue which contributes to the development of type 2 diabetes (T2D). Adipose tissue-resident immune cells maintain homeostasis in lean patients and regulate or exacerbate inflammation in obese patients. Understanding the role of these immune cells will be critical for treating metabolic disease. iNKT cells and cytokine-producing B cells are enriched in white adipose tissue as compared with spleen, and these populations change during obesity. We determined that cytokine-producing B cells in adipose tissue share phenotypic traits with memory- or alternatively activated B cells, including T–bet and CD11c expression. We find that these and other B cells in VAT are supported by iNKT cells in both lean and obese states. Specifically, obese mice showed a significant drop in their frequency of IL-10+ adipose B cells and a concomitant increase in frequency of Tbet+CD11c+CD11b+ adipose B cells, as compared to lean mice. We find the same pattern of B cell activation and a decrease in IL-10+ B cells in human adipose tissue from overweight/obese patients as compared to lean patients. We also find that glycolipid activation of iNKT cells skews adipose tissue B cells to produce anti-inflammatory cytokines, suggesting iNKT activation may provide a therapeutic approach to restore regulatory B cell homeostasis in adipose tissue of obese subjects and reduce metabolic symptoms associated with T2D.

32

iNKT cells require TCR and cytokine signals from distinct APC to support IL-4 production and shape thymic development

Kristin Hogquist1, Haiguang Wang1, Corey N Miller2, Elise R Breed1, You Jeong Lee1, Mark S Anderson3 1. University of Minnesota, Minneapolis, MN, United States

2. University of California San Francisco, San Francisco, California, USA

3. University of California San Francisco, San Fransico, California, USA

Steady state IL-4 production by a unique subset of iNKT cells (NKT2) in the thymus conditions CD8+ T cells to become “memory like”. However, the signals that cause NKT2 cells to constitutively produce IL-4 and what additional influence this has in the thymus are poorly defined. Using histocytometry, IL-4 producing NKT2 cells were localized to the thymic medulla, and depletion of medullary thymic epithelial cells (mTEC) using Aire-DTR mice resulted in a dramatic loss of IL-4 production. NKT2 cells express and require IL-17RB, the receptor for IL-25. Thus we examined mTEC using IL-25 reporter “FLARE” mice, and observed a distinct sub-population of chemosensory “tuft-like” cells that constitutively express IL-25 in the medulla. Using pouf3-/- and DLCKCreRosaDTA mice, we showed these cells are required for IL-4 production in NKT2 cells. We also show here that NKT2 cells require TCR stimulation for continuous IL-4 production, as NKT2 cells lost Nur77GFP and IL-4 production when intra-thymically transferred to CD1d deficient hosts. However, in bone marrow chimeric hosts, only hematopoietic, not stromal APC, provided such stimulation. These data suggest that NKT2 cells require factors from multiple APC in the thymic medulla for IL-4 production. In addition to conditioning CD8+ T cells, we show that NKT-derived IL-4 influences class switching in thymic B cells, medullary organization, and the composition of thymic macrophages. The challenge going forward is to understand what type of ligands NKT2 respond to and why this response dramatically shapes lymphocyte development.


33

How E and Id proteins regulate iNKT cell development, subset differentiation and function

Louise DCruz1, Heather M Buechel1, Adolfo B Frias1, Lisa Beppu1 1. University of Pittsburgh, Pittsburgh, PA, United States

E protein transcription factors and their negative regulators the Id proteins have been implicated in many different aspects of invariant Natural Killer T (iNKT) cell biology. Previously, we have shown the E protein transcription factor, HEB, is essential for the earliest stages of iNKT cell thymic development and that the Id protein, Id2, is required for iNKT cell peripheral survival. Additionally, we have shown that E proteins positively regulate PLZF expression in iNKT cells during thymic development and that both Id2 and Id3 were required for differentiation of specific iNKT cell subsets. Our most recent data demonstrated that Id2 was required for regulating hyporesponsive iNKT cells indicating these transcriptional regulators can affect iNKT cell function as well as differentiation and survival. Here we present new preliminary data showing Id3 is highly expressed by a subset of iNKT cells in adipose tissue. Conditional loss of Id3 resulted in significant loss of activated adipose-resident iNKT cells, while loss of both Id2 and Id3 led to complete loss of adipose resident iNKT cells. We are currently exploring how E and Id proteins together regulate adipose resident iNKT cell differentiation and function.

34

Development of functional MAIT cells.

Hui-Fern Koay1, 3, 2, Shian Su4, Daniela Zalcenstein4, Cyril Seillet4, Matthew Ritchie4, Shalin Naik4, Adam Uldrich1, 2, James

McCluskey2, Gabrielle Belz4, Daniel Pellicci1, 2, Dale Godfrey1, 2 1. Australian Research Council Centre of Excellence for Advanced Molecular Imaging, University of Melbourne, Parkville, VIC, Australia

2. Microbiology and Immunology,, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC, Australia

3. The University of Melbourne, Melbourne, VIC, Australia

4. The Walter And Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia

Mucosal-associated-invariant T (MAIT) cells detect microbial vitamin-B2 derivatives presented by the antigen-presenting molecule, MR1, and represent the largest population of ab T cells with a single specificity within the human body. Yet, MAIT cell numbers vary widely between individuals, thus it is important we understand the factors that regulate their development and function. We recently mapped the developmental pathway by identifying new populations of thymic MAIT cells in humans and mice, and the multiple checkpoints that control the generation of functional MAIT cells#. Transition through each checkpoint is regulated by MR1 and the final checkpoint that generates mature functional MAIT cells is controlled by multiple factors, including the transcription factor PLZF and microbial colonisation. MAIT cell maturation can also occur after thymic emigration of immature MAIT cells. We have now examined these MAIT cell maturation subsets from mice and humans, by RNA-sequencing in combination with flow cytometry and real time PCR to decipher the factors that are critical for this progression. In addition to validating the known cell surface profile, we also demonstrate the regulation of MAIT cells by key genetic factors involved in modulating T cell development. Notably, there is sharp downregulation of LEF1 and SATB1; and upregulation Id2, IL23R, and ICOS; between stage 1 and 3 of MAIT cell maturation. We go on to demonstrate that several of these factors play a significant role in MAIT cell maturation. This provides detailed transcriptome analysis to the mechanisms in which MAIT cells develop and the candidates that may underpin variations in the numbers of these cells.


35

A novel alternative pathway of V14+ NKT cell development directly from CD4-CD8- double-negative thymocytes, bypassing the CD4+CD8+ double-positive stage

Masaru Taniguchi1 1. Laboratory for Immune Regulation, RIKEN Center for Integrative Medical Sciences, Kanagawa, Japan

Although all NKT cells belonging to either the CD4-CD8- (DN) or CD4+ population express an invariant TCR with the same specificity, they comprise an amazing array of functionally distinct subsets classified as NKT1, NKT2, NKT17 and NKT10. Each subset has different cytokine secretion profiles and is defined by the expression of different arrays of cytokine receptors and characteristic transcription factors. In both human and mouse, the CD4-CD8- double-negative (DN) NKT cells, in striking contrast to CD4+ NKT cells, are known to elicit strong antitumor activity and Th1-type responses. These functional differences between DN and CD4+ NKT cells cannot, however, be adequately explained by the currently accepted “mainstream” or “CD4+CD8+ (DP) pathway” model of NKT cell development. Therefore, questions regarding the developmental origin of DN NKT cells remain unresolved.

Here we provide definitive genetic evidence by fate-mapping or conditional gene ablation of Rag2 gene approaches controlled by the DP stage-specific expression of E8IIICre recombinase that supports the existence of an alternative developmental pathway through which a fraction of DN NKT cells develops from late DN stage thymocytes, bypassing the DP stage. Importantly, the DN pathway preferentially gives rise to Th1-NKT cells with strong cytotoxic potential and distinct distribution patterns in peripheral tissues. These observations add new insights into our understanding of the development of NKT cells, as it appears that the differentiation stage as well as the microenvironmental niche of precursor cells undergoing positive selection may play an important role in determining their further developmental programs, ultimately leading to the emergence of various functional NKT cell subsets.

36

Are fat NKT cells born that way?

Joshua A Vieth1, Joy Das1, Fanomezana M Ranaivoson1, Davide Comoletti1, Lisa K Denzin1, Derek B Sant'Angelo1 1. Child Health Institute, Rutgers Robert Wood Johnson Medical School, New Brunswick, NEW JERSEY, United States

We pursued the concept that the TCR expressed by invariant NKT cells is functionally analogous to an innate immune receptor. As such, we considered the possibility that conserved regions of the heterodimer might control binding. Molecular modeling suggested that a hydrophobic patch, created upon TCR pairing, might have such a function. We find that that disruption of this patch by mutation of the TCRb chain ablated recognition of CD1d by the NKT TCR, but retained the capacity to functionally interact with MHCI and MHCII. Partial disruption of this region, however, did not detectably alter recognition of CD1d:aGal Cer or CD1d:OCH ligands. Unexpectedly, development of NKT cells expressing this partially disrupted TCR was substantially altered. In particular, PLZF was expressed at very low levels and the transcription factor Nfil3 (E4BP4) was induced. This expression pattern was similar to what is found in adipose resident NKT cells (arNKT cells). Indeed, we found that NKT cells bearing this mutant TCR selectively accumulated in the adipose tissue. Collectively, our data imply that developing NKT cells are directed into the arNKT cell lineage during thymic development as opposed to acquiring their distinct phenotype due to external cues in the adipose microenvironment.


44

Resolvins & pro-resolving mediator signals in resolution of inflammation

Charles Serhan1 1. Brigham and Women's Hospital, Harvard University, Boston, MA, United States

Uncontrolled inflammation is now known to be a component of many of the widely occurring chronic diseases such as arthritis, periodontal disease, asthma, cardiovascular diseases and neurodegenerative diseases. Using a systems approach with self-limited inflammatory infectious exudates to map tissue events, cell traffic and identification of protein and chemical mediators, we identified 3 structurally separate families of potent n-3 essential fatty acid-derived (EPA, DPA, DHA) novel mediators, termed resolvins, protectins and maresins. Complete structural elucidation and total organic synthesis of these new molecules demonstrated their functions in vivo in the resolution of acute inflammation in many animal models. Each member of this super-family is chemically distinct and functions as a pro-resolving local mediator that controls the duration and magnitude of acute inflammatory responses with actions in pico- to nanogram range in animal disease models. The biosynthetic pathways and potent mediators from the resolvin, protectin and maresin bioactive metabolomes are coined specialized pro-resolving mediators (SPM). Mapping of these resolution circuits provides new avenues to probe the molecular basis of many widely occurring diseases (CN Serhan, Nature 2014)1. This presentation shall focus on our recent advances in the biosynthesis and functions of specialized pro-resolving mediators (SPM), stereochemical assignments, total organic synthesis of new resolvins, SPM and their actions in counter-regulation of pro-inflammatory cytokines (TNFa, IL-6) and pro-inflammatory eicosanoids. SPM possess potent multi-pronged anti-inflammatory, pro-resolving, and anti-microbial actions in animal models. We use LC-MS-MS mediator-metabololipidomics to profile SPM in human tissues (serum, plasma2, breast milk3, adipose and brain) uncovering new pathways that stimulate tissue regeneration and bacterial clearance4-6. Several SPM are in clinical development and in ongoing clinical trials in humans. Identification of SPM during self-limited inflammations resolution phase indicates that resolution is an active programmed process challenging the old concept that resolution is a passive process where chemotactic molecules dilute and simply wane to resolve the local leukocyte exudates. Together these findings indicate that endogenous resolution pathways may underlie prevalent diseases associated with uncontrolled inflammation and open the potential for resolution-based pharmacology.

45

Defining a human T cell innateness gradient

Maria Gutierrez-Arcelus1, 2, 3, Alex R. Mola1, Susan K. Hannes1, 2, Nikola C. Teslovich4, Watts F.M. Watts1, Michael B. Brenner1,

Soumya Raychaudhuri1, 2, 3, 5, Patrick J. Brennan1 1. Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA

2. Division of Genetics, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

3. Program in Medical and Population Genetics, Broad Institute of Harvard and MIT, Cambridge, MA, USA

4. Stanford University School of Medicine, Stanford, CA, USA

5. Faculty of Medical and Human Sciences, University of Manchester, Manchester, UK

The cell types that are the focus of our field, iNKT cells, MAIT cells, and γδ T cells, blur the line between innate and adaptive immunity, manifesting innate-like rapid responses in diverse inflammatory contexts, while also using adaptive-like antigen receptors generated in the thymus through somatic recombination to recognize defined antigens. Understanding these cells that do not fit neatly into pre-existing notions of innate and adaptive immunity remains an interesting puzzle with only some pieces in place, and a fundamental question in our field.

To define the nature of human innate T cells, we undertook immunophenotyping and ultra-low-input, high-depth RNA-Seq analysis of iNKT cells, MAIT cells, Vδ1, Vδ2, Vδ3, and δ/αβ T cells, as well as adaptive and innate comparator cell populations, with the goal of identifying the transcriptional basis of T cell ‘innateness.’ To assess the global transcriptional relationships between cell types, we performed a series of bioinformatic analyses including comparative gene expression analysis using linear mixed models, principal component analysis, gene set enrichment analysis, and promoter/enhancer regulator prediction. Findings were confirmed with extensive validation and functional perturbation of key pathways.

Our quantitative assessment of global transcriptional relationships revealed an innateness gradient in which innate T cells clustered distinctly between prototypical innate and adaptive populations. Identification of the gene signatures that positively and negatively associated with this gradient allowed us to define a transcriptional basis for lymphocyte innateness, including PLZF-associated and PLZF-independent dimensions. Key pathways operating in innate T cells included cytotoxicity, chemokine and cytokine networks, redox regulation, unique mechanisms of priming, and a myc-associated altered translational state defined by regulated ribosomal biogenesis. Together, these studies demonstrate that innate T cells are a distinct arm of the immune system defined by the steady-state expression of shared transcriptional programming uncovered by studying these populations collectively.


46

iNKT cell – DC cross-talk after microbial PAMP encounter

Michael Brenner1, 2, Carlos Donado1, 2, Patrick Brennan1, 2 1. Brigham and Women's Hospital, Boston, MA, USA

2. Harvard Medical School, Boston, MA, USA

iNKT cells operate within bi-directional cellular networks to alter the quality of immune responses through both cytokine production and direct cell-cell interactions. DCs are a part of this iNKT cell network, functioning as sentinels for microbial invasion and potent activators of iNKT cells. Following exposure of DCs to microbes or their PAMPs, iNKT cells are rapidly activated. Once activated, iNKT cells in turn activate various DC effector functions, thereby enhancing downstream immune responses. During bacterial or fungal infection, we and others characterized iNKT cell-expressed IFNg and CD40L drive DC maturation and instruction, including enhanced antigen presentation, IL-12 secretion, and subsequent Th1 responses. As iNKT cells enhance immune responses in many inflammatory settings, we hypothesized that they might also modulate the cytosolic surveillance pathways involved in the assembly of inflammasomes and production of IL-1b, key regulators of innate and adaptive inflammatory responses. Here, we describe how iNKT cell cognate help directs DCs to produce high levels of IL-1b. The process requires the expression of CD1d by DCs and can occur after DC exposure to TLR agonists, even in the absence of exogenous lipid antigens. While iNKT cells trigger activation of the NLRP3 inflammasome in DCs, the majority of the IL-1β released is inflammasome-independent and occurs in the absence of cell death. Direct cell-cell interactions between iNKT cells and DCs drive early IL-1β through the NLRP3 inflammasome, and sustained IL-1b production through an inflammasome-independent mechanism. We are determining the molecular players that mediate this pathway, and its role in inflammation and host defense.

47

Using single cell analysis to understand the thymic selection of NKT cells

Lisa Kain1, 2, 3, Marie Holt1, 2, 3, Louis Gioia1, 2, 3, Steve Head1, 2, 3, Paul Savage1, 2, 3, Florian Winau1, 2, 3, Andrew Su1, 2, 3, Luc

Teyton1, 2, 3 1. The Scripps Research Institute, La Jolla, CA, United States

2. Harvard Medical School Boston, Massachusetts, USA

3. Brigham Young University Provo, Utah, USA

We have gathered several lines of evidence that show that NKT cells are selected in the thymus on two ligands, a-glucosylceramide and a-galactosylceramide. In fetal organ cultures, we could demonstrate that these two ligands were remarkably similar in their selecting abilities, both being capable of supporting positive and negative selection, depending of the dose and nature of the ligand used. As a-glucosylceramide and a-galactosylceramide distribute to the cortex and the medulla, respectively, the question we need to answer is whether the two pathways of selection operate independently or together. In order to do so, we have used knockouts of acid a-glucosidase and acid a-galactosidase, the two selective enzymes that inactivate the terminal products of degradation of each pathway, a-glucosylpsychosine and a-psychosine, respectively. We will discuss in that context the advantages and limitations of using single cell analysis technology to access T cell repertoire and gene expression profiles and assign the characteristics of the two pathways.


48

Stem Cell-Engineered iNKT Cells for Cancer Immunotherapy

Lili Yang1 1. University of California, Los Angeles, Los Angeles, CA, United States

Invariant natural killer T (iNKT) cells comprise a small population of αβ T lymphocytes. They bridge the innate and adaptive immune systems and mediate strong and rapid responses to many diseases, including cancer, infections, allergies and autoimmunity. However, the study of iNKT cell biology and the therapeutic applications of these cells are greatly limited bytheir small numbers in vivo (~0.01-1% in mouse and human blood). Here, we report a new method to generate large numbers of iNKT cells in mice through T cell receptor (TCR) gene engineering of hematopoietic stem cells (HSCs). We showed that iNKT TCR-engineered HSCs could generate a clonal population of iNKT cells. These HSC-engineered iNKT cells displayed the typical iNKT cell phenotype and functionality. They followed a two-stage developmental path, first in thymus and then in the periphery, resembling that of endogenous iNKT cells. When tested in a mouse melanoma lung metastasis model, the HSC-engineered iNKT cells effectively protected mice from tumor metastasis. Using a humanized BLT (human bone marrow-liver-thymus engrafted) mouse model, we further proved that engineering human CD34+ HSCs with human iNKT TCR gene can effectively program these cells to develop into human iNKT cells. Therefore, the HSC-iNKT method provides a powerful and high-throughput tool to investigate the in vivo development and functionality of clonal mouse and human iNKT cells. More importantly, this method takes advantage of the self-renewal and longevity of HSCs to generate a long-term supply of engineered iNKT cells, thus opening up a new avenue for iNKT cell-based immunotherapy.

49

iNKT cell emigration out of the lung vasculature during inflammation requires neutrophils and monocyte-derived dendritic cells

Ajitha Thanabalasuriar1, Arpan S Neupane1, Jing Wang1, Matthew F Krummel1, Paul Kubes1 1. University of Calgary, Calgary, Canada

Invariant natural killer T (iNKT) cells are a subset of innate T cells that recognize glycolipids presented on CD1d molecules. These cells are crucial for protection against bacterial infections, including respiratory Streptococcous pneumoniae infection. The lung harbors a resident population of iNKT cells, although these cells have been implicated to play a role in many pulmonary disease models, little is known about their behavior, localization, and function. Using lung intravital microscopy, we examined the behavior and mechanism of pulmonary iNKT cell activation in response to the specific iNKT cell ligand a-galactosylceramide or S. pneumoniae infection. In naive mice, the major fraction of iNKT cells resided in the vasculature, but a small critical population resided in the extravascular space. Administration of either a-GalCer or S. pneumoniae into the airways induced CD1d-dependent rapid recruitment of neutrophils out of the vasculature. These neutrophils guided iNKT cells from the lung vasculature via CCL17. Depletion of monocyte-derived DCs abrogated both the neutrophil and subsequent iNKT cell extravasation and activation. Moreover, impairing pulmonary iNKT cell extravasation by blocking CCL17 increased susceptibility to S. pneumoniae infection, suggesting a critical role for specifically pulmonary iNKT cell extravasation and activation in host defense.


50

Induction of anti-inflammatory purinergic signaling in activated human iNKT cells

Joel Linden1, Jennifer C. Yu2, Gene Lin1 1. La Jolla Institute for Allergy and Immunology, La Jolla, CALIFORNIA, United States

2. Pediatric Hematology/Oncology, University of California/Rady Children's Hospital, San Diego, California, United States

CD1d-restricted lipid antigens, or IL-12 and IL-18 activate invariant natural killer T (iNKT) cells that rapidly produce both Th1 and Th2 cytokines. iNKT cells collected during vaso-occlusive episodes from patients with sickle cell disease displayed strong sterile activation with elevated expression of phospho-NF-kB p65 (Ser276), Tbet, IFN-γ, IL-13 as well as anti-inflammatory adenosine A2A receptors (A2ARs) and the ecto-ATPase CD39, that dephosphorylates extracellular ATP and ADP. We hypothesized that induction of purinergic signaling molecules upon activation of iNKT cells gradually modifies their cytokine production. Activation of cultured human iNKT cells with α-CD3/α-CD28 induced mRNAs for pannexin1 and the P2X7 receptor (these molecules can dimerize to form a channel that releases ATP and NAD to the extracellular space); the equilibrative nucleoside transporter, ENT1; ecto-enzymes CD73, CD38 and CD39 (that together convert extracellular ATP or NAD to adenosine); and A2ARs. Transcript for adenosine deaminase (ADA), which degrades adenosine, was reduced. Exposure of iNKT cells to the A2AR agonist regadenoson, or the adenylyl cyclase activator forskolin during iNKT cell activation reduced production of IFN-γ and enhanced production of IL-13 and CD39. Based on these findings we define "purinergic cytokine bias" in activated iNKT cells as a gradual increase in the expression of purinergic signaling molecules (P2X7R/PANX1, CD38 CD39, CD73 and A2AR) and a decrease in ADA expression, to enhance ATP/NAD release and metabolism and adenosine signaling, and to increase the ratio of Th2/Th1 cytokine production. The data suggest that a coordinated purinergic response functions to limit the extent and duration of iNKT cell activation.

51

Exogenous glycolipids override a regulatory role of iNKT cells during auto-inflammation

Mikael Karlsson1 1. Karolinska Institutet, Stockholm, STOCKHOLM, Sweden

Invariant natural killer T (iNKT) cells serve as early rapid responders in the innate recognition of both self and foreign antigens. iNKT cells have been demonstrated to both enhance and negatively regulate B cell activation. The negative regulation has been shown to be induced during auto-inflammation in response to syngeneic apoptotic cells and inflammasome activation in response to the innate cytokine IL-18. Mechanistically, IL-18 activated iNKT cells regulate self-reactive B cell activation by preventing B cell entry into the germinal center. We have recently shown that this pathway involves neutrophils that license the iNKT cells to regulate B cells through FAS ligand mediated killing. Since activation of iNKT cells using exogenous glycolipid agonists has shown great clinical potential, we investigated whether iNKT cells exert their regulatory function and how iNKT cells respond to exogenous glycolipids during inflammation in response to IL-18. We find that exogenous glycolipids disrupt the regulatory cellular cooperation between neutrophils and iNKT cells and that iNKT cells get activated to become iNKT follicular helper cells. This in effect enhances the autoreactive B cell response including production of auto-antibodies. These findings have relevance for autoimmune diseases where infections and vaccination using glycolipids that provide ligands for iNKT cells could disrupt a potentially important negative regulatory loop and allow for autoreactive B cells to enter the germinal center.


52

Leptin receptor antagonism of iNKT cell function: a novel strategy to combat multiple myeloma

Koen Venken1, 2, Mérédis Favreau2, 3, Eline Menu3, Djoere Gaublomme1, 2, Karin Vanderkerken3, Sylvia Faict3, Ken Maes3, Elke De Bruyne3, Srinath Govindarajan1, 2, Michael Drennan1, 2, Xavier Leleu4, Kristin Jochmans3, Lennart Zabeau5, Jan Tavernier5,

Dirk Elewaut1, 2 1. VIB Inflammation Research Center, Zwijnaarde, Belgium

2. Rheumatology, Ghent University, Ghent, Belgium

3. Department of Hematology and Immunology, Myeloma Center Brussels, Vrije Universiteit Brussel (VUB), Brussels, Belgium

4. Service d’Hématologie et Thérapie Cellulaire, Pôle Régional de Cancérologie, Hospital de la Miléterie, Poitiers, France

5. VIB Medical Biotechnology Center, Faculty of Medicine and Health Sciences, University Ghent - VIB, Ghent, Belgium

A hallmark of bone marrow changes with aging is the increase in adipocyte composition, but how this impacts development of multiple myeloma (MM) is unknown. Leptin, an adipokine released by adipocytes plays a crucial influence on energy homeostasis but also displays important immune modulatory properties. In this study, we investigated whether leptin has a role in iNKT cell mediated anti-tumor immunity. We observed a marked and progressive increase in leptin levels and upregulated leptin receptor expression levels on iNKT cells during multiple myeloma progression, both in human disease and the 5T33 myeloma animal model. Moreover, iNKT cells (and also MAIT cells) are numerically impaired in blood and bone marrow samples of newly diagnosed MM patients and showed elevated PD-1 expression levels linked to disease progression. MM cells and leptin synergistically counteracted the anti-tumor function of both murine and human iNKT cells in vitro, and in vivo blockade of leptin receptor signaling in combination with iNKT stimulation resulted in superior tumor protection. The effects of LR blockade on MM development were lacking in the CD1d-/- myeloma mice, underscoring a major role for leptin in modulating iNKT cell dependent anti-myeloma effects. Additionally, in the presence of leptin receptor antagonism repeated α-GalCer administration in myeloma mice did not lead to profound iNKT cell anergy, as observed by a strikingly persistent IFN-γ secretion. A partial rescue of iNKT cell functionality could also be observed in non-diseased mice. Moreover, by means of intravital dual-photon microscopy in Cxcr6GFP/+ mice we could demonstrate a restoration of liver iNKT cell dynamics in vivo upon leptin antagonism unlike the unresponsiveness seen in anergic conditions. Altogether, these data support the novel concept that iNKT anergy upon TCR stimulation, a major drawback in iNKT based therapies, is modulated by blocking Leptin receptor signaling.

53

NKT cell-adipocyte interactions play an important role in the development of obesity

Kazuya Iwabuchi, Koki Fujita1, Misao Iizuka1, Luc Van Kaer2, Masashi Satoh1 1. Department of Immunology, School of Medicine Kitasato University , Sagamihara, Japan

2. Department of Pathology, Microbiology and Immunology, School of Medicine, Vanderbilt University, Nashville, TN, USA

The development of obesity, which is caused by adipocyte hypertrophy, is influenced by interactions within the adipose tissue between various cells of the immune system such as macrophages, T cells, B cells, innate lymphoid cells, and eosinophils. Natural killer T (NKT) cells are a subset of T lymphocytes that recognize lipid antigens in the context of the CD1d protein and are found in various tissues including adipose tissue. Using CD1d knockout (KO), we previously demonstrated that the CD1d-NKT cell axis plays an important role in the development of high-fat diet-obesity. However, the CD1d+ cell type important for interacting with NKT cells during obesity has remained elusive. Since adipocytes as well immune cells in the stromal vascular fraction express CD1d, we considered that adipocytes might be able to present lipid antigens to NKT cells. Indeed, such an interaction was demonstrated between mature adipocytes and NKT cells by several labs, including ours. To determine whether NKT cell-adipocyte interactions affect the development of obesity, we generated mice with an adipocyte-specific deletion of CD1d (Adipoq-cre-Cd1d1f/f; CD1d-AdKO), and fed these animals with a high-fat diet. CD1d-AdKO mice showed smaller body weight gain and improved insulin sensitivity as compared with control mice. In adipose tissue of CD1d-AdKO mice, the expression of the pro-inflammatory cytokine IFN-g was reduced whereas expression of the anti-inflammatory adipokine adiponectin was increased. Moreover, accumulation of macrophages was markedly reduced in adipose tissue of CD1d-AdKO mice compared with control mice. These results suggested that the interactions between adipocytes and NKT cells through a presentation of endogenous lipid antigen(s) in the context of CD1d induces production of pro-inflammatory cytokines such as IFN-g which promotes adipose tissue inflammation that contributes to obesity. Results for adipocyte-specific CD1d-AdKO mice and findings obtained with other tissue-specific CD1d-KO mice will be discussed.


54

Adipose tissue resident innate PLZF+ gd T cells are key players in immunometabolic homeostasis

Lydia Lynch2, 1, Ayano C Kohlgruber2, Michael B Brenner2 1. Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland

2. Harvard Medical School, Boston, MA, United States

Adipose tissue harbors a unique and diverse immune compartment that is important for physiologic responses to fasting and feeding, and more recently, regulation of body weight and thermogenesis. Much of what we know about the adipose tissue immune system comes from obesity studies where perturbations in immune cells or signaling molecules can either protect or contribute to local and systemic inflammation and subsequent insulin resistance. However, the immune compartment of adipose tissue from lean healthy mice and humans is substantial and little is known about its role at steady state. Approximately 80-90% of the adipose immune system is innate, and enriched for ‘unconventional’ invariant T cells, including iNKT cells, MAIT cells and gd T cells. Here we show an enriched, tissue-resident population of γδ T cells in the adipose tissue that control core body temperature in response to temperature fluctuations. They do this through cross-talk with stromal cells that provide critical cues in thermogenic regulation. While γδ T cells are thought is as the guardians at barrier sites, less is known about their steady state role in non-barrier tissues. Our findings unravel an unexpected physiological role for γδ T cells as an important communication bridge for the induction of adaptive thermogenesis.

55

RORγt inhibition selectively targets pathogenic human iNKT and γδ-T cells enriched in Spondyloarthritis while preserving IL-22 responses

Dirk Elewaut1, Koen Venken2 1. VIB Inflammation Research Center, Ghent University, Ghent, Belgium

2. Laboratory for Molecular Immunology and Inflammation, Department of Rheumatology, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium

Dysregulated IL-23/IL-17 responses have been linked to inflammatory diseases including psoriasis, psoriatic arthritis and other forms of spondyloarthritides (SpA). IL-23/IL-17 inflammation is controlled by RORγt, the key Thelper17 (Th17) cell transcriptional regulator. RORγt is also expressed by subsets of innate-like T cells, including invariant natural killer T (iNKT) and γδ-T cells, but how this contributes to disorders such as SpA is still unclear. Here we describe a unique population of RORγt+T-betloPLZF- iNKT and γδ-hi T cells present in healthy peripheral blood. iNKT and γδ-hi T cells showed profound IL-23 mediated Th17-like immune responses and were clearly enriched within inflamed joints. Unsupervised clustering analyses revealed a marked heterogeneity of human blood iNKT and γδ-T cells which seemed skewed in SpA patients. Strikingly, RORγt inhibition blocked γδ17 and iNKT17 cell function while selectively sparing IL-22+ subsets. Overall, these findings highlight a unique diversity of human RORγt+ T cells and underscore the potential of RORγt antagonism to modulate aberrant type 17 responses.


56

A functionally diverse population of human T cells recognizes non-microbial antigens presented by MR1

Marco Lepore1, Lucia Mori1, Gennaro De Libero1 1. University of Basel, 4031 Basel, Switzerland

MR1 presents riboflavin-related microbial metabolites and folate-derivatives to MAIT-cells, but it was unknown whether MR1 could present alternative antigens to other T cell lineages. We identified a novel population of human T cells (which we called MR1T cells), present in genetically different healthy individuals, displaying diverse TCR α and β chains and recognizing MR1-expressing cells in the absence of microbial ligands. Analysis of MR1T cell clones revealed specificity for distinct cell-derived antigens and alternative transcriptional strategies for metabolic programming, cell cycle control and functional polarization following antigen stimulation. Phenotypic and functional characterization of MR1T cell clones showed multiple chemokine receptor expression profiles and secretion of diverse effector molecules, suggesting functional heterogeneity. Accordingly, MR1T cells exhibited distinct T helper-like capacities upon MR1-dependent recognition of target cells expressing physiological levels of surface MR1. These data extend the role of MR1 beyond microbial antigen presentation and indicate MR1T cells are a normal part of the human T cell repertoire.

57

Regulatory B cells control the differentiation of suppressive iNKTcells via CD1d.

Claudia Mauri1, Kristine Oleinika1, Elizabeth Rosser1 1. Medicine, University College London, London, United Kingdom

Regulatory B cells (Bregs) express high levels of CD1d, however, its role in Breg biology and contribution to iNKT cell function during inflammation remains unknown. I will present data showing how Bregs “bridge" the innate and adaptive immune responses through the generation of iNKT cells with suppressive function. Mice lacking CD1d-expressing B cells developed exacerbated arthritis compared to wild-type mice and failed to respond to α-galactosylceramide treatment. In these mice we show that CD1d lipid presentation by B cells is critical for the induction of iNKT cells that in turn down-regulate Th1 and Th17 adaptive immune responses and arthritis. We will discuss novel data showing how Bregs via lipid presentation, in an IL-10 independent manner, control and restrain excessive inflammation in autoimmunity.


58

Towards the definition of the role of MAIT cells in ulcerative colitis by single cell studies

Siddhartha Sharma1, Brian Abe1, Anne Costanzo1, Marie Holt1, Gauree Konijeti1, 2, Luc Teyton1 1. The Scripps Research Institute, La Jolla, CALIFORNIA, United States

2. Scripps Green Hospital, La Jolla, California, USA

Ulcerative colitis (UC) is an increasingly prevalent form of inflammatory bowel disease. UC is characterized by a chronic T cell mediated inflammation of colonic mucosa that can cause bloody diarrhea, abdominal and rectal pain, weight loss and fatigue. Currently, it is thought that a confluence of immune dysregulation, genetics, the microbiome, and environmental factors contribute to the colonic inflammation. One immune cell subtype that may be a “link” between microbial antigens, environmental factors and chronic inflammation are the mucosal associated invariant T (MAIT) cells as they represent a significant proportion of CD8+ T cells in peripheral blood and gut mucosa. We are in the process of examining a cohort of 24 newly diagnosed UC patients, prior to treatment, using MR1 tetramers and single cell analysis to examine MAIT cells in blood, normal and inflamed colonic mucosa. After 9 patients, we have confirmed the decrease in MAIT cell frequency in peripheral blood of UC patients relative to normal blood donors (NBD) but gene expression (GE) profiling was similar for all. Locally, frequency of MAIT cells was increased 6 fold in inflamed colonic mucosa relative to patient matched uninflamed mucosa, while GE profiles revealed differences with peripheral blood MAIT cells and a unique pattern for MAIT cells isolated from inflamed mucosa. The paired TCR sequencing of cells isolated from blood and mucosa is ongoing and will reveal whether different Vb and/or CDRs sequences are correlated to the various activation profiles. Single cell RNA-sequencing will be used to detail the various GE profiles of MAIT cells in all three sites and help us understand the function of these cells in UC lesions. These preliminary results are supportive of a role of MAIT cells in the pathology of UC.

59

Cytotoxic and regulatory role of mucosal-associated invariant T cells in type 1 diabetes

Agnes Lehuen1 1. Institut Cochin, Paris, FRANCE, France

Type 1 diabetes (T1D) is an autoimmune disease resulting from the destruction of pancreatic-β cells by the immune system involving innate and adaptive immune cells. Mucosal-associated invariant T (MAIT) cells are innate-like T-cells recognizing bacterial riboflavin-precursor derivatives presented by the MHC-I related molecule, MR1. Since T1D is associated with gut microbiota modification, we investigated MAIT cells in this pathology. In T1D patients and non-obese (NOD) diabetic mice, we detected MAIT cell alterations, including increased granzyme B production, which occur before disease onset. Analysis of NOD mice deficient for MR1, and therefore lacking MAIT cells, revealed a loss of gut integrity and increased anti-islet responses associated with exacerbated diabetes. Altogether our data highlight the role of MAIT cells in the maintenance of gut integrity and the control of anti-islet autoimmune responses. MAIT cell monitoring could represent a new


60

NKT cell-targeted immunotherapy for non-small cell lung cancer

Shinichiro Motohashi1 1. Department of Medical Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan

Recent advances in cancer immunotherapy have clearly shown dramatic clinical benefits in some advanced cancer patients. The clinical benefit of immune checkpoint inhibitors, not only in immune responsive tumors, such as melanoma and renal cell carcinoma, but also in non-small cell lung cancer (NSCLC) which was previously considered to be non-responsive, has thus provided proof of the concept for the efficacy of immunotherapy. At the same time, only a limited number of patients successfully responded to immunotherapy and therefore it remains insufficiently effective for many patients. Therefore, combination therapies will likely be required to enhance the anti-tumor activity of immune therapy.

Currently, we focus our attention on the development of invariant natural killer T (iNKT) cell-targeted immunotherapy for NSCLC and head and neck squamous cell carcinoma (HNSCC) to improve the clinical outcome. Previous clinical studies have shown the intravenous injection of a-galactosylceramide (aGalCer)-pulsed antigen presenting cells (APCs) induced the activation of endogenous iNKT cells and iNKT cell-dependent responses. Moreover, an increase in the number of IFN-g producing cells in peripheral blood mononuclear cells has been shown to be associated with a prolonged survival. A dramatic infiltration of iNKT cells and accumulation of conventional T cells in the tumor microenvironment was also observed after aGalCer-pulsed APCs. Based on these results, the phase II clinical trials of aGalCer-pulsed APCs for NSCLC was designed. The protocol treatment in patients with advanced or recurrent NSCLC who received the first line chemotherapy and the follow-up period has now been completed. Data analyses are now planned after the fixation of all data.

The potential of such combination therapies targeting both iNKT cells and other immunotherapies, such as PD-1/PD-L1 inhibitors or tumor antigen specific therapies, has been demonstrated in the preclinical setting. The strategies and future perspectives for iNKT cell-targeting immunotherapy will also be discussed.

61

Immunity to the same tumor in lungs vs skin is differentially regulated by NKT cells or Treg cells and is asymmetrically cross-protective

Jay Berzofsky1, Jessica J O'Konek1, Elena Ambrosino1, Anja C Bloom1, Zheng Xia1, Masaki Terabe1 1. National Cancer Institute, NIH, Bethesda, MARYLAND, United States

It has been assumed that T cells induced at one tumor site can travel to distant tumor sites to mediate regression and that the immunity induced and its immune regulation are both determined by the biology of the tumor’s internal microenvironment, not its anatom ic location. The role of different tissue locations in immune regulation and protections, and the crosstalk between different tissue sites, is not well explored. Here, we examined the immune regulation and T cell protective mechanisms when the same tumor, the BALB/c colon carcinoma CT26, is in the lungs versus subcutaneous sites in the same mouse. We find differences in both immune regulation and immunity arising when that regulation is removed. Lack of NKT cells in CD1d-/- mice leads to tumor rejection in the lungs, but not in the skin, whereas depletion of CD25+ Treg cells protects in the skin but not in the lungs. Furthermore, the T cells induced after Treg depletion in the presence of subcutaneous tumor can also prevent tumor growth in the lungs of that mouse, but the converse is not true: the T cells that protect the lungs when NKT cells are absent do not protect against tumors in the skin of the same mouse. Also, splenic CD4+ and/or CD8+ cells from Treg-depleted subcutaneous tumor-bearing mice protect RAG-/- mice from tumors at both sites, whereas T cells from CD1d-/- mice rejecting lung tumors protect RAG-/- recipients from lung but not subcutaneous tumors. Thus, there is asymmetry in the memory T-cell cross-talk between these sites. This work demonstrates the surprising importance of tissue location, even when the tumor and mouse are identical, in determining what regulatory mechanisms are dominant and what spectrum of T cell immunity is induced. This has important implications for immunotherapy of primary and metastatic tumors.


62

Retargeting T cells against leukemia by lipid-specific TCR transfer

Michela Consonni1, Claudio Garavaglia1, Claudia de Lalla1, Alessandra Bigi1, Monica Casucci2, Attilio Bondanza2, Marco

Lepore3, Lucia Mori3, Gennaro De Libero3, Fabio Ciceri4, Paolo Dellabona1, Giulia Casorati1 1. Division of Immunology, Transplantation, and Infectious Diseases; Experimental immunology Unit, San Raffaele Scientific Institute, Milan, Italy

2. Division of Immunology, Transplantation, and Infectious Diseases, Innovative immunotherapies Unit, San Raffaele Scientific Institute, Milan, Italy

3. Experimental Immunology, Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland

4. Division of Regenerative medicine, Stem cells, and Gene therapy, Hematology and hematopoietic stem cell transplantation Unit, San Raffaele Scientific Institute, Milan, Italy

CD1-restricted T cells specific for self-lipids could play a role in cancer immune surveillance, where self-antigens are the targets of immune responses. We showed that primary acute myelogenous (AML) and B-lymphoblastic (B-ALL) leukemia blasts express CD1c and are recognized by a group of CD1c self-reactive T cells specific for methyl-lysophosphatidic acids (mLPAs), a novel class of self-lipid antigens that accumulate in malignant cells, which control leukemia growth in vitro and in vivo. These findings point to CD1c and self-lipids as new potential targets for leukemia immunotherapy. The little polymorphism of CD1 molecules and their expression on mature leukocytes are indeed highly attractive for adoptive cell therapy (ACT) with such T cells in the context of stem cell transplantation for hematological malignancies. To assess the feasibility of ACT for acute leukemia with mLPA-specific T cells, we generated a library of lentiviral vectors encoding a panel of human mLPA-specific TCRs. Upon TCR transduction, either Jurkat T cells or human primary T cells were specifically retargeted against CD1c-expressing malignant targets in vitro, highlighting a lead mLPA-specific TCR suitable for adoptive immunotherapy. Primary T cells transduced with this TCR killed CD1c-expressing malignant targets in vitro and significantly delayed leukemia progression in NSG mice. To gain further insight into the efficacy and safety of mLPA-specific ACT, we generated transgenic mice expressing CD1c with a pattern similar to the human one, which harbored functional APCs recognized by mLPA-specific T cells and selected a CD1c self-reactive peripheral T cell repertoire. Leukemia immunotherapy by lipid-specific T cells is currently investigated in the CD1c Tg mice with two T cell models: (I) retrogenic mice bearing a monoclonal T cell population expressing the human lead mLPA-specific TCR; and (II) transferred peripheral T cells engineered with the same TCR.

63

Investigating anti-lymphoma effects of human CD4+ iNKT cells in vivo: effectors or adjuvants?

Nicholas A Zumwalde1, Akshat Sharma1, Xuequn Xu1, Shannon C Kenney1, Jenny E Gumperz1 1. University of Wisconsin School of Medicine and Public Health, Madison, WI, United States

Tumor immunotherapy currently centers on two main approaches. The first is the use of "checkpoint" blockade antibodies to relieve PD-1 and CTLA-4 mediated immunosuppression of a patient's T cells, so that they become able to kill the tumor. This strategy essentially provides an adjuvant that unlocks the endogenous anti-tumor immune response. The second approach, cellular immunotherapy, involves administering cytolytic lymphocytes that act directly as anti-tumor effectors. Most prominent in this category is the use of chimeric antigen receptor (CAR) T cells, that have been genetically modified to specifically target the patient's tumor. The remarkable success of these approaches for treating a variety of cancers has generated tremendous hope and excitement, yet a number of issues remain. For example, checkpoint blockade is not always effective, or in some cases can result in highly damaging immune attacks on healthy organs. There are also concerns about the time required to generate genetically modified CAR-T cells that are individually tailored for each patient, as well as the cost and long-term safety of this strategy. Here we have investigated using CD4+ iNKT cells as a cellular immunotherapy, since prior studies in murine models have suggested that these cells have powerful adjuvant-like functions that can activate antigen-specific MHC-restricted T cell responses. To do this, we used a pre-clinical model of human B lymphomagenesis in vivo, in which human umbilical cord blood cells are transferred into immune-deficient NSG mice, and infected with the Epstein-Barr virus (EBV). This results in the de novo formation of human B lymphomas in the mice over a period of 3-4 weeks. Importantly, lymphomagenesis occurs in the presence of cognate human T cells that invade the tumors, but that usually fail to control them due to inhibition via PD-1 and CTLA-4. This system thus allows for the analysis of iNKT cell immunotherapy in the context of an immunosuppressive environment that has silenced the endogenous T cells. Administration of a single dose of CD4+ iNKT cells, with no added antigen, results in significantly reduced tumor burden in this model. Remarkably, the iNKT cell immunotherapy is effective at late time points (i.e. after tumor masses are already well-established and infiltrated by T lymphocytes), but shows little or no effect if the iNKT cells are administered at earlier time points (i.e. when EBV-infected cells are present but before tumor masses are evident). Further supporting an adjuvant-like effect by the iNKT cell immunotherapy, splenic T cells harvested from iNKT-treated but not PBS-treated control mice showed EBV-specific responses upon re-challenge with synthetic peptides in vitro. These results suggest that CD4+ iNKT cells, which typically show much less cytotoxic function than their double-negative iNKT counterparts, nevertheless may mediate potent anti-tumor responses by activating endogenous effectors. The adjuvant-like functions of CD4+ iNKT cells may thus provide an option for cellular immunotherapy that is independent of genetic modification, and that may synergize with other immunotherapeutic methods.


64

Profiling NKT cell responses in cancer patients to identify immune biomarkers

Tonya Webb1 1. University of Maryland Sch of Med, Baltimore, MD, United States

Natural killer T (NKT) cells play a critical role in anti-tumor immunity. While clinical trials focused on NKT cell modulation have had limited efficacy, the best patient responders to NKT cell-based immunotherapeutic strategies are those with detectable levels of functional NKT cells. Therefore, a specific and highly sensitive assessment of NKT cell function should be performed prior to the initiation of immunotherapy. We have developed a novel cell-based molecular diagnostic for quantitation of a patient’s NKT cell activity by monitoring IFN-γ induction. We have measured NKT cell number and function in healthy donors, breast cancer patients, and lymphoma patients. We found that in certain types of cancer, NKT cell number does not correlate with function. In addition, we have identified immune gene signatures and serum biomarkers that correlate with a reduction in circulating NKT cell function. Collectively, these data highlight the need for assessing immune cell function preceding treatment, and further suggest that NKT cell-based immunotherapeutic strategies may be more effective in specific types of cancer.

65

Harnessing natural and engineered properties of NKT cells for adoptive cancer immunotherapy

Leonid Metelitsa1 1. Baylor College of Medicine, Houston, TX, United States

T cells engineered to express chimeric antigen receptors (CARs) to CD19 produced high rates of complete responses in patients with B-cell malignancies. However, CAR T cell immunotherapy of solid tumors remains largely ineffective. Besides the differences in target antigens and tumor biology, the type of effector cells could play a major role in determining the efficacy of CAR-redirected immunotherapy. My presentation will elucidate the potential advantages of Vα24-invariant NKT cells (iNKTs) compared with conventional T cells as a carrier of CARs or recombinant TCRs for immunotherapy of solid tumors. First, human iNKTs and CAR iNKTs better localize to the tumor site compared with T and CAR T cells from the same PBMC preparations in a xenogeneic model of neuroblastoma in NSG mice. Second, in addition to direct killing of tumor cells via a tumor-specific CAR, iNKTs can kill or functionally reprogram tumor-supportive M2-like macrophages in a CD1d-dependent manner. In contrast, polyclonal T cells are devoid of CAR-independent anti-tumor activities. Third, unlike T cells, iNKTs from allogeneic donors are not expected to induce graft-versus-host disease in HLA-mismatched recipients. Moreover, we recently engineered CAR constructs that co-express shRNA targeting expression of HLA molecules on the cell surface of iNKTs without making them susceptible to NK-cell cytotoxicity. The resultant CAR iNKTs can be universally tolerated and produced in bulk from selected healthy volunteers for a cost-effective “off-the-shelf” cancer immunotherapy.


66

AhR regulation of CD1d function in the intestinal epithelium

Richard Blumberg1, Shankar S Iyer1, Thomas Gensollen1, Amit Gandhi1, Sungwhan F Oh1, Amadeu Llebaria2, Anthony

Maxwell3 1. Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA

2. Laboratory of Medicinal Chemistry, Department of Biomedicinal Chemistry, Institute of Advanced Chemistry of Catalonia IQAC-CSIC, Barcelona, Spain

3. Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, UK

iNKT cells have been recognized to play an important role in defining host commensalism in mucosal surfaces and the response to mucosal pathogen exposures. At the same time, iNKT cells are under the influence of the commensal microbiota such that in the germ-free (GF) state iNKT cell quantity is increased in the intestines and lungs and can only be normalized when microbes are present during an early life “window of opportunity” when the host is responsive to microbial signals such as those derived from sphingolipid producing bacteria such Bacteroides fragilis. Interestingly, oxazolone-induced colitis occurs and with increased severity in the GF state. Normalization of responses to oxazolone in fact only occur if microbial commensalism is established in early, but not later life in association with normalization of iNKT levels to that observed under SPF conditions. This suggests that a set-point is established in early life that determines CD1d-restricted iNKT cell responses to environmental factors capable of stimulating iNKT cell-mediated inflammation, as modeled with oxazolone as an agent of exposure. This is interesting in light of current hypotheses for IBD pathogenesis wherein environmental triggers activate inflammation in a susceptible host. This lead us to hypothesize that oxazolone may be an example of a much larger collection of environmental chemical moieties capable of triggering CD1d-restricted iNKT cell responses. We have thus sought evidence for the existence of oxazolone-related chemicals in the environment and microbiota capable of inducing intestinal inflammation. In so doing, we have identified environmental mimetics of oxazolone that are derived from dietary or microbial sources which are characterized by the presence of a 5-membered oxazole ring and capable of driving CD1d-dependent inflammation through activation of the aryl hydrocarbon receptor pathway within IECs of the colon. As efforts to define and evaluate natural and synthetic compounds from dietary, industrial and agricultural sources, in either homeostatic or disease contexts have generally been elusive, these results have important and broad disease implications.

67

iNKT cell suppression of tumor immunity in intestinal polyposis

Susanna Cardell1 1. Department of Microbiology and Immunology, University of Gothenburg, Gothenburg, Sweden

CD1d-restricted invariant natural killer T (iNKT) cells are known as potent early regulatory cells of immune responses. Besides the established roles in the regulation of inflammation and autoimmune disease, many studies have shown that iNKT cells have important roles in tumor surveillance and the control of tumor metastasis. In contrast, we demonstrate that absence of iNKT cells dramatically decreased the number of intestinal polyps in APCMin/+ mice, a model for colorectal cancer. In this model, regulatory iNKT cells promoted intestinal polyp formation by locally enhancing Treg cells and anti-inflammatory M2 macrophages, and immunosuppression of anti-tumor TH1-immunity. Polyp iNKT cells were enriched for IL-10 and IL-17 producing cells, showed a distinct phenotype, and they were negative for the NKT cell transcription factor PLZF. To investigate whether iNKT cell directed therapy could subvert the tumor promoting function of iNKT cells and reduce tumor growth, we performed preclinical therapeutic studies using different iNKT cell ligands. iNKT cell directed immunotherapy could reduce the number and size of polyps, however, the choice of activating ligand, and timing of treatment, was essential.


68

CD1a: a key player in inflammatory skin diseases

Ji Hyung Kim1, 2, Yu Hu1, 2, Tang Yongqing3, Jessica Kim1, 2, Victoria A. Hughes3, Jérôme Le Nours3, Elsa A. Marquez3,

Anthony W. Purcell3, Qi Wan1, 2, Masahiko Sugita4, Jamie Rossjohn3, Florian Winau1, 2 1. Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, Massachusetts, United States

2. Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, United States

3. Infection and Immunity Program & Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia

4. Institute for Virus Research, Kyoto University, Kyoto, Japan

In contrast to conventional T cells that recognize peptides on MHC proteins, CD1 molecules present lipid antigens to T lymphocytes. The abundant expression of CD1a hallmarks Langerhans cells in the skin, a subtype of dendritic cell (DC) with antigen-presenting functions. CD1a can bind and display a broad spectrum of lipid antigens derived from exogenous sources, such as bacteria, or host origin. The intricate immune system of the skin is critically involved in responses to extrinsic insults like allergens, as well as in autoimmune diseases, such as psoriasis. However, the in vivo role of CD1a on Langerhans cells remains unclear, principally because CD1a is expressed in humans but lacking in mice. To overcome this obstacle, we generated human CD1a-transgenic mice and investigated the impact of CD1a on skin inflammation. Here we show that the lipidic molecule urushiol from the plant poison ivy induces severe skin inflammation in a CD1a-dependent fashion. The immune response is exclusively driven by CD1a-expressing Langerhans cells that elicit the generation of CD4 T cells, producing the inflammatory cytokines IL-17 and IL-22. Notably, human subjects with poison ivy dermatitis showed a similar cytokine signature following CD1a-mediated urushiol recognition. Among different urushiol congeners, we identified diunsaturated pentadecylcatechol (C15:2) as the dominant antigen for CD1a-restricted T cells. We determined the crystal structure of the CD1a-urushiol (C15:2) complex to 1.9Å resolution, demonstrating the molecular basis of urushiol interaction with the antigen-binding cleft of CD1a. In a model for psoriasis, CD1a massively amplified inflammation mediated by Th17 cells reactive with self lipid antigens from skin. Strikingly, treatment with blocking antibodies against CD1a fully abrogated skin inflammation. Patients suffering from psoriasis showed strong inflammatory T cell activation in response to CD1a. Thus, we propose CD1a as a novel target for future therapeutic strategies against inflammatory skin diseases.

69

Viruses and MAIT cells

Paul Klenerman1 1. University Of Oxford, Oxford, UK

MAIT cells can respond to MR1 and microbially-derived ligand via their TCR. They can also be readily activated in a TCR-independent manner, through sensing of inflammatory cues. This behaviour potentially broadens their involvement in host defence and inflammation. We have previously explored this and shown that in humans, MAIT cells (and related cell types) are readily activated in vivo in response to diverse virus infections via cytokines including IL-18 and Type I interferon. In subsequent studies, my group have collaborated with James McCluskey’s team working with murine MAIT cells and have now addressed whether such viral sensing has any protective or pathogenic role in vivo. In this talk I will describe some of the key findings regarding activation and protection using intact C57BL/6, knockout and transfer models. Since MAIT cells are highly abundant in humans, and since they are readily activated by a variety of significant viral pathogens, such proof-of-principle is important in understanding their role in disease. This non-TCR dependent behaviour may also be relevant in bacterial infections, even for pathogens with an intact riboflavin synthesis pathway.


70

Colonic iNKT cell establishment is specifically regulated by macrophages during early life.

Thomas Gensollen1, 2 1. Brigham and Women's Hospital, Boston, MA, United States

2. Harvard Medical School, Boston, MA, United States

Substantial evidence exists that the interactions between the commensal microbiota and the immune system of intestinal tissues during early life may set the stage for later development of the immune system and susceptibility to inflammatory bowel diseases (IBD). Among the numerous immune cells that are involved in the pathogenesis of IBD, invariant natural killer T (iNKT) cells are increasingly recognized to play an important role. The absence of microbiota during early life but not thereafter leads to iNKT cell accumulation in the colon and incurs later life susceptibility to colitis initiated by environmental triggers. The mechanisms underlying this accumulation of iNKT cells and how they are regulated by the microbiota exclusively during the “neonatal window of opportunity” remains elusive. Consistent with previous studies, we used parabiotic mice to demonstrate that iNKT cells are tissue resident cells in adult life. This suggests that the iNKT cell niche is therefore established in early life. Therefore, we used a specific model of transgenic mice that allows for inducible depletion of Csf1r and Lyz2 expressing macrophages and showed that macrophages can regulate iNKT cell numbers in peripheral tissues in a very limited period of time in the colon but not other tissues such as spleen and thymus in a pathway that is CD1d-independent. We show that regulation of iNKT cells by macrophages during this specific window of time during early life can influence later life activation of colonic iNKT cells. Further, early life colonic macrophages are regulated by microbiota and produce signal(s) that are currently under investigation using RNA-Seq that may be responsible for the establishment of iNKT cells in the colon. In summary, we have identified colonic macrophages as regulators of iNKT cell residence specifically during the “neonatal window of opportunity” and describe signals regulated by microbiota that may support this process.

71

Inhibition of endocytic lipid antigen presentation by common lipophilic environmental pollutants

Manju Sharma1, Xiang Zhang1, Shuangmin Zhang1, Liang Niu1, Shuk-mei Ho1, Aimin Chen1, Shouxiong Huang1 1. University of Cincinnati College of Medicine, Cincinnati, OH, USA, United States

Air pollution is a major global health threat contributing to millions of annual deaths. Air pollutants as non-heritable factors are now recognized as triggers for multiple human inflammatory disorders involving T cells. We postulate that lipid antigen presentation for T cell activation is susceptible to lipophilic air pollutants, such as polycyclic aromatic hydrocarbons. To test this hypothesis, we determined whether the common polycyclic aromatic hydrocarbons, benzo[a]pyrene and diesel exhaust particles, impact on the activation of lipid-specific T cells. Our results demonstrated that the expression of CD1a and CD1d proteins, and the activation of CD1a- and CD1d-restricted T cells were sensitively inhibited by benzo[a]pyrene even at the low concentrations detectable in exposed human populations. Similarly, diesel exhaust particles showed a marginal inhibitory effect. Using transcriptomic profiling, we discovered that the gene expression for regulating the endocytic function, lipid metabolism, and the production of cytokines and chemokines was perturbed by benzo[a]pyrene. Imaging flow cytometry also showed that CD1a and CD1d proteins were retained in early and late endosomal compartments, respectively, supporting an impaired endocytic lipid antigen presentation for T cell activation upon benzo[a]pyrene exposure. This work conceptually demonstrates that lipid antigen presentation for T cell activation is inhibited by lipophilic pollutants through profound interference with gene expression and endocytic function, likely further disrupting regulatory cytokine secretion and ultimately exacerbating inflammatory diseases.


72

Ontogeny of human mucosal-associated invariant T cells and related T cell subsets

Olivier Lantz1, Ghada B Youssef2, Marie Tourret2, Marion Salou1, Liana Ghazarian2, Sophie Caillat-Zucman2, 3 1. Institut Curie, PSL Research University, Inserm, Paris, France

2. Institut national de recherche médicale (INSERM), Center for Research on Inflammation; Paris Diderot University, Paris, France

3. Laboratoire d’Immunologie, Hôpital Saint-Louis, Paris, France

Human and murine MAIT cells share many characteristics (specificity, phenotype and effector activities) with the exception that 1) murine MAIT cells already acquire a memory (CD44hi) phenotype in the thymus and do not expand much in the periphery while human MAIT cells are naïve at birth in the cord blood before acquiring a memory phenotype and expanding to reach high clonal size in adulthood. 2) In mice, MAIT cells can be divided into two subsets expressing either T-bet or RORgt, the latter being the most abundant. In contrast, human MAIT cells simultaneously express T-bet, RORgt, EOMES and IKZF2 (Helios).

In human cord blood, Va7.2-CD161high T cells are more abundant than the Va7.2+ subset. Measurements of Va7.2+CD161high and related subsets (Va7.2-CD161high and NKT) in infants of different gestational ages indicate that Va7.2+ and Va7.2- CD161high T cells undergo an early wave of thymic development during gestation, and likely share a common prenatal developmental program. CD161high T cell (either Va7.2+ or Va7.2-) frequencies in the cord blood are highly correlated between twins irrespective of the homo or hetero-zygotic status, indicating that prenatal environmental factors may control their development. Contrary to adult blood, in which virtually all Va7.2+ CD161high T cells are labeled with MR1:5-OP-RU tet, only a small proportion of Va7.2+ CD161high T cells in cord blood are stained by the tetramer. Interestingly, these cells display a TRAV and TRBV repertoire very similar to adult MAIT cells. A few days after birth, all Va7.2+ CD161high T cells acquire a memory phenotype and few week later, the great majoriy is labeled with MR1:5-OP-RU tetramer. This suggests that only the MR1:5-OP-RU-reactive cells expand in the periphery, diluting out other Va7.2+CD161high and Va7.2- CD161high populations. Still, it takes five to six years to reach adult MAIT cells frequencies. Our data suggest that the high clonal size of adult MAITs is antigen-driven and likely due to the fine specificity of the TCR a and b chains allowing recognition of MR1-restricted microbial antigens.

Altogether, our data emphasize the importance of environmental factors in shaping MAIT cell development. Moreover, the two-step development pathway of MAIT cells occurs in the thymus in mice while the final maturation step occurs in the periphery after birth in humans.

73

Possible protective roles of MAIT cells and environmental iNKT cell antigens in asthma in inner-city children.

Gerhard Wingender1, Shilpi Chandra, Jason A Greenbaum, Archana Khurana, Amin M Gholami, Michael Rosenbach, Katy Jaffee, James E Gern, Robert Wood, George O'Connor, Megan Sandel, Meyer Kattan, Leonard Bacharier, Alkis Togias,

Anthony A Horner, Mitchell Kronenberg 1. Izmir Biomedicine and Genome Center (IBG), Balcova/Izmir, N/A, Turkey

In human asthma the involvement of iNKT cells is still controversial, while there has been little analysis of MAIT cells. Using samples from the URban Environment and Childhood Asthma (URECA) birth cohort study, we carried out a comprehensive analysis of iNKT cells, MAIT cells and mainstream CD4+ T cells from the peripheral blood, and of the iNKT cell antigenic activity in house dust samples. These values were correlated with clinical outcomes, including aeroallergen sensitization and recurrent wheeze at 3 and asthma at age 7. Our results indicate that the iNKT cell frequency in the blood at one year of age was not correlated with the development of allergic sensitization or asthma. However, an increased content of iNKT cell antigens in the house dust was associated with protection from asthma. Furthermore, the frequency of MAIT cells was associated with increased production of IFNg by activated CD4+ T cells from children that did not go on to develop asthma. Moreover, an increased MAIT cell frequency early in life was associated with a decreased risk of asthma. Together, these data suggest that MAIT cell frequency and iNKT cell antigen exposure early in life correlates with protection from asthma development.


74

Homeostatic immunity and the microbiota

Yasmine Belkaid1 1. National Institute of Health, Rockville Pike, United States

Content not available at time of print.

200

iNKT cells in intestinal immunity

Patricia Barral1 1. King's College London, London, United Kingdom

The mammalian gastrointestinal mucosa is colonized by a highly complex mixture of microorganisms that establish a mutualistic relationship with the host. The defence system of the intestine comprises an epithelial layer and a plethora of immune cells that restrict commensals within the gut while preserving their number and diversity. Conversely, commensals are required for the development of a fully functional immune system. There is growing evidence for a key role for iNKT cells, CD1d expression and lipid presentation in the modulation of intestinal immunity. iNKT cell recognise lipids from commensals and many mucosal cell populations (dendritic cells, B cells, intestinal epithelial cells, innate lymphoid cells) are able to internalise and load lipids on CD1d, yet the contribution of the distinct CD1d+ populations to intestinal immunity remains poorly understood. We have recently investigated how CD1d+ cells contribute to the presentation of intestinal lipids, the effect that they exert on mucosal iNKT cells and ultimately their role in the regulation of intestinal homeostasis.

201

Regulation of CD1a-dependent T cell activation in human skin

Annemieke de Jong, G C Monnot1, C Tejeda1, A Chang1, H F Jiang2, 3, W Zeng1, C Rhode4, S Cremers2, 3, 5 1. Department of Dermatology, Columbia University Medical Center, New York, NY, USA

2. Department of Pathology & Cell Biology, Columbia University Medical Center, New York, NY, USA

3. Department of Medicine, Columbia University Medical Center, New York, NY, USA

4. Department of Surgery, Columbia University Medical Center, New York, NY, USA

5. The Irving Institute for Clinical and Translational Medicine, Columbia University Medical Center, New York, NY, USA

Beyond providing barrier function, extracellular lipids in human skin are thought to play an immunological role, since we have previously shown that small hydrophobic lipids present in sebaceous glands and stratum corneum can function as antigens for CD1a-restricted T cells. Recent publications showing a central role for CD1a in skin inflammatory conditions such as psoriasis and allergic contact dermatitis, suggest that CD1a-restricted T cells can have an immunopathogenic role. Yet, their presence in healthy skin, together with CD1ahigh Langerhans cells and an abundance of antigenic lipids, suggests that under homeostatic conditions, mechanisms must be in place to regulate the activation of CD1a-restricted T cells. Our research focuses on the nature of potential regulatory mechanisms, including influence of 1) relative amounts of antigenic lipids and non-antigenic lipids competing for CD1a binding 2) the effects of common skin commensals/pathogens, and 3) expression of inhibitory receptors on CD1a-autoreactive T cells. Addressing the first mechanism using cell-based and cell-free plate-bound CD1a protein and CD1a-restricted T cell lines, we have preliminary evidence that ceramides, which are the most abundant extracellular lipids in human skin, can displace antigenic lipids from CD1a, thereby inhibiting lipid-specific T cell activation. Ceramides with long acyl chains more effectively displaced the antigenic lipids than shorter chain analogs. This is potentially relevant in atopic dermatitis, where not only the total amount of ceramides, but also specifically longer chain ceramides are reduced. Our recently initiated investigation in atopic dermatitis patients and controls, aims to determine if a relative reduction in non-antigenic ceramides in stratum corneum is correlated with increased frequencies and activation of CD1a-autoreactive T cells in the skin. This would support the notion that the shifts in skin lipid composition observed in this inflammatory skin disease, in particular a decrease in non-antigenic CD1a binding lipids, may partially underlie increased T cell activation in the skin.


212

Epigenetic regulation of NKT cell development

Florian Winau1 1. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA

Invariant Natural Killer T (iNKT) cells recognize lipid antigens presented on CD1d molecules and protect the host against numerous diseases. Transcription factors, such as T-bet and PLZF, participate in iNKT cell development in the thymus, inducing expression of target genes that enable iNKT cells to perform unique effector functions. However, it is unclear how a multitude of transcription factors is orchestrated within an epigenetic framework that controls lineage-specific gene expression. Transcription of lineage-specific genes is regulated by dynamic change of the histone methylation state. In particular, enzymatic removal of methyl groups by the H3K27-specific demethylases UTX and JMJD3 facilitates gene transcription. While widely studied in stem cell biology, little is known about this epigenetic regulation in leukocyte development. Here, we find that H3K27-specific demethylases are essential cell-intrinsic factors for iNKT cell development. UTX-deficient iNKT cells exhibit impaired expression of iNKT signature genes, including NK cell receptors and the transcription factor Tbx21. Genome-wide epigenetic profiling uncovers a decrease in activation-associated H3K4me3 and a reciprocal increase in repressive H3K27me3 marks within the promoters of genes that are downregulated in UTX-deficient iNKT cells. UTX binds to these promoters and its enzymatic demethylase activity is vital for establishing the iNKT gene expression program. Moreover, we identify the AP-1 transcription factor JunB as novel master regulator of the iNKT cell gene signature, highlighted by its molecular interaction with UTX. In addition, we discovered that iNKT cells harbor super-enhancers for their key transcription factors T-bet, PLZF, and JunB, underlining their role in lineage specification. Taken together, we propose a dual mode of action in which UTX binds to the promoters of hallmark genes and in parallel interacts with key iNKT cell transcription factors to regulate gene expression. These findings have profound implications for our understanding how T cell development is controlled by sophisticated epigenetic mechanisms.

213

The differing roles of CD1d2 and CD1d1 proteins in type I Natural Killer T cell development and function

Laurent Gapin1, Srinivasan Sundararaj2, Jingjing Zhang1, Sai Harsha Krovi1, Romain Bedel1, Kathryn Tuttle1, Natacha

Veerapen3, Gurdyal S Besra3, Jerome Le Nours2, Jennifer Matsuda1, 4, Jamie Rossjohn2, 5 1. University of Colorado, Aurora, CO, United States

2. Monash University, Melbourne, Australia

3. University of Birmingham, Birmingham, UK

4. National Jewish Health, Denver, CO, USA

5. Cardiff University, Cardiff, United Kingdom

Major histocompatibility complex class I-like CD1 molecules have evolved to present lipid-based antigens to T cells. Differences in the antigen-binding clefts of the CD1 family members determine the conformation and size of the lipids that are presented, although the factors that shape CD1 diversity remain unclear. In mice, two homologous genes, CD1D1 and CD1D2, encode the CD1d protein, which is essential to the development and function of Natural Killer T (NKT) cells. However, it remains unclear whether both CD1d isoforms are equivalent in their antigen presentation capacity and functions. CD1d2 molecules can be expressed in the thymus of some mouse strains where they select functional type I NKT cells. Intriguingly, the TCR repertoire and phenotype of CD1d2-selected type I iNKT cells in CD1D1-/- mice differs from CD1d1-selected iNKT cells. The structure of CD1d2 revealed that the A´ pocket of its antigen-binding groove is markedly restricted in size compared to CD1d1 molecules. Accordingly, CD1d2 molecules cannot present glycolipid antigens with long acyl chains efficiently, favoring the presentation of short acyl chains antigens. These results indicate that the two CD1d molecules present different sets of self-antigen(s) in the mouse thymus, thereby potentially impacting the development of iNKT cells.


RAPID FIRE ABSTRACTS

202

Mucosal-associated invariant T cells augment immunopathology in chronic Helicobacter pylori infection

Criselle D'Souza1, 2, Troi Pediongco1, Huimeng Wang1, Jean-Pierre Y Scheerlinck2, Lyudmila Kostenko1, Robyn Esterbauer1, Andrew Stent2, Sidonia BG Eckle1, Bronwyn S Meehan1, Richard A Strugnell1, Hanwei Cao1, Ligong Liu3, 4, Jeffrey YM Mak3, 4, George Lovrecz5, Louis Lu5, David P Fairlie3, 4, Jamie Rossjohn6, 8, 7, James McCluskey1, Alison L Every2, Zhenjun

Chen1, Alexandra J Corbett1 1. Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Melbourne, VIc, Australia

2. Centre for Animal Biotechnology, Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Melbourne, Vic, Australia

3. Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld, Australia

4. Australian Research Council Centre of Excellence in Advanced Molecular Imaging, The University of Queensland, Brisbane, Qld, Australia

5. BioMedical Manufacturing, CSIRO, Melbourne, Vic, Australia

6. Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Melbourne, Vic, Australia

7. Institute of Infection and Immunity, Cardiff University School of Medicine, Cardiff, United Kingdom

8. ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, Vic, Australia

Mucosal-associated invariant T (MAIT) cells produce inflammatory cytokines (IL-17, IFNγ, TNF), and cytotoxic granzymes in response to by-products of microbial riboflavin (vitamin B2) synthesis. Although MAIT cells are protective against some pathogens, we reasoned that they might contribute to pathology in chronic bacterial infection. MAIT cells could be detected in human gastric tissue using MR1-tetramers. By immunofluorescent staining we observed MAIT cells in proximity to Helicobacter pylori bacilli in gastric tissue from three individuals. In order to determine whether MAIT cells contribute to chronic inflammation and gastritis following Helicobacter infection, we examined the MAIT cell response in a mouse H. pylori SS1 infection model. Following infection, MAIT cells accumulated to high numbers in the gastric mucosa of both MAIT TCR transgenic mice and wild-type C57BL/6 mice with pre-boosted MAIT cells, as well as a proportion of wild-type mice that had no pre-boosting. Gastric MAIT cells possessed an effector memory Tc1/Tc17 phenotype, and were associated with accelerated gastritis characterised by augmented recruitment of neutrophils, macrophages, dendritic cells, eosinophils and non-MAIT T cells. These changes were accompanied by marked gastric atrophy. Interestingly, one long-term infected MAIT TCR transgenic mouse developed gastric lymphoma. Thus, we demonstrate a pathogenic role for MAIT cells in Helicobacter-associated inflammation, revealing a broader potential role for MAIT cell-driven immunopathology in chronic bacterial infection.

203

Natural Killer T cells are master regulators of post-sepsis immunosuppression

Edy Kim1, Hadas Ner-gaon2, Ji Young Choi1, Jing Yu Guo1, Christophe Benoist3, Tal Shay2, Michael B Brenner1 1. Brigham & Women's Hospital, Cambridge, MA, United States

2. Dept. of Life Science, Ben-Gurion University of the Negev, Beersheba, Israel

3. Dept. of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA

Publish consent withheld


204

The TCR signal strength influences the specification of iNKT subsets

Sai Harsha Krovi1, Kathryn D Tuttle1, Jingjing Zhang1, Romain Bedel, Lisa K Peterson, Leonard L Dragone, Kent Riemondy1,

Jay Hesselberth1, James Scott-Browne, Laurent Gapin1 1. University of Colorado Denver, Aurora, COLORADO, United States

Invariant Natural Killer T (iNKT) cells play a central role in several immune responses in diverse biological contexts ranging from autoimmunity, injury and infections to pregnancy and cancer. Three major phenotypically and functionally distinct iNKT cell subpopulations, each with a propensity to traffic to different tissues and to secrete different cytokines upon activation, have been identified. These fate assignments are already evident in the thymus, suggesting that they are conferred upon iNKT cells during selection, although the cues that direct these decisions remain poorly understood. Since the strength of signal propagated through the T cell antigen receptor (TCR) has been demonstrated to influence T cell fate both in the thymus and periphery, we explored the contribution of the TCR to iNKT subset diversification. Here, we show that in wildtype mice, the avidity of the iNKT TCR correlates with iNKT cell subsets and the differential expression of various markers reflecting strength of signaling during selection. Interestingly, the TCRβ chains utilized by the iNKT cells are unique to each iNKT cell subset. Additionally, altering the signal transduced through the TCR during selection, either through the use of a fixed TCRβ chain or a mouse containing a hypomorphic TCR signaling kinase (Zap70), dramatically affected the ability of iNKT cells to properly diversify, exemplified by the preferential reduction of the PLZFhi and RORγt+ subsets. These data provide support for the idea that the TCR signaling event perceived by each iNKT precursor during selection serves as a branching point for the different cells, each of which then follows a distinct developmental program culminating in differentiation into one of the three lineages.

205

A dominant immune mechanism mediated by type I NKT cell subsets in different phases of non-alcoholic fatty liver disease

Idania Marrero1, Igor Maricic1, Akiko Eguchi1, Surya Dasgupta1, Ariel Feldstein1, Carolyn Hernandez1, Rohit Loomba1, Vipin

Kumar1 1. University of California San Diego, La Jolla, CALIFORNIA, United States


206

MAIT and iNKT cell subsets share similar tissue residency programs

Marion Salou1, Anne-Gaëlle Goubet1, Virginie Premel1, Aurélie Darbois1, Ruby Alonso1, Francois Legoux1, Olivier Lantz1 1. Institut Curie, Paris, FRANCE, France


207


TET proteins control iNKT cell lineage specification and TCR mediated expansion

Ageliki Tsagaratou 1, Sini Rautio 2, Edahi Gonzalez Avalos1, James Scott-Browne 1, Susan Togher 1, Lukas Chavez3, Ellen V

Rothenberg 4, Harri Lähdesmäki3, Anjana Rao1 1. La Jolla Institute, LA JOLLA, CA, United States

2. Department of Information and Computer Science, Aalto University School of Science, Aalto, Finland

3. Computational Oncoepigenomics Group, Division of Pediatric Neurooncology , German Cancer Research Center (DKFZ), Heidelberg, Germany

4. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA

TET proteins are 2-oxoglutarate- and Fe(II) dependent dioxygenases that catalyze the hydroxylation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidation products (5-formylcytosine and 5-carboxylcytosine) in DNA. They can act as mediators of “active” (replication-independent) DNA demethylation, achieved through excision of 5fC and 5caC by thymine DNA glycosylase (TDG) followed by replacement with an unmethylated cytosine by base excision repair. They are also novel epigenetic marks that are recognized specifically by readers.

The role of TET proteins in developing thymocytes remains elusive. In the present study, we show that simultaneous deletion of TET2 and TET3 in mice (DKO) results in aberrant development of invariant NKT cells that are skewed towards the NKT17 lineage. We also found upregulation of proliferation controlling genes as well as genes that are normally expressed in precursor cells. This results in an unprecedented expansion of iNKT cells, leading to lethality. The expansion is driven by antigen recognition, since the disease is effectively transmitted to fully immunocompetent recipient mice only if they express CD1d, a non-classical MHC protein that presents lipid antigens to iNKT cells. Molecular analysis revealed that TET2 and TET3 drive iNKT lineage specific DNA demethylation and control gene expression via DNA demethylation and/or chromatin accessibility of key lineage specifying factors such as RORgt, Tbet and ThPOK.

Collectively, TET2 and TET3 are fundamental regulators that seal iNKT lineage fate, ensure proper development and maturation and safeguard aberrant TCR mediated expansion.

208

Defective NKT cell development in BRCA-1 mutant mice

Susannah C Shissler1, Tonya J Webb1 1. University of Maryland, Baltimore, Baltimore, MD, United States

Breast cancer accounts for 250,000 new cancer diagnoses annually – making it a significant health burden. Inherited mutations in Breast Cancer susceptibility (BRCA)-1 or 2 genes significantly increase the likelihood of cancer development. Upon diagnosis, women with BRCA-1 mutations are more likely to present at a younger age and with a more aggressive phenotype. Despite advances in screening and treatment, breast cancer incidence and mortality rates have remained constant over the past three decades, underscoring the need for innovative treatment strategies, particularly immunotherapy. In order for immunotherapy to be effective, one must have a functioning immune system. Preliminary studies indicate a profound reduction in natural killer T (NKT) cells in women with BRCA mutations. Similarly, in a BRCA-1 mutant mouse model, there is a 70-85% decrease in NKT cells in the periphery and in the thymus. NKT cells play a critical role in cancer immune surveillance, thus we hypothesize that mutations in BRCA lead to impaired NKT cell development and this loss contributes to cancer development. NKT cell development diverges from conventional T cells at the double positive stage where they divert into a developmental pathway characterized by four distinct stages. This study aims to determine at which stage in thymic development NKT cells are lost in BRCA-1 mutant mice, and the mechanism accounting for the paucity of NKT cells in the periphery. BRCA-1 mutant NKT cells are primarily restrained in stage 2 of development with very few progressing to stage 3. Importantly, this research identifies a major deficit in host anti-tumor immunity. This work will aid in understanding the mechanisms by which BRCA-1 regulates NKT cell development, and may lead to the development of novel immunotherapeutic strategies.

209


Bacteria license antigen presenting cells for MR1-mediated MAIT cell activation

Tom Williams1, Rajesh Lamichhane1, Sara de la Harpe2, Joel Tyndall2, Andrea Vernall2, James Ussher1 1. Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand

2. School of Pharmacy, University of Otago, Dunedin, New Zealand

MAIT cells are antibacterial T cells that are abundant in liver and mucosal surfaces. The MAIT cell T cell receptor is restricted by MR1, which presents antigens derived from 5-amino-6-d-ribitylaminouracil (5-A-RU), a metabolic precursor of riboflavin synthesis. Many different bacterial species, both pathogenic and commensal, can produce 5-A-RU, and the antigens derived from 5-A-RU can be found in the supernatant of bacterial cultures. Given the abundance and anatomical location of MAIT cells and the multiple sources of 5-A-RU, MR1-mediated MAIT cell activation must be tightly regulated to prevent immunopathology.

Previous work has suggested that TLR agonists and NFkB may regulate MR1 surface expression and E. coli-induced MAIT cell activation1. In this study, we have dissected the regulation of MR1-mediated MAIT cell activation by bacteria. Using THP1.hMR1 cells, we found that non-ligand producing bacteria (E. coli ΔribD and Enterococcus faecalis) and TLR-1 and -6 agonists were able to increase MR1 surface expression in the absence of 5-A-RU. When combined with 5-A-RU, non-ligand-producing bacteria strongly enhanced MR1 surface expression compared with 5-A-RU alone; in contrast, no enhancement was seen with TLR agonists or with lysed bacteria. Inhibiting phagocytosis with cytochalasin D significantly reduced E. coli ΔribD + 5-A-RU-mediated MR1 upregulation. Inhibiting ER-Golgi transport with brefeldin A completely inhibited MR1 upregulation to all stimuli. Finally, we confirmed that MAIT cell activation in response to 5-A-RU was enhanced by non-ligand producing bacteria.

We propose a model whereby MAIT cell activation is enhanced in the presence of bacteria, both through antigen-presenting cell (APC) licensing via pattern recognition receptors and through delivery of 5-A-RU to the phagosome. From the phagosome, 5-A-RU (or the pyrimidine ligand that is derived from it) accesses the ER by an unknown mechanism before trafficking to the cell surface. Therefore, robust MR1-mediated MAIT cell activation requires phagocytosis of tissue-invasive bacteria by APCs.

1. 1. Ussher JE, van Wilgenburg B, Hannaway RF, Ruustal K, Phalora P, Kurioka A, Hansen TH, Willberg CB, Phillips RE, Klenerman P (2016) TLR signaling in human antigen-presenting cells regulates MR1-dependent activation of MAIT cells. Eur J Immunol 46: 1600-1614.

210

Oral administration of iNKT cell ligand OCH induces anti-inflammatory immune responses in

healthy human subjects and multiple sclerosis：Results of investigator-initiated, first-in-human

phase 1 study

Takashi Yamamura1 1. National Institute of Neuroscience, NCNP, Kodaira, TOKYO, Japan

Multiple sclerosis (MS) is a putative autoimmune disease of the central nervous system (CNS), of which pathogenesis could be linked with dysbiosis in gut microbiota. Works have demonstrated that regulatory cell populations, including iNKT cells, MAIT cells and regulatory T cells (Treg), are significantly altered in MS. It is possible that disrupted balance between autoreactive and regulatory cells may be caused by dysbiosis at least partially. We previously reported that oral administration of a sphingosine-truncated analog of α-galactosylceramide, OCH, would selectively induce IL-4 production from iNKT cells, thereby preventing the development of experimental autoimmune encephalomyelitis (EAE) (Nature 2001). To explore the potential value of OCH as a therapeutic agent for MS, we have completed a first-in-human phase 1 study of OCH in 15 healthy subjects (HS) and 9 patients with MS. To evaluate the effect of OCH in vivo, flow cytometer and DNA microarray analyses were conducted for peripheral blood samples obtained at various time points after oral OCH treatment. Unexpectedly high concentrations of OCH were detected in the recipients’ blood, indicating absorption of OCH from gut is better in human than rodents. Subsequently, we have observed a number of favorable changes in the blood of the HS and MS, which included (i) a significant increase of T reg [CD45RA-Foxp3+ effector/activated regulatory T cells] at 6 h, (ii) upregulation of immunoregulatory genes (MAFB and IL4I1) at 6 and 24 h, (iii) downregulation of immune-activating genes (NR4A2, FOS, and FOSB) at 6 and 24 h, and (iv) downregulation of GZMB and killer cell immunoglobulin-like receptors. Three of the patients received weekly oral administration of OCH for three months without any adverse events. The results are promising and would validate a need for phase 2 study.

211


Cerebrospinal fluid and serum lipoprotein distribution affects the antigenicity of sulfatide for human iNKT cells

Peter van den Elzen1, Jessica Tuengel1, Dongjun Zheng1, William Panenka1, Annelein Stax1 1. University of British Columbia, Vancouver, BRITISH COLUMBIA, Canada

We recently demonstrated that human, but not mouse, invariant NKT cells recognize sulfatide presented by CD1d. Sulfatide derived from myelin or from human cerebrospinal fluid (CSF) apolipoprotein E (apoE) is capable of activating human iNKT cells. We considered the pathophysiologic contexts in which CSF apoE-bound sulfatide may be capable of activating iNKT cells, namely upon disruption of the blood-brain barrier (BBB). We found that serum lipoproteins are present in the brain of MS patients in active and pre-active lesions where there is BBB disruption. In vitro, while sulfatide exclusively resides in apoE fractions within CSF, it more broadly distributes in lipoprotein fractions in human serum and its ability to activate iNKT cells is enhanced. The addition of serum VLDL-apoE to CSF also enhances the antigenicity of sulfatide. The differential ability of different lipoprotein fractions in the CSF and the serum to affect sulfatide is likely to be of importance to neuroinflammatory conditions such as multiple sclerosis (MS) and traumatice brain injury (TBI).

1. 1. Stax AM, Tuengel J, Girardi E, Kitano N, Allan LL, Liu V, Zheng D, Panenka WJ, Guillaume J, Wong CH, van Calenbergh S, Zajonc DM, van den Elzen P. (2017) Autoreactivity to Sulfatide by Human Invariant NKT Cells. Journal of Immunology. 2017 July 1; 199(1): 97-106


Poster Session 1 Listing - Odd Numbers

abs# 75 - Anna Helena Jonsson M.F. Gurish, P.J. Brennan, D.A. Rao, J. McCluskey, P.A. Nigrovic, M.B. Brenner MAIT cells and other innate-like lymphocytes in blood and synovial fluid of rheumatoid arthritis and related autoimmune arthritis

abs# 77 - Edwin Leeansyah M. Gulam, N. Abdullah, A. Kamarulzaman, S.R. Lewin, J.K. Sandberg, R. Rajasuriar The relationship between IL-7Rα polymorphisms and MAIT cell dysfunction in treated HIV-1 infection

abs# 79 - Amy L Ellis A.J. Balgeman, M.A. Rodgers, C. Updike, T. Baranowski, C.A. Scanga, S.L. O'Connor Characterization of MR-1 tetramer-positive cells in a cynomolgus macaque model of M. tuberculosis/SIV coinfection

abs# 81 - Huimeng Wang C. D'Souza, X. Lim, L. Kostenko, T. Pediongco, S. Eckle, B. Meehan, N. Wang, L. Liu, J. Mak, D. Fairlie, Y. Iwakura, J. Gunnersen, A. Stent, J. Rossjohn, G. Westall, L. Kjer-Nielsen, R. Strugnell, J. McCluskey, A. Corbett, T. Hinks, Z. Chen MAIT cells protect against fatal pulmonary infection by Legionella species via IFNg and can be augmented by synthetic ligands.

abs# 83 - Zhenjun Chen H. Wang, L. Kjer-Nielsen, C. D’Souza, T.S. Hinks, L. Kostenko, X. Lim, S.B. Eckle, B.S. Meehan, D.C. Jackson, L. Liu, M. Teng, V.K. Kuchroo, D. Yu, B. Fazekas, D.P. Fairlie, J. McCluskey, R.A. Strugnell, A.J. Corbett MAIT cells’ in vivo activation requirements

abs# 85 - Daniel T Leung A.M. Spivak, M.S. Bennett, M.L. Coletti, S. Trivedi, F.A. Barrios, M.T. Rondina Anti-HIV-1 effector potential of mucosal-associated invariant T (MAIT) cells

abs# 87 - Tan-Yun Cheng D.G. Pellicci, P.J. Brennan, S. Huang, R.W. Birkinshaw, A. Shahine, K. Wun, S. Gras, M.B. Brenner, J. Rossjohn, D.I. Godfrey, D. Moody The TCR Trap: detecting the number and structure of lipids in CD1-TCR complexes

abs# 89 - Amadeu Llebaria R. Borràs, Y. Harrak, L. Usero, H. Koay, . Pellicci, J. Le Nours, R. Price, T. Yongqing, A. Sánchez, D.I. Godfrey, J. Rossjohn, C. Roura-Mir Sugar mimetics in α-galactosylceramide analogues: Potent immunostimulating agents via iNKT activation

abs# 91 - Piotr Humeniuk W. Paster, S. Geiselhart, T. Webb, P. Steinberger, K. Hoffmann-Sommergruber A Jurkat based NFκB-eGFP iNKT reporter cell line to evaluate the interaction of food-derived lipids with iNKT cell receptors


abs# 93 - Lise Pasquet K. Camara, A.C. Bloom, S.K. Richardson, A.R. Howell, M. Terabe, J.A. Berzofsky The ceramide structure of sulfatide-analogues influences the functional activity of type II NKT cells

abs# 95 - Charlotte A. James K.K. Quan, M. Gilleron, J. Prandi, V.R. Yedulla, Z.Z. Moleda, E. Diamanti, M. Khan, V.K. Aggarwal, P. Reinink, S. Lenz, R.O. Emerson, T.J. Scriba, M.N. Souter, D.I. Godfrey, D.G. Pellicci, D. Moody, A.J. Minnaard, C. Seshadri, I. Van Rhijn CD1b tetramers identify T cells that recognize natural and synthetic diacylated sulfoglycolipids from Mycobacterium tuberculosis

abs# 97 - Erik D Layton M. Smith, K.K. Quan, T.J. Scriba, C. Seshadri A multi-tetramer flow cytometry panel for studying the phenotypes of human donor-unrestricted T cells in clinical studies

abs# 99 - Catarina F Almeida S. Sundararaj, J. Le Nours, O. Patel, B. Cao, D. Pellicci, S. Williams, J. Rossjohn, A. Uldrich, D. Godfrey Recognition of a microbial glycolipid antigen by both Type 1 and Type 2 NKT cells

abs# 103 - Weiming Yuan X. Wen, S. Kim, M. Li, P. Rao, O. Akbari, R. Bosselut, P. Dellabona, G. Casorati, M. Exley Development of humanized mouse models for in vivo studies of human iNKT cells

abs# 107 - Michael S Lee T.J. Webb Targeting S1P to restore NKT cell responses to lymphoma

abs# 109 - Prabhjeet Phalora S. Hester, E. Marchi, J. Ussher, S. Mohammed, P. Klenerman A proteomics screen to identify MR1 associated proteins and implications for MR1 function

abs# 111 - Jacinta M Wubben H.E. McWilliam, J. Le Nours, J. McCluskey, J.A. Villadangos, J. Rossjohn A Tail of MR1 Internalization

abs# 113 - Shilpi Chandra J. Gray, W.B. Kiosses, A. Khurana, K. Hitomi, C. Crosby, Z. Fu, M. Zhao, N. Veerapen, S.K. Richardson, S. . Porcelli, G. Besra, A.R. Howell, S. Sharma, B. Peters, M. Kronenberg A whole genome mouse siRNA screen to identify novel genes involved in lipid antigen presentation

abs# 115 - Erik von Seth C. Zimmer, M. Reuterwall-Hansson, A. Barakat, U. Arnelo, A. Bergquist, M. Ivarsson, N. Björkström Redistribution of functionally impaired MAIT cells to bile ducts in primary sclerosing cholangitis


abs# 117 - Olivier Gasser K. Sharples, C. Barrow, G. Williams, E. Bauer, C. Wood, B. Mester, M. Dzhelali, G. Caygill, J. Jones, C. Hayman, V. Hinder, J. Macapagal, M. McCusker, R. Weinkove, G. Painter, M. Brimble, M. Findlay, R. Dunbar, I. Hermans A phase I vaccination study with dendritic cells loaded with NY-ESO-1 and a-galactosylceramide: induction of polyfunctional T cells in high-risk melanoma patients

abs# 119 - Niklas K Bjorkstrom C.L. Zimmer, L. Berglin, M. Cornillet, O. Strauss, E. von Seth, E.C. Ellis, A. Bergquist Loss and exhaustion of MAIT cells in cholangiocarcinoma

abs# 121 - Ho Ngai G. Tian, A.N. Courtney, E. Marinova, W. Huang, L. Guo, L.S. Metelitsa IL-21 maintains CD62L expression during NKT-cell ex vivo expansion and enhances antitumor activity of NKT cell therapy in vivo

abs# 123 - Nicholas A Zumwalde J.D. Haag, M.N. Gould, J.E. Gumperz Investigating the role of human MAIT cells in breast duct carcinogenesis

abs# 125 - Nicholas M. Provine A. Amini, L. Garner, M. Esposito, S.S. Slevin, M. FitzPatrick, K.D. Williamson, H. Ferry, L. Lee, S.K. Chinnakannan, S. Capone, A. Folgori, E. Barnes, P. Klenerman Activation of MAIT cells by adenovirus vectors

abs# 127 - Joshua Lange R. Anderson, O. Gasser, G. Painter, I. Hermans Novel mucosal associated invariant T (MAIT) cell agonists to explore the function of MAIT cells as cellular adjuvants

abs# 129 - Marco ML Lepore J.J. Pentier Harnessing unconventional T cells and their targets for immunotherapy with high affinity bi-specific TCRs

abs# 131 - Amy N Courtney G. Tian, L. Guo, E. Marinova, J. Jin, X. Gao, J. Wei, A. Heczey, K.B. Ghaghada, L.S. Metelitsa NKT cells control tumor associated macrophages and metastatic growth in neuroblastoma

abs# 133 - Alessandra Bigi C. de Lalla, M. Consonni, A. Mancino, C. Garavaglia, V. Malacarne, M. Lepore, L. Mori, G. De Libero, A. Graziani, P. Dellabona, G. Casorati Understanding the generation of CD1c-restricted self-lipid antigen in leukemia cells

abs# 135 - Jingling Jin W. Huang, D. Liu, L. Guo, M. Wood, B. Liu, E. Marinova, G. Dotti, L. Metelitsa NKT cells expressing a GD2-specific chimeric antigen receptor with CD28 endodomain and IL-15 undergo dramatic in vivo expansion and mediate long-term tumor control in a metastatic model of neuroblastoma


abs# 137 - Derek G Doherty Dockry, S. O'Leary, L. Gleeson, J. Lyons, J. Keane, S.G. Gray Epigenetic induction of CD1d expression primes lung cancer cells for killing by invariant NKT cells

abs# 139 - Gloria Galvan S. Dorta-Estremera, J.K. Sastry Curative efficacy of intranasal HPV vaccine supplemented with aGalCer adjuvant in preclinical mouse vaginal HPV tumor model

abs# 143 - Krystle K Quan D. Wilburn, W. Swanson, C. Seshadri The conserved nature of CD1 and T-cell receptor interactions between humans and non-human primates

abs# 145 - Srinath Govindarajan D. Gaublomme, R. Van Der Cruyssen, E. Verheugen, S. Van Gassen, Y. Saeys, T. Iwawaki, S. Janssens, B. Lambrecht, M. Drennan, D. Elewaut IRE1alpha Regulate Cytokine mRNAs Stabilization Within Invariant NKT1 and 17 Cells

abs# 147 - Caroline Boulouis J. Dias, J. Gorin, R.H. van den Biggelaar, A. Gibbs, L. Loh, M. Gulam, D.F. Nixon, K. Broliden, A. Tjernlund, J.K. Sandberg, E. Leeansyah Human CD8-negative MAIT cells are functionally distinct from CD8-positive MAIT cells

abs# 149 - Haiguang Wang K. Hogquist CCR7 defines a multipotent progenitor for iNKT cells in thymus and periphery

abs# 151 - Patrick W Darcy D.B. Sant'Angelo Ectopic expression of PLZF results in an RORγt positive cytotoxic T cell subset

abs# 153 - Liang Cao X. Weng, A. Kumar, Y. He, J. Zhao, L.A. Sena, S. Weinberg, N.S. Chandel, C. Wang Dysfunction of mitochondrial complex III preferentially affects the development of CD1d-restricted NKT cells and Mucosal associated invariant T (MAIT) cells

abs# 155 - Katharine J Goodall A. Nguyen, A. Matsumoto, S. Sant, J. McMullen, K. Kedzierska, J. Trapani, M. Degli-Esposti, J. Rossjohn, L.C. Sullivan, D.M. Andrews Differential glycosylation of the CD8a homodimer regulates binding to the non-classical MHC I, H2-Q10

abs# 157 - Cole Anderson A. Iyer , R. O'Connell , D. Leung Development and function of mucosal-associated invariant T cells are regulated by the micro-RNA miR-155.


abs# 159 - Thomas Hägglöf A. Kumagai, W.J. Kaiser, E.A. Leadbetter RIP inflammatory pathways are required to maintain iNKT cell homeostasis and enable cognate neutrophil-iNKT cell interaction

abs# 161 - Rajesh Lamichhane S. de la Harpe, T. Harrop, A. Vernall, J. Tyndall, P. Dearden, J. Kirman, J. Ussher Differences in the effector functions of MR1- and cytokine stimulated MAIT cells

abs# 163 - Isaac Engel G. Seumois, M. Paynich, S. Liang, S. Rosales, P. Vijayanand, M. Kronenberg Transcriptional profiling of peripheral invariant NKT subsets

abs# 165 - Catriona V Nguyen-Robertson M.N. Souter, S.J. Reddiex, J.M. Cheng, A.P. Uldrich, J. Rossjohn, I. Van Rhijn, S.J. Williams, D. Moody, D.I. Godfrey, D.G. Pellicci Characterization of CD1a-restricted T cells using CD1a-lipid tetramers

abs# 167 Asako Chiba T. Nagaishi, S. Miyake MAIT cells exacerbate the disease course of oxazolone-induced colitis.

abs# 169 - Andrew E Hogan A. O'Brien, L.M. Tobin, L. Lynch, D. O'Shea Altered cellular metabolism in Mucosal-associated invariant T cells from obese patients

abs# 171 - Amine Toubal B. Kiaf, M. Rhimi, L. Beaudoin, L. Cagninacci, O. Lantz, J. Rossjhon, J. McCluskley, E. Maguin, P. Lesnik, A. Lehuen Impact of MAIT cells in obesity and Type 2 Diabetes

abs# 173 - Edy Kim K. Ikeda, K. Hayashida, J. Choi, J. Guo, F. Ichinose, M.B. Brenner Diverse Natural Killer T cells regulate inflammation, neurological injury and mortality in cardiac arrest and resuscitation.

abs# 175 - Maroua Ferhat A. Robin, S. Giraud, J. Gombert, A. Thierry, A. Herbelin The possible role of the IL-33/iNKT cell axis during kidney ischemia-reperfusion injury

abs# 177 - Shelby O'Connor A. Ellis, N. Kannal, A. Balgeman Detection of polymorphisms in nonhuman primate MR-1 alleles

abs# 179 - Fatima Macedo C.S. Pereira, H.S. Ribeiro, B. Perez-Cabezas The GM2 ganglioside inhibits iNKT cell responses in a CD1d-dependent manner


abs# 181 - Yasmeen G Ghnewa S.E. Khorsandi, X. Huang, L. Ramsay, W. Jassem, N. Heaton, Y. Ma Innate T cells are significantly altered in the livers of patients with alcoholic liver disease

abs# 183 - Kerri G. Lal E. Leeansyah, J. Dias, M. Creegan, M.C. Costanzo, L. Eller, S. Krebs, H. Kibuuka, L. Maganga, S. Nitayapan, F. Sawe, J. Ake, N.L. Michael, M.L. Robb, M.A. Eller, J.K. Sandberg Mucosal associated invariant T (MAIT) cell dynamics during acute HIV-1 infection

abs# 185 - Michal J Sobkowiak H. Davanian, A. Tjernlund, A. Gibbs, S. Nayak, J. Dias, S. Aleman, C. Krüger-Weiner, M. Moll, R. Heymann, E. Leeansyah, M. Sällberg Chen, J.K. Sandberg MAIT cells in the human oral mucosa exhibit a CD69+/CD103+ tissue resident memory phenotype and lowered cytolytic potential

abs# 187 - Ayano C Kohlgruber S. Gal-Oz, N. LaMarche, D. Duquette, H. Nguyen, A. Mina, M. Shimazaki, D. Sant'Angelo, A. Tavakkoli, U. von Andrian, A.S. Banks, T. Shay, M.B. Brenner, L. Lynch Gamma delta T cells producing IL-17A regulate adipose Treg homeostasis and thermogenesis

abs# 189 - Laura Valestrand N.L. Berntsen, E. Schrumpf, T.H. Karlsen, R.S. Blumberg, E. Melum Lipid antigens in bile from patients with liver diseases activate NKT cells

abs# 193 - David Lewinsohn E. Karamooz 6-FP Pretreatment Demonstrates an MR1 Recycling Pathway

abs# 201 - Annemieke de Jong G.C. Monnot, C. Tejeda, A. Chang, H.F. Jiang, W. Zeng, C. Rhode, S. Cremers Regulation of CD1a-dependent T cell activation in human skin

abs# 203 - Edy Kim H. Ner-gaon, J. Choi, J. Guo, C. Benoist, T. Shay, M.B. Brenner Natural Killer T cells are master regulators of post-sepsis immunosuppression

abs# 205 - Vipin Kumar I. Marrero, I. Maricic, A. Eguchi, S. Dasgupta, A. Feldstein, C. Hernandez, R. Loomba A dominant immune mechanism mediated by type I NKT cell subsets in different phases of non-alcoholic fatty liver disease

abs# 207 - Ageliki Tsagaratou S. Rautio , E. Gonzalez Avalos, J. Scott-Browne , S. Togher , L. Chavez, E.V. Rothenberg , H. Lähdesmäki, A. Rao TET proteins control iNKT cell lineage specification and TCR mediated expansion

abs# 209 - James Ussher T. Williams, R. Lamichhane, S. de la Harpe, J. Tyndall, A. Vernall Bacteria license antigen presenting cells for MR1-mediated MAIT cell activation


abs# 211 - Peter van den Elzen J. Tuengel, D. Zheng, W. Panenka, A. Stax Cerebrospinal fluid and serum lipoprotein distribution affects the antigenicity of sulfatide for human iNKT cells

abs# 213 - Laurent Gapin S. Sundararaj, J. Zhang, S. Krovi, R. Bedel, K. Tuttle, N. Veerapen, G.S. Besra, J. Le Nours, J. Matsuda, J. Rossjohn The differing roles of CD1d2 and CD1d1 proteins in type I Natural Killer T cell development and function

Poster Session 2 Listing - Even Numbers

abs# 76 - Kimia T. Maleki J. Tauriainen, N.K. Bjorkstrom, K. Blom, J.K. Sandberg, C. Ahlm, J. Klingström Peripheral blood MAIT cells are activated and reduced in frequency during human hantavirus infection

abs# 78 - Jean-Baptiste Gorin M. Moll, L. Zimmermann, E. Leeansyah, J.K. Sandberg HIV-1 Vpu and Nef proteins interfere with MR1 surface expression

abs# 80 - Martine Gilleron N. Mebarek, A. Vercellone, T. Cheng, J. Nigou, D. Moody MPLA vaccine adjuvant induces group 1 CD1 protein expression on human monocytes

abs# 82 - Jennifer A Juno S. Eckle, K. Wragg, B. Meehan, T. Haripriya Amarasena, J. Mak, L. Liu, D. Fairlie, J. McCluskey, S.J. Kent Progressive Simian-Human Immunodeficiency Virus (SHIV) infection alters expression of key surface and transcriptional proteins on macaque MAIT cells

abs# 84 - Cole Anderson S. Trivedi, R. Campbell, M. Rondina , D. Leung Mucosal associated invariant T (MAIT) cells in experimental and clinical sepsis.

abs# 86 Johan K. Sandberg O. Sortino, E. Richards, J. Dias, E. Leeansyah, I. Sereti Interleukin-7 as a potential therapeutic strategy to reconstitute MAIT cells

abs# 88 - Andrew Chancellor A. Tocheva, C. Cave-Ayland, L. Tezera, A. White, J.A. Dulayymi, J. Bridgeman, S. Wilson, M. Tebruegge, B. Marshall, S. Sharpe, T. Elliott, C. Skylaris, J.W. Essex, M. Baird, S. Gadola, P. Elkington, S. Mansour Mycobacterium tuberculosis mycolic acid lipid tails govern GEM T cell responses

abs# 90 - Masaki Terabe K. Clark, A. Bloom, D. Venzon, M. Suzuki, L. Pasquet, B.J. Compton, S. Cardell, S.A. Porcelli,


J.A. Berzofsky Characterizing β-Mannosylceramide presented by CD1d

abs# 92 - Josephine F Reijneveld T. Cheng, K. Wun, S. Gras, J. Rossjohn, B. Moody, I. Van Rhijn Human CD1c-autoreactive T cells recognize monoacylglycerol and diverse self lipids using an ‘absence of interference’ mechanism

abs# 94 - Peter Reinink M. Souter, T. Cheng, J. Kubler-Kielb, K. Strle, A. Steere, B. Moody, D. Pellicci, D. Godfrey, I. van Rhijn CD1b presents Borrelia burgdorferi glycolipid to human T cells.

abs# 96 - Maren Thomaier Y. Wang, E. Huc-Claustre, L. Trautenberg, S. Baltzer, L. Dai, G. Grotenberg, A. Shevchenko, S. Zeissig Sortase-A-based strategy for the analysis of CD1d-associated lipids

abs# 98 - Xuefen Yang L. Fox, A. Sharma, Z. Jiang, J.E. Gumperz Serum albumin delivers LPC to DCs resulting in enhanced autoreactive activation of human iNKT cells

abs# 100 - Jeffrey YW Mak W. Xu, R.C. Reid, A.J. Corbett, B.S. Meehan, H. Wang, Z. Chen, J. Rossjohn, J. McCluskey, L. Liu, D.P. Fairlie Chemical insights and new antigens for MAIT cell activation

abs# 102 - Hamish EG McWilliam M. Zorkau, S. Wormald, J. Mintern, J. McCluskey, J. Rossjohn, J. Villadangos Unravelling the molecular mechanisms of how MR1 presents a pathogen metabolic signature to MAIT cells

abs# 104 - Rebeca Jimeno J. Saez de Guinoa, N. Farhadi, P. Jervis, L.R. Cox, G.S. Besra, P. Barral Innate lymphoid cells modulate iNKT cell immunity

abs# 106 - Gitanjali A Narayanan E. Karamooz, M.J. Harriff, D.M. Lewinsohn The role of MR1 isoforms in antigen presentation to MAIT cells

abs# 108 - Sara Suliman T. Amariuta, S. Asgari, I. Van Rhijn, M.B. Murray, S. Raychaudhuri, D.B. Moody Vitamin A-Mediated Regulation of CD1 Expression During Human Dendritic Cell Differentiation

abs# 110 - Dilip Shrestha S. Strauss, M. Fritzsche, M. Salio, V. Cerundolo, C. Eggeling Role of cellular actin in CD1d-mediated antigen presentation


abs# 112 - Corinna A Kulicke E. De Zan, M. Salio, P. Klenerman, S.M. Nijman, V. Cerundolo Identification of novel modulators of MR1 trafficking using a gene trap screen in haploid cells

abs# 114 - Goh Murayama A. Chiba, K. Yamaji, N. Tamura, S. Miyake Role of mucosal-associated invariant T cells in lupus pathogenesis.

abs# 116 - Abel Trujillo-ocampo H. Cho, W. Ruiz-Vazquez, J. Molldrem, J. Im Ex Vivo expansion of iNKT cells for adoptive cell therapy.

abs# 118 - Mariko Takami F. Ihara, T. Kamata, S. Motohashi A role of AhR signaling in NKT cell immunotherapy

abs# 120 - A. Raul Castano I. Esteban, A. Saavedra, A. Elisenda Th1 biased iNKT-cell activation as an effective anti-tumor response

abs# 122 - Fumie Ihara D. Sakurai, M. Takami, Y. Okamoto, S. Motohashi Regulatory T cells increased in advanced head and neck cancer patients suppress NKT cell function and correlate with disease progression

abs# 124 - Ellie-May Jarvis S. Collings, A. Authier-Hall, N. Dasyam, A. Maxwell, J. Lowe, C. Barrow, B. Luey, J. Nacey, B. Delahunt, O. Gasser, I. Hermans, R. Weinkove Profiling human innate-like T cells in cancer and checkpoint blockade

abs# 126 - Dominique Bollino R. Muthuswamy, K. Odunsi, T.J. Webb Interrogating the VEGF immunosuppressive axis in ovarian cancer

abs# 128 - Marco ML Lepore J.J. Pentier Harnessing unconventional T cells and their targets for immunotherapy with high affinity bi-specific TCRs

abs# 130 - Andreas Weigert D. Sekar, S. Wallner, S. Zukunft, N. Ferreirós, H. Stark, I. Fleming, B. Brüne Ether-linked phosphatidylserine species as novel mammary tumor-enriched antigens for type II NKT cells

abs# 132 - Gloria Delfanti F. Cortesi, G. Antonini, C. Garavaglia, M. Consonni, G. Casorati, P. Dellabona Therapeutic reprogramming of the tumor microenvironment by iNKT cells


abs# 134 - Chiara Ceriotti M. Jaeger, E. Huc-Claustre, S. Zeissig The role of CD1d in intestinal tumor development

abs# 136 - Nyambayar Dashtsoodol T. Shigeura, T. Tashiro, M. Taniguchi NKT cell-targeted cancer vaccine mediating long-term memory responses and strong antitumor activity

abs# 138 - Juan JU Umana T.T. Mallevaey TCR-engineered iNKT cells directed towards tumour-associated antigens for cancer immunotherapy

abs# 140 - Ashanty M Melo M. Conroy, E. Foley, J.V. Reynolds, J. Lysaght, D.G. Doherty Depletions of invariant natural killer T cells in blood and omentum from patients with upper gastrointestinal cancer

abs# 142 - Maureen Banach E. Edholm, J. Robert Exploring the evolutionary relevance of MHC class I-like molecules and innate-like T cells in tumor immunity.

abs# 144 - Eva-Stina Edholm K. Rhoo, J. Robert A new distinct MHC class I-like restricted invariant T cell lineage at the forefront of mycobacterial immunity uncovered by reverse genetics in the amphibian Xenopus

abs# 146 - Jeanne Perroteau L. Hesnard, M. Devilder, L. Gapin, E. Scotet, X. Saulquin, L. Gautreau-Rolland Identification and thymic development of human autoreactive iNKT cells

abs# 148 - Francois Legoux Y. El Morr, M. Salou, A. Darbois, O. Lantz MAIT and NKT cell development is controlled by the same pathways and results in identical transcriptional programs

abs# 150 - Joshua A Vieth J. Das, F.M. Ranaivoson, D. Comoletti, L.K. Denzin, D.B. Sant'Angelo NKT TCR domains unrelated to ligand binding instruct the thymic development of an adipose resident subset

abs# 152 - Vipin Kumar H. Sheng, I. Marrero, I. Maricic, S. Fanchiang, S. Zhang, S. Dasgupta, D. Sant’Angelo A novel population of liver-associated innate-like PLZF+CD8ααTCRαβ T cells with immune regulatory properties


abs# 154 - Angela Nguyen K. Goodall, D. Andrews Characterisation of the biochemical signature regulating interaction between CD8aa and the liver expressed non-classical MHC molecule, H2-Q10

abs# 156 - You Jeong Lee PLZF regulates effector differentiation of gd T cells

abs# 158 - Mallory L Paynich J.P. Scott-Browne, I. Engel, M. Kronenberg Assessment of global chromatin accessibility in peripheral iNKT cell subsets

abs# 162 - Erin W Meermeier N.L. Stucky, C.L. Zheng, G.M. Swarbrick, J. Tran, A. Worley, D.M. Lewinsohn Elucidation of Antibacterial Functions of MAIT Cells in Human Lung Mucosa: A Transcriptomics Study

abs# 164 - Julia D Berkson C. Slichter, H. Oberbillig, M. Prlic Human mucosal-associated invariant T (MAIT) cells in inflamed tissues

abs# 166 - Jianyun Liu H. Nan Circulating Mucosal-Associated Invariant T Cells are Reduced in Obesity

abs# 170 - Hiroshi Watarai Y. Ren, E. Sekine-Kondo, R. Shibata A Novel Mouse Model of iNKT Cell-deficiency Generated by CRISPR/Cas9 Technology Reveals a Pathogenic Role of iNKT Cells in Metabolic Disease.

abs# 172 - Nelson M. LaMarche R.V. Tatituri, L. Lynch, M.B. Brenner Identification of Adipose Tissue Factors that Induce Regulatory iNKT Cells

abs# 174 - Carlos Donado D. Simmons, P. Brennan, M. Brenner iNKT cells catalyze a potent and sustained IL-1β secretion pathway.

abs# 176 - Akimichi Saito N. Ishimori, Y. Obata, S. Kinugawa, N. Satoh, K. Iwabuchi, S. Motohashi, K. Ohtani, T. Ide, H. Tsutsui Development of novel therapeutic strategy for heart failure via activating invariant natural killer T cells

abs# 178 - Bradley W. Bolling R. Pei, D.A. Martin Aronia berry consumption inhibits T cell adoptive transfer colitis in mice by modulating Th17 and Treg populations


abs# 180 - Yasmeen G Ghnewa M. Yuksel, S.E. Khorsandi, X. Huang, L. Ramsay, W. Jassem, N. Heaton, Y. Ma The association between mucosal associated invariant T (MAIT) cells and liver graft quality

abs# 182 - Akshat Sharma S.M. Lawry, N.A. Zumwalde, B.S. Klein, N.M. Sherer, J.E. Gumperz LFA-1 signaling drives TCR- and cytokine-independent IFNγ production by innate T-lymphocytes

abs# 184 - Rachel Cotton T. Cheng, I. Van Rhijn, R.A. Clark, B. Moody Capturing the tissue-resident CD1a-reactive T cell population from human skin

abs# 186 - Laura Valestrand N.L. Berntsen, F. Zheng, B. Fosby, T.H. Karlsen, P. Line, X. Jiang, E. Melum Natural killer T cells promote cholestatic liver disease in bile duct ligated mice

abs# 188 - Clare S Hardman Y. Chen, M. Salimi, R. Jarrett, D. Johnson, V. Jarvinen, R. Owens, E. Repapi, D. Cousins, J. Barlow, A. McKenzie, G. Ogg CD1a presentation of endogenous antigens by group 2 innate lymphoid cells

abs# 190 - Elodie Macho-Fernandez N. Barton, M. Halimani, C. Nunes-Alves, M.H. Young, N. Veerapen, S.M. Behar, G. Besra, L. Gapin, M. Brigl Function of MR1-Restricted Mucosal-Associated Invariant T Cells during Citrobacter rodentium infection

abs# 192 - Jason Kelly B. Toohey, D. Gray, G. Kannourakis, S.P. Berzins Characterizing MAIT cells in human mucosal cancers

abs# 200 - Patricia Barral iNKT cells in intestinal immunity

abs# 202 - Alexandra J Corbett C. D'Souza, T. Pediongco, H. Wang, J.Y. Scheerlinck, L. Kostenko, R. Esterbauer, A. Stent, S.B. Eckle, B.S. Meehan, R.A. Strugnell, H. Cao, L. Liu, J.Y. Mak, G. Lovrecz, L. Lu, D.P. Fairlie, J. Rossjohn, J. McCluskey, A.L. Every, Z. Chen Mucosal-associated invariant T cells augment immunopathology in chronic Helicobacter pylori infection

abs# 204 - Sai Harsha Krovi K.D. Tuttle, J. Zhang, R. Bedel, L.K. Peterson, L.L. Dragone, K. Riemondy, J. Hesselberth, J. Scott-Browne, L. Gapin The TCR signal strength influences the specification of iNKT subsets

abs# 206 - Marion Salou A. Goubet, V. Premel, A. Darbois, R. Alonso, F. Legoux, O. Lantz MAIT and iNKT cell subsets share similar tissue residency programs


abs# 208 - Susannah C Shissler T.J. Webb Defective NKT cell development in BRCA-1 mutant mice

abs# 210 - Takashi Yamamura Oral administration of iNKT cell ligand OCH induces anti-inflammatory immune responses in

healthy human subjects and multiple sclerosis：Results of investigator-initiated, first-in-human

phase 1 study


POSTER ABSTRACTS

75

MAIT cells and other innate-like lymphocytes in blood and synovial fluid of rheumatoid arthritis and related autoimmune arthritis

Anna Helena Jonsson1, Michael F Gurish1, Patrick J Brennan1, Deepak A Rao1, James McCluskey2, Peter A Nigrovic1, 3,

Michael B Brenner1 1. Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, Boston, MA, USA

2. Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia

3. Division of Immunology, Boston Children's Hospital, Boston, MA, USA

MAIT cells, iNKT cells, and γδT cells respond to non-peptide antigens and exhibit a poised state at baseline, enabling rapid activation of cytokine production and cytotoxic activity upon stimulation. However, the role(s) innate-like lymphocytes may play in autoimmune diseases are not well understood. We assessed the frequencies and properties of innate-like lymphocytes in patients with seropositive rheumatoid arthritis (RA) and several other types of autoimmune arthritis (seronegative RA, juvenile idiopathic arthritis, and spondyloarthritis). Interestingly, the frequency of MAIT cells is sharply reduced in the blood of patients with seropositive rheumatoid arthritis, similar to published observations in chronic infections such as tuberculosis and hepatitis C virus. Importantly, MAIT cells are not enriched in synovial fluid aspirated from actively inflamed joints. Nonetheless, MAIT cells in synovial fluid are activated, as evidenced by expression of HLA-DR and PD-1, and they produce IFNγ, TNF, and IL-17A upon stimulation. We hypothesized that MAIT cell numbers might be low due to increased cell death. Indeed, MAIT cells have higher levels of activated caspases than other T cell subsets, and their numbers drop drastically when cultured in the presence or absence of CD3- and CD28-mediated stimulation, despite proliferation at nearly the same rate as CD4 and CD8 T cells. The mechanism of this process is not yet clear, but we suspect that it may explain the low frequency of MAIT cells in patients with RA. Additional studies are in progress to further characterize MAIT cells in RA, including mechanisms that may result in MAIT cell depletion in RA.

76

Peripheral blood MAIT cells are activated and reduced in frequency during human hantavirus infection

Kimia T. Maleki1, Johanna Tauriainen1, Niklas K. Bjorkstrom1, Kim Blom1, Johan K. Sandberg1, Clas Ahlm2, Jonas

Klingström1 1. Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden

2. Division of Infectious Diseases, Department of Clinical Microbiology, Umeå University, Umeå, Sweden

While MAIT cells are innate-like T cells specifically responding to microbial vitamin metabolites presented by MR1 molecules, it was recently shown that IL-18 produced during viral infections can contribute to MAIT cell activation. Hantaviruses cause two severe human diseases; hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia and hantavirus pulmonary syndrome (HPS) in the Americas, with case-fatality rates of up to 10% and 40%, respectively. Hantavirus infection of humans is characterized by increased vascular leakage and strong inflammatory responses displayed by high levels of pro-inflammatory cytokines and vigorous NK cell, B cell, and CD8 T cell responses. The human immune response upon hantavirus infection is believed to be an important factor in the disease pathogenesis, yet the mechanistic background to what is driving the inflammatory responses is largely undescribed. We and others recently observed that systemic IL-18 levels are increased in HFRS and HPS patients. Here, we performed a phenotypic characterization of peripheral MAIT cells in 25 Swedish HFRS patients, during acute and convalescent phase. The frequency of MR1 tetramer-defined MAIT cells was reduced during acute HFRS, as compared to uninfected controls. The residual MAIT cells still present were characterized by an activated phenotype with increased expression of CD69, CD38, and granzyme B. In addition, MAIT cells of HFRS patients showed high expression of Ki67. These data suggest that hantavirus infection induces activation and proliferation of peripheral MAIT cells. Reduced expression of the gut homing receptors α4β7 and CCR6 further suggested that MAIT cells might have been sequestered away from circulation to mucosal sites. Continued studies will aim at investigating possible effects of hantavirus on the MAIT cell phenotype and function in vitro, and at exploring the potential role for MAIT cells in hantavirus immunopathogenesis.


77

The relationship between IL-7Rα polymorphisms and MAIT cell dysfunction in treated HIV-1 infection

Muhammad Yaaseen Gulam1, Noor Kamila Abdullah2, Adeeba Kamarulzaman2, Sharon R. Lewin3, 4, Johan K. Sandberg5,

Reena Rajasuriar2, 3, Edwin Leeansyah5, 1 1. Program in Emerging Infectious Diseases, Duke-National University of Singapore Medical School, Singapore

2. Centre of Excellence for Research in AIDS (CERIA), University of Malaya, Kuala Lumpur, Malaysia

3. Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Parkville, Victoria, Australia

4. Department of Infectious Diseases, Alfred Health and Monash University, Melbourne, Australia

5. Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden

MAIT cells were persistently depleted and functionally exhausted in HIV-infected patients despite long-term antiretroviral therapy (ART). Furthermore, we have shown that IL-7 could rescue functionality in MAIT cells from HIV-infected patients in vitro. Single-nucleotide polymorphisms (SNPs) of the IL-7 receptor α-subunit (IL-7Rα/CD127), which modulate soluble(s)IL-7Rα levels, influence bioavailability of circulating IL-7 and affect subsequent downstream signalling. Given the strong effects of IL-7 on MAIT cells, we therefore investigated the potential role of IL-7Rα polymorphisms on MAIT cell numbers and function in healthy control (HC) subjects and HIV-1-infected patients on long-term ART.

The IL-7Rα promoter haplotypes were identified by haplotype-tagging SNPs from a cohort of 22 HC and 36 HIV-1-infected patients. The number and phenotype of MAIT cells, levels of the MAIT cell classical transcription factors (TFs), and MAIT cell function following bacterial stimulation were measured by flow cytometry.

We found MAIT cell levels and functions in infected patients were still significantly lower than those of HC despite an average of 7 years on suppressive ART. Interestingly, HC with IL-7Rα haplotype 2, defined as T-allele carriers at the tagging SNP rs6897932, had higher levels of MAIT cells in circulation than HC with non-haplotype 2 polymorphisms. These MAIT cells also expressed higher levels of classical TFs and appeared to have better functionality. sIL-7Rα levels were lower in HC harbouring haplotype 2, and there was a trend towards an inverse correlation between sIL-7Rα levels and MAIT cell frequency. These associations were not observed in HIV-infected patients, despite sIL-7Rα levels were still significantly lower in haplotype 2 patients. Notably, there was a significant correlation between MAIT cell levels and ART duration that was observed only in patients harbouring IL-7Rα haplotype 2. These results indicate that IL-7Rα polymorphism may play a significant role in MAIT cell biology and influence MAIT cells recovery in HIV-1 infection.

78

HIV-1 Vpu and Nef proteins interfere with MR1 surface expression

Jean-Baptiste Gorin1, Markus Moll1, Louisa Zimmermann1, Edwin Leeansyah1, Johan K Sandberg1 1. Karolinska Institutet, Stockholm, STOCKHOLMS LäN, Sweden


79

Characterization of MR-1 tetramer-positive cells in a cynomolgus macaque model of M. tuberculosis/SIV coinfection

Amy L Ellis1, Alexis J Balgeman1, Mark A Rodgers2, Cassaundra Updike2, Tonilynn Baranowski2, Charles A Scanga2, Shelby

L O'Connor1 1. University of Wisconsin-Madison, Madison, WI, United States

2. University of Pittsburgh, Pittsburgh, Pennsylvania, USA

In HIV+ individuals, the most common cause of morbidity and mortality is co-infection with Mycobacterium tuberculosis (Mtb). Mucosal Associated Invariant T (MAIT) cells detect Mtb-derived metabolites presented by MR-1 molecules. The contribution of MAIT cells to controlling Mtb replication or whether their function is compromised by co-infection with HIV is relatively understudied. HIV/Mtb co-infections can be modeled in SIV/Mtb co-infected non-human primates, facilitating longitudinal analysis of MAIT cell phenotypes and functionality in blood and bronchoalveolar lavage fluid (BALF). Furthermore, individual TB granulomas can be collected at necropsy and assessed for the presence of MAIT cells as well. In the present study, we infected SIV+ or SIV-naïve Mauritian cynomolgus macaques with Mtb and tested the hypothesis that SIV infection could alter the frequency and phenotypes of MR-1 restricted T cells in the peripheral blood. Animals were infected with a low dose (~10 CFU) Mtb Erdman strain for six weeks. The phenotype of peripheral blood MAIT cells was analyzed at this time. Animals co-infected with SIV had slightly higher frequencies of MAIT cells expressing the inhibitory markers PD1 and TIGIT, and lower frequencies of MAIT cells expressing the proliferation marker Ki-67. While this was a relatively small study, ongoing and future projects will elucidate changes in MAIT cells at higher doses of Mtb infection, as well as changes to MAIT cells in the BALF.


80

MPLA vaccine adjuvant induces group 1 CD1 protein expression on human monocytes

Naila Mebarek1, Alain Vercellone1, Tan-Yun Cheng2, Jérôme Nigou1, D. Branch Moody2, Martine Gilleron1 1. CNRS, TOULOUSE, *, France

2. Brigham and Women’s Hospital, Boston

Developing vaccines based on lipid antigens requires defining appropriate adjuvants. Whereas CD1d is constitutively expressed on antigen presenting cells (APC), CD1a, CD1b and CD1c are not. Therefore, one desired property of adjuvants would be to induce group 1 CD1 protein expression. Activation of some Toll-Like Receptors (TLR) induces group 1 CD1 protein expression on human monocytes, converting myeloid precursors into competent APC. Indeed, ligands of TLR2/1 (19 kDa lipoprotein, Pam3CSK4), TLR2/6 (Malp-2, Pam2CSK4), TLR4 (LPS) and TLR5 (flagellin) trigger expression of CD1b. In the context of Mycobacterium tuberculosis and Borrelia burgdorferi infections, TLR2-mediated induction of group 1 CD1 protein expression was found to be caused by the release of a soluble factor identified as the cytokine IL1β.

Synthetic monophosphoryl lipid A (MPLA), a nontoxic derivative of the endotoxin lipopolysaccharide, has been approved by the USDA as the first TLR agonist for use as a vaccine adjuvant (MPL®), based on its ability to direct adaptive immune responses with little toxicity. The latter has been associated with a bias toward TRIF signaling and low or no production of mature IL1β. Surprisingly, we found that MPLA is able to induce the expression of CD1a, measured as a surrogate of the group 1 CD1 proteins. Moreover, CD1a expression was inhibited by treating monocytes by Z-VAD-FMK, a potent inhibitor of caspase-1, the enzyme required for the cleavage of pro-IL1β into the mature form. Z-VAD-FMK treatment concomitantly reduced the production of IL1β measured in the cell lysate, although we do not know at this stage whether it is pro- or mature IL1β.

Altogether, our data show that MPLA induces group 1 CD1 protein expression on APC by a mechanism that is under investigation, and would be appropriate for development as an adjuvant to be incorporated in vaccines made of lipid antigens.

81

MAIT cells protect against fatal pulmonary infection by Legionella species via IFNg and can be augmented by synthetic ligands.

Huimeng Wang1, Criselle D'Souza1, Xin Yi Lim1, Lyudmila Kostenko1, Troi Pediongco1, Sidonia Eckle1, Bronwyn Meehan1, Nancy Wang1, Ligong Liu2, Jeffrey Mak2, David Fairlie2, Yoichiro Iwakura3, Jenny Gunnersen1, Andrew Stent1, Jamie Rossjohn4, 5, Glen Westall6, Lars Kjer-Nielsen1, Richard Strugnell1, James McCluskey1, Alexandra Corbett1, Timothy Hinks1, 7,

Zhenjun Chen1 1. University of Melbourne, Melbourne, VIC, Australia

2. The University of Queensland, Brisbane, Australia

3. Tokyo University of Science, Tokyo, Japan

4. Monash University, Melbourne, Australia

5. Cardiff University, Cardiff, UK

6. Alfred Hospital, Melbourne, Australia

7. University of Oxford, Oxford, UK

Mucosal Associated Invariant T (MAIT) cells are an abundant subset of immune cells. MAIT cells are found to recognize metabolites derived from bacterial riboflavin synthesis presented by MHC-related 1 (MR1) molecule, which suggests that they play a critical role in immunity against bacterial infections. By using a murine pulmonary infection model with Legionella longbeachae, we found MAIT cells accumulated robustly at the site of infection and produced several critical cytokines to control the infection, including IFN-g, GM-CSF and IL-17A. We were able to show for the first time with a clinically relevant human pathogen that MAIT cells contributed to bacterial clearance in wild type mice and this protection role became more apparent in the absence of CD4+ T cells. Notably, adoptively transferred MAIT cells provide Rag2GammaC knockout mice with full protection against lethal Legionella infection in an IFN-g dependent manner. These findings suggest that MAIT cells are important to control Legionella longbeachae; a finding of potentially wider relevance to other intracellular infections.


82

Progressive Simian-Human Immunodeficiency Virus (SHIV) infection alters expression of key surface and transcriptional proteins on macaque MAIT cells

Jennifer A Juno1, Sidonia Eckle1, Kathleen Wragg1, Bronwyn Meehan1, Thakshila Haripriya Amarasena1, Jeffrey Mak2, Ligong

Liu2, David Fairlie2, James McCluskey1, Stephen J Kent1 1. University of Melbourne, Melbourne, VIC, Australia

2. Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia

Background: MR1-restricted T cells (or MAIT cells) are depleted from the peripheral blood and exhibit functional defects during HIV infection. The mechanisms responsible for this depletion and the significance of MAIT cell dysfunction during HIV infection are not fully understood. To address this gap in knowledge in a relevant animal model, we produced Macaca nemestrina-specific, 5-OP-RU-loaded MR1 tetramers to identify pigtail macaque (PTM) MAIT cells and to characterize their phenotype and function in naïve and SHIV-infected macaques.

Results: MAIT cells were readily identified in PTM PBMC and share many phenotypic similarities with human MAIT cells, including expression of the Vα7.2 TCR, CCR6, IL-18Ra, CCR5, CXCR3 and the transcription factors PLZF and Eomes, although they express relatively low levels of Tbet. PTM MAITs exhibit robust expression of IFNγ, TNFα, GM-CSF and IL-17 following PMA/Io stimulation, but their ex vivo responses to direct 5-OP-RU stimulation are substantially weaker than human MAIT cells. Following SHIV challenge, PTM MAIT frequency is relatively stable for as long as 52 weeks post-infection and does not decline in a manner analogous to that observed in HIV infection. Progressive SHIV infection is, however, associated with phenotypic perturbation of MAIT cells, including rapid loss of CXCR3 expression, decreased CCR5 surface density, and loss of PLZF expression. Interestingly, while the majority of conventional T cells expressing the mucosal homing marker and HIV/SIV binding protein α4β7 are depleted during SHIV infection, MAIT cell a4b7 expression is maintained.

Conclusions: The production of M. nemestrina-specific MR1 tetramers has allowed for the identification and characterization of MAIT cells in PTMs for the first time. The lack of MAIT cell depletion during progressive SHIV infection is notably distinct from human HIV infection, and differences in macaque and human α4β7 expression and functional profile may point to mechanisms underpinning the HIV-specific depletion of these cells.

83

MAIT cells’ in vivo activation requirements

Huimeng Wang1, Lars Kjer-Nielsen1, Criselle D’Souza1, Timothy SC Hinks1, Lyudmila Kostenko1, XinYi Lim1, Sidonia BG Eckle1, Bronwyn S Meehan1, David C Jackson1, Ligong Liu2, Michele Teng2, Vijay K Kuchroo3, Di Yu4, Barbara Fazekas5,

David P Fairlie2, James McCluskey1, Richard A Strugnell1, Alexandra J Corbett1, Zhenjun Chen1 1. Peter Doherty Institute,University of Melbourne, Melbourne, VIC, Australia

2. Institute for Molecular Bioscience, The University of Queensland, Brisban, Queensland, Australia

3. Harvard Medical School, Brigham and Women's Hospital, Boston, Massachusetts, The United States

4. Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia

5. Sydney Medical School, The university of Sydney, Sydney, NSW, Australia

Mucosal associated invariant T (MAIT) cells detect the class I-like molecule MR1 complexed with conserved microbial riboflavin metabolites leading to subsequent MAIT cell activation and antibacterial host defence. The nature of the antigen presenting cells that activate MAIT cells and the factors that dictate their ultimate effector function are still not clear, therefore we established mouse models of different bacterial infections to evaluate these determinants. We observed that both classical haemopoietic APCs and non-bone marrow-derived APC could present antigens to MAIT cells and activate them efficiently, but the dominant APC was pathogen-dependent. In shaping effector function, the cytokines IL-12 and IL-23 both played key roles such that IL-12 tended to skew MAIT cells towards a Th1 profile whereas IL-23 directed MAIT cell effector function towards a Th17 profile simultaneously promoting MAIT cell proliferation. In addition, we found that the co-stimulating receptor, ICOS was critical for optimal MAIT cell activation. Our findings suggest how MAIT cell function might be modulated for therapeutic interventions.


84

Mucosal associated invariant T (MAIT) cells in experimental and clinical sepsis.

Shubhanshi Trivedi1, Cole Anderson1, Robert Campbell3, 2, Matthew Rondina 3, 2, Daniel Leung1 1. Division of Infectious Disease, Department of Internal Medicine, University of Utah School of Medicine , Salt Lake City , Utah

2. George E. Wahlen VAMC GRECC, Salt Lake City , Utah

3. Department of Internal Medicine, University of Utah School of Medicine , Salt Lake City

Sepsis is an acute systemic inflammatory response to infection associated with high morbidity and mortality. Recent studies have indicated that depletion of MAIT cells is associated with the development of secondary infections in septic patients. Here, we aimed to investigate the mechanisms by which MAIT cells contribute to sepsis pathology. We evaluated immune responses in C57BL/6 wild type (WT) or MHC-related-molecule I whole body knock-out (MR1-KO) mice using the cecal ligation and puncture (CLP) model of polymicrobial sepsis. We found that sepsis-induced mortality was significantly increased in MR1-KO mice compared to WT mice (p = 0.003). When lung and serum cytokine levels were assessed in these mice, levels of IL-1α, IL-1β, TNF-α, IL-27, IL-17A, IFN-β and GM-CSF in lungs, but not serum, were found to be lower in MR1-KO mice compared to WT mice. Our data suggest that MAITs may be important for protection against sepsis and that MAITs may contribute to lung-specific cytokine responses and protection against secondary infections. Furthermore, our studies in humans indicates that IFN-γ and IL-17A cytokine expression in peripheral MAIT cells are decreased in patients with severe sepsis at day 1 of illness as compared to that of day 90 and age-matched healthy controls. Insights from both mice and human studies will improve our understanding of MAIT cell function during sepsis, and highlight therapeutic potentials of MAIT cells in sepsis.

85

Anti-HIV-1 effector potential of mucosal-associated invariant T (MAIT) cells

Adam M Spivak1, Michael S Bennett1, McKenna L Coletti1, Shubhanshi Trivedi1, Fabio AP Barrios1, Matthew T

Rondina2, Daniel T Leung1 1. Division of Infectious Diseases, University of Utah School of Medicine, Salt Lake City, UT, United States

2. Internal Medicine, University of Utah School of Medicine, Salt Lake City, UT, USA

In persons living with HIV infection, antiretroviral therapy (ART) is not curative due to the persistence of replication-competent proviruses primarily in long-lived memory CD4+ T cells. One strategy to eradicate this latent reservoir, termed ‘shock and kill,’ involves administration of compounds able to reverse proviral latency in order for the immune system to target these cells. In a recent clinical trial of panobinostat, the frequency and activity of NK cells and plasmacytoid dendritic cells, but not HIV-1-specific CD8+ T cells, were associated with decreased HIV-1 proviral DNA. This in vivo data identifies the cytotoxic antiviral response of innate lymphocytes as a highly promising means to target the latent reservoir. Recent reports have identified that MAIT cells are able to target virally infected cells. Thus, we hypothesize that MAIT cells are uniquely-armed to aid in the clearance of the HIV-1 reservoir in the setting of latency reversal. To investigate this, we isolated peripheral blood mononuclear cells (PBMCs) from HIV-1-infected individuals and stimulated with microbial antigen. Using single-cell paired clonality-functionality sequencing, we show that antigen-stimulated MAIT cells have a unique clonal distribution in HIV-1-infected individuals who control viremia and maintain normal T cell counts without ART (known as elite controllers), distinct from both aviremic patients on ART and uninfected individuals. We also observed that MAIT clones expanded from PBMCs, when activated in both TCR-dependent and TCR-independent manners, exhibit potent cytotoxic activity against HIV-1-infected infected cells in vitro. In conclusion, MAIT cells represent a previously unexplored effector cell population that has the potential to contribute to recognition and clearance of HIV-1-infected cells in the context of latency reversal.

86

Interleukin-7 as a potential therapeutic strategy to reconstitute MAIT cells

Ornella Sortino1, Elizabeth Richards1, Joana Dias2, Edwin Leeansyah2, Johan K. Sandberg2, Irini Sereti1 1. NIAID, National Institutes of Health, Bethesda, Maryland, USA

2. Karolinska Institutet, Stockholm, Sweden

Mucosa-associated invariant T (MAIT) cells are antimicrobial innate-like T cells present in peripheral blood, lung, liver and mucosal tissues of humans. MAIT cells are defined by a semi-invariant T cell receptor (TCR) a-chain, are restricted by the major histocompatibility complex (MHC)-related protein 1 (MR1). Individuals with chronic HIV-1 infection have low frequencies and functional impairment of MAIT cells, and these deficiencies are not restored with antiretroviral therapy (ART). Notably, MAIT cells express high levels of IL-7R (CD127), and IL-7 dramatically enhances the function of MAIT cells from both healthy individuals and HIV-infected patients in vitro. We hypothesized that in vivo administration of IL-7 in chronically HIV-1 infected patients on ART may overcome the inability of long-term ART alone to restore MAIT cells. To address this hypothesis, we evaluated the numbers, phenotype, and functionality of MAIT cells before and after IL-7 administration in chronically HIV-1 infected individuals on ART. Seven HIV+ subjects were treated with three subcutaneous injections of IL-7 and PBMCs were collected at baseline and at 12 weeks post IL-7 treatment. IL-7 led to an increase in the frequency of MAIT cells within total lymphocytes, compared to baseline. Furthermore, the absolute number of MAIT cells among the CD3+ cell population significantly increased at week 12 compared to baseline. Notably, there was a preferential expansion of the


CD8+ MAIT cell subset. These results suggest that IL-7 may represent a therapeutic intervention for the restoration of MAIT cells in chronic HIV-1 infection and in other conditions associated with MAIT cell loss.

87

The TCR Trap: detecting the number and structure of lipids in CD1-TCR complexes

Tan-Yun Cheng1, Daniel G. Pellicci2, 3, Patrick J. Brennan1, Shouxiong Huang4, Richard W. Birkinshaw5, 6, Adam Shahine5, 6,

Kwok Wun5, 6, Stephanie Gras5, 6, Michael B. Brenner1, Jamie Rossjohn5, 6, 7, Dale I. Godfrey2, 3, D. Branch Moody1 1. Department of Medicine, Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

2. Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC, Australia

3. ARC Centre of Excellence in Advanced Molecular Imaging, University of Melbourne, Parkville, VIC, Australia

4. Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, OH, USA

5. Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria, Australia

6. ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria, Australia

7. Institute of Infection and Immunity, Cardiff University, School of Medicine, Heath Park, Cardiff, UK

The major challenge in CD1 antigen discovery is designing a system to unequivocally identify rare antigenic lipids from among abundant and structurally similar endogenous or exogenous lipid ligands that also bind CD1 proteins. We invented the T cell receptor (TCR) Trap, which takes advantage of specific TCR binding to the CD1-antigen complexes to pull out and then sensitively characterize antigenic lipids by mass spectrometry methods. Applying this method to CD1a autoreactive T cells, we discovered a highly unexpected antigen recognition pattern whereby many structurally unrelated lipids could mediate CD1a and TCR binding, leading to the absence of interference model. For CD1d-iNKT cells, the TCR trap identified a single type of α-linked monohexosylceramide in the TCR-lipid-CD1d ternary complex, bypassing the usual two-stage discovery method in which compounds are first tested for T cell activation and then later for biochemical content. Here we characterize CD1b ligands, which are all molecules eluted from cellular CD1b as well as the subset of ligands that allow ternary complexes of CD1b-lipid-TCRs to form. First, we developed a quantitative mass spectrometry method to determine the number and absolute quantities of lipid ligands bound to CD1b. Based on nominal mass and collisional pattern, we next deduced the molecular structure and exact masses of the lipid ions, emphasizing the role of phosphatidylglycerol and other phospholipids. Third, we confirmed the identity of each lipid using a quadrupole time-of-flight mass spectrometer equipped with high performance liquid chromatography, which provides a retention time and the accurate mass. Our data demonstrate a clear correlation between endogenous lipids eluted from CD1b and the cellular lipids of the expression system. Our method provides for the first time, a quantitative approach to determine the absolute amount of ligands bound to CD1b and antigens trapped in TCR and CD1 complexes.

88

Mycobacterium tuberculosis mycolic acid lipid tails govern GEM T cell responses

Andrew Chancellor1, Anna Tocheva1, Chris Cave-Ayland2, Liku Tezera1, Andrew White3, Juma Al Dulayymi4, John Bridgeman5, Susan Wilson6, 1, Marc Tebruegge8, 7, 1, Ben Marshall8, 9, 1, Sally Sharpe10, Tim Elliott11, 9, Chris-Krinton Skylaris2, 9,

Johnathan W Essex2, 9, Mark Baird4, Stephan Gadola12, 9, 1, Paul Elkington9, 7, 1, Salah Mansour9, 1 1. Academic Unit of clinical and experimental Sciences, University of Southampton, Southampton, UK

2. School of Chemistry , University of Southampton, Southampton, UK

3. National Infections Service, Public Health England, Salisbury, UK

4. School of Chemistry, Bangor University, Bangor, UK

5. Cellular Therapeutics Ltd, Manchester, UK

6. Immunohistochemistry Unit , University of Southampton, Southampton, UK

7. Global Health Research Institute, University of Southampton, Southampton, UK

8. NIHR Southampton Respiratory Biomedical Research Unit, Southampton, UJ

9. Institute for Life Sciences, University of Southampton, Southampton, UK

10. Public Health England, Salisbury, UK

11. Cancer Sciences Unit, University of Southampton, Southampton, UK

12. F.Hoffmann-La Roche Ltd, Basel, Switzerland

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major human pandemic that urgently requires new diagnostic and vaccination approaches. The lipid rich envelope of Mtb consists of diverse mycolic acids that are presented by non-polymorphic CD1b molecules to donor-unrestricted germline-encoded mycolyl lipid-reactive (GEM) T cells. However, the molecular requirements governing mycolic acid antigenicity for GEM T cell receptor (TCR) are unknown. Using a large panel of synthetic mycolates, we demonstrate that structural alterations in mycolic acid meromycolate chains that are buried deeply within the CD1b protein, are antigenic determinants that influence GEM-TCR activity. We demonstrate the antigenicity of meromycolate chains both in vitroand in ex vivo T cell responses from human TB patients, and reveal marked functional differences on GEM-TCR responses.


Meromycolate chain functional group type, position and stereochemistry were critical for mycolate antigenic potency. Computational simulations suggested that meromycolate chains regulate mycolate head group movement, directly modulating GEM-TCR activity. Finally, we demonstrate CD1b expression within human lung TB granulomas, indicating a role for CD1b-lipid antigen presentation at the site of infection. Our results have significant implications for the design of novel vaccines that target GEM T cells.

89

Sugar mimetics in α-galactosylceramide analogues: Potent immunostimulating agents via iNKT activation

Amadeu Llebaria1, Roser Borràs1, Youssef Harrak1, Lorena Usero2, Hui-Fern Koay3, 4, Daniel Pellicci3, 4, Jerome Le Nours5, 6,

Rhys Price5, 6, Tang Yongqing5, 6, Ana Sánchez2, Dale I. Godfrey3, 4, Jamie Rossjohn5, 6, Carme Roura-Mir2 1. IQAC-CSIC, Barcelona, Spain

2. Cellular Immunology Lab, Institut de Biotecnologia I Biomedicina, Universitat Autonoma de Barcelona, Bellaterra, Barcelona, Spain

3. Dept. of Microbiology and Immunology , Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Australia

4. ARC Center of Excellence in Advanced Molecular Imaging, University of Melbourne, Parkville, Australia

5. Dept. of Biochemistry and Molecular Biology, School of Biomedicical Sciences, Monash University, Clayton, Australia

6. ARC Centre of Excellence in Advanced Molecular Imaging, Monash University , Clayton, Australia

The design and synthesis of aminocyclitol moiety with control of sterochemistry and the attachment to phytoceramide backbone results in several analogues of α-galactosylceramide (aGalCer) [1, 2], a potent glycolipid antigen acting on invariant Natural Killer T (iNKT) cells, binding to the CD1d protein in presenting cells and being recognized by iNKT TCR. iNKT cells are a unique subtype of T lymphocytes expressing surface markers either of T-cells (TCR) and Natural Killer (NK1.1 in mouse and CD161 in human) cells, this singular combination of properties confer to iNKT the capability to act either in innate or adaptive immune responses. They are believed to be involved in several diseases such as type I diabetes, asthma, allergy or cancer among others. The activation of iNKT cells by aGalCer triggers a strong release of cytokines without selectivity between Th1 or Th2 response, and in some cases, after a first administration induces iNKT cells anergy. These limitations, together with the promising role of iNKT as immunotherapeutic tool, stimulate the search of new analogues with improved biological profiles, constituting an interesting and challenging research objective. Our aminocyclitol-ceramide analogues and some new structural concepts introduced have led to potent activity in vitro and in vivo. Moreover, structural studies on CD1d-Ag-TCR complex point out a binding mode similar to that of aGalCer [3, 4].

[1] Harrak, Y.; et al. ChemMedChem 2009, 4, 1608.

[2] Harrak, Y.; et al. Journal of the American Chemical Society 2011, 133, 12079.

[3] Patel, O.; et al. The Journal of Immunology 2011, 187, 4705.

[4] Kerzerho, J.; et al. The Journal of Immunology 2012, 188, 2254.

90

Characterizing β-Mannosylceramide presented by CD1d

Katharine Clark1, Anja Bloom1, David Venzon1, Motoshi Suzuki2, Lise Pasquet1, Benjamin J Compton3, Susanna Cardell4,

Steven A Porcelli5, Jay A Berzofsky1, Masaki Terabe1 1. NIH/NCI, Bethesda, MARYLAND, United States

2. NIH/NHLBI, Bethesda, Maryland, United States

3. Victoria University of Wellington, Wellington, New Zealand

4. Goteborg University, Goteborg, Sweden

5. Albert Einstein College of Medicine, New York

β-Mannosylceramide (b-ManCer) is an iNKT cell agonist that induces protection against tumors through a TNF-α-NOS dependent pathway, distinct from the IFN-Υ-dependent pathway induced by a-GalCer and other α-linked ligands.. One enigma remaining is how this agonist with β-linked sugar moiety is presented by CD1d. In this study, we asked whether monoclonal antibodies, L363 and L317, specific for an α-GalCer-CD1d complex, could bind to a β-ManCer-CD1d complex. Surprisingly both antibodies recognized β-ManCer-CD1d complexes. The antibodies also inhibited activation of both iNKT cell hybridomas and primary splenic iNKT cells. The magnitude of inhibition of β-ManCer-induced stimulation by L363 was greater than that of α-GalCer. In contrast, L363 did not bind to a sulfatide-CD1d complex or inhibit activation of a type II NKT cell hybridoma. Distinct from the iNKT cell TCR that can reorient the glycosyl head of glycolipids protruding from the CD1d groove to assume a structure like that of α-GalCer-CD1d, L363 has been reported not to be able to reorient the glycosyl head. Thus, these results suggest that the β-ManCer-CD1d complex has a three-dimensional structure similar to that of the α-GalCer-CD1d complex despite its beta-linked sugar moiety.


91

A Jurkat based NFκB-eGFP iNKT reporter cell line to evaluate the interaction of food-derived lipids with iNKT cell receptors

Piotr Humeniuk1, Wolfgang Paster2, Sabine Geiselhart1, Tonya Webb3, Peter Steinberger2, Karin Hoffmann-Sommergruber1 1. Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria

2. Institute of Immunology, Medical University of Vienna, Vienna, Austria

3. Department of Microbiology & Immunology, University of Maryland School of Medicine, Baltimore, USA

Background: In contrast to conventional T lymphocytes invariant natural killer T cells (iNKTs) recognize lipid-based antigens presented by the class I MHC homolog CD1d. Upon activation, iNKTs can affect immune responses by promoting the secretion of Th1, Th2 or Th17 immune regulatory cytokine patterns. So far, iNKTs have been identified as important players in different types of immune responses. However, the role of iNKTs in a food allergic reaction is not well understood.

Methods: An iNKT reporter system was engineered by introducing the human receptor into a human leukemic Jurkat T cell line carrying an NF-kB-driven fluorescent transcriptional reporter construct (Jkt-iNKT). Antigen presenting cells were generated by expression of human CD1d in the murine thymoma cell BW (BW-CD1d). Reporter induction (NF-kB-driven eGFP-expression) was measured by flow cytometry. The specificity and sensitivity of our system was compared with the murine DN32 hybridoma iNKT cell line based assays. Food-derived lipid extracts and separated lipid fractions were obtained by Folch extraction and preparative thin layer chromatography, respectively. Lipid samples were screened applying a plate bound assay with recombinant CD1d as well as co-culture assays with Jkt-iNKT and BW-CD1d cell lines.

Results: Jurkat cells stably expressing the human iNKT TCR receptor (Jkt-iNKT) were generated and shown to specifically react with iNKT antigens presented in the context of CD1d. Detection limit for well-known iNKT cells antigens (α-GalCer and OCH) were similar for Jkt-iNKT and DN32 cell lines. Different food-derived lipid fractions from hazelnut, walnut, sunflower, and buckwheat were separated and analyzed by TLC.

Conclusions: Our Jurkat-based iNKT cell reporter cell line proved to be a useful tool (regarding readout and feasibility) to study the activation capacity of lipid molecules to activate human iNKT receptors. In addition, our reporter system is faster and cheaper as compared to assays based on murine hybridoma iNKT cell lines.

92

Human CD1c-autoreactive T cells recognize monoacylglycerol and diverse self lipids using an ‘absence of interference’ mechanism

Josephine F Reijneveld1, 2, Tan-Yun Cheng1, Kwok Wun3, Stephanie Gras3, 4, Jamie Rossjohn3, 4, Branch Moody1, 5, Ildiko Van

Rhijn1, 5, 2 1. Brigham and Women's Hospital, Boston, MA, United States

2. School of Veterinary Medicine, Utrecht University, Utrecht, Netherlands

3. Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia

4. ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Australia

5. Harvard Medical School, Boston, MASSACHUSETTS, United States

CD1 research often focuses on finding new bacterial lipid antigens. However, autoreactivity has frequently been detected in the CD1 system. For CD1a, small headless lipids permit docking of autoreactive T cell receptors (TCRs) that bind CD1a but not the carried lipid. This ‘absence of interference’ mechanism broadly predicts features of antigen recognition: low structural specificity for the antigen carried, TCR binding to CD1 proteins carrying many diverse lipids and lipid ligands with small head groups. For CD1b and CD1d, most known TCRs show direct contact with phospholipid and glycolipid head groups that protrude from the groove. The ‘head group’ recognition model predicts high specificity for one type of lipid ligand that has a defined hydrophilic head group.

Owing to the small number of known self antigens and the lack of any ternary crystal structures, it is currently unknown whether CD1c uses one or both of these two antigen recognition mechanisms. We isolated polyclonal T cells from a healthy donor using CD1c tetramers loaded with the small, non-polar model lipid, monoacylglycerol (MAG). Several CD1c-MAG tetramer-positive subpopulations also bound CD1c tetramers that carried diverse endogenous self ligands (CD1c-endo). An IFN-y ELISPOT assay showed that CD1c-expressing antigen presenting cells activated the isolated T cells with or without the presence of MAG, but CD1a or CD1b expressing antigen presenting cells did not, confirming CD1c autoreactivity. These results demonstrate the low specificity for multiple self ligands, which are the functional hallmarks of ‘absence of interference.’ Further we detect CD1c-endo tetramer staining on T cells in many human blood donors, suggesting that this recognition pattern is common. Overall, this work identifies a new self antigen for the CD1c system and points toward broad use of a mechanism that shows an absolute requirement for CD1c but low specificity for the self lipid carried.


93

The ceramide structure of sulfatide-analogues influences the functional activity of type II NKT cells

Lise Pasquet1, Kaddy Camara2, Anja C. Bloom1, Stewart K. Richardson2, Amy R. Howell2, Masaki Terabe1, Jay A. Berzofsky1 1. NCI/NIH, Bethesda, MD, United States

2. Department of chemistry, University of Connecticut, Storrs, CT, United States

Type II NKT cells are CD1d-restricted lymphocytes that, in contrast to type I NKT cells, do not express a semi-invariant TCR, but a diverse repertoire. Whereas all type I NKT cells recognize α-galactosylceramide, type II NKT cells react against different lipids and one subset is specific for sulfatide. Sulfatide (C24:1) is composed of a galactose head carrying a sulfate group and β-linked to a ceramide portion formed by a 24-carbon fatty acid chain with 1 double bound and an 18-carbon sphingosine chain. When loaded into CD1d, the ceramide portion is inserted in the CD1d binding grove leaving the sugar moiety protruding to be recognized by the TCR. It is described that the structure of the ceramide influences the interaction of the TCR with the lipid-CD1d complex and determines the reactivity to the lipid. We synthesized diverse sulfatide-analogues varying in the number of double bonds in the fatty acid and in the number of hydroxyl groups and double bonds in the sphingoid base and evaluated, in vitro and in vivo, the reactivity of type II NKT cells with these analogues. We used fluorescently labeled, analogue-loaded-CD1d tetramers to stain lung mononuclear cells and identified sulfatide (C24:1), phyto-C24:1, C24:2 and phyto-C24:2 reactive NKT cells. All the analogue-reactive cells also reacted with sulfatide, but did not recognize α-GalCer. Therefore, analogue-specific cells were distinct from type I NKT cells and were sulfatide-reactive type II NKT cells. In vitro, all analogues induced a CD1d-dependent IL-13 production but at different levels. In vivo, analogues had variable effects on the number of liver or lung metastases in mouse tumor models. It is known that sulfatide-reactive type II NKT cells have a regulatory role in several diseases. The identification of new ligands inducing altered functional activity of these cells might be of great interest for developing new anti-tumor immunotherapies.

94

CD1b presents Borrelia burgdorferi glycolipid to human T cells.

Peter Reinink2, 1, Michael Souter3, 4, Tan-Yun Cheng1, Joanna Kubler-Kielb5, Klemen Strle6, Allen Steere6, Branch Moody1, Dan

Pellicci3, 4, Dale Godfrey3, 4, Ildiko van Rhijn2, 1 1. Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital and Harvard Medical School, Boston, Boston, MA, USA

2. Department of Infectious Diseases and Immunology , Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands

3. Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Australia

4. Australian Research Council Centre of Excellence for Advanced Molecular Imaging, University of Melbourne, Melbourne, Australia

5. National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA

6. Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Boston, MA, USA

Lyme disease is caused by the spirochete Borrelia burgdorferi. Two ubiquitous lipids of Borrelia burgdorferi, BBGL1 and BBGL2, comprise 35% of the total lipid mass of the bacteria and are specifically synthesized by pathogenic Borrelia spp. It is known from previous studies that BBGL2 can bind to CD1d and activate CD1d restricted NKT cells. In this study, we carried out FACS-sorting with CD1b-BBGL2 tetramers, to obtain a T cell line from a Lyme disease patient that recognizes BBGL2 presented by CD1b. The T cell clone binds to CD1b-BBGL2 tetramers but not to mock loaded CD1b tetramers or CD1b loaded with the negative control lipid phosphatidylglycerol. Although there is specific recognition of the CD1b-BBGL2 complex using tetramers, the primary T cells produce comparable levels of IFN-γ in an ELISPOT assay when stimulated with CD1b-expressing antigen presenting cells in the presence or absence of BBGL2 lipid. The TCR sequence was determined by single cell sequence and by transduction of the TCR we demonstrated that BBGL2 specificity was mediated by the TCR. Whereas the primary T cells preferably bind to CD1b tetramers loaded with BBGL2, we think that the activation by antigen presenting cells without the addition of BBGL2 is caused by a combination of a low affinity interaction with endogenous lipid-loaded CD1b and a high expression level of CD1b on the antigen presenting cells. We call this phenomenon “antigen-modulated autoreactivity” against the CD1b molecule, where there is an increased reaction to an antigen over a baseline autoreactivity of the T cell towards CD1b. Currently, more Lyme disease patients are being screened for the presence and frequency of CD1b-BBGL2 specific T cells using CD1b tetramers.


95

CD1b tetramers identify T cells that recognize natural and synthetic diacylated sulfoglycolipids from Mycobacterium tuberculosis

Charlotte A. James1, Krystle K. Quan1, Martine Gilleron2, Jacques Prandi2, Vijayendar R. Yedulla3, Zuzanna Z. Moleda3, Eleonora Diamanti3, Momin Khan4, Varinder K. Aggarwal4, Peter Reinink5, Stefanie Lenz5, Ryan O. Emerson6, Thomas J. Scriba7, Michael N. T. Souter8, Dale I. Godfrey8, Daniel G. Pellicci8, D. Branch Moody9, Adriaan J. Minnaard3, Chetan

Seshadri1, Ildiko Van Rhijn5, 9 1. Department of Medicine, University of Washington, Seattle, WA, United States

2. Institut de Pharmacologie et Biologie Structurale, Université de Toulouse, Toulouse, France

3. Stratingh Institute for Chemistry, University of Groningen, Groningen, The Netherlands

4. School of Chemistry, University of Bristol, Bristol, United Kingdom

5. Department of Infectious Diseases and Immunology, School of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

6. Adaptive Biotechnologies, Seattle, Washington, United States

7. South African Tuberculosis Vaccine Initiative and Institute of Infectious Disease and Molecular Medicine, Division of Immunology, Department of Pathology, University of Cape Town, Cape Town, South Africa

8. Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, and Australian Research Council Centre of Excellence for Advanced Molecular Imaging, The University of Melbourne, Melbourne, Australia

9. Department of Rheumatology, Allergy and Immunology, Brigham and Women’s Hospital, Boston, Massachusetts, United States

Diacylated sulfoglycolipids (Ac2SGL) are a family of cell wall lipids that have only been detected in virulent strains of Mycobacterium tuberculosis. T cells are activated by Ac2SGL when the lipids are bound to CD1 molecules, but tools to study Ac2SGL-specific T cell responses in humans are lacking. To study the molecular requirements for T cell receptor (TCR) binding and T cell activation, we generated CD1b tetramers loaded with natural or synthetic Ac2SGL analogs. Due to the relatively non-polymorphic nature of CD1 among human populations these tetramers can be applied to samples independently of the donor’s genetic background. T cell l ines derived using natural Ac2SGL are activated by synthetic analogs in a manner that is relatively independent of lipid chain length and hydroxylation but sensitive to saturation status. By contrast, two T cell lines derived using a tetramer loaded with unsaturated Ac2SGL are not activated by the natural antigen. Notably, the TCRs of T cell clones specific for the natural antigen share no similarity to the TCRs of the T cell clones that are only activated by unsaturated Ac2SGL analogs. As contrasted with other systems in which the naturally occurring lipid variation does not control T cell responses, these data show that each molecular variant within a class of naturally occurring lipids represents a distinct antigen. This principle will guide the emerging use these tetramers in large-scale translational studies investigating the diagnostic potential of SGL-specific T cell responses and SGL-based vaccine strategies. It is imperative to first understand whether synthetic analogs with a precise chemical definition are bioequivalent to the mixture of antigens that comprise natural Ac2SGL. These data provide a hierarchy of synthetic Ac2SGL analogs, and analogs with a high bioequivalence to the natural mixture can be used to generate tetramers for such studies.

96

Sortase-A-based strategy for the analysis of CD1d-associated lipids

Maren Thomaier1, Yuting Wang1, Emilie Huc-Claustre2, Laura Trautenberg1, Sandrine Baltzer1, Lingyun Dai3, Gijs

Grotenberg3, Andrej Shevchenko4, Sebastian Zeissig1, 5 1. DFG-Center for Regenerative Therapies Dresden Technische Universität Dresden, Dresden, SAXONY, Germany

2. CRTC Cancer Research Centre Toulouse, Toulouse, France

3. Singapore-MIT Alliance Department, National University of Singapore, Singapore

4. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

5. Universitäts Klinkum Karl Gustav Carus, Dresden, Germany

The analysis of lipids associated with CD1 is technically challenging as detergents required for the extraction of CD1 from membranes lead to the loss of bound lipids. The majority of studies that previously analyzed CD1-associated lipids therefore used genetically modified CD1 proteins lacking the transmembrane domain and that are directly secreted into the cell culture supernatant after passage through the secretory pathway1-3. These studies gave important information on the endogenous lipids bound to CD11-3. However, lack of the cytoplasmic tail of soluble CD1 proteins is associated with defect endolysosomal trafficking and thus does not allow the study of lipids that associate with CD1 in these compartments. We therefore aimed to build on recent studies that developed cleavable CD1 proteins with cytoplasmic tails3, 4 to study CD1-associated lipids.

To this end, we developed an engineered CD1d protein carrying the recognition motif of Sortase-A (SortA) from S. aureus in its extracellular juxtamembrane domain. The transpeptidase SortA cleaves its recognition sequence, which is not present in eukaryotes, forming an acyl-enzyme intermediate and subsequently links the cleaved protein covalently to an N-terminal triglycine motif6, 7. This strategy leads to a surface cleavable CD1d protein with a C-terminal tag allowing for detergent-free extraction and tag-based purification of CD1d that is suitable for the analysis of associated lipids using shotgun and HPLC-MS based lipidomics.

Mouse CD1d carrying the SortA recognition motif could be expressed in various cell lines, showed expression levels comparable to wildtype CD1d and was able to load and present exogenous lipid antigens to NKT cells. Moreover, nearly complete shaving of cell-surface CD1d was achieved without alterations in cell viability. CD1d could be coupled to various tags and purified using affinity


chromatography. As such, SortA-based strategies allow for detergent-free, enzymatic harvest of CD1d proteins from the cell surface for subsequent analysis of associated lipids.

1. 1) Cox, D., Fox, L., Tian, R., Bardet, W., Skaley, M., Mojsilovic, D., Gumperz, J. & Hildebrand, W. Determination of cellular lipids bound to human CD1d molecules. PLoS One 4, e5325 (2009).

2. 2) Huang, S., Cheng, T.Y., Young, D.C., Layre, E., Madigan, C.A., Shires, J., Cerundolo, V., Altman, J.D. & Moody, D.B. Discovery of deoxyceramides and diacylglycerols as CD1b scaffold lipids among diverse groove-blocking lipids of the human CD1 system. Proc Natl Acad Sci U S A 108, 19335-19340 (2011).

3. 3) Yuan, W., Kang, S.J., Evans, J.E. & Cresswell, P. Natural lipid ligands associated with human CD1d targeted to different subcellular compartments. J Immunol 182, 4784-4791 (2009).

4. 4) Muindi, K., Cernadas, M., Watts, G.F., Royle, L., Neville, D.C., Dwek, R.A., Besra, G.S., Rudd, P.M., Butters, T.D. & Brenner, M.B. Activation state and intracellular trafficking contribute to the repertoire of endogenous glycosphingolipids presented by CD1d [corrected]. Proc Natl Acad Sci U S A 107, 3052-3057 (2010)

5. 5) Olszak T, Neves JF, Dowds CM, Baker K, Glickman J, Davidson NO, Lin CS, Jobin C, Brand S, Sotlar K, Wada K, Katayama K, Nakajima A, Mizuguchi H, Kawasaki K, Nagata K, Müller W, Snapper SB, Schreiber S, Kaser A, Zeissig S, Blumberg RS. Protective mucosal immunity mediated by epithelial CD1d and IL-10. Nature. 2014 May 22;509(7501):497-502.

6. 6) Popp, M.W., Antos, J.M., Grotenbreg, G.M., Spooner, E. & Ploegh, H.L. Sortagging: a versatile method for protein labeling. Nat Chem Biol 3, 707-708 (2007).

7. Witte, M.D., Wu, T., Guimaraes, C.P., Theile, C.S., Blom, A.E.M., Ingram, J.R., Li, Z., Kundrat, L., Goldberg, S.D. & Ploegh, H.L. Site-specific protein modification using immobilized sortase in batch and continuous-flow systems. Nat. Protocols 10, 508-516 (2015).

97

A multi-tetramer flow cytometry panel for studying the phenotypes of human donor-unrestricted T cells in clinical studies

Erik D Layton1, Malisa Smith1, Krystle K Quan1, Thomas J Scriba2, Chetan Seshadri1 1. Department of Medicine, University of Washington, Seattl, Washington, United States of America

2. Department of Pathology, South African Tuberculosis Vaccine Initiative (SATVI) and Institute of Infectious Disease and Molecular Medicine, Division of Immunology, University of Cape Town, Cape Town, South Africa

The non-polymorphic antigen presenting molecules CD1, MR1, BTN3A1 are used by antigen presenting cells to present non-peptide antigens to T cells. These “donor-unrestricted T cells” (DURTs) have not been extensively evaluated in human clinical studies unlike T-cells specific for peptide antigens. To address this, we developed a multi-parameter flow cytometry panel aimed at characterizing and quantifying the phenotypes of DURTs in clinical specimens. The core of this panel consists of the T-cell lineage (CD3, CD4, and CD8), memory (CD45RA, and CCR7) markers, as well as markers for δ1 and δ2 expressing γδ T cells. 5-OP-RU loaded MR1 tetramer and α-galactosylceramide (α-GalCer) loaded CD1d tetramer identify canonical populations of mucosal associated invariant T (MAIT) cells and invariant NKT cells respectively. Anti-TRAV1-2 helps define MAIT cells and germline-encoded mycolyl reactive (GEM) T cells. GEM T cells are specific for the immunodominant antigen, glucose monomycolate (GMM), which is presented by CD1b and common to many mycobacteria. In contrast with the CD1b GMM tetramer, CD1b diacylated sulfoglycolipid (Ac2SGL) tetramer distinguishes T cells specific for virulent mycobacteria. Optimization of this panel was done through antibody titrations and fluorescence minus one (FMO) experiments using T-cell lines specific for each of the tetramer reagents. Tetramers were qualified with respect to linearity, range, limit of detection, limit of quantification, reproducibility, repeatability, intermediate precision, and accuracy. Finally, we used this panel to perform a cross-sectional study of a well characterized cohort of South African adolescents with and without latent tuberculosis infection (LTBI). We used t-Distributed Stochastic Neighbor Embedding (t-SNE) to visualize the data, and we found qualitative differences among cells specific to CD1b-GMM and CD1b-Ac2SGL in the presence of LTBI. This panel is easily modified to include additional tetramers or phenotypic markers to characterize DURTs in studies of mycobacterial infection, disease, and vaccination.

98

Serum albumin delivers LPC to DCs resulting in enhanced autoreactive activation of human iNKT cells

Xuefen Yang1, Lisa Fox2, Akshat Sharma2, ZY Jiang1, Jenny E. Gumperz2 1. Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou, GUANGDONG, China

2. Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Madison, WI

Human invariant natural killer T (iNKT) cells have been shown to recognize lysophosphatidylcholine (LPC) as an antigen presented by CD1d. However, the physiological sources of LPC that activate iNKT cells remain unclear, since LPC and related lysophospholipids are produced both intracellularly and extracellularly through the action of a variety of phospholipase A2 enzymes. The major extracellular repositories and transporters of lysophospholipids are apolipoproteins and serum albumin. We found that pre-exposing human monocyte-derived DCs to medium containing human albumin, but not human apolipoproteins, resulted in an enhanced ability to activate iNKT cell IFN-g secretion. Pre-exposing DCs to de-lipidated human albumin failed to improve their ability to stimulate the iNKT cells. The activity of the de-lipidated albumin was recovered when it was pre-treated with synthetic LPC, but not when it was pre-


treated with phosphatidylcholine (PC) or oxidized PC. Mass spectrometric analysis identified a C18:1 form of LPC among the compounds in an organic extract of human serum albumin. DCs exposed to LPC-loaded albumin showed upregulated expression of the adhesion ligand ICAM-1, and demonstrated increased conjugation with iNKT cells. iNKT cell IFN-γ secretion via this pathway appeared to be partially dependent on CD1d recognition and TCR signaling, and was also dependent on IL-12 stimulation and JAK signaling. Since addition of an anti-CD40 blocking mAb abrogated the iNKT cell IFN-γ secretion, we hypothesize that the iNKT cell response is a result of a feedback interaction in which the iNKT cells engage CD40 on the DCs, which stimulates the DCs to produce IL-12 that in turn co-stimulates iNKT cell IFN-γ secretion. These results show for the first time that extracellular LPC is delivered to DCs via serum albumin, resulting in enhanced autoreactive activation of human iNKT cells.

99

Recognition of a microbial glycolipid antigen by both Type 1 and Type 2 NKT cells

Catarina F Almeida1, 2, Srinivasan Sundararaj3, 4, Jerome Le Nours3, 4, Onisha Patel3, 4, Benjamin Cao5, Daniel Pellicci1, 2,

Spencer Williams5, Jamie Rossjohn3, 4, 6, Adam Uldrich1, 2, Dale Godfrey1, 2 1. Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne , Melbourne, VIC, Australia

2. Australian Research Council Centre of Excellence in Advanced Molecular Imaging, The University of Melbourne, Melbourne, VIC, Australia

3. Department of Biochemistry and Molecular Biology, School of Biomedical Sciences , Monash University, Melbourne, Clayton, Australia

4. Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Monash, VIC, Australia

5. School of Chemistry , Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC, Australia

6. Institute of Infection and Immunity, Cardiff University, School of Medicine, Heath Park, Cardiff, UK

Natural Killer T (NKT) cells are T cell lymphocytes that recognise lipid antigens presented by the MHC Class I-like molecule CD1d. There are two broad types of NKT cells: Type 1 NKT cells, which display limited T cell receptor (TCR) diversity and recognise a common lipid-Ag: α-Galactosylceramide α-GalCer), while Type 2 NKT cells express diverse TCRs and do not recognise α-GalCer. It is generally considered that there is no overlap between the lipid antigens that these two types of NKT cells recognize. However, here we demonstrate that the microbial-derived lipid-Ag α-glucuronosyl-diacylglycerol-(α-GlcA-DAG) can be recognized by subsets of both Type 1 and Type 2 NKT cells as well as a population of Vα10Jα50 T cells that fall between Type 1 and Type 2 NKT cells, as we have previously described. Single-cell TCR sequencing and the generation of TCR-transduced cell lines confirmed the diversity of TCRs that are capable of recognizing and responding to this antigen. Furthermore, through examination of the interaction with a panel of CD1d mutant molecules, combined with x-ray crystallographic analysis we revealed that Type 2 NKT cell TCRs can interact with CD1d plus α-GlcA-DAG in a similar manner to the highly conserved docking mode displayed by Type 1 NKT TCRs. Taken together, these data show that Type 2 NKT cells can detect a spectrum of lipid antigens presented by CD1d using a range of CD1d-docking modes.

100

Chemical insights and new antigens for MAIT cell activation

Jeffrey YW Mak1, 2, Weijun Xu1, 2, Robert C Reid1, 2, Alexandra J Corbett3, Bronwyn S. Meehan3, Huimeng Wang3, Zhenjun

Chen3, Jamie Rossjohn4, 5, 6, James McCluskey3, Ligong Liu1, 2, David P Fairlie1, 2 1. ARC Centre of Excellence in Advanced Molecular Imaging, Brisbane, Queensland, Australia

2. Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland, Australia

3. Department of Microbiology & Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia

4. Infection and Immunity Program & Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia

5. ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, Victoria, Australia

6. Institute of Infection and Immunity, Cardiff University, Cardiff, United Kingdom

Mucosal associated invariant T cells are activated by chemically unstable antigens (e.g. 5-OP-RU and 5-OE-RU). Here we compare and rationalize antigen instability, and develop a superior preparation of antigens with vastly improved purity and stability compared to current preparations. Secondly, we have created a functionally equivalent analogue of 5-OP-RU that is completely stable in water. Its MAIT cell activation potency correlates with its computationally predicted conformation. This analogue upregulates surface expression of human MR1, enables MR1 refolding to form MR1 tetramers that detect MAIT cells in human PBMCs, and stimulates cytokine expression (IFNγ, TNF) by human MAIT cells. The analogue can also induce MAIT cell accumulation in mice lungs after administration with a co-stimulant. These antigens are useful for in vitro and in vivo MAIT cell studies, and their chemical and biological properties will be described.


1. Mak, J. Y. W. et al. Stabilizing short-lived Schiff base derivatives of 5-aminouracils that activate mucosal-associated invariant T cells. Nat. Commun. 8, 14599

102

Unravelling the molecular mechanisms of how MR1 presents a pathogen metabolic signature to MAIT cells

Hamish EG McWilliam1, Matthew Zorkau1, Sam Wormald2, Justine Mintern3, James McCluskey1, Jamie Rossjohn4, Jose

Villadangos3, 1 1. The Peter Doherty Institute, University of Melbourne, Melbourne, Victoria, Australia

2. The Walter and Eliza Hall Institute, Melbourne, Victoria, Australia

3. Bio21, University of Melbourne, Parkville

4. Monash University, Clayton, Victoria, Australia

MR1 is the only antigen presenting molecule that displays a pathogen metabolic signature, the Vitamin B-related antigens (VitBAg). This allows the MR1-restricted MAIT cells to detect infection even if the antigen presenting cell has not physically encountered the pathogen. Given the distinct nature of MR1 ligands and their role, the MR1 presentation pathway is expected to possess unique features absent in other pathways of antigen presentation. Indeed, we recently showed that MR1 is the only human antigen presenting molecule that does not constitutively present self-ligands. Instead the majority of MR1 accumulates in the endoplasmic reticulum (ER) in a pool of empty molecules until a VitBAg forms a covalent bond (Schiff base) with a Lys residue in the antigen binding site of MR1. This acts as a “molecular switch” that triggers complete folding and egress of MR1 molecules from the ER to the cell surface (1). We hypothesised that MR1 requires molecular machinery along this pathway, therefore we aimed to characterise these accessory proteins that enable MR1 presentation of VBAg. Using both a genome-wide CRISPR-Cas9 screen and a proteomics approach we identified chaperones required for MR1 stabilisation, folding, and trafficking to and from the cell surface. We found that tapasin and tapasin-related protein both bound to ER-resident MR1, and the deletion of these resulted in a loss of the majority of the ligand-receptive MR1 pool due to faster degradation of MR1. While nascent molecules could still load and present VitBAg without these chaperones, the loss of the MR1 pool resulted in a significantly reduced ability to present VitBAg. Therefore, these chaperones play a key role in stabilising empty MR1 in the ER for the rapid presentation of bacterial metabolites, and illustrates a role for these chaperones beyond facilitating MHC class I peptide loading.

1. McWilliam et al, Nat Immunol 2016

103

Development of humanized mouse models for in vivo studies of human iNKT cells

Xiangshu Wen1, Seil Kim1, Michelle Li1, Ping Rao1, Omid Akbari1, Remy Bosselut2, Paolo Dellabona3, Giulia Casorati3, Mark

Exley4, Weiming Yuan1, 5 1. University of Southern California, Los Angeles, CA, United States

2. Center for Cancer Research National Cancer Institute, Bethesda, MA, USA

3. Experimental Immunology Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy

4. Agenus, Inc and Harvard Medical School, Boston, MA, USA

5. Molecular Microbiology and Immunology, University of Southern California, Los Angeles, CA, 90033

Invariant NKT (iNKT) cells are innate-like T cells, which demonstrate potent anti-tumor function in mouse models. In sharp contrast, iNKT cell ligands have shown limited efficacy in human anti-tumor clinics, mostly due to the significant differences in CD1d lipid presentation properties and profound disparity in the composition and abundance of iNKT cells between human and mice. To build more relevant in vivo models for studying human iNKT cells, we have developed a CD1d-humanized mouse model (hCD1d-KI) with human CD1d knocked in (Proc. Natl. Acad. Aci. USA 110: 2963-8, 2013). To further humanize the mouse model, we introduced human invariant TCRa chain (Va24Ja18) into the hCD1d-KI mice. Interestingly, we observed a human-like iNKT cell abundance and co-receptor expression pattern in the humanized mouse models. In particular, we detected a substantial subset of iNKT cells expressing CD8ab (J. Immunol. 195:1459-69, 2015). The CD8ab+ iNKT cells show a strong Th1-biased cytokine response and potent cytotoxicity upon activation. The lower binding of iNKT TCRs to human CD1d/lipid complex than that of mouse counterparts as well as higher prevalence of Vb7 TCRb in these CD8+ iNKT cells suggested a low avidity-based developmental program for these iNKT cells which includes the suppression of the transcriptional factor, Th-POK. Using a Th-POK-transgenic mouse, we demonstrated that the suppression of Th-POK is essential for the development of CD8ab+ iNKT cells. Our establishment of the new humanized mouse models will facilitate the investigation of in vivo functional properties of human iNKT cells and identification of optimal glycolipid ligands for iNKT cell-based immunotherapies.


104

Innate lymphoid cells modulate iNKT cell immunity

Rebeca Jimeno1, Julia Saez de Guinoa1, Nazanin Farhadi1, Peter Jervis2, Liam R Cox3, Gurdyal S Besra2, Patricia Barral1 1. KINGS COLLEGE LONDON, LONDON, United Kingdom

2. School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, Uk

3. School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

Innate lymphoid cells (ILCs) are a family of immune cells that function as critical orchestrators of immune responses at mucosal surfaces. They can be classified into three different populations based on the expression of canonical transcription factors ILC1 (T-bet+), ILC2 (GATA3+) and ILC3 (RORγt+). ILCs are emerging as central regulators of immunity as they are able to control the homeostasis and activation of a broad range of cells including B cells, dendritic cells, intestinal epithelial cells or conventional T cells. However, their possible contribution to lipid-dependent immune responses has never been explored. We have investigated whether ILCs participate in the regulation of iNKT cell immunity. We have found that murine ILCs from various tissues express CD1d, being ILC3 the population that express the highest levels of CD1d. ILC3s are able to internalize and present lipids on CD1d to iNKT cells inducing their activation. Conversely, crosslink of CD1d in vitro and administration of lipid antigen in vivo results in ILC3 activation and cytokine production. Our data identifies a novel ILC3-iNKT cell axis, which could function in a variety of immune responses where CD1d-dependent immunity plays a central role.

106

The role of MR1 isoforms in antigen presentation to MAIT cells

Gitanjali A Narayanan1, Elham Karamooz2, 3, Melanie J Harriff2, 3, David M Lewinsohn2, 3 1. Department of Biomedical Engineering, Oregon Health and Science University, Portland, OR, USA

2. Department of Pulmonary and Critical Care Medicine, Oregon Health and Science University, Portland, OR, USA

3. Portland VA Medical Center, Portland, OR, United States

Mucosal-associated invariant T (MAIT) cells are highly enriched in the lung and play a key role in early immune defense against a broad array of pathogens, including mycobacteria. MR1 is a Class I-related molecule that presents metabolites of the riboflavin synthesis pathway to activate MAIT cells. MR1 is unusual amongst MHC molecules as MR1 pre-mRNA undergoes alternative splicing to produce multiple isoforms. Only the full length isoform, MR1A, is known to activate MAIT cells. The other isoforms, MR1B and MR1C, are not well characterized, although MR1B is thought to activate MAIT cells. To test the function of MR1 isoforms, we transfected MR1-deficient A549 cells with plasmids expressing each of the MR1 isoforms. We then infected these cells with M. smegmatis and tested their ability to activate MAIT cells. Cells expressing MR1A activated MAIT cells, but neither MR1B nor MR1C could do so. When coexpressed with MR1A, MR1B inhibited MAIT cell activation following infection. We further show that MR1A and MR1B colocalize inside intracellular vesicles. We hypothesize that MR1B prevents MR1A loading with ligand or trafficking to the cell surface. These data suggest that MR1A mediated activation of MAIT cells may be regulated by MR1B, thereby avoiding inappropriate MAIT cell activation in the absence of mycobacterial infection.

107

Targeting S1P to restore NKT cell responses to lymphoma

Michael S Lee1, Tonya J Webb1 1. University of Maryland, Baltimore, Baltimore, MD, United States

Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin’s lymphoma, which encompasses a group of cancers derived primarily from B cells that have been increasing in incidence over the past 30 years. Despite being initially responsive to combination chemotherapy, median survival remains around 5 years due to high rates of relapse. Natural Killer T (NKT) cells play an important role in cancer surveillance and can reduce MCL tumor burden in vivo; however, NKT cells are reduced in number and function in MCL patients compared to healthy donors. Sphingolipid metabolism plays an important role in regulating MCL survival and proliferation. We found that sphingosine-1-phosphate (S1P) is upregulated in MCL cells. Knockdown of sphingosine kinase 1 (SK1), the enzyme responsible for generating S1P, in human MCL cells results in a significant increase in CD1d-mediated NKT cell activation, as assessed by cytokine production and cytotoxicity. Lipidomic studies identified cardiolipin as being upregulated in SK1 knockdown cells. Cardiolipin has been reported to bind to CD1d molecules. Thus, to test if cardiolipin could induce NKT cell activation, we pulsed antigen-presenting cells with cardiolipin and then co-cultured the cells with a panel of NKT cell hybridomas. We found that pretreatment with cardiolipin leads to increased cytokine production by NKT hybridomas. Cardiolipin is typically sequestered in the mitochondrion; therefore, future studies will focus on identifying factors required for CD1d-mediated processing and presentation of cardiolipin. Collectively, these studies will delineate novel pathways important for immune recognition of malignant cells and could lead to the development of new treatments for lymphoma.


108

Vitamin A-Mediated Regulation of CD1 Expression During Human Dendritic Cell Differentiation

Sara Suliman1, Tiffany Amariuta2, 3, Samira Asgari2, 3, Ildiko Van Rhijn1, Megan B Murray4, Soumya Raychaudhuri2, 3, D Branch

Moody1 1. Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women's Hospital, Boston, MA, United States

2. Division of Biomedical Informatics, Harvard Medical School, Boston, MA, United States

3. Program in Medical and Population Genetics, Broad Institute, Cambridge, MA, United States

4. Department of Global Health and Social Medicine, Harvard Medical School, Boston, MA, United States

While most work has focused on CD1 proteins as antigen presenting molecules, CD1 proteins also serve as defining features of human dendritic cell (DC) differentiation. Prior gene expression studies in response to bacterial factors highlighted opposing regulation of group 1 CD1 (CD1a, CD1b and CD1c) and group 2 (CD1d), where CD1a, CD1b and CD1c were thought to be concomitantly upregulated and undertake parallel functions in presenting foreign lipid antigens to T cells. CD1a expression in monocyte derived DCs was previously shown to be inhibited by all-trans-retinoic-acid, a precursor of which is vitamin A. Interestingly, vitamin A deficiency was recently reported to be a strong predictor of progression to active tuberculosis in patients in Peru, and thus providing a previously unknown perspective on the role of a common nutrient in tuberculosis outcomes. We hypothesized that risk of tuberculosis progression can be partly mediated by vitamin A effects on DC maturation, tracked by upregulation of CD1 proteins.

We used multi-parameter flow cytometry to broadly determine expression patterns of CD1 and other markers on monocytes differentiated with IL-4 and GM-CSF. CD1c and HLA-DR were almost universally expressed on these cultured DCs by the second day. Subsequently, DCs upregulated CD1b expression by day 3, and fully differentiated into CD1a+CD1b+ cells by day 6. All-trans-retinoic-acid selectively inhibited transition from the CD1a-CD1b+ into CD1a+CD1b+ DC stages in a dose-dependent manner. Conversely, chemical inhibition of all-trans-retinoic-acid reversed this effect. These results demonstrate orchestrated step-wise up-regulation of CD1 proteins during in vitro differentiation of DCs, leading to ongoing efforts to define transcriptional factors that control CD1 protein expression and DC differentiation. These findings will shed light on how a common micronutrient regulates CD1 expression as a proxy for differentiation of DCs, which are currently used as agents of immunotherapy and vaccination.

109

A proteomics screen to identify MR1 associated proteins and implications for MR1 function

Prabhjeet Phalora1, Svenja Hester1, Emanuele Marchi1, James Ussher2, Shabaz Mohammed1, Paul Klenerman1 1. University of Oxford, Oxford, OXFORDSHIRE, United Kingdom

2. Department of Microbiology and Immunology, University of Otago, Otago, Dunedin, New Zealand


110

Role of cellular actin in CD1d-mediated antigen presentation

Dilip Shrestha1, Sebastian Strauss2, Marco Fritzsche1, Mariolina Salio1, Vincenzo Cerundolo1, Christian Eggeling1 1. Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom

2. Max Planck Institute of Biochemistry, Munich, Germany

Adaptive immunity is critical for distinguishing self from non-self threats. However, how our immune system is able to respond to dangers and spare self entities remains a subject of immense research. Due to its non-polymorphic nature and its capability to present diverse lipids to T cells, featuring polarizing immunomodulatory functions, the antigen-presenting molecule CD1d has been a focal point in vaccine development in the last decade. Consequently, there is a fair understanding of the relationship between the types of lipids presented by CD1d and its associated T cell-mediated biological functions. The molecular mechanisms in antigen-presenting cells (APCs) governing the presentation of these antigens to T cells however remain relatively less known, yet they are important for guiding the antigen-related effector functions of T cells. Here, using advanced live-cell optical microscopy, we studied the role of the intracellular cortical actin cytoskeleton in CD1d-mediated lipid antigen presentation. We used the drugs known to specifically interfere with actin polymerization and compared the effect they had on the lateral diffusion of plasma membrane CD1d and on their efficiency to activate T cells. These experiments uncovered a crucial role for the actin cortical meshwork in APCs in fine-tuning lipid antigen presentation. We also found a decrease in CD1d mobility upon lipid antigen loading which is potentially a result of an accompanying extensive reorganization of actin cytoskeleton.


111

A Tail of MR1 Internalization

Jacinta M Wubben1, 2, 3, Hamish EG McWilliam4, Jerome Le Nours1, 2, 3, James McCluskey4, Jose A Villadangos4, 5, Jamie

Rossjohn1, 2, 3, 6 1. Infection and Immunity Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia

2. Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia

3. Australian Research Council (ARC) Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, VIC, Australia

4. Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Parkville, VIC, Australia

5. Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, VIC, Australia

6. Institute of Infection and Immunity, Cardiff University School of Medicine, Cardiff, United Kingdom

MR1 is a MHC-I like molecule that is ubiquitously expressed in a number of cell types and presents exogenous vitamin B metabolites to MAIT cells. Recently, it has been determined that MR1 resides in the endoplasmic reticulum until it binds its antigen, then is trafficked through the Golgi to be presented at the cell surface. After cell surface presentation, approximately 65% of MR1 is internalized within 4 hours and is degraded via endocytosis. Nonetheless, a small percentage (~5%) is recycled from the endosome to the cell surface, which provides an opportunity for ligand exchange to occur.

While the cellular pathway of internalization and recycling of MR1 is now known, the molecular mechanism of internalization is still unclear. Consequently, the aim of this study was to identify the sequence motifs within the cytoplasmic tail of MR1 that is required for internalization. To investigate this, a series of stable C1R cell lines overexpressing mutants of the cytoplasmic tail of MR1 were created via retroviral transduction and characterized for the ability of these forms to traffic to and from the cell surface. No significant difference was observed in the ability of the mutant MR1 molecules to traffic to the cell surface compared to the wild-type MR1. However, different mutants of the cytoplasmic tail resulted in variations in the rate of internalization of MR1. These findings suggest that there are multiple mechanisms contributing to the internalization of MR1 from the cell surface, which will need to be further investigated.

112

Identification of novel modulators of MR1 trafficking using a gene trap screen in haploid cells

Corinna A Kulicke1, 2, Erica De Zan3, Mariolina Salio1, Paul Klenerman4, 2, Sebastian M Nijman3, Vincenzo Cerundolo1 1. MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK

2. Peter Medawar Building for Pathogen Research, Nuffield Department of Medicine, University of Oxford, Oxford, UK

3. Ludwig Institute for Cancer Research Ltd. and Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK

4. Translational Gastroenterology Unit, John Radcliffe Hospital, Headington, Oxford, UK

The monomorphic MHC-I-related protein 1 (MR1) presents vitamin metabolites to mucosal-associated invariant T (MAIT) cells, an innate-like subset of T lymphocytes. The best characterised MAIT-activating MR1 ligands are unstable pyrimidine intermediates of riboflavin synthesis, a pathway specific to certain fungi and bacteria and, thus, intrinsically non-self for humans. While it has been reported that soluble, extracellular ligands encounter MR1 in an incompletely folded, ligand-receptive state in the endoplasmic reticulum (ER), it remains to be determined how the antigenic metabolites initially gain access to the cytosol and, ultimately, reach the ER. Here, we use a functional genetic screening technique based on insertional mutagenesis of the near-haploid human cell line HAP1 to discover novel players in MR1 antigen presentation and trafficking. A HAP1 clone overexpressing human MR1 was transduced with a gene trap virus to inactivate genes in an unbiased manner. Subsequently, the mutagenised population was incubated with the MR1-stabilising ligand Acetyl-6-Formylpterin, stained for MR1 surface expression, and analysed by flow cytometry. The tails of the distribution were FACS sorted to enrich for cells in which positive or negative regulators of MR1 were inactivated. Mapping of the viral insertion sites by Illumina deep sequencing allowed identification of genes statistically overrepresented in either of the two sorted populations which constitute putative modulators of MR1 intracellular trafficking or MR1 stability. Among the most significant positive regulators of MR1 surface expression identified in our screen was β2-microglobulin, binding of which is a known requirement for the surface translocation of MR1. A number of putative regulators of MR1 trafficking were validated using a CRISPR/Cas9 approach and tested in functional and biochemical assays.


113

A whole genome mouse siRNA screen to identify novel genes involved in lipid antigen presentation

Shilpi Chandra1, James Gray2, William B Kiosses1, Archana Khurana1, Kaori Hitomi1, Catherine Crosby1, Zheng Fu3, Meng Zhao1, Natacha Veerapen4, Stewart K. Richardson5, Steven A. Porcelli6, Gurdyal Besra4, Amy R. Howell5, Sonia Sharma2, 7,

Bjoern Peters8, Mitchell Kronenberg1, 9 1. Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, LA Jolla, CA, United States

2. The Functional Genomics Center, La Jolla Institute for Allergy and Immunology, La Jolla, CA, United States

3. Bioinformatics Core, La Jolla Institute for Allergy and Immunology, La Jolla, CA, United States

4. School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK

5. Department of Chemistry, University of Connecticut, Storrs, CT, USA

6. Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, United States

7. Division of Cellular Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, United States

8. Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, United States

9. Division of Biological Sciences, University of California, San Diego, La Jolla, CA, United States

The pathways governing lipid antigen uptake and presentation by CD1 proteins remain incompletely defined. We have carried out a whole genome siRNA screen in a macrophage cell line for genes that affect the presentation of a potent glycosphingolipid antigen, GalGalCer, to iNKT cells. This antigen requires internalization and lysosomal carbohydrate antigen processing to remove the terminal galactose. After several rounds of validation, functional classification and gene expression analysis, we identified a set of 57 genes that lead to decreased CD1d antigen presentation. The majority of the genes we identified, including members of the HOPS and ESCRT complexes, did not perturb surface CD1d expression, but we demonstrate instead that they effected the formation of surface CD1d complexes with the stimulating glycolipid, either by altering antigen localization, CD1d traffic, or both. Interestingly, our data show that Abcc1 and several other ABC family transporters are involved in lipid antigen presentation by CD1d. Mice deficient for Abcc1 had reduced iNKT cell responses to glycolipid lipid antigens, and decreased survival following infection with Streptococcus pneumoniae, which has an antigen that stimulates iNKT cells. Although the CD1d and MHC-II antigen presentation pathways both depend on antigen loading in the lysosome, the majority of genes did not affect MHC-II antigen presentation, indicating a clear distinction between these two pathways.

Supported by NIH grants AI 71922, AI 105215 and U01 GM111849

114

Role of mucosal-associated invariant T cells in lupus pathogenesis.

Goh Murayama1, Asako Chiba1, Ken Yamaji1, Naoto Tamura1, Sachiko Miyake1 1. JUNTENDO UNIVERSITY SCHOOL OF MEDICINE, TOKYO, JAPAN, Bunkyo-ku, TOKYO, Japan

Background: Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the production of autoantibodies. Previously we reported association of MAIT cell activation with disease activity in patients with systemic lupus erythematosis. In this study, We set out to clarify functions of MAIT cells in a lupus model by using FcγRIIB-/- Yaa mice.

Methods: FcγRIIB-/-Yaa mice were crossed to MR1 deficient mice lacking MAIT cells, and disease progression was compared between MR1KO FcγRIIB-/- Yaa and MR1+/+ FcγRIIB-/- Yaa mice at 1-4 months of age. T follicular helper cells (TFH cells) and regulatory T cells (Tregs) among splenocytes from these mice were analyzed by using flowcytometer.

Results: MR1KO FcγRIIB-/-Yaa mice showed a reduction of serum anti-dsDNA antibody levels and an increase of survival rate. There was a trend of less proteinuria and less severe nephritis in MR1KO FcγRIIB-/-Yaa mice. MR1KO FcγRIIB-/-Yaa mice developed exacerbated inflammation in the skin lesions with a higher histopathological dermatitis score compared to MR1+/+ FcγRIIB-/-Yaamice. Flow cytometric analysis of splenocytes revealed that the proportion of TFH cells was decreased but that of Tregs was increased in MR1KO FcγRIIb-/-Yaa mice compared to MR1+/+ FcγRIIb-/-Yaa mice.

Conclusions: These data suggests that MAIT cells exhibit dual roles in lupus pathogenesis. MAIT cells enhance autoantibody production and the disease severity of nephritis, but have a suppressive effect on dermatitis. Further studies are under going to uncover the mechanisms by which MAIT cells are involved in each target tissues.

115

Redistribution of functionally impaired MAIT cells to bile ducts in primary sclerosing cholangitis

Erik von Seth1, Christine Zimmer1, Marcus Reuterwall-Hansson1, Ammar Barakat1, Urban Arnelo1, Annika Bergquist, Martin

Ivarsson1, Niklas Björkström1 1. Karolinska Institutet, Stockholm, STOCKHOLM, Sweden

Primary sclerosing cholangitis (PSC) is chronic liver disease characterized by inflammation and fibrosis of small and large bile ducts commonly associated with inflammatory bowel disease (IBD). MAIT cells are abundant in the human liver and have been shown to locate around bile ducts. Interestingly biliary epithelial cells can present antigen via MR1 and activate MAIT cells. However, the role of


MAIT cells in PSC remains unclear. We used PBMCs to characterize MAIT cells in PSC patients comparing them to healthy individuals and patients with IBD and primary biliary cholangitis (PBC) as controls. In a first step we noted a reduced MAIT cell frequency in PSC patients, as well as in diseased controls, compared to healthy individuals with preferential loss of CD8+ MAIT cells. Further phenotypical analysis showed a slight increase in the expression of activation markers in PSC patients and reduced expression of CXCR6+ as compared to healthy individuals. In contrast, diseased controls exhibited a more activated phenotype. In line with the noted phenotypic alterations, MAIT cell function was reduced in response to E.coli or cytokine stimulation in both PSC and IBD patients whereas PBC disease controls presented with intact function. Finally, when investigating whether MAIT cells localized to bile ducts during PSC, we noted a tenfold increase in the number of the MAIT cells residing in PSC patient bile ducts compared to controls. These findings suggest a redistribution of functionally impaired MAIT cells to the compartment affected by chronic inflammation in PSC.

116

Ex Vivo expansion of iNKT cells for adoptive cell therapy.

Abel Trujillo-ocampo1, Hyun-Woo Cho1, Wilfredo Ruiz-Vazquez1, Jeffrey Molldrem1, Jin S. Im1 1. The University of Texas M.D. Anderson Cancer Center, Texas Medical Center, HOUSTON, United States

Invariant Natural Killer T (iNKT) cells are unique subset of innate T cells that regulate several immune responses, thus have potential for novel immunotherapeutics to modulate adaptive immunity in certain diseases such allergy, infectious, autoimmune disease and cancer. One of the limitations for developing iNKT cell therapy is difficulty to acquire clinical meaningful number of iNKT cells as they are extremely rare population comprising only less than 0.1% of circulating T cells. Here, we developed an effective strategy to expand iNKT cells in Ex Vivo for adoptive cell therapy. First, iNKT cells from adult peripheral blood mononuclear cells (PBMC) and cord blood mononuclear cells (CBMC) were enriched by MACS with anti-iNKT microbeads, and subsequently co-cultured with allogeneic dendritic cells in the presence of αGalCer and IL-2. After single antigen specific stimulation for 10-14 days, we obtained 2.8x10e7 iNKT cells (range: 0.1-7.0x10e7) from 12 consecutive 5x10e8 adult PMBC, and 1.2x10e7 iNKT cells (range: 0.4-4.9x10e7) from 10 consecutive 5x10e8 CBMC. Although the lower absolute number of iNKT cells was present in cord blood, cord iNKT cells underwent a drastic expansion by 493.7 folds (range:200-1684) compared to 71.2 folds (range:22-3937) for adult iNKT cells. The purity of expanded iNKT cells were consistently greater than 90%. Expanded cord iNKT cells were exclusively CD4+ (97%, range:96.5-99.79%), while adult iNKT cells contained varying degree of CD4+(68.8%, range:38-89%), CD4-CD8alpha-(22.8%, range:4.0-47.9%), and CD8alpha+ (6.2%, range:0.73-17.80%). Expanded cord CD4+iNKT cells showed a profound polarization towards Th2- functional phenotype compared to expanded adult CD4+iNKT cells, and expanded adult CD4-iNKT cells displayed polarizationtowards Th-1 functional phenotype and expressed higher degrees of various natural killer receptors. In summary, we demonstrated that it is feasible to acquire highly pure, and highly polarized iNKT cells through single antigenic expansion in clinically meaningful number for adoptive cell therapy to modulate adaptive immunity in immune-microenvironment.

117

A phase I vaccination study with dendritic cells loaded with NY-ESO-1 and a-galactosylceramide: induction of polyfunctional T cells in high-risk melanoma patients

Olivier Gasser1, Katrina Sharples2, Catherine Barrow3, Geoffrey Williams4, 5, Evelyn Bauer1, Catherine Wood1, 3, Brigitta Mester1, Marina Dzhelali3, Graham Caygill6, Jeremy Jones6, Colin Hayman7, Victoria Hinder8, Jerome Macapagal8, Monica

McCusker8, Robert Weinkove1, Gavin Painter7, Margaret Brimble4, 5, Michael Findlay8, Rod Dunbar4, 5, Ian Hermans1 1. Malaghan Institute of Medical Research, Wellington, New Zealand

2. University of Otago, Dunedin, New Zealand

3. Capital and Coast District Health Board, Wellington, New Zealand

4. University of Auckland, Auckland, New Zealand

5. Maurice Wilkins Center for Molecular Biodiscovery, Auckland, New Zealand

6. Glycosyn, Wellington, New Zealand

7. Ferrier Institute, Victoria University of Wellington, Wellington, New Zealand

8. Cancer Trials New Zealand, University of Auckland, Auckland, New Zealand

Vaccines that elicit targeted tumor antigen-specific T cell responses have the potential to be used as adjuvant therapy in patients with high risk of relapse. However, the responses induced by vaccines in cancer patients have generally been disappointing. In order to improve vaccine function, we investigated the possibility of exploiting the immunostimulatory capacity of type 1 Natural Killer T (NKT) cells, a cell type enriched in lymphoid tissues that can trigger improved antigen-presenting function in dendritic cells (DCs). In this phase I dose-escalation study, we treated eight patients with high-risk surgically resected stage II-IV melanoma with intravenous autologous monocyte-derived DCs loaded with the NKT cell agonist α-GalCer and peptides derived from the cancer testis antigen NY-ESO-1. Two synthetic long peptides spanning defined immunogenic regions of the NY-ESO-1 sequence were used. This therapy proved to be safe and immunologically effective, inducing increases in circulating NY-ESO-1-specific T cells that could be detected directly ex vivo in seven out of eight patients. These responses were achieved using as few as 5 x 105 peptide-loaded cells per dose. Analysis after in vitro restimulation showed increases in polyfunctional CD4+ and CD8+ T cells that were capable of manufacturing two or more cytokines simultaneously. Evidence of NKT cell proliferation and/or NKT cell-associated cytokine secretion was seen in most patients. In light of these strong responses, the concept of including NKT cell agonists in vaccine design requires further investigation.


118

A role of AhR signaling in NKT cell immunotherapy

Mariko Takami1, Fumie Ihara1, Toshiko Kamata1, 2, Shinichiro Motohashi1 1. Department of Medical Immunology, Graduate School of Medicine, Chiba University, Chiba-shi, CHIBA, Japan

2. Department of Thoracic Surgery, Kimitsu Central Hospital, Kisarazu-shi, CHIBA, Japan

Invariant natural killer T (iNKT) cells express T-cell receptor, which recognizes α-galactosylceramide (α-GalCer) presented on CD1d, and they also play an essential role in antitumor immunity. We previously developed NKT cell immunotherapy, which adoptively transfers α-GalCer pulsed dendritic cells (DCs) to activate iNKT cells followed by the activation of other immune cells for treating lung cancer patients. Since some patients responded to the treatment and showed an increased IFN-γ production with a prolonged survival whereas others did not, it is important to understand the mechanism by which NKT cells are activated by DCs in order to improve the current therapy. Aryl hydrocarbon receptor (AhR) is a transcription factor that translocates from the cytoplasm to the nucleus upon ligand binding. AhR signaling is known to modulate multiple immune cell functions and differentiation. However, whether or not AhR signaling plays any role in NKT cell immunotherapy still remains unclear. We therefore sought to determine whether AhR signaling affects the NKT cell responses in antitumor immunity. We found that monocyte derived DCs (moDCs) treated with the AhR ligand FICZ had the ability to induce greater cytokine production, including IFN-γ and TNF-α, by iNKT cells in vitro. Furthermore, the expression of the immune checkpoint molecules PD-L1 and PD-L2 was downregulated on these moDCs. These data suggest that the AhR ligand may be potentially useful for next-generation NKT cell immunotherapy. We are currently investigating the mechanism underlying PD-L downregulation by AhR signaling.

119

Loss and exhaustion of MAIT cells in cholangiocarcinoma

Christine L Zimmer1, Lena Berglin1, Martin Cornillet1, Otto Strauss1, Erik von Seth2, Ewa C Ellis3, Annika Bergquist2, Niklas K

Bjorkstrom1 1. Center for Infectious Medicine, Karolinska Institutet, Stockholm, Sweden

2. Center for Digestive Diseases, Karolinska University Hospital, Stockholm, Sweden

3. Division of Transplantation Surgery, CLINTEC, Karolinska Institutet, Stockholm, Sweden

Cholangiocarcinoma (CCA) is a malignancy with dismal prognosis that arises from biliary epithelial cells (BECs) lining intra- and extra-hepatic bile ducts. BECs possess antigen presentation capacity and can activate MAIT cells via MR1. MAIT cells are known to be enriched in human liver and locate around bile ducts. Thus, MAIT cells may affect antitumor immune responses in CCA. However, little is known regarding MAIT cell presence and function in tumors. When characterizing MAIT cells in non-tumorous tissues, we noted a specific enrichment of the cells within the biliary tract system. Assessing CCA, the tumor microenvironment was characterized by a retained presence of cytotoxic T cells and high numbers of regulatory T cells. However, a specific loss of MAIT cells was noted within tumors as compared to surrounding non-affected liver tissue. The residual intratumoral MAIT cell population was enriched for cells expressing tissue residency markers but displayed an exhausted phenotype. Intriguingly, the loss of MAIT cells was accompanied by high expression of MR1 within tumors. This expression was primarily confined to myeloid cells suggesting an MR1-dependent mechanism driving the loss and exhaustion of intratumoral MAIT cells. It still remains to be explored whether the residual MAIT cells possess the capacity to target and kill tumor cells and whether such targeting is beneficial for the patients.

120

Th1 biased iNKT-cell activation as an effective anti-tumor response

Ignasi Esteban1, Alejandra Saavedra, Alari-Pahissa Elisenda, A. Raul Castano1 1. Universidad Autónoma de Barcelona, Cerdanyola Del Vallés, SPAIN

Activation of iNKT cells is a potentially potent immunotherapeutic tool, but the excessive potency of the prototypic α-GalCer agonist with simultaneous production of pro- and anti-inflammatory cytokines hampers its clinical use. α-GalCer analogs may overcome these limitations if capable of directing the response to the appropriate effector brand.

We previously showed how a synthetic analog with a modified polar head induced a strong Th1 response and an efficient iNKT cell dependent antitumor response in a lung metastases model. A new analog analyzed induced a stronger and absolutely specific Th1 response, with no traces of Th2 cytokines. These in vivo characteristics of iNKT agonists were not anticipated by in vitro functional assays. The characteristic cytokine storm produced by α-GalCer was not induced, which translates in increased antitumor response both in preventive and therapeutic settings, more efficiently controlling the establishment of lung metastases. Other tumor models, as survival experiments after i.p. administration of tumor lines, also showed a more potent antitumor response.

Secondary transactivation of adaptive and innate effector cells is potently induced, with strong mobilization and activation. In vivo cytotoxicity experiments show differential responses, both quantitative and qualitatively, against NK-sensitive or fully competent targets raised by the different agonists. In vivo depletion experiments demonstrate involvement of NK cells and macrophages in tumor killing, which varies depending on the target tumor and the iNKT agonist. Furthermore, differential activation kinetics and cytokine and chemokine induction, may underline mechanistic differences in the antitumor response.


Additionally, adjuvancy functionality in the induction of humoral response against model antigen is qualitatively different depending on the agonist, with an immunoglobulin subtype change toward opsonizing antibodies, precisely those that mediate anti-pathogenic effector cellular responses. As this new agonist also activates human iNKT cells, it may be an advantageous reagent for future translation to the clinic.

121

IL-21 maintains CD62L expression during NKT-cell ex vivo expansion and enhances antitumor activity of NKT cell therapy in vivo

Ho Ngai1, Gengwen Tian1, Amy N Courtney1, Ekaterina Marinova1, Wei Huang1, Linjie Guo1, Leonid S Metelitsa1 1. Baylor College of Medicine, Houston, TX, United States

Vα24-invariant Natural Killer T cells (NKTs) have potent antitumor properties and are being developed for cellular immunotherapy of cancer. Such therapy requires extensive ex vivo expansion of primary NKTs while preserving their longevity and function. In a recent report from our group, CD62L+ subset of NKTs has been shown to have longer persistence in vivo and stronger antitumor activity than CD62L- counterpart. However, the requirements for the preservation of CD62L+ NKTs during ex vivo expansion remain largely unknown. Comparative gene expression analysis of CD62L+ and CD62L- NKT subsets revealed a significantly higher expression of IL-21R in the former, which was confirmed at the protein level by flow cytometry. Hence, we hypothesized that IL-21 preferentially supports CD62L+ NKTs. To test this hypothesis, we expanded primary human peripheral blood NKTs using in vitro stimulation with their cognate antigen, α-galactosylceramide. The culture was supplemented with IL-2, IL-21, or both cytokines. We found that in contrast to IL-2, IL-21 alone failed to support NKT-cell expansion. However, a combined treatment with IL-2 and IL-21 produced more NKTs compared with IL-2 alone. Moreover, the former condition significantly increased frequency of CD62L+ NKTs that was associated with the selective downregulation of a pro-apoptotic gene BCL2L11 in the CD62L+ NKT-cell subset. We also found that IL-2/IL-21-expanded NKTs were more cytotoxic against lymphoma cells that correlated with enhanced expression of granzyme B. Following adoptive transfer to NSG mice, IL-2/IL-21-expanded NKTs persisted significantly longer and had higher therapeutic efficacy in a lymphoma model compared with IL-2-expanded NKTs. Our results instruct inclusion of IL-21 in the NKT-cell expansion protocols for cancer immunotherapy applications.

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Regulatory T cells increased in advanced head and neck cancer patients suppress NKT cell function and correlate with disease progression

Fumie Ihara1, Daiju Sakurai1, Mariko Takami1, Yoshitaka Okamoto1, Shinichiro Motohashi1 1. Chiba University, Chiba, CHIBA, Japan

Background: We have been developing NKT cell-based immunotherapy for head and neck cancer (HNC) patients who achieved complete response after standard therapy. However, the existence of immunosuppressive cells, including regulatory T cells (Tregs), could be an important factor limiting the efficacy of NKT cell-based immunotherapy for advanced cancer-bearing patients. Therefore, we examined the association between the frequency of peripheral blood Tregs and the clinical outcome of HNC patients, as well as the influence of Tregs on NKT cell function.

Methods: Forty-six HNC patients who received standard therapy at Chiba University Hospital, 23 benign tumor patients, and 4 healthy volunteers were enrolled. We examined the frequencies of functional Treg subsets based on the expressions of Foxp3, CD4, and CD45RA in peripheral blood mononuclear cells of these patients during standard treatment.

NKT cells obtained from healthy volunteers were cultured in the presence of α-GalCer and IL-2 for 7 days. Tregs from Naïve CD4+ T cells were cultured in the presence of IL-2, TGF-b and anti-CD3/CD28 beads for 7 days. These induced NKT cells and Tregs were co-cultured with or without use of a Transwell chamber, and NKT cell function was analyzed. We also examined the effect of paclitaxel on the suppressive activity of Tregs on NKT cells.

Results: Treg frequency was significantly higher in HNC patients than that in benign tumor patients. Increased frequency of Tregs was decreased after the standard curative treatment which, in turn, increased again during the early phase of recurrence. Functional analysis in vitro indicated that Tregs inhibited the function of NKT cells by cell-to-cell contact. Paclitaxel restored the impaired NKT cell function by inhibiting Tregs proliferation.

Conclusion: Increased Tregs are associated with the clinical outcome of HNC patients. Although Tregs inhibited the NKT cell function, paclitaxel restored NKT cell activation.


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Investigating the role of human MAIT cells in breast duct carcinogenesis

Nicholas A Zumwalde1, Jill D Haag1, Michael N Gould1, Jenny E Gumperz1 1. University of Wisconsin - Madison, Madison, WI, United States

Most human breast malignancies arise in the ductal epithelium, which is colonized by a unique microbiome, yet the role of innate T lymphocytes that recognize microbial products in breast carcinogenesis is not known. We recently characterized the major immune subsets associated with primary human breast epithelial ducts and identified for the first time the presence of Va7.2+ mucosal-associated invariant T (MAIT) cells. The majority of breast-derived MAIT cells appeared Th1-biased after PMA/ionomycin stimulation, however, a substantial fraction produced IL-17A but not IFNg. Exposure to E. coli treated breast carcinoma cell lines potently induced MAIT cell cytokine production, and also led to surface expression of LAMP-1, suggesting cytolytic degranulation. Addition of an anti-MR1 antibody almost completely blocked MAIT cell TNFa production, and also partially blocked IFNg and degranulation, in response to bacterially treated breast carcinoma cells. However, a portion of the MAIT cell effector responses may be mediated by expression of counter-ligands for the NKG2D receptor on breast carcinoma lines. Thus, MAIT cells may influence breast duct carcinogenesis in multiple ways. Their IL-17A production may ultimately play a pro-tumorigenic role; however, since it is also known to provide epithelial barrier integrity, it may hinder malignancy at earlier stages. Moreover, MAIT cells may carry out immunosurveillance of nascently transformed cells by either T cell receptor-dependent or innate (e.g. NKG2D-mediated) pathways. A key future question is whether imbalances within the breast duct microbiome promote cancer by influencing the functions of breast MAIT cells.

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Profiling human innate-like T cells in cancer and checkpoint blockade

Ellie-May Jarvis2, 1, 3, Shaun Collings3, Astrid Authier-Hall1, Nathaniel Dasyam1, Alice Maxwell4, Jessica Lowe4, Catherine

Barrow4, Brendan Luey4, John Nacey1, 3, Brett Delahunt2, Olivier Gasser1, Ian Hermans1, 5, 6, Robert Weinkove2, 1, 4 1. Malaghan Institute of Medical Research, Wellington, New Zealand

2. Department of Pathology and Molecular Medicine , University of Otago Wellington, Wellington, New Zealand

3. Wellington School of Medicine , University of Otago Wellington, Wellington, New Zealand

4. Wellington Blood and Cancer Centre, Capital and Coast District Health Board, Wellington, New Zealand

5. Maurice Wilkins Centre, Auckland, New Zealand

6. School of Biological Sciences, Victoria University, Wellington, New Zealand

Innate-like T (ILT) cells, such as iNKT, MAIT and Vγ9Vδ2 T cells, have been exploited both as effectors and as adjuvants in cancer immunotherapy studies, including in a clinical trial at our own centre. Advanced malignancies are often associated with immune suppression, so knowledge of the function of ILTs in patients with cancer, and the impact of immune-modulatory treatments such as checkpoint blockade, is important to inform the design of future ILT cell-activating immunotherapies.

To address this, we are conducting three complementary studies: 1. a cross-sectional study to assess the frequency and function of ILTs in men with localised and advanced prostate cancer, compared to age-matched male controls; 2. in vitro investigations using healthy human blood cells to assess the expression and function of immune checkpoints on ILTs, and their amenability to blockade by clinically-available inhibitors; and 3. a longitudinal study examining ILT frequency and function before and during PD1 blockade for metastatic melanoma.

To date, we have recruited 36 men with prostate cancer (both low grade, indolent disease and metastatic, castrate-resistant disease), 20 healthy male age-matched controls and 10 patients receiving pembrolizumab for melanoma. We have developed a suite of assays to characterise ILT frequency and function from peripheral blood mononuclear cells, preliminary results of which indicate a functional impairment of blood iNKT cells from men with prostate cancer. Finally, our in vitro assays indicate that iNKT and MAIT cells express PD1, and that PD1 ligation inhibits their function.

We expect these studies to contribute to the development of ILT cell-targeted immunotherapies in patients with cancer, in terms of patient selection and to inform the rational design of combination trials with checkpoint blockade.

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Activation of MAIT cells by adenovirus vectors

Nicholas M. Provine1, Ali Amini1, Lucy Garner1, Maria Luisa Esposito2, Stephanie S.M. Slevin1, Michael FitzPatrick1, Kate D. Williamson1, Helen Ferry1, Lian Ni Lee3, Senthil K. Chinnakannan3, Stefania Capone2, Antonella Folgori2, Eleanor Barnes3, 4,

Paul Klenerman1, 3, 4 1. Translational Gastroenterology Unit, NDM Experimental Medicine, University of Oxford, Oxford, Oxfordshire, United Kingdom

2. ReiThera, Srl, Rome, Italy

3. Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, Oxfordshire, United Kingdom

4. NIHR Biomedical Research Centre, Oxford, Oxfordshire, United Kingdom

Adenovirus-derived (Ad) vectors are being developed for use as anti-cancer vaccines and as oncolytics. Additionally, Ad vectors are undergoing testing as vaccine vectors against every major human pathogen. It is now appreciated that different Ad vector serotypes induce distinct innate immune responses in vivo and in vitro, in addition to circumventing pre-existing immunity. This includes differential induction of IFN-γ by T cells. However, the processes driving this robust IFN-γ production remain unexplained. We thus hypothesized that (1) Ad vectors activate MAIT cells, and (2) that distinct serotypes of Ad vectors differ in their capacity to activate MAIT cells.

To test these hypotheses, human PBMCs were transduced with Ad serotype 5 (Ad5) vectors and ChAdOX1 vectors. After 24 hours, T cell activation was assessed by expression of CD69, Granzyme B, and IFN-γ. Consistent with our hypotheses, ChAdOX1 robustly induced IFN-γ production by MAIT cells (mean of 49.7%) at an MOI of 103 VP. By contrast, Ad5 induced negligible production of IFN-γ (mean of 0.5%). Similar differential induction of Granzyme B and CD69 was also observed. Mechanistically, the differential capacity of ChAdOX1 and Ad5 to activate MAIT cells was due to two factors: (1) ChAdOX1 robustly induced IL-18 production by monocytes, while Ad5 induced no detectable IL-18; and (2) ChAdOX1 transduced pDCs and induced production of IFN-α/β, while Ad5 failed to transduce pDCs. Finally, we determined that Cathepsin B-mediated activation of the NLRP3 inflammasome was critical for the production of IL-18 and activation of MAIT cells in response to ChAdOX1 stimulation.

In summary, we demonstrate for the first time that a virus-derived, candidate oncolytic/vaccine vector platform can potently activate innate-like T cells, and distinct vectors display differential activation capacity. These data can inform improved, rational design of adenovirus vectors as therapeutics for cancer and infectious diseases.

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Interrogating the VEGF immunosuppressive axis in ovarian cancer

Dominique Bollino1, Ravikumar Muthuswamy2, Kunle Odunsi2, Tonya J Webb1 1. Microbiology and Immunology, University of Maryland , Baltimore, Maryland, USA

2. Gynecologic Oncology and Immunology, Roswell Park Cancer Institute, Buffalo, New York, USA

Ovarian cancer accounts for more deaths worldwide than any other gynecologic malignancy. Despite advances in treatment, mortality rates have remained steady, underscoring the need for new therapeutic strategies. A significant challenge for current therapies is overcoming tumor immunosuppression, therefore the identification of immunosuppressive factors produced within the tumor microenvironment, and the ability to target these factors could enhance anti-tumor immune responses. We have previously shown that ovarian cancers shed the ganglioside GD3, which binds with high affinity to CD1d molecules, thereby suppressing NKT cell activation. In addition to GD3, we have identified vascular endothelial growth factor (VEGF) as an immunosuppressive factor secreted by ovarian cancers. Importantly, the level of VEGF in ovarian cancer serum and ascites fluid has been reported to directly correlate with disease burden, and is inversely correlated with survival. We found that VEGF inhibition results in decreased GD3 levels in human ovarian cancer cell lines and a concomitant restoration of NKT cell responses. To determine the mechanism by which VEGF signaling induces GD3 shedding by ovarian cancer cells and to elucidate the effect of VEGF on NKT cell function in vivo, we utilized the ID8 ovarian cancer mouse model. We found that ID8 cells overexpressing VEGF (ID8-VEGF) shed higher levels of GD3, and tumor-associated lymphocytes from ID8-VEGF tumor-bearing mice have decreased immune function. In good agreement with our preclinical studies, we found that higher VEGF levels in the ascites of ovarian cancer patients correlates with decreased immune cell function in lymphocytes isolated from both the tumor microenvironment and the periphery. These data indicate that ovarian cancers may use VEGF to inhibit NKT cell responses as an early mechanism of tumor immune evasion, and suggest that targeting the VEGF/GD3 pathway may have a significant impact on anti-tumor immune responses.

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Novel agonists to explore the function of mucosal-associated invariant T (MAIT) cells as cellular adjuvants

Joshua Lange1, Regan Anderson2, Olivier Gasser1, Gavin Painter2, Ian Hermans1 1. Malaghan Institute of Medical Research, Wellington, WELLINGTON, New Zealand

2. Ferrier Institute, Wellington, New Zealand

Invariant natural killer T (iNKT) cells act as cellular adjuvants by providing co-stimulation to antigen presenting cells (APCs) upon recognition of the glycolipid, a-galactosylceramide (aGalCer), in the context of CD1d. Thus, when aGalCer is co-administered with soluble peptide antigens, iNKT cells are able to license APCs, thereby enhancing the immunogenic presentation of peptide:MHC complexes to CD8+ T cells, leading to increased antigen-specific immunity. However, in mice, iNKT cells represent ~1% of all circulating T cells while in humans iNKT cells are of a much lower frequency (~0.01 - 0.1% of all circulating T cells). Whether this significant discrepancy between mouse and human iNKT cell frequency will limit their capacity to act as cellular adjuvants in clinical applications is unknown.

Another innate-like T cell population, mucosal associated invariant T (MAIT) cells, has recently been recently described to play key roles in both sterile and non-sterile pathologies. Indeed, MAIT cells can rapidly exert effector functions upon TCR dependent recognition of riboflavin metabolites, presented in the context of MR1, or in a TCR independent manner via cytokines. Importantly, and in contrast to iNKT cells, they represent an abundant population of circulating human T cells (1-10%). Using novel chemistry, we have generated synthetic MAIT cell agonists to determine whether, similarly to iNKT cells, MAIT cells can be harnessed as immunotherapeutic adjuvants.

128

Harnessing unconventional T cells and their targets for immunotherapy with high affinity bi-specific TCRs

Marco ML Lepore1, Johanne JP Pentier1 1. Immunocore Ltd, Abingdon, OXON, United Kingdom

T cells have the capacity to detect an extremely large variety of antigens and mount robust inflammatory and cytotoxic responses against transformed or infected cells. Because of these unique properties, T cells are subjects of intense translational research activity, which aim to develop effective immunotherapy approaches to treat cancer and infections. These studies focus on “classical” MHC- restricted T cells recognizing peptide presented by cancer or infected cells.

In addition to peptides, T cells also recognize other classes of antigens, including lipids and metabolites displayed by non-polymorphic antigen presenting molecules such as CD1, MR1 and BTN3A1. These T cells, defined as “unconventional”, comprise heterogeneous populations with diverse roles in cancer, infections and autoimmunity, and therefore display a broad immunotherapeutic potential, as suggested by a number of recent proof-of principle studies.

At Immunocore, we developed a novel class of soluble bi-specific biologics, ImmTACTM/V, consisting of affinity-enhanced TCRs recognizing selected tumor or microbial antigens presented by specific HLAs, fused to an anti-CD3 effector domain. The TCR domain strongly binds cells expressing both the target antigen and the specific HLA type, while the anti-CD3 domain induces activation of the patient’s T cells, thus redirecting their response toward malignant or infected cells.

We aim to apply our ImmTAC/V technology to also target disease-associated non-peptide molecules recognized by unconventional T cells. As lipid and metabolite antigens are subjected to a low-mutation rate and are presented by non-polymorphic proteins, this approach may overcome the therapeutic limitations imposed by the occurrence of immune-escape variants and the high polymorphism of HLAs.

129

Harnessing unconventional T cells and their targets for immunotherapy with high affinity bi-specific TCRs

Johanne JP Pentier1, Marco ML Lepore1 1. Immunocore Ltd, Abingdon, OXON, United Kingdom

T cells have the capacity to detect an extremely large variety of antigens and mount robust inflammatory and cytotoxic responses against transformed or infected cells. Because of these unique properties, T cells are subjects of intense translational research activity, which aim to develop effective immunotherapy approaches to treat cancer and infections. These studies focus on “classical” MHC- restricted T cells recognizing peptide presented by cancer or infected cells.

In addition to peptides, T cells also recognize other classes of antigens, including lipids and metabolites displayed by non-polymorphic antigen presenting molecules such as CD1, MR1 and BTN3A1. These T cells, defined as “unconventional”, comprise heterogeneous populations with diverse roles in cancer, infections and autoimmunity, and therefore display a broad immunotherapeutic potential, as suggested by a number of recent proof-of principle studies.

At Immunocore, we developed a novel class of soluble bi-specific biologics, ImmTACTM/V, consisting of affinity-enhanced TCRs recognizing selected tumor or microbial antigens presented by specific HLAs, fused to an anti-CD3 effector domain. The TCR domain


strongly binds cells expressing both the target antigen and the specific HLA type, while the anti-CD3 domain induces activation of the patient’s T cells, thus redirecting their response toward malignant or infected cells.

We aim to apply our ImmTAC/V technology to also target disease-associated non-peptide molecules recognized by unconventional T cells. As lipid and metabolite antigens are subjected to a low-mutation rate and are presented by non-polymorphic proteins, this approach may overcome the therapeutic limitations imposed by the occurrence of immune-escape variants and the high polymorphism of HLAs.

130

Ether-linked phosphatidylserine species as novel mammary tumor-enriched antigens for type II NKT cells

Divya Sekar1, Stefan Wallner2, Sven Zukunft1, Nerea Ferreirós1, Holger Stark3, Ingrid Fleming1, Bernhard Brüne1, Andreas

Weigert1 1. Goethe-University Frankfurt, Frankfurt, HESSE, Germany

2. Institute of Clinical Chemistry and Laboratory Medicine, University Hospital, Regensburg, Germany

3. Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University , Düsseldorf, Germany

Control of tumor growth by adaptive immune cells requires the recognition of tumor antigens. Beside an altered proteome that provides peptide neo-antigens, tumors show an altered lipidome, potentially supplying novel or overexpressed lipid antigens that are recognized by natural killer T cells (NKT cells). NKT cells are a heterogeneous population of immunoregulatory lymphocytes that recognize foreign as well as self-lipids that are presented on the surface of antigen-presenting cells (APC) via CD1d molecules. Whereas type I NKT cells mainly induce protective immunity, type II NKT cells suppress anti-umor immune responses. We therefore hypothesized the presence of type II NKT antigens in tumors that limit anti-tumor immunity. To identify such tumor-enriched lipid antigens we performed a comparative mass spectrometric screening of CD1d-binding lipids from autochthonous mouse mammary tumors and normal mammary glands. Of 31 CD1d-bound lipids that were enriched in mammary tumors compared to normal tissue a surprisingly high proportion were ether lipids, most of them exhibiting a serine headgroup. These ether-linked phosphatidylserine (ePS) species showed the highest overabundance in murine mammary carcinomas. Accordingly, PS synthesis pathways are overexpressed in breast cancer. Moreover, enhanced expression of phosphatidylserine synthase 1 (PTDSS1) correlates with a poor prognosis for breast cancer patients. By using ether-PS-loaded CD1d tetramers, a new subset of type II NKT cells recognizing the serine headgroup was identified. Such NKT cells recognizing ePS showed an intrinsically reduced inflammatory potential. Analysis of PTDSS1-knockdown tumors grown in syngeneic mice confirmed a role for PS in anti-tumor immunity. In conclusion, we provide evidence for the existence of tumor-enriched lipid antigens that impact tumor development by affecting anti-tumor immunity.

131

NKT cells control tumor associated macrophages and metastatic growth in neuroblastoma

Amy N Courtney1, Gengwen Tian2, Linjie Guo1, Ekaterina Marinova1, Jingling Jin1, Xiuhua Gao1, Jie Wei1, Andras Heczey1,

Ketan B Ghaghada3, Leonid S Metelitsa1, 2, 4 1. Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA

2. Department of Pathology and Immunology, Baylor College of Medicine, Houston, Tx, USA

3. Department of Pediatric Radiology, Texas Children's Hospital, Houston, TX, USA

4. Center for Cell and Gene Therapy, Department of Medicine, Baylor College of Medicine, Houston, TX, USA

Vα24-invariant NKT cells (NKTs) control tumor growth via poorly understood interactions with CD1d-positive tumor-associated macrophages (TAMs). TAMs comprise M1- and M2-like subsets, but only CD163high M2-like TAMs are associated with poor outcome in neuroblastoma (NB) patients. Here, we demonstrate that NKTs selectively target M2-like TAMs via contact-dependent and independent mechanisms. Upon direct contact with antigen-pulsed M1 or M2, NKTs selectively kill the latter. Additionally, we found that antigen-activated NKTs could reprogram M2 into functional M1-like macrophages via GM-CSF production. Furthermore, adoptive transfer of human NKTs resulted in M1-like polarization of TAMs in metastatic NB xenografts in humanized NSG mice. To further explore the role of NKT–TAM axis in tumor immune surveillance, we examined the effect of NKT deficiency on tumor progression and TAM accumulation in NB-Tag transgenic model of NB. Mice lacking either type I (Jα18-/-NB-Tag) or all (CD1d-/-NB-Tag) NKTs had shortened survival compared with NB-Tag mice (P < 0.0002). At four month of age we observed an increase of CD11b+Ly6G-Ly6C-F4/80+ TAMs in primary tumors of NKT deficient groups compared with NB-Tag mice. Despite no difference in the size of primary adrenal tumors between groups, the increase of TAM frequency coincided with metastatic spread in NKT deficient groups as detected by CT imaging and confirmed by pathological analysis. By five months, nearly all mice in NKT deficient groups had distant metastases in liver and lungs but none of the NKT replete mice had detectable distant metastases. Thus, our results reveal a novel mechanism of immune regulation, in which NKTs selectively control M2-like TAMs and suppress tumor metastasis.

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Therapeutic reprogramming of the tumor microenvironment by iNKT cells

Gloria Delfanti1, Filippo Cortesi1, Gaia Antonini1, Claudio Garavaglia1, Michela Consonni1, Giulia Casorati1, Paolo Dellabona1 1. Experimental Immunology Unit, Division of Immunology; Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, ITALY, Italy

CD1d-restricted invariant (i)NKT cells participate in cancer immune surveillance but their site and mechanisms of action are incompletely understood. We investigated the oncogene induced mouse prostate cancer (PC) model TRAMP, in which the absence iNKT cell resulted in more aggressive tumors and decreased survival. iNKT cells actively remodelled the TRAMP tumor microenvironment (TME). iNKT cell deficiency subverted the tumor microenvironment, promoting pro-angiogenic and immunosuppressive programs in infiltrating CD45+ cells, and resulted in a significant expansion of pro-angiogenic tumor associated macrophages (TEMs), and reduction of the pro-inflammatory ones. Reduced iNKT cells, and increased TEM and pro-angiogenic signatures, characterized also aggressive human PCs. iNKT cells localized in the tumor stroma in direct contact with macrophages and their transfer in tumor-bearing mice restricted TEMs, restored the tumor associated macrophage (TAM) balance, and delayed tumor progression. TAM modulation by iNKT cells required the combinatorial engagement of CD1d, CD40 and Fas, which promoted selective killing of the pro-angiogenic population and survival of the pro-inflammatory one. These results suggested that iNKT cells may be utilized as a cellular platform to therapeutically reprogram the TME and enforce tumor-opposing functions, which is critical for the successful outcome of the current immunotherapy approaches. To this aim, we sought to engineering iNKT cells with TCRs specific for MHC-restricted peptides derived from tumor-associate antigens (TAAs), in order to achieve dual targeting of TEMs and cancer cells with a single immune effector upon adoptive immunotherapy. We could transduce mouse iNKT cells in vitro with exogenous TCRs specific for epitopes derived from gp100, Ova or SV40LT tumor antigens which are variably co-expressed with the endogenous invariant TCR. Engineered iNKT cells are dual functional and specifically recognize CD1- and MHC-restricted ligands in vitro. Their therapeutic activity is currently being investigated in vivo against different types of transplantable mouse tumors expressing the cognate antigens.

133

Understanding the generation of CD1c-restricted self-lipid antigen in leukemia cells

Alessandra Bigi1, Claudia de Lalla1, Michela Consonni1, Alessandra Mancino1, Claudio Garavaglia1, Valeria Malacarne2, 3,

Marco Lepore4, Lucia Mori4, Gennaro De Libero4, Andrea Graziani2, 3, Paolo Dellabona1, Giulia Casorati1 1. Experimental Immunology Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy

2. Department of Translational Medicine and Institute for Research and Cure of Autoimmune Diseases, University of Piemonte Orientale, Novara, Italy

3. Lipid Signaling in Cancer and Metabolism Unit, Division of Experimental Oncology, San Raffaele Scientific Institute, Milan, Italy

4. Experimental Immunology, Department of Biomedicine, University Hospital Basel and University of Basel, Basel, Switzerland

CD1 antigen presenting molecules are expressed in mature lympho-hematopoietic cells. Recently, we found that CD1c-restricted T cells recognized the methyl lysophosphatidic acid (mLPA) as self-lipid antigen and efficiently killed CD1c-expressing acute leukemia blasts. mLPA is an ether-lipid, which hallmarks peroxisome-derived biosynthesis, and was found accumulated in acute leukemia cells, whereas it was poorly present in normal hematopoietic cells. Nothing is known about the control of mLPA synthesis in malignant versus normal myeloid/lymphoid cells. This is critical to understand the pathophysiology of CD1 self-reactive T cell responses, as well as to improve leukemia recognition by CD1c-restricted T cells. We are currently trying to assess the role of different enzymes that may be implicated in mLPA biosynthesis, and are also found upregulated in most human tumors and involved in maintaining the malignant phenotype: 1. Alkylglycerone phosphate synthase (AGPS), the peroxisomal enzyme specifically responsible for ether-lipid synthesis; 2. Phospholipase A1/A2, which are involved in lysophospholipid generation; 3. Diacylglycerol kinase α (DGKα), a switch enzyme that can phosphorylate ether-lysoglycerolipids to generate ether-phospholipids. The expression of these enzymes was specifically knocked-down by lentivirus-mediated shRNA transfer, or their activity was inhibited by pharmacological treatments. Preliminary data were obtained with DGKα. Efficient and stable protein knock-down was obtained in Molt4 ALL cells, without affecting CD1c expression, which resulted in a significantly reduced recognition by mLPA-specific T cells. Interference of DGKα and the other enzymes is currently being investigated in Molt4 and THP1 acute leukemia cell lines.

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The role of CD1d in intestinal tumor development


Chiara Ceriotti1, Melanie Jaeger1, Emilie Huc-Claustre2, Sebastian Zeissig1, 3 1. DFG Center for Regenerative Therapies Dresden, Dresden, Germany

2. CRCT Cancer Research Center of Toulouse, Toulouse, France

3. Universitätsklinikum Carl Gustav Carus, Dresden, Germany

CD1d-dependent natural killer T (NKT) cell regulate tumor development and both the promotion of intestinal tumor growth as well as its inhibition by NKT cells has been described (1–7). While these divergent effects at least partially originate from cell-specific roles of type I and type II NKT cell subsets (8–10), it is not known whether antigen presenting cells (APCs) also contribute in a cell-specific manner to NKT cell-dependent regulation of intestinal tumor development. We have recently demonstrated opposing roles of intestinal epithelial cells (IECs) and myeloid cells in CD1d- and NKT cell-mediated regulation of intestinal inflammation (11). We therefore investigated in the present study whether similar cell type-specific effects of CD1d exist in the control of intestinal tumor development.

The Cre/loxP system was used to generate conditional cell-specific CD1d knockout (KO) mice that lack CD1d in IECs or myeloid cells in models of spontaneous, orthotopic intestinal tumor development (Apc Min/+ and Apc flox mice). Neither mice with individual deletion of CD1d in myeloid cells nor those with IEC-specific CD1d deletion showed significant difference in tumor growth and multiplicity compared to CD1d-sufficient littermates. Constitutive Cd1d1-/- mice showed a mild reduction in tumor size compared to Cd1d1+/- littermates, while tumor multiplicity was unaltered.

Together, these data show that in the specific microbial and nutritional context provided in our facility, individual deletion of CD1d in IECs or myeloid cells has little impact on intestinal tumor development, while constitutive deletion of CD1d is associated with reduced tumor growth. Differences in the extent of CD1d- and NKT cell-dependent promotion of tumor growth between similar mouse strains maintained in different facilities (10) further suggests that CD1d-mediated control of tumor development is dependent on environmental factors such as the microbiota and/or nutrition.

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2. M. Terabe et al., Transforming Growth Factor-β Production and Myeloid Cells Are an Effector Mechanism through Which CD1d-restricted T Cells Block Cytotoxic T Lymphocyte–mediated Tumor Immunosurveillance. J. Exp. Med. 198, 1741–1752 (2003).

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4. M. Terabe et al., A nonclassical non-Valpha14Jalpha18 CD1d-restricted (type II) NKT cell is sufficient for down-regulation of tumor immunosurveillance. J. Exp. Med. 202, 1627–33 (2005).

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8. R. L. Bjordahl, L. Gapin, P. Marrack, Y. Refaeli, iNKT Cells Suppress the CD8+ T Cell Response to a Murine Burkitt’s-Like B Cell Lymphoma. PLoS One. 7, e42635 (2012).

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11. T. Olszak et al., Protective mucosal immunity mediated by epithelial CD1d and IL-10. Nature. 509, 497–502 (2014).

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NKT cells expressing a GD2-specific chimeric antigen receptor with CD28 endodomain and IL-15 undergo dramatic in vivo expansion and mediate long-term tumor control in a metastatic model of neuroblastoma

Jingling Jin1, Wei Huang1, Daofeng Liu1, Linjie Guo1, Michael Wood1, Bin Liu1, Ekaterina Marinova1, Gianpietro Dotti1, 2,

Leonid Metelitsa1 1. Department of Pediatrics Center for Cell and Gene Therapy Texas Children’s Cancer Center, Baylor college of medicine, Houston, TX, United States

2. Department of Microbiology and Immunology, Baylor College of Medicine, Houston, TX, United States

Vα24-invariant Natural Killer T cells (NKTs) preferentially localize to the tumor site in neuroblastoma and other types of cancer and have natural antitumor properties that make them attractive as a carrier of tumor-specific chimeric antigen receptors (CARs). We previously demonstrated that adoptively transferred NKTs expressing GD2-specific CARs (CAR.GD2) can effectively localize to the tumor site and mediate antitumor activity in a xenogenic model of neuroblastoma in NSG mice. In this study, we explored whether expression of IL-15, the main homeostatic cytokine for NKTs, within CAR.GD2 would further enhance NKT-cell in vivo persistence and therapeutic efficacy. So, we synthesized CAR.GD2 constructs with a costimulatory CD28 or 41BB endodomain with or without IL-15. NKTs that were transduced with CD28/IL-15 and 41BB/IL-15 CARs secreted similar levels of IL-15 and significantly improved NKT-cell in vitro expansion compared with IL-15-less CARs in response to repeated stimulation with neuroblastoma cells. After transfer to NSG mice with human neuroblastoma xenografts, NKTs expressing IL-15-containing CARs persisted significantly longer compared with those expressing IL-15-less CARs. NKTs expressing CD28/IL-15 CAR underwent a progressive in vivo expansion at the sites of


neuroblastoma metastases. Indeed, the frequency of CD28/IL-15 CAR NKTs reached 30% of bone marrow cells two months after a single injection. Nonetheless, human NKTs did not accumulate in normal murine tissues and did not induce xeno-GvHD. Treatment with CD28/IL-15 CAR NKTs on day 7 after tumor injection resulted in the median survival of 70 days compared to the range of 42 - 53 days in untreated control and groups treated with unmodified NKTs or NKTs expressing other CAR.GD2 constructs (P < 0.001). Thus, a combined use of CD28 costimulatory endodomain and IL-15 in the CAR design enables potent in vivo expansion and anti-tumor activity of CAR.GD2 NKTs cells that should be considered for immunotherapy of neuroblastoma and other solid tumors.

136

NKT cell-targeted cancer vaccine mediating long-term memory responses and strong antitumor activity

Nyambayar Dashtsoodol1, 2, Tomokuni Shigeura1, Takuya Tashiro3, Masaru Taniguchi1 1. Center for Integrative Medical Sciences, Laboratory for Immune Regulation, RIKEN, Yokohama, Japan

2. Mongolian National University of Medical Sciences, Ulaanbaatar, Mongolia

3. Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan

Current tumor therapies, including immunotherapies, focus on passive eradication or at least reduction of the tumor mass. However, cancer patients quite often suffer from tumor relapse or metastasis after such treatments. To overcome these problems, we have developed an NKT cell-targeted immunotherapy focusing on active engagement of the patient’s immune system, but not directly targeting the tumor cells themselves. Natural Killer T (NKT) cells express an invariant antigen receptor a chain encoded by Trav11 (Vα14)-Traj18 (Jα18) gene segments in mice and TRAV10 (Vα24)-TRAJ18 (Jα18) in humans and recognize glycolipid ligand in conjunction with a monomorphic CD1d molecule. The NKT cells play a pivotal role in the orchestration of antitumor immune responses by mediating adjuvant effects that activate various antitumor effector cells of both innate and adaptive immune systems and also aid in establishing a long-term memory response. Here, we established NKT cell-targeted therapy using a newly discovered NKT cell glycolipid ligand, RK, which has a stronger capacity to stimulate both human and mouse NKT cells compared to previous NKT cell ligand. Moreover, RK mediates strong adjuvant effects in activating various effector cell types and establishes long-term memory responses, resulting in the continuous attack on the tumor that confers long-lasting and potent antitumor effects. Since the NKT cell ligand presented by the monomorphic CD1d can be used for all humans irrespective of HLA types, and also because NKT cell-targeted therapy does not directly target tumor cells, this therapy can potentially be applied to all cancer patients and any tumor types.

137

Epigenetic induction of CD1d expression primes lung cancer cells for killing by invariant NKT cells

Éilis Dockry1, Seonadh O'Leary1, Laura Gleeson1, Judith Lyons1, Joseph Keane1, Steven G Gray1, Derek G Doherty1 1. Trinity College Dublin, Dublin, CO. DUBLIN, Ireland

Immunotherapies that target CD1d-restricted invariant NKT (iNKT) cells can prevent tumor growth in murine models but trials in humans have shown limited clinical efficacy. Here, we show that iNKT cells are depleted from blood and bronchial lavage samples from patients with non-small cell lung cancer (NSCLC) suggesting a role for these cells in immunity against NSCLC. We also interrogated the Kaplan-Meier Plotter database of 1926 NSCLC patients and found that low expression of CD1d mRNA is significantly associated with poor patient survival. We hypothesized that CD1d expression in NSCLC is epigenetically regulated and can be modulated using epigenetic targeting therapies. Treatment of the CD1d-negative NSCLC cell lines, A549 and SK-MES-1, with DNA methyltransferase inhibitors and histone deacetylase inhibitors resulted in a dose-dependent induction of CD1d mRNA and protein expression. Chromatin immunoprecipitation analysis indicated that this induction of CD1d expression directly involved chromatin remodelling. Treatment of the NSCLC cell lines with DNA methyltransferase inhibitors and histone deacetylase inhibitors at therapeutic low doses made them targets for iNKT cell-mediated cytolytic degranulation. Thus, epigenetic manipulation of CD1d expression may augment the efficacy of iNKT cell-based immunotherapies for NSCLC.

138

TCR-engineered iNKT cells directed towards tumour-associated antigens for cancer immunotherapy

Juan JU Umana1, Thierry TM Mallevaey1 1. University of Toronto, Toronto, ON, Canada


Activated iNKT cells exert both cytotoxic and helper functions. Importantly, they activate/recruit immune cells and have been implicated in tumour rejection and tumour immuno-surveillance. Our goal is to exploit iNKT cell anti-tumour properties in adoptive cell therapy and redirect their specificity towards tumour-associated antigens (TAAs) through T cell receptor (TCR) engineering. We hypothesize that engineered iNKT cells will be more effective than conventional CD8 T cells and provide long-term immunity.

We currently use the OT-I model TCR, which recognizes a peptide from the chicken ovalbumin (OVA) presented by MHC class-I. Expanded CD8 T cells and iNKT cells were transduced with retroviruses encoding the OT-I TCR. Engineered iNKT cells, but not CD8 T cells, showed an early robust response to OVA peptide presented by dendritic cells (DCs) by producing IFN-γ and TNF-α. Moreover, engineered iNKT cells also produce granzyme B and upregulate FasL, indicative of their cytotoxic properties. The kinetics and magnitude of their response towards OVA peptide were similar to their response towards cognate lipid antigens, suggesting that their innate functions were preserved. We are conducting in vitro cytotoxic assays to demonstrate direct cytotoxic function of our engineered iNKT cells to OVA-expressing B16F10 melanoma and EL4 lymphoma cell lines.

In adoptive cell therapy, protection correlates with the persistence of transferred cells in the host and their ability to traffic to tumour sites. Adoptive transfer experiments demonstrate that engineered iNKT cells not only persist in the host, but they also traffic to various organs. Moving forward, we will assess the short and long-term response of engineered iNKTs in vivo, as well as their ability to promote the rejection of B16F10-OVA or EL4-OVA cells and compare to that of engineered CD8 T cells.

Ultimately, we propose TCR-engineered iNKT cells will constitute a safe and more effective therapy for cancer treatment.

139

Curative efficacy of intranasal HPV vaccine supplemented with aGalCer adjuvant in preclinical mouse vaginal HPV tumor model

Gloria Galvan1, Stephanie Dorta-Estremera1, Jagannadha K Sastry1 1. MD Anderson Graduate School of Biomedical Sciences, Houston, TX, United States

The use of immune checkpoint modulation to enhance anti-tumor immunity while minimizing immune suppressive responses within tumors has proven to be highly successful for the treatment of many cancers, however, toxic side effects and financial constraints limit its wider clinical application. Using preclinical models of human papillomavirus (HPV) cancers, we tested whether vaccination employing adjuvants with potent immune enhancing potentials and clinical safety profiles could alleviate this concern for achieving anti-tumor efficacy. Since most HPV tumors occur at mucosal tissues, we incorporated mucosal intranasal vaccination with E6 and E7 peptides representing tumor-specific antigens to treat vaginal HPV+ TC-1 tumors. We observed that vaccine formulation incorporating the CpG-containing oligo-deoxynucleotide (CpG-ODN), which activates antigen-presenting cells (APCs) through the TLR9 pathway, was effective in reducing tumor growth relative to untreated controls, however, complete and durable regression in the majority of mice required supplementing the vaccine with the α-galactosylceramide (αGalCer) adjuvant to enhance APCs activation through the engagement of natural killer T (NKT) cells. The improved efficacy achieved by incorporating aGalCer adjuvant in addition to CpG-ODN correlated with doubling of CD8 T cells exhibiting enhanced cytotoxic potential (54% increase in granzyme B) within the tumor. Furthermore, the ratio of CD8 T cells to regulatory T cells was augmented by 84% in mice treated with the combination adjuvants versus CpG-ODN alone. Intranasal vaccination delivering α-GalCer, to allow for anergy-free stimulation of NKT cells and the alternate licensing of dendritic cells, which complements the classical licensing afforded by the CpG-ODN signaling to attract diverse populations of CD8+ CTL, preferentially to the female reproductive site, is the hypothesized mechanism underlying the superior efficacy of the HPV vaccine incorporating the two adjuvants.

140

Depletions of invariant natural killer T cells in blood and omentum from patients with upper gastrointestinal cancer

Ashanty M Melo1, Melissa Conroy1, Emma Foley1, John V Reynolds1, Joanne Lysaght1, Derek G Doherty1 1. Trinity College Dublin, Dublin, DUBLIN, Ireland

Background: Oesophageal and gastric cancers cause over 1.1 million deaths annually. Current therapeutic regimes focus on chemo-radiotherapy prior to surgery. However, only 20-30% of patients respond to treatment, therefore, new treatments are urgently required. iNKT cells are innate T cells with antitumour activity. In humans, they account for <1% of peripheral T cells but comprise ~10% of omental adipose tissue lymphocytes. We investigated the presence of iNKT cells in blood and omental tissue from gastrointestinal cancer patients. We also investigated if systemic chemotherapies have adverse effects on iNKT cell functions.

Methods: iNKT cells were quantified in pre- and post-treatment blood and omentum from 89 patients with gastric cancer, SCC and OAC using flow cytometry. iNKT cells were isolated from human blood samples and expanded in vitro. iNKT cells from 4 donors were treated with various concentrations of cisplatin, carboplatin, paclitaxel and 5-fluorouracil, for 24 and 48 hrs. Cells were stained with annexin V and propidium iodide for cell death assays, and co-cultured with CD1d transfected HeLa cells pulsed with α-GalCer, for assays of cytolytic degranulation and intracellular cytokine production.

Results: iNKT cells were found to be depleted from blood and omentum of cancer patients. No significant differences in iNKT cell numbers were observed between pre-treatment and post-treatment blood, from patients with OAC, SSC or gastric cancer. Cisplatin, 5-Fluorouralcil, carboplatin and paclitaxel exhibited a dose-dependent inhibition of iNKT cell viability. 5–fluorouracil and carboplatin


inhibited degranulation by iNKT cells in co-culture with glycolipid-pulsed CD1d transfected HeLa cells. These doses of 5-fluorouracil and carboplatin also affected IFN-γ, but not IL-4 expression.

Discussion: Low iNKT cell numbers may predispose individuals to upper gastrointestinal cancers. Boosting of iNKT cell numbers may have therapeutic value. However, exposure to systemic chemotherapy can negatively affect their functions and should be considered when developing iNKT cell-based immunotherapies.

142

Exploring the evolutionary relevance of MHC class I-like molecules and innate-like T cells in tumor immunity.

Maureen Banach1, Eva-Stina Edholm1, Jacques Robert1 1. University of Rochester Medical Center, Rochester, NY, United States

Tumors and cancers are found across all vertebrates. Likewise, the detection and destruction of cancerous cells is a function conserved in non-mammalian vertebrates. In human and murine cancer, the relevance of natural killer T (NKT) in tumor immunity is an active research area. Notably, the conditions that lead to either anti- or pro-tumoral activities of these cells remain partially understood. To help resolve this issue, we are taking a comparative and evolutionary approach that relies on a tumor immunity model in the amphibian Xenopus. Despite phylogenetic distance, the immune system between amphibians and mammals is fundamentally similar. Using RNA interference (RNAi), we demonstrated that one of Xenopus MHC class I-like (XNC) genes, XNC10, is critical for the development and tumor-related function of Vα6 innate-like T (iT) cell subset, expressing an invariant T cell receptor (iTCR) α chain rearrangement – Vα6-Jα1.43. Our recent study suggests that an additional iT cell subset (Vα22 iT), characterized by Vα22-Jα1.32 iTCR α chain, is specifically involved in the immunity against Xenopus thymic lymphoid tumors. The intraperitoneal transplantation of Xenopus tumor cells into syngeneic tadpoles results in significant early infiltration of both Vα6 and Vα22 iT cells, concomitant with the decrease of these subsets from spleen and thymus. We hypothesize that the recruitment and anti-tumor activity of the two iT cell subsets can be manipulated by impairing the tumor expression of XNC10 and/or other XNC genes. To explore this possibility, we are developing a reverse genetic approach with RNAi and three-component CRISPR/Cas9 system to generate transgenic animals deficient in either Vα6 or Vα22 iT cells and tumor lines stably deficient in specific XNC gene.

143

The conserved nature of CD1 and T-cell receptor interactions between humans and non-human primates

Krystle K Quan1, Damien Wilburn2, Willie Swanson2, Chetan Seshadri1 1. Medicine, University of Washington, Seattle, Washington, United States

2. Genome Sciences, University of Washington, Seattle, Washington, United States

Human T cells evolved to recognize peptide and non-peptide antigens produced by pathogens such as Mycobacterium tuberculosis (M.tb). The human genome contains five CD1 genes that vary in the types of lipids they bind as well as their patterns of cellular expression and intracellular trafficking. We conducted an evolutionary genetic analysis of CD1 genes in humans and non-human primates. Apart from CD1A in olive baboon, each paralog exists as a single gene copy in each primate species and forms a phylogenetically distinct group that generally matches the species phylogeny. These data suggest a lack of diversifying selection over 50 million years of evolution, which stands in stark contrast to the history of the major histocompatibility complex system for presenting peptide antigens to T cells. We found evidence of positive selection in CD1A, CD1C, and CD1E, and such rapidly evolving sites overwhelmingly clustered in the ligand-binding groove. Because CD1D was the most conserved antigen-presenting molecule, we next explored whether molecular interactions between CD1D and T-cell receptors (TCR) were also conserved across species. We used human CD1D tetramers loaded with α-galactosylceramide (α-GalCer) to sort invariant NKT (iNKT) cells from a healthy rhesus macaque. We derived an iNKT-cell line, which bound the tetramer with high avidity and produced IFN-ɣ and TNF-α when restimulated with α-GalCer-loaded human CD1D-transfectants. The dominant TCR-α and TCR-β chains of the rhesus iNKT cell line showed high homology to the human iNKT TCR, including residues known to be critical for binding CD1D-α-GalCer. These data show that the interaction of the iNKT TCR and CD1D-α-GalCer is nearly uniform between humans and non-human primates. Our genetic analysis and molecular studies also provide proof-of-concept that human CD1 tetramers can be repurposed to probe the basic biology of CD1-restricted T-cells in non-human primate models of disease.

144

A new distinct MHC class I-like restricted invariant T cell lineage at the forefront of mycobacterial immunity uncovered by reverse genetics in the amphibian Xenopus

Eva-Stina Edholm1, Kun Hyoe Rhoo1, Jacques Robert1 1. University of Rochester Medical Center, Rochester, NEW YORK, United States


The amphibian Xenopus laevis is to date the only species outside mammals where an MHC class I-like restricted innate-like T cell subset (iVa6-Ja1.43) reminiscent of iNKT cells has been identified and functionally characterized. Here, we report the identification of a distinct iT cell subset (Va45-Ja1.14) requiring a different MHC class I-like molecule (XNC4) for its development and function. We used two complementary reverse genetic approaches by transgenesis: RNAi to either impair either XNC4 or the Va45-Ja1.14 rearrangement and CRISPR-Cas9 to disrupt the Ja1.14 gene segment, to demonstrate that this innate-like immune surveillance system is critical for host resistance to mycobacteria infection. Both XNC4 deficiency that prevents iVa45-Ja1.14 T cell development, and direct disruption of the iVa45-Ja1.14 rearrangement dramatically impair tadpole immune response and resistance to M. marinum infection. In contrast, iVa45-Ja1.14 deficient tadpoles remain competent against ranavirus infection, unlike iVa6 iT cell deficient tadpoles that are highly susceptible to ranavirus infection. These data suggest that amphibians, which are evolutionary separated from mammals for more than 350 million years, have independently diversified a prominent and convergent MHC-like restricted innate-like T cell system.

145

IRE1alpha Regulate Cytokine mRNAs Stabilization Within Invariant NKT1 and 17 Cells

Srinath Govindarajan1, 2, Djoere Gaublomme1, 2, Renee Van Der Cruyssen1, 2, Eveline Verheugen1, 2, Sofie Van Gassen1, 3, Yvan

Saeys1, 3, Takao Iwawaki4, Sophie Janssens1, 5, Bart Lambrecht1, 5, Michael Drennan1, 2, Dirk Elewaut1, 2 1. VIB-UGENT, Center for Inflammation Research, Ghent, BELGIUM, Belgium

2. Unit for Molecular Immunology and Inflammation, Department of Rheumatology , VIB-UGENT, Center for Inflammation research, Ghent, Belgium

3. Unit of Information Technology, Department of Respiratory Medicine, VIB-UGENT, Center for Inflammation Reesearch, Ghent, Belgium

4. Advanced Scientific Research Leaders Development Unit, Gunma University, Maebashi, Gunma, , Japan

5. Department of Respiratory Medicine, VIB-UGENT, Center for Inflammation Research, Ghent, Belgium

The inositol-requiring enzyme 1a (IRE-1a) is a type I endoplasmic reticulum (ER) transmembrane protein containing both kinase and sequence-specific endoribonuclease (RNase) activities. Upon ER stress, activation of IRE-1a results in the cleavage of an 26 base pair intron from mRNA coding for X box binding protein 1 (Xbp-1). Spliced Xbp-1, a potent transcription factor transactivates a number of genes involved in restoring cellular homeostasis. In this study, we show that T cell receptor (TCR)-dependent activation of the invariant natural killer T (iNKT) cell lineage results in splicing of Xbp-1 mRNA as well as the upregulation of genes associated with induction of the unfolded protein response (UPR). A subsequent analysis of iNKT cell lineage development in T cell-specific IRE-1a knock-out mice revealed comparable tissue distribution and maturation profiles when compared to controls, however, TCR-dependent cytokine production by the NKT1 and NKT17 cell lineage was severely impaired in the absence of IRE-1a. In this context, impaired cytokine production by IRE-1a deficient NKT1 and NKT17 cells was due to reduced mRNA stability for cytokines such as IL-2, IL-4, IL-6, IL-13, TNFa, IFNg and 1L-17A. Furthermore, we show that T cell-specific IRE-1a knock-out mice are protected from oxazolone-induced ulcerative colitis, suggesting that IRE-1a activity within the iNKT cell lineage may be a therapeutic target for treatment of the disease.

146

Identification and thymic development of human autoreactive iNKT cells

Jeanne Perroteau1, Leslie HESNARD1, Marie-Claire DEVILDER1, Laurent GAPIN2, Emmanuel SCOTET1, Xavier SAULQUIN1,

Laetitia GAUTREAU-ROLLAND1 1. Centre de Recherche en Cancérologie et Immunologie Nantes-Angers (CRCINA) - UMR Inserm 1232 - ERL CNRS 6001 - Université de Nantes, Nantes, FRANCE, France

2. National Jewish Health and University of Colorado, Denver, Colorado, USA

Invariant Natural Killer T (iNKT) lymphocytes express both NK receptors and a semi-invariant αβ TCR (T Cell Receptor), which is restricted by the CD1d molecule presenting glycolipid antigens. Among them, αGalactosylCeramid (αGalCer) is a potent ligand of all iNKT cells. In some contexts, iNKT cells are also able to detect endogenous glycolipids, which suggests an autoreactive potential of some of them. However, the mechanisms that regulate this autoreactivity are poorly understood, especially in humans, because of the difficulty to detect and isolate autoreactive iNKT cells ex vivo.

We have previously studied the thymic maturation of human antigen-specific conventional T cells (Hesnard et al., 2016), using a highly sensitive tetramer-based immunomagnetic cell separation approach (Legoux et al., 2010). In this study, we extended this strategy to the analysis of human iNKT lymphocytes. αGalCer-CD1d tetramers were used to identify the total iNKT cell population, while ones loaded with the partial agonist α-linked glycolipid OCH detected putative autoreactive iNKT cells, as documented by the group of S. Gadola (Matulis et al., 2010). Phenotypic and frequency analyses of iNKT stained by αGalCer- and/or OCH-CD1d tetramers allowed us to decipher intrathymic selection mechanisms that occur during human iNKT cell development. Moreover, we generated iNKT cell lines from the peripheral blood of healthy donors and identified some human iNKT cell lines showing a strong autoreactive activity against target cells expressing endogenous glycolipids-CD1d complexes. The cloning of cells from these autoreactive lines evidenced various autoreactivity levels, that suggest a TCR variability, which is currently analyzed.

Altogether, our results indicate the existence of autoreactive subsets of human iNKT cells in the peripheral blood in healthy donors. Importantly, we show for the first time that these lymphocytes have probably circumvented thymic selection processes and can be involved in various physiopathological contexts such as autoimmune diseases or cancers.


147

Human CD8-negative MAIT cells are functionally distinct from CD8-positive MAIT cells

Joana Dias1, Caroline Boulouis1, Jean-Baptiste Gorin1, Robin H. G. A. van den Biggelaar1, 2, Anna Gibbs3, Liyen Loh5, 4, Muhammad Yaaseen Gulam6, Douglas F. Nixon4, 7, Kristina Broliden3, Annelie Tjernlund3, Johan K. Sandberg1, Edwin

Leeansyah1, 6 1. Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden

2. Department of Infectious Diseases and Immunology, Universiteit Utrecht, Utrecht, the Netherlands

3. Unit of Infectious Diseases, Department of Medicine Solna, Center for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden

4. Division of Experimental Medicine, Department of Medicine, University of California San Francisco, San Francisco, California, United States of America

5. Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria, Australia

6. Program in Emerging Infectious Diseases, Duke-National University of Singapore Medical School, Singapore

7. Department of Microbiology, Immunology, and Tropical Medicine, George Washington University, Washington, D. C., United States of America

Mucosa-associated invariant T (MAIT) cells are a large subset of unconventional T cells that recognise microbial riboflavin metabolites presented by the MHC class I-like protein MR1. In humans, they express the invariant TCR Vα7.2 segment, coupled with restricted Jα segments and limited Vβ repertoires, and consist of two subsets defined by the expression of the CD8α co-receptor, namely CD4- CD8+ (CD8+) and CD4- CD8- (DN) MAIT cells. There is currently scant knowledge on the biology of these two MAIT cell subsets and their relationship to each other.

In the present study, we found that these subsets displayed limited differences in their surface immunoproteome, with higher expression levels of co-stimulatory molecules on CD8+ MAIT cells being the most notable difference. However, CD8+ MAIT cells had higher basal levels of cytolytic proteins, and distinct expression patterns of classical T cell transcription factors. Such differences were more pronounced in mucosal tissue-derived CD8+ MAIT cells, and appeared linked to enhanced functionality of CD8+ MAIT cells following stimulations with riboflavin-autotrophic and -auxotrophic Escherichia coli strains and mitogens. Interestingly, DN MAIT cells had higher propensity for apoptosis both at resting state and following such stimulations.

Activation of CD8+ MAIT cells through TCR and bacterial stimulations led to the downregulation of CD8α and induced the appearance of DN MAIT cells. This process appeared to depend on presentation of riboflavin metabolite antigen by MR1. Finally, DN MAIT cells from human fetal tissues displayed a more mature phenotype and accumulated over gestational time, with reciprocal contraction of CD8+ MAIT cells during in utero development. In adult peripheral blood, DN MAIT cells expressed a more restricted Vβ repertoire and belonged as a subset of CD8+ MAIT cells’ Vβ repertoire. Overall, our study defined key biological differences of the two major subsets of human MAIT cells and their apparent derivative relationship.

148

MAIT and NKT cell development is controlled by the same pathways and results in identical transcriptional programs

Francois Legoux1, Yara El Morr, Marion Salou, Aurélie Darbois, Olivier Lantz 1. Institut Curie, Paris, FRANCE, France


149

CCR7 defines a multipotent progenitor for iNKT cells in thymus and periphery

Haiguang Wang1, Kristin Hogquist1 1. University of Minnesota, Minneapolis, MINNESOTA, United States

Invariant natural killer T (iNKT) cells comprise three major effector subsets (NKT1, NKT2 and NKT17) defined by distinct transcription factors and cytokine producing potential. However, the precise steps of their differentiation in the thymus and periphery have been controversial. We demonstrate here that the small proportion of thymic iNKT and mucosal associated invariant T (MAIT) cells that express CCR7 are at early stage of development and serve as multipotent progenitors giving rise to effector subsets within the thymus. In particular, amongst PLZFhi iNKT cells, CCR7 and PD-1 distinguished multipotent progenitors from IL-4 producing NKT2 effectors.


Using intra-thymic labeling and transfer, we also showed that CCR7+ iNKT cells emigrate from the thymus in a KLF2 dependent manner, and undergo further maturation after reaching the periphery. These and parabiosis studies showed that thymic NKT1 and NKT17 were largely resident—they were not derived from the peripheral pool and did not contribute to the peripheral pool. Finally, each thymic iNKT effector subset produces distinct factors that influence T cell development. Our findings demonstrate how the thymus is both a source of iNKT progenitors and a unique site of tissue dependent effector cell differentiation.

150

NKT TCR domains unrelated to ligand binding instruct the thymic development of an adipose resident subset

Joshua A Vieth2, 1, Joy Das3, Fanomezana M Ranaivoson2, 1, Davide Comoletti2, 4, 1, Lisa K Denzin2, 5, 6, 7, 1, Derek B

Sant'Angelo2, 5, 6, 7, 1 1. The Child Health Institute of NJ, Rutgers University, New Brunswick, NJ, USA

2. Rutgers Robert Wood Johnson Medical School, Rutgers University, New Brunswick, NJ, USA

3. Immunology Program, Sloan Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

4. Department of Neuroscience and Cell Biology, Rutgers University, New Brunswick, NJ, USA

5. Rutgers Graduate School of Biomedical Sciences, Rutgers University, New Brunswick, NJ, USA

6. Department of Pediatrics, Rutgers University, New Brunswick, NJ, USA

7. Department of Pharmacology, Rutgers University, New Brunswick, NJ, USA

The role of natural killer T cells (NKT cells) in adipose tissue is a topic of much interest, as reports have suggested a correlation between depletion of the adipose-resident NKT (arNKT) cell population and chronic obesity. It has been shown that arNKT cells are unique in terms of their function and phenotype; they produce anti-inflammatory and regulatory cytokines, and also induce the accumulation of FoxP3-expressing Tregs in adipose tissue. There has been much speculation about whether these arNKT cells are a unique subset of the NKT cell repertoire or whether NKT cells take on a regulatory phenotype due to external cues in the adipose microenvironment. Our recently published work focused on the uniquely conserved NKT T cell antigen receptor (TCR) and the role its seemingly rigid structure plays in the selection and development of NKT cells. In fact, the interaction between the NKT TCR and the antigen-presenting molecule CD1d is distinct from interactions between the TCR and major histocompatibility complex (MHC) in many ways, including decreased total buried surface area, less post-ligation conformational changes, and yet increased overall avidity. Our molecular

modeling suggested that a hydrophobic patch created after TCRα –TCRβ pairing has a role in maintaining the conformation ofthe NKT

cell TCR. Disruption of this patch ablated recognition of CD1d by the NKT cell TCR but not interactions of the TCR with MHC. Partial disruption of the patch, while permissive to the recognition of CD1d, significantly altered NKT cell development, which resulted in the selective accumulation of adipose-tissue-resident NKT cells. These results indicate that a key component of the TCR is essential for the development of a distinct population of NKT cells, and provide the first data evidencing that arNKT cells are a distinct lineage of NKT cells that develop in the thymus.

151

Ectopic expression of PLZF results in an RORγt positive cytotoxic T cell subset

Patrick W Darcy2, 1, Derek B Sant'Angelo2, 1, 3, 4, 5 1. Rutgers Robert Wood Johnson Medical School, Rutgers University , New Brunswick , New Jersey, USA

2. The Child Health Insitiute of New Jersey, Rutgers University, New Brunswick, NEW JERSEY, United States

3. Department of Pediatrics, Rutgers University, New Brunswick, New Jersey, USA

4. Rutgers Graduate School of Biomedical Sciences, Rutgers University, New Brunswick, New Jersey, USA

5. Department of Pharmacology , Rutgers University, New Brunswick, New Jersey, USA

Promyelotic Leukemia Zinc Finger (PLZF), a member of the BTB-ZF transcription factor family, is a critical regulator of Natural Killer T (NKT) cell and Mucosal Associated Invariant T (MAIT) cell biology. In the absence of PLZF, the development of both T cell subsets is stunted. In addition, PLZF expression is required for the unique cytokine production capabilities of NKT cells and MAIT cells. We investigated PLZF's role in T cell development and function using a mouse model in which conventional T cells expressed PLZF under the Lck promoter. Specifically, we focused on a population of cytotoxic T cells that expressed the transcription factor RORγt upon ectopic expression of PLZF. We were intrigued by this population for three reasons. (1) RORγt marks cells primed to produce IL-17. (2) Subsets of NKT cells (NKT17s) and MAIT cells express RORγt and produce IL-17 following primary activation. (3) Naive wild-type cytotoxic T cells do not express RORγt. Initially we established that the Lck.PLZF transgenic, RORγt expressing cytotoxic T cells were not an NKT cell or MAIT cell population by demonstrating that the Lck.PLZF transgenic RORγt expressing cytotoxic T cells possessed an unbiased TCR repertoire. Lck.PLZF transgenic, RORγt expressing cytotoxic T cells displayed an NKT17/MAIT cell phenotype primed to produce IL-17 in response to activation, evidenced by the presence of IL-17 transcript within the cells in the absence of activation and by the production of IL-17 protein following primary activation. In addition, the Lck.PLZF transgenic, RORγt expressing cytotoxic T cells expressed an array of cell surface molecules characteristic of NKT17s and MAIT cells. Finally, Lck.PLZF transgenic, RORγt expressing cytotoxic T cells accumulated in non-lymphoid tissues, a phenotype associated with NKT17s and MAIT cells. These results support the argument that co-expression of PLZF and RORγt confers a distinct phenotype upon T cells.


152

A novel population of liver-associated innate-like PLZF+CD8ααTCRαβ T cells with immune regulatory properties

Huiming Sheng1, Idania Marrero1, Igor Maricic1, Stephen Fanchiang1, Sai Zhang2, Surya Dasgupta1, Derek Sant’Angelo2, Vipin

Kumar1 1. University of California San Diego, La Jolla, CALIFORNIA, United States

2. Rutgers University, New Brunswick, NJ, United States


153

Dysfunction of mitochondrial complex III preferentially affects the development of CD1d-restricted NKT cells and Mucosal associated invariant T (MAIT) cells

Liang Cao1, Xiufang Weng1, Amrendra Kumar1, Ying He1, Jie Zhao1, Laura A. Sena1, 2, Sam Weinberg1, 2, Navdeep S. Chandel1,

2, Chyung-Ru Wang1 1. Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States

2. Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States

T cell fate and function are determined by intricate coordination between cellular signaling and metabolic pathways, but the precise metabolic determinants for iNKT cell development are unknown. We found that iNKT cell development requires intact mitochondrial metabolism. Mice with a T cell-specific deletion of Ubiquinol-Cytochrome C Reductase Rieske Iron-Sulphur Polypeptide 1 (T-Uqcrfs1-/-

), a mitochondrial complex III protein, had a cell-intrinsic defect in iNKT cell development while thymic development of conventional T cells proceeded normally. Residual iNKT cells in T-Uqcrfs1-/- mice retained the ability to proliferate in vivo but exhibited increased apoptosis, diminished T-bet levels and impaired responsiveness to TCR and IL-15 stimulation. In the iNKT cell hybridoma cell line DN32.D3 with knockdown of Uqcrfs1, the production of IL-2 was decreased; this was associated with a reduced level and impaired nuclear translocation of NFAT. Interestingly, the development of MAIT cells is also impaired in T-Uqcrfs1-/- mice. Our study highlights the critical role played by mitochondrial metabolism in providing signaling intermediates that modulate TCR signaling in vivoand their effects on iNKT and MAIT cell development and function.

154

Characterisation of the biochemical signature regulating interaction between CD8aa and the liver expressed non-classical MHC molecule, H2-Q10

Angela Nguyen1, Katharine Goodall1, Dan Andrews1 1. Monash University, Melbourne, VICTORIA, Australia

Functioning as a key component of antigen presentation, the major histocompatibility complex (MHC) is crucial for the elicitation of immune responses. Understanding the biology and interactions of these molecules is key in understanding immunity as a whole. Although not as well understood as their classical counterparts, non-classical MHC molecules have only been recently shown to be immunologically relevant due to their tissue restriction and highly specialised functions. Our group has recently characterised H2-Q10, a murine non-classical MHC molecule, which shows overexpression in the liver. Of particular interest is a novel interaction that occurs between H2-Q10 and the CD8aa homodimer expressed on gamma delta T cells. We have shown that this interaction is dependent on the glycosylation signature of CD8aa, with N-glycosylation implicated as the key pathway controlling recognition. We have also observed that the a3 domain of H2-Q10 is the point of contact for CD8aa binding and further determined key residues within the a3 domain that regulate this interaction. Our results have provided insight into the biochemical signature governing the novel interaction between H2-Q10 and CD8aa and opens up further studies data into elucidating the immunological consequences of this interaction on gamma delta T cells and immunity.

155

Differential glycosylation of the CD8a homodimer regulates binding to the non-classical MHC I, H2-Q10

Katharine J Goodall1, Angela Nguyen1, Aya Matsumoto2, Sneha Sant3, Julie McMullen2, Katherine Kedzierska3, Joseph

Trapani4, Mariapia Degli-Esposti5, Jamie Rossjohn6, 7, 8, 9, Lucy C Sullivan10, Dan M Andrews1 1. Immunology and Pathology, Monash University, Melbourne, Victoria, Australia

2. Baker Heart & Diabetes Institute, Melbourne, Victoria, Australia


3. Immunology, Peter Doherty Institute, Melbourne, Victoria, Australia

4. Cancer Immunology, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia

5. Lions Eye Institute, Perth, Western Australia, Melbourne

6. Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia

7. Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, Victoria, Australia

8. Infection and Immunity Program, Biomedicine Discovery Institute, Melbourne, Victoria, Australia

9. Institute of Infection and Immunity, Cardiff University School of Medicine, Cardiff, Wales

10. Microbiology, Peter Doherty Institute, Melbourne, Vic, Australia

Gamma delta T cells are conserved across ~500 million years of evolution, highlighting their importance to immunity. Despite this, the identification of receptor ligand interactions that control their development has not been largely explored. In this study we demonstrate that the class Ib MHC, H2-Q10, binds to the CD8αα homodimer found on gamma delta T cells. The importance of this interaction is underscored by our observation that recognition of H2-Q10 is controlled by differential glycosylation between gamma delta and alpha beta T cells. We also demonstrate that H2-Q10 and CD8αα expressing gamma delta T cells are resident in the liver and their interaction is important to cellular development. These results provide the first evidence that glycosylation is differentially regulated amongst gamma delta and alpha beta T cells. Furthermore, they also suggest that co-evolution between class Ib MHC and gamma delta T cells may play an important role in their biology.

156

PLZF regulates effector differentiation of gd T cells

You Jeong Lee2, 1 1. Academia of Immunology and Microbiology (AIM), Institute for Basic Science (IBS), Daejeon, Korea

2. POSTECH, 77 Cheongam-ro, Namgu, Pohang, GYOUNGBUK, Korea

gd T cells develop in the thymus and are enriched in intraepithelial layer of intestine. The development and differentiation of gd T cells were previously thought to be dependent on their TCR usage, and V1 (Vd6.3–Vg1.1+), V6 (Vd6.3+Vg1.1+) and V2 (Vg2+) gd T cells represented IFNg, IL-4 and IL17 producing subsets respectively. Here we show that, regardless of their TCRs, CD24hi gd T cells are immature and CD24low gd T cells expressed PLZF and differentiated into effector lineages expressing TBET (PLZFlow), GATA3 (PLZFhi) and RORgt (PLZFint). In the thymic medulla, these cells produced IFNg, IL-4 and IL17 respectively and transcription factors, rather than their TCR usage, determined the cytokine nature of gd T cells. In the gut, intraepithelial gd T cells were uniformly PLZF– TBET+ expressing V5 (Vg5+) TCR, but derived from previously PLZF+ cells. The development of intraepithelial gd T cells were not influenced by intestinal microbiome, but their activation was influenced by food antigens, suggesting their potential regulatory role. Remarkably, CD24low V5, V6 and V2 gd T cells shared transcriptional nature with NKT1, NKT2 and NKT17 cells respectively, indicating PLZF regulates effector differentiation of both iNKT and gd T cells.

157

Development and function of mucosal-associated invariant T cells are regulated by the micro-RNA miR-155.

Cole Anderson1, Anita Iyer 2, Ryan O'Connell 3, Daniel Leung 1 1. Division of Infectious Disease, Department of Internal Medicine, University of Utah School of Medicine , Salt Lake City , Utah

2. Medicine , Massachusetts General Hospital , Boston

3. Division of Microbiology and Immunology, Department of Pathology , University of Utah School of Medicine , Salt Lake City , Utah

Mucosal-associated invariant T (MAIT) cells are non-classical T cells that are enriched in mucosal tissues and rapidly produce inflammatory cytokines in response to activation by microbial metabolites. MAIT cells have been shown to play a key role in the response to acute respiratory, mycobacterial and HIV infections, but the regulatory mechanisms that control the effector function of MAITs are not well understood. Here, we show that the proinflammatory micro-RNA, miR-155, is important for MAIT cell development and their ability to secrete IFN-γ upon stimulation. In stimulated MAITs, miR-155 expression was significantly up regulated, and lung derived MAITs from mice lacking miR-155 had a diminished IFN-γ response upon stimulation. Lastly, the MAIT cell compartment in miR-155 deficient mice was considerably reduced compared to wild-type counterparts. These results demonstrate the importance of micro-RNAs in MAIT cell biology, and provide direction for future studies involving the regulatory networks controlling MAIT cell development and function.

158

Assessment of global chromatin accessibility in peripheral iNKT cell subsets

Mallory L Paynich1, James P Scott-Browne1, Isaac Engel1, Mitchell Kronenberg1


1. La Jolla Institute for Allergy and Immunology, La Jolla, CA, United States

iNKT cells differentiate into three effector cell subsets in the thymus, NKT1, NKT2, and NKT17, which closely resemble subsets of Th1, Th2, and Th17 CD4+ T cells, as well as ILC1, ILC2, and ILC3 innate lymphoid cells, respectively. Recent work from our lab and others recently determined that thymic iNKT cell subsets have a highly divergent epigenetic landscape and gene program. Following egress from the thymus, iNKT cells localize to tissues throughout the body and many do not recirculate. Although the phenotype and localization of iNKT cells in the periphery has been established, how tissue localization impacts the epigenetic landscape of each subset is not known. Here, we assess global chromatin accessibility in iNKT cell subsets in peripheral tissues including the spleen, lymph nodes, and liver, as well as the lung, where they provide vital immunity to bacterial infections. To do this, we utilized an assay for transposase-accessible chromatin using sequencing (ATAC-seq). With ATAC-seq, we are identifying regions of open chromatin and nucleosome-bound and nucleosome-free positions in regulatory regions. Future experiments will determine the impact of tissue localization on the gene regulatory landscape of iNKT cell subsets, and the degree to which regulatory elements established in the thymus carry over into tissues. Further, we will determine if the tissue microenvironment contributes to priming or activation of these cells.

Supported by NIH R01 AI71922 and T32 AI 125179

159

RIP inflammatory pathways are required to maintain iNKT cell homeostasis and enable cognate neutrophil-iNKT cell interaction

Thomas Hägglöf1, Abigail Kumagai1, William J Kaiser1, Elizabeth A Leadbetter1 1. Microbiology, Immunology, and Molecular Genetics, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA

Apoptosis and programmed necrosis (necroptosis) are cell death pathways with common signaling molecules, including Receptor-interacting protein kinase (RIP) 1, RIP3, and Caspase-8, which are critical for death receptor-induced apoptosis as well as necroptosis. Necroptosis is crucial during development and in response to viral infections. Here, we found a decreased frequency of iNKT cells in RIP3-/- Casp8-/- double-deficient (DKO) and RIP1-/- RIP3-/- Casp8-/- triple-deficient (TKO) mice in spleen as compared to RIP3-deficient or wild type mice. Thymic iNKT cell development was normal in RIP3-deficient and TKO mice when compared to wild type mice and so was iNKT cell activation as measured by CD69 and T-bet expression. However, we found a reduced proportion of NKT2 cells in the thymus of both RIP3-deficient and TKO mice as compared to wild type controls. In peripheral organs, TKO mice had a significant decrease in the proportion of iNKT cells, and remaining iNKT cells expressed lower levels of CD69 and T-bet, as compared to wild type or RIP3-deficient mice. Splenic neutrophils from TKO mice expressed significantly lower levels of CD1d, as compared to RIP3-/-, DKO, or wild type mice, indicating that RIP1 is required to equip neutrophils for cognate engagement of iNKT cells. In summary, our data suggests that apoptosis is required to maintain thymic iNKT subsets as well as peripheral iNKT cell homeostasis and activation. Additionally, RIP1 is specifically required for neutrophil CD1d expression, thereby enabling interaction with iNKT cells.

161

Differences in the effector functions of MR1- and cytokine stimulated MAIT cells

Rajesh Lamichhane1, Sara de la Harpe2, Thomas Harrop3, Andrea Vernall2, Joel Tyndall2, Peter Dearden3, Joanna Kirman1,

James Ussher1 1. Department of Microbiology and Immunology, University of Otago, Dunedin, Otago, New Zealand

2. School of Pharmacy, University of Otago, Dunedin, Otago, New Zealand

3. Department of Biochemistry, University of Otago, Dunedin, Otago, New Zealand

MAIT cells are a recently recognised innate-like T-cell subset which express a semi-invariant T-cell receptor (Vα7.2-Jα12/20/33) and are restricted to antigen presented by the evolutionary conserved, non-polymorphic MHC class I related protein, MR1. MR1 presents unstable derivatives of precursors of riboflavin, triggering MAIT cell activation via their T-cell receptor (TCR). They can also be activated via their cytokine receptors, mainly by IL-12 and IL-18. However, it is unclear how the effector function of human MAIT cells differ in response to different stimuli. In this study, we investigated the effector function of MAIT cells to E. coli, soluble ligand (5-A-RU) and cytokines (IL-12+IL-18). We observed rapid activation of human MAIT cells and significant cytokine production against all stimuli, although significant TNF-α production was only observed when MAIT cells were activated via their TCR by E. coli or 5-A-RU. Differences in timing of maximal activation were seen with different stimuli, both at the protein and transcriptomic levels. To explore the effector functions of activated MAIT cells and further outline the differences between the two modes of activation, RNA sequencing was performed on MAIT cells stimulated with E. coli, 5-A-RU or IL-12+IL-18. Differences in MAIT cell response were observed in cytokine and chemokine production and the profile of cytotoxic molecules expressed. Overall, this suggests that the effector function of MAIT cells differs depending upon the mode of activation.

162


Elucidation of Antibacterial Functions of MAIT Cells in Human Lung Mucosa: A Transcriptomics Study

Erin W Meermeier1, Nick L Stucky1, Christina L Zheng1, Gwendolyn M Swarbrick1, Jessica Tran1, Aneta Worley1, David M

Lewinsohn1 1. Oregon Health & Science University, Portland, OREGON, United States

MR1-restricted MAIT cells are a T cell subset that recognize infection by a broad array of microbial pathogens important to human health, including respiratory pathogens like Mycobacterium tuberculosis (Mtb). MAIT cells are defined by the use of a semi-invariant T cell receptor which recognizes microbial small molecules derived from the riboflavin biosynthesis pathway presented on the HLA-Ib molecule MR1. Upon activation, MAIT cells have effector capacity that has been associated with the control of intracellular infection with Mtb (IFN-γ, TNF, and cytolytic capacity). Murine models of infection have shown that MAIT cells are important in the first days of an infection, and in shaping the subsequent adaptive immune response. At present, whether MAIT cells play a unique role in human pulmonary defense is unknown. Here we use whole-transcriptome RNA-sequencing to map the phenotype and function of MAIT cells in human lungs and blood. We map distinct gene sets unique to MAIT cells in comparison to conventional CD8+ T cells in each tissue. We anticipate that these gene sets will provide our field with a comparative MAIT cell transcriptome resource library. Here, we identify unexpected gene expression associated with antibacterial immunity that have not been previously associated with CD8+ T cells. We use controlled ex vivo assays to validate significant protein expression and analyze mycobacterial growth inhibition. Using an MR1/Ag-tetramer, we characterize the in situ organization of MAIT cells in the human respiratory tract. Our data suggest that mucosal MAIT cells form a functionally distinct subtype in comparison to those in the blood. We postulate that MAIT cells have additional antibacterial capabilities than conventional CD8+ T cells and serve as a targeted immune defense barrier at the mucosal interface. Our study highlights the relevance of analyzing human cell function at barrier tissues as an exploration into the breadth of antibacterial immunity.

163

Transcriptional profiling of peripheral invariant NKT subsets

Isaac Engel1, Gregory Seumois2, Mallory Paynich2, Shu Liang2, Sandy Rosales2, Pandurangan Vijayanand2, Mitchell

Kronenberg2 1. La Jolla Institute for Allergy & Immunology, La Jolla, CA, United States

2. La Jolla Institute for Allergy & Immunology, La Jolla, CA, United States

Invariant natural killer T cells (iNKT cells) have immune stimulatory or inhibitory effects on the immune response that are context-dependent, which is due in part to the existence of functional iNKT cell subsets dedicated to producing certain cytokines. We have previously conducted a characterization of iNKT subsets in the thymus, including NKT1, NKT2 and NKT17 cells, through fluorescent cell sorting and RNAseq. The data gleaned from this study demonstrated that iNKT subsets were highly divergent from each other, and allowed us to identify a number of novel markers for these subsets. These data led us to consider how the transcriptional signatures of iNKT subsets in peripheral organs compare to those in the thymus. This question is particularly relevant given that the final maturation of at least some of the peripheral subsets takes place after egress from the thymus. We have now initiated transcriptomic analyses of iNKT subsets in peripheral organs, employing a recently developed variation of RNAseq in which relatively small numbers of cells (typically 400) are collected by fluorescence sorting and then reverse transcription of RNA is performed directly on cell lysates, followed by PCR amplification and sequencing. We will present data from studies of several peripheral iNKT subsets obtained from the spleen, liver and lung. We will also describe modifications to our sorting protocol that allow us to distinguish NKT2 cells from a separate iNKT population that expresses a mix of NKT1 and NKT2 markers.

164

Human mucosal-associated invariant T (MAIT) cells in inflamed tissues

Julia D Berkson1, Chloe Slichter1, Hayley Oberbillig1, Martin Prlic1 1. University of Washington, Seattle, SEATTLE, United States

Mucosal-associated invariant T (MAIT) cells recognize bacterial metabolites via a semi-invariant T cell receptor (TCR) presented by MHC-like molecule MR1. Once activated, MAIT cells are a potent effector population with cytotoxic and proinflammatory properties. We recently reported that a TCR signal is not sufficient to elicit effector function, instead proinflammatory cytokines in addition to a TCR signal are required for sustained MAIT cell effector functions. We proposed that this requirement allows MAIT cells to distinguish between commensal (TCR signal only) and pathogenic bacteria (TCR signal + inflammation). We next wanted to address how MAIT cells function within an inflamed tissue environment. Specifically, if inflammation resulted in increased MAIT effector function or an exhausted phenotype because of the sustained stimuli. We obtained gingival tissue and matched blood from donors undergoing periodontic surgery for various inflammation-related diseases. MAIT cells within inflamed mucosa showed increased effector function


directly ex vivo compared to blood including expression of granzyme B and TNFa. RNAseq analysis of MAIT cells from inflamed mucosal tissue revealed a distinct transcriptional signature compared to MAIT cells in the blood. We observed a significant upregulation of chemotaxis and activation genes as well as inhibitory genes from multiple immune-modulatory pathways. Our data suggest that MAIT cells in inflamed tissues can maintain effector function, but also express immunomodulatory genes that presumably serve to avoid unwanted tissue damage.

165

Characterization of CD1a-restricted T cells using CD1a-lipid tetramers

Catriona V Nguyen-Robertson1, 2, Michael NT Souter1, 2, Scott JJ Reddiex1, 2, Janice MH Cheng1, 2, 3, Adam P Uldrich1, 2, Jamie

Rossjohn4, 5, Ildiko Van Rhijn6, Spencer J Williams3, D Branch Moody6, Dale I Godfrey1, 2, Daniel G Pellicci1, 2 1. The Department of Microbiology and Immunology, The Doherty Institute, The University of Melbourne, Parkville, Victoria, Australia

2. ARC Centre of Excellence in Advanced Molecular Imaging at The University of Melbourne, Parkville, Victoria, Australia

3. School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia

4. ARC Centre of Excellence in Advanced Molecular Imaging at Monash University, Clayton, Victoria, Australia

5. Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia

6. Brigham and Women's Hospital Division of Rheumatology, Immunology and Allergy and Harvard Medical School, Boston, MA, USA

In contrast to conventional T cells that recognize peptide antigens presented by proteins encoded in the major histocompatibility complex (MHC), other T cells recognize lipid antigens presented by MHC-like CD1 family members, CD1a, CD1b, CD1c and CD1d. Recent studies have suggested that CD1a-restricted T cells comprise a unique T cell population in human blood and may also play a unique functional role in skin. Here, we have produced mammalian CD1a tetramers to investigate the phenotype and function of human CD1a-restricted T cells directly ex vivo. Interestingly, we have shown that CD1a-restricted T cells that recognize non-self lipid antigens can also be self-reactive. Additionally, we have defined the T cell receptor (TCR) usage of both self- and foreign-lipid-reactive CD1a-restricted T cells, demonstrating that while they exhibit a diverse TCR repertoire, there is some biased variable gene usage. Experiments with CD1a mutant cell lines revealed that different TCRs can bind across the entire binding cleft of CD1a, which is likely to increase the diversity of lipid antigens that can be recognised by CD1a-restricted T cells. Phenotypic analyses of these cells revealed that they are negative for IL-18R and CD161, markers typically used to define other unconventional, innate-like T cells, such as type I NKT cells and MAIT cells, thereby distinguishing CD1a-restricted T cells from other unconventional T cells. Collectively, these studies represent an important step forward in characterizing CD1a-restricted T cells, and further understanding their role in human health and disease.

166

Circulating Mucosal-Associated Invariant T Cells are Reduced in Obesity

Jianyun Liu1, Hongmei Nan2 1. Indiana University, School of Medicine, Indianapolis, INDIANA, United States

2. Epidemiology, Richard M. Fairbanks School of Public Health, Indiana University, Indianapolis, IN, USA

Obesity-associated inflammation with altered immune responses significantly contributes to obesity-related diseases, but the underlying mechanisms are largely unknown. Recent studies have revealed that the gut microbiota also contributes to obesity and the pathology of obesity-related diseases. Mucosal-associated invariant T (MAIT) cells are a unique subpopulation of T cells characterized by the expression of a semi-invariant TCR α chain (Vα19 in mice; Vα7.2 in humans). The development and maturation of MAIT cells require the gut microbiota and antigen-presenting molecule MR1. However, the actual function of MAIT cells has not been well studied. Here we find that obese patients have fewer circulating MAIT cells than age- and gender-matched healthy-weight donors. We also determined the expression levels of the invariant TCR of MAIT cells and classical T cell transcription factors using real-time PCR. We found that Vα7.2 mRNA expression in samples from obese patients was similar to that of healthy donors. Interestingly, mRNA expression of the Vα7.2 TCR was positively correlated with the transcription factor TBX21 (found in T helper 1 cells) and BCL6 (found in follicular helper T cells), regardless of whether the samples were from healthy donors or obese patients. Our study suggests that obesity alters the number of MAIT cells. In addition, the correlation between MAIT cell TCR gene expression and transcription factors of classical effector T cells suggests the existence of transcriptional pathways linking MAIT cells to the function of conventional T cells.

167

MAIT cells exacerbate the disease course of oxazolone-induced colitis.

Asako Chiba1, Takashi Nagaishi2, Sachiko Miyake1 1. Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan

2. Department of Gastroenterology, Tokyo Medical and Dental University, Tokyo, Japan


Background: Ulcerative colitis (UC) is a chronic, relapsing-remitting, inflammatory disorder of the large intestine in humans. Previously, we have reported that mucosal associated invariant T (MAIT) cells were activated in the peripheral blood and accumulated in the inflamed mucosa in the patients with UC. This study aimed at investigating the role of these cells in the pathogenesis of oxazolone-induced colitis, a murine model of UC.

Methods: Oxazolone colitis was induced in MR1-/- mice with C57BL/6 background and its littermate MR1+/+controls. Mice were presensitized by application of 3% oxazolone in 100% ethanol to the shaved skin. Five days later, 1% oxazolone solution in 50% ethanol was administered intra-rectally. Mice were examined daily for body weight, stool consistency, hematochezia, and rectal bleeding, and the disease activity index (DAI) were scored by using these parameters. On day 9, mice were euthanized to assess colon length and histology. The severity of colitis was assessed by using histological injury score (Kennedy RJ et al., Br J Surg. 2000).

Results: Even though both MR1-/- and MR1+/+ mice developed colitis, MR1-/- mice exhibited higher survival rate and lower DAI score compared to MR1+/+ mice. The colon length was reduced in MR1+/+ mice. The histological injury score was lower in MR1-/- mice compared to that of MR1+/+mice.

Conclusions: MR1-/- mice developed less severe colitis clinically and histologically compared to MR1+/+ mice. These findings indicate that MAIT cells exacerbate the disease course of oxazolone colitis. Together with previous studies of UC patients by our and other groups, MAIT cells may be playing an important role in inflammatory bowel diseases especially UC.

169

Altered cellular metabolism in Mucosal-associated invariant T cells from obese patients

Aisling O'Brien1, 2, Laura M Tobin2, Lydia Lynch3, Donal O'Shea2, Andrew E Hogan1, 4 1. National Childrens Research Centre, Crumlin, DUBLIN, Ireland

2. St Vincent's University Hospital, Dublin, Ireland

3. Trinity College Dublin, Dublin, Ireland

4. Maynooth University, Maynooth, Co. Kildare, Ireland

Obesity is a global epidemic and responsible for the development of numerous chronic diseases including type II diabetes mellitus and cardiovascular disease. It is well established that obesity negatively alters circulating and tissue resident immune cell frequencies and functions. Mucosal associated invariant T (MAIT) cells are a population of innate T cells, which express an invariant T cell receptor, restricted by the MHC like molecule MR1. Upon activation MAIT cells can produce several cytokines including IFN-g, TNF-a and IL-17. We have previously reported that MAIT cells are dysregulated in obesity, with altered circulating and adipose tissue frequencies and a reduction in IFN-g production. The mechanisms driving their defective cytokine production are currently unknown. We performed RNA sequencing of MAIT cells isolated from obese adults and reveal extensive alterations in metabolic pathways compared to lean controls. Upon activation, immune cells undergo metabolic reprogramming, this allows for the increased demand for energy and biological intermediates required for the formation of effector proteins such as cytokines. Metabolic reprogramming can also determine the type of immune response elicited by specific immune cells. The metabolic pathways utilized by MAIT cells are currently unknown. In this report, we demonstrate that both cellular glycolysis and oxidative phosphorylation are important for MAIT cell cytokine production. We show that obesity induces defects in glucose consumption, mTOR activation and glycolytic metabolism, which explains the loss of IFN-g production previously reported in obesity. Collectively our data shows the intrinsic metabolic pathways controlling MAIT cell cytokine production and highlight a mechanism for the altered MAIT cell responses reported in obesity.

170

A Novel Mouse Model of iNKT Cell-deficiency Generated by CRISPR/Cas9 Technology Reveals a Pathogenic Role of iNKT Cells in Metabolic Disease.

Yue Ren1, Etsuko Sekine-Kondo1, Risa Shibata1, Hiroshi Watarai1 1. Division of Stem Cell Cellomics, Institute of Medical Science, The University of Tokyo, Tokyo, Japan

iNKT cells play important roles in immune regulation by bridging the innate and acquired immune systems. The functions of iNKT cells have been investigated in mice lacking the Traj18 gene segment that were generated by traditional embryonic stem cell technology, but these animals contain a biased T cell receptor (TCR) repertoire that might affect immune responses. To circumvent this confounding factor, we have generated a new strain of iNKT cell-deficient mice by deleting the Traj18 locus using CRISPR/Cas9 technology, and these animals contain an unbiased TCR repertoire. We employed these mice to investigate the contribution of iNKT cells to metabolic disease and found that obese Traj18−/− mice show reduced weight gain and ameliorated metabolic parameters, thus indicating a pathological role of iNKT cells in the development of obesity-associated disorders including insulin resistance and glucose tolerance. The new Traj18-deficient mouse strain will assist in and contribute to the studies of iNKT cell biology.

171

Impact of MAIT cells in obesity and Type 2 Diabetes


Amine Toubal1, Badr Kiaf1, Moez Rhimi2, Lucie Beaudoin1, Lucie Cagninacci1, Olivier Lantz3, Jamie Rossjhon4, James

McCluskley5, Emmanuelle Maguin2, Philippe Lesnik6, Agnès Lehuen1 1. Institut Cochin, U1016, I.N.S.E.R.M, Paris, PARIS, France

2. Micalis Institut, INRA, Jouy en Josas, France

3. Institut Curie, INSERM, Paris, France

4. DBMB, Monash University, Clayton, Australia

5. DMI, University of Melbourn, Parkville, Australia

6. ICAN, INSERM, Paris, France


172

Identification of Adipose Tissue Factors that Induce Regulatory iNKT Cells

Nelson M. LaMarche1, Raju V. V. Tatituri1, Lydia Lynch1, Michael B. Brenner1 1. Brigham and Women's Hospital / Harvard Medical School, Boston, MASSACHUSETTS, United States

Analogous to the classical Th1, Th2, and Th17 helper T cell subsets, NKT1, NKT2, and NKT17 cells have been described with stereotypical cytokine production profiles, transcription factor expression, and tissue localization. Many reports have described iNKT cells in the context of proinflammatory immune responses where they respond to danger signals and microbial lipid antigens to mediate host defense. In contrast, we recently identified a distinct role of iNKT cells in adipose tissues of mice and humans where they display a unique regulatory phenotype. Adipose iNKT cells produce high levels of IL-2 and IL-10, which drive the expansion of Tregs and M2 macrophages, respectively, and have a unique transcriptional profile that sets them apart from iNKT cells in other organs. How these iNKT cells arise, however, is incompletely understood. We performed coculture of splenic and thymic iNKT cells with adipose tissue, and found that they rapidly adopted many of the transcription factor, surface marker, and cytokine production profiles characteristic of adipose iNKT cells, including an upregulation of E4BP4 and a downregulation of PLZF. We then performed in vitro and in vivo analyses to identify the cellular and molecular inducers of regulatory iNKT cells in adipose tissue. Our work highlights the importance of the adipose microenvironment in contributing to the unique phenotype of adipose iNKT cells.

173

Diverse Natural Killer T cells regulate inflammation, neurological injury and mortality in cardiac arrest and resuscitation.

Edy Kim1, Kohei Ikeda2, Kei Hayashida2, Ji Young Choi1, Jing Yu Guo1, Fumito Ichinose2, Michael B Brenner1 1. Brigham & Women's Hospital, Cambridge, MA, United States

2. Dept. of Anesthesia, Critical Care and Pain Medicine , Massachusetts General Hospital, Boston, MA, USA


174

iNKT cells catalyze a potent and sustained IL-1β secretion pathway.

Carlos Donado1, Daimon Simmons1, Patrick Brennan1, Michael Brenner1 1. Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA


175

The possible role of the IL-33/iNKT cell axis during kidney ischemia-reperfusion injury


Maroua Ferhat1, 2, 4, 3, Aurelie Robin1, 4, 3, Sebastien Giraud1, 4, 3, Jean-Marc Gombert1, 5, 2, 3, Antoine Thierry1, 6, 2, 3, André

Herbelin1, 2, 4, 3 1. INSERM, Ss, DD, France

2. University of Poitiers, Poitiers, France

3. INSERM U1082, Poitiers, France

4. CHU of Poitiers, Poitiers, France

5. Laboratory of Immunology, CHU of Poitiers, Poitiers, France

6. Department of Nephrology and Transplantation, CHU of Poitiers, Poitiers, France

Inflammation is a prominent feature of renal ischemia-reperfusion (IR) injury characterized by leukocyte infiltration and renal tubular injury. Although invariant natural killer T (iNKT) cells are known for their deleterious role during renal IR injury, the signals that initiate their recruitment and functions in this inflammatory situation remain poorly understood. Assuming on the one hand that alarmin release by necrotic cells during IR injury may be critical (Thierry et al., 2014) and on the other hand that the alarmin IL-33 targets iNKT cells (Bourgeois et al., 2009&2011), we hypothesized that IL-33 might play a part in kidney IR injury by recruiting iNKT cells. Here, we addressed this issue by using C57BL/6 mice lacking IL-33 (IL-33Gt/Gt). Induction of IR injury was performed by unilateral clamping of the renal pedicle for 32 min after contralateral nephrectomy. We observed release of IL-33 shortly after kidney IR concomitantly with an increase in IL-33 plasma levels within one hour of reperfusion. Compared to wild-type control mice, IL-33-deficient mice exhibited reduced renal IR-induced injury, as was attested by function preservation, reduced histological change and attenuation of neutrophil recruitment. This was associated with the loss of IFN-γ/IL-17A-producing iNKT cell recruitment. Yet, even though iNKT cell-deficient (Jα18KO) mice were protected against IR injury, their circulating IL-33 continued to be increased in this experimental setup similarly to that of their wild-type counterparts. This finding, along with the in vitro observation that IL-33 targets iNKT cells by inducing both IFN-γ and IL-17A led us to put forward the hypothesis that endogenous IL-33 contributes to kidney IRI by promoting iNKT cell recruitment and cytokine production, resulting in neutrophil infiltration and activation at the injury site. This study supports the notion that the IL-33/iNKT cell axis represents a new general physiopathological mechanism involved in sterile inflammation associated with tissue damage.

176

Development of novel therapeutic strategy for heart failure via activating invariant natural killer T cells

Akimichi Saito1, Naoki Ishimori1, Yoshikuni Obata1, Shintaro Kinugawa1, Norihiro Satoh2, Kazuya Iwabuchi3, Shinichiro

Motohashi4, Kishio Ohtani5, Tomomi Ide5, Hiroyuki Tsutsui5 1. Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan

2. Hokkaido University Hospital, Sapporo, Japan

3. Kitasato University School of Medicine, Sagamihara, Japan

4. Chiba University Hospital Center for Advanced Medicine, Chiba, Japan

5. Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan

Inflammatory mediators play a crucial role in the development of heart failure (HF). Previous basic and clinical research has advanced the modern treatment of HF, however, its efficacy is still limited. Invariant natural killer T (iNKT) cells, a unique subset of T lymphocytes, play an important role in regulating tissue inflammation. We have demonstrated that the administration of α-galactosylceramide (α-GalCer), which specifically activates iNKT cells, could protect the heart against left ventricular (LV) remodeling and failure after myocardial infarction (MI). However, it activates iNKT cells temporarily and induces their anergy after repeated administration. In contrast, α-GalCer-pulsed human dendritic cells (α-GalCer/DC) can activate iNKT cells without inducing anergy even after repeated administration and have been investigated in the treatment of lung cancer.

To obtain α-GalCer/DC, peripheral blood mononuclear cells were separated from donors by apheresis, cultured with GM-CSF (800 U/mL) and IL-2 (100 U/mL) for 6 days, and pulsed with α-GalCer (100 ng/mL). Non-clinical studies have demonstrated that, similar to α-GalCer itself, intravenous administration of α-GalCer/DC (3.0×106 cells, 1 and 4 days after the creation MI) into the MI mice could also efficiently activate iNKT cells and had a protective effect against LV remodeling and failure. Furthermore, they had no toxicity when administered to nude mice under the GLP standard. Now, we are planning Phase I/II clinical trial of iNKT cell-targeted therapy to determine its clinical safety and efficacy in HF patients on top of the standard pharmacological and non-pharmacological treatments.

177

Detection of polymorphisms in nonhuman primate MR-1 alleles

Shelby O'Connor1, Amy Ellis1, Nadean Kannal1, Alexis Balgeman1 1. University of Wisconsin-Madison, Madison, WI, United States

The MHC-like molecule, MR-1, presents vitamin metabolites to Mucosal Associated Invariant T cells. It has been described that MR-1 has limited described polymorphisms among humans. However, it is known that the sequence of MR-1 is different in nonhuman primates, compared to humans. In this study, we wanted to identify polymorphisms in MR-1 alleles from nonhuman primates. We sequenced MR-1 alleles from different populations of cynomolgus macaques (n=25), rhesus macaques (n=9), and marmosets. We


found 8 different alleles across all of these populations. We expressed these alleles in 293T cells and each allele could be loaded with 6-formylpterin (6-FP) and presented on the surface. Although the differences across alleles were not in TCR contact or antigen binding sites, these variant sites could have other functional implications when loaded with ligands from different pathogens. In conclusion, we find that MR-1 does have different allele variants, and their function should be explored further in the future.

178

Aronia berry consumption inhibits T cell adoptive transfer colitis in mice by modulating Th17 and Treg populations

Ruisong Pei1, Derek A. Martin1, Bradley W. Bolling1 1. University of Wisconsin-Madison, Madison, WISCONSIN, United States

Increased fruit consumption is associated with reduced risk of inflammatory bowel disease. Little is known about how diet affects T cells during development of colitis. Aronia (Aronia mitschurinii ‘Viking’) berries are rich in polyphenols that putatively modulate inflammatory cytokines. Our objective was to identify the effects of aronia berry consumption on T cell populations in the mouse adoptive transfer model of colitis. Colonic inflammation was induced in recombinase activating gene-1 (RAG)-/- mice by transfer of CD4+CD62L+ cells (naïve T cells) from syngenic C57BL6J donors. Upon transfer, RAG-/- consumed a control diet or diets fortified with 4.5% w/w lyophilized ‘Viking’ aronia berry. Mice were sacrificed 5 or 7 weeks after adoptive transfer to characterize T cell populations by flow cytometery immunohistochemistry. Aronia consumption inhibited colitis-induced wasting at weeks 5-7 after adoptive transfer. At week 4 after transfer, adoptive transfer increased pro-inflammatory T cell populations and increased colonic cytokines. Aronia consumption inhibited colonic IFN-γ and IL-6. Furthermore, aronia increased lamina propria Treg (CD3+CD4+FoxP3+) and Th17 (CD3+CD4+IL17A+) populations. The Th17 population had greater proportions of IL-10+ and IL-22+ producing cells. Total colonic CD4+ was reduced in aronia-fed mice as determined by immunohistochemistry. At 7-8 weeks after transfer, levels of mesenteric lymph node Th17 had normalized between the control and aronia groups, but increased Treg populations persisted in the aronia groups. Aronia berry consumption appears to inhibit the initiation of adoptive transfer colitis by the modulation of T cells. These changes occur early after adoptive transfer of naïve T cells and prior to extensive colitic weight loss. Increasing Treg and anti-inflammatory Th17 may be a potential dietary strategy to inhibit the onset of colitis.

179

The GM2 ganglioside inhibits iNKT cell responses in a CD1d-dependent manner

Catia S Pereira1, Helena S Ribeiro1, Begona Perez-Cabezas1, Fatima Macedo1 1. i3S - Instituto de Investigacao e Inovacao em Saude, Porto University, Porto, Portugal

GM2 is a glycosphingolipid present in cellular membranes that progressively accumulates in GM2 gangliosidoses patients. GM2 gangliosidoses are rare genetic diseases caused by a defective activity of the lysosomal enzyme β-hexosaminidase A. iNKT cell numerical defects have been described in several mouse models of LSDs, including mouse models of GM2 gangliosidoses. Here we analyzed iNKT cell frequency and phenotype in GM2 gangliosidoses patients and analyzed, in vitro, the effect of GM2 on CD1d-mediated iNKT cell activation.

GM2 gangliosidoses patients (n=5) tended to have a lower frequency of iNKT cells than age matched healthy subjects (n=14), although this difference was not statistically significant. Notwithstanding, patients show a clear increase in the percentage of iNKT cells expressing CD4 and a decrease in iNKT cells expressing CD161.

To analyze the direct effect of GM2 in iNKT cell activation, we cultured monocytes or total peripheral blood mononuclear cells (PBMC) with GM2 alone or with GM2 and the iNKT cell antigen α-Galactosylceramide (α-GalCer). GM2 alone did not induce activation of iNKT cells, implying that this lipid is not antigenic for human iNKT cells. However, simultaneous incubation of the cells with GM2 and α-GalCer led to a reduction of iNKT cell activation in a dose dependent manner and a concomitant decrease of iNKT cell expansion from PBMCs. This effect was exerted by a reduction in the amount of stimulatory CD1d:α-GalCer complexes in the presence of GM2, as demonstrated by the use of an antibody specific for this complex.

The analysis of iNKT cells on sphingolipidoses highlights the complexity of iNKT cell activation and the importance of non-antigenic glycosphingolipids in the modulation of this process.

180

The association between mucosal associated invariant T (MAIT) cells and liver graft quality

Yasmeen G Ghnewa1, Muhammed Yuksel1, Shirin E Khorsandi2, Xiaohong Huang1, Laura Ramsay2, Wayel Jassem1, Nigel

Heaton2, Yun Ma1 1. Liver studies, King's College Hospital, King's College London, London, UK

2. Liver studies, , King's College Hospital, London, UK

Mucosa-associated invariant T (MAIT) cells are a novel subset of innate-like T cells. They are the most abundant T cell in human liver tissue, representing as much as 50% of all hepatic T cells, and found mainly around the portal tract. Recently MAIT cells have been


dubbed “new guardians of the liver”, with potential roles in protecting against infection, cancer and autoimmunity. However, these liver-abundant cells have not been described in the setting of liver transplantation. It is unknown whether these cells have the potential to induce graft acceptance or rejection in three types of major liver grafts from DBD, DCD and living related donors (LRD).

Using flow cytometry we defined MAIT cells by assessing the expression of CD3, CD4, CD8, CD161 and the Vα7.2 invariant TCR chain on intrahepatic immune cells isolated from perfusates of 22 DBD, 16 DCD and 6 LRD livers. Peak levels of peripheral blood aspartate aminotransferase (AST), a surrogate marker for organ rejection, and histological signed of acute rejection post transplant were recorded.

Following transplantation, livers displaying early signs of acute rejection had significantly higher proportions of MAIT cells than those without any signs of rejection (P = 0.026). Furthermore, peak AST levels during the 7-day period post transplantation, correlated positively with CD4posCD8pos (r = 0.443, P = 0.01) MAIT cells and negatively with CD4negCD8neg MAIT cells (r = - 0.435, P = 0.01). Furthermore, CD4negCD8neg MAIT cells were significantly reduced in DCD compared to DBD and LRD (P < 0.05), and CD4negCD8neg MAIT cells were enriched in DCD grafts.

We have found a link between MAIT cells and allograft function, where high proportions of MAIT cells, in particular CD4posCD8pos MAIT cells, is associated with a higher risk of organ rejection. However, such a risk is reduced with higher proportions of CD4negCD8neg MAIT cells.

181

Innate T cells are significantly altered in the livers of patients with alcoholic liver disease

Yasmeen G Ghnewa1, Shirin E Khorsandi2, Xiaohong Huang1, Laura Ramsay2, Wayel Jassem1, Nigel Heaton2, Yun Ma1 1. Liver studies, King's College Hospital, King's College London, London, UK

2. Liver studies, , King's College Hospital, London, UK

Innate T cells include natural killer T (NKT) cells, mucosa-associated invariant T (MAIT) cells, and gamma delta (γδ) T cells. Innate T cells are enriched in the human liver, with MAIT cells making up to 50% of hepatic T cells. It's been reported recently that the frequencies of innate T cells was reduced in the liver of mixed chronic liver disease cohort compared to healthy controls (HC). However, little is known about the affects of alcoholic liver disease (ALD) on liver-resident human innate T cells. We aimed to characterize these cells in the liver of patients with ALD.

Intrahepatic mononuclear cells (HMC) from liver perfusates of 10 explanted ALD livers and 10 healthy controls (HC) were analysed by flow cytometry for the expression of CD3, Vα24-Jα18, Vα7.2, CD161, γδ-TCR, CD19, CD4, CD8, CD40, MHC-II, and CD1d.

The frequency of T cells within HMC was significantly reduced in patients with ALD compared to HC (P=0.0036). The frequencies of MAIT cells and γδ T cells as a proportion of total T cells were significantly reduced (P=0.004) and (P=0.01) respectively. The proportions of total T cell subsets based on CD4 and CD8 expression were significantly skewed in patients with ALD. CD4+ T cells were significantly increased (P=0.0001), while CD8+ T cells (P=0.01) and CD4-CD8- T cells (P=0.0003) were significantly reduced. B cells and monocytes from ALD patients showed a significant up regulation of CD40 and CD1d, but not MHC class II compared to HC.

MAIT cells and γδ T cells are reduced in frequency in the liver of patients with ALD. The selective up regulation of CD1d and CD40 may be relevant to the cause of depletion in the innate T cell pool . These findings warrant further investigation into the role of MAIT and γδ T cells in the pathogenesis of ALD.

182

LFA-1 signaling drives TCR- and cytokine-independent IFNγ production by innate T-lymphocytes

Akshat Sharma1, Stephanie M Lawry1, Nicholas A Zumwalde1, Bruce S Klein1, Nathan M Sherer1, Jenny E Gumperz1 1. University of Wisconsin Madison, Madison, WI, United States

PLZF, the signature transcription factor of innate T-lymphocytes, has been shown to confer elevated cell-surface expression of the integrin, LFA-1. While integrins are known to mediate both adhesive and signaling activities, the functional impact of LFA-1 expression by innate T lymphocytes remains poorly understood. Here we show that exposing human iNKT cells to high valencies of ICAM-1 (an adhesion ligand for LFA-1) is sufficient to induce their secretion of IFNγ in the absence of TCR or cytokine stimulation. iNKT cell IFNγ secretion via this LFA-1-mediated pathway required mRNA transcription, and depended on calcium and MAPK signaling, but JAK-STAT activation was dispensible. Exposing primary human blood lymphocytes to high valency ICAM-1 revealed a responding T cell population that was highly enriched for PLZF+ cells, including iNKT cells, MAIT cells, and Vδ2+ T cells. Thus, this pathway of IFNγ production appears to be a shared feature of several human innate T-lymphocyte populations. These results indicate that innate T lymphocytes may contribute IFNγ in inflammatory contexts where ICAM-1 is upregulated e.g. multiple sclerosis, atherosclerosis and sepsis. Moreover, since this pathway is dependent on neither foreign antigens nor inflammatory cytokines, it may represent a mechanism for the production of "early" IFNγ that is thought to play a key role in defining the course of the subsequent response.

183

Mucosal associated invariant T (MAIT) cell dynamics during acute HIV-1 infection


Kerri G. Lal1, 2, 3, Edwin Leeansyah1, 4, Joana Dias1, Matthew Creegan2, 3, Margaret C. Costanzo2, 3, Leigh Anne Eller2, 3, Shelly Krebs2, 3, Hannah Kibuuka5, Lucas Maganga6, Sorachai Nitayapan7, Fredrick Sawe8, 2, Julie Ake2, Nelson L. Michael2, Merlin L.

Robb2, 3, Michael A. Eller2, 3, Johan K. Sandberg1 1. Medicine, Karolinska Institutet, Stockholm, Sweden

2. U.S. Military HIV Research Program, Silver Spring, MD, USA

3. Henry M. Jackson Foundation, Bethesda, MD, USA

4. Program in Emerging Infectious Diseases, Duke-National University of Singapore Medical School, Singapore 169587, Singapore

5. Makerere University Walter Reed Project, Kampala, Uganda

6. Walter Reed Program- Tanzania, Mbeya, Tanzania

7. Royal Thai Army Component, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand

8. Kenya Medical Research Institute/Walter Reed Project, Kericho, Kenya

In chronic HIV-1 infection, MAIT cells are lower in frequency, have increased expression of activation markers, and are less functional compared to healthy controls. The temporal dynamics of MAIT cell loss and dysfunction in HIV-1 infection is not known. To better understand MAIT cell dynamics in early HIV-1 infection, cryopreserved PBMC from 20 donors followed from pre-infection through early acute infection were studied for changes in MAIT cell frequency, phenotype, and function prior to infection and at 3 time points out to 3 months post-infection. Donor PBMC were from women in East Africa and men in Thailand. Baseline pre-infection frequencies of MAIT cells differed between the two regions at pre-infection, with East Africans having lower baseline frequencies of MAIT cells compared to Thais (median=0.737% and 1.93% of CD3+ T cells, respectively). Surprisingly, MAIT cell frequencies did not change significantly during acute HIV-1 infection compared to pre-infection. However, MAIT cells showed an increased expression of activation markers CD38, HLA-DR, PD-1, and TIGIT at peak viral load (median 16 days since first positive test for HIV-1 RNA), and remained elevated three months post-infection (median 84.5 days since first positive test for HIV-1 RNA). Expression of CD38 and HLA-DR correlate positively with plasma levels of sCD14 (r=0.727 and r=0.807, respectively), a marker of monocyte activation and microbial translocation. Functionally, MAIT cells displayed reduced production of TNFα and IFNγ after in vitro stimulation with PMA/ionomycin at the 3 months post-infection time point compared to pre-infection. Together, these data are consistent with a model where MAIT cells become highly activated early in HIV-1 infection, possibly due to translocation of microbial products across a compromised gut barrier. In this model, chronic exposure to antigen may lead to activation-induced cell death and slow progressive decline in MAIT cell frequency and functionality over time.

184

Capturing the tissue-resident CD1a-reactive T cell population from human skin

Rachel Cotton1, 2, Tan-Yun Cheng2, Ildiko Van Rhijn2, Rachael A Clark1, 3, Branch Moody1, 2 1. Harvard University, Cambridge, MA, United States

2. Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Boston, MA, USA

3. Department of Dermatology, Brigham and Women's Hospital, Boston, MA, USA

CD1a-reactive T cells bind CD1a without directly contacting the cargo in the CD1a pocket. Altered-self lipids, environmental lipids, or small molecules in CD1a fine-tune the activation of these T cells by interfering with or permitting CD1a-TCR contacts. The CD1a system in humans is uniquely concentrated in skin: CD1a has constitutively high surface expression on epidermal Langerhans cells; epidermal lipids are permissive for CD1a-TCR binding; and CD1a-reactive T cells in circulation have a skin-homing Th22-like signature. Studies from our group and others implicate CD1a-autoreactive T cells in allergic and inflammatory skin diseases. However, capturing CD1a-reactive T cells from their tissue microenvironment remains a major gap. There are no known surface markers or TCR gene patterns to discriminate CD1a-reactive cells from other skin-resident T cells. To obtain skin T cells from healthy individuals, we cultured discarded skin from cosmetic surgeries on 3D matrices with IL-2 and IL-15, recovering millions of T cells migrating from the skin per donor. To enrich CD1a-dependent responses, we co-cultured skin T cells with in vitro derived CD1a++ Langerhans’-like cells. We measured the frequency of CD1a-depdendent cells based on IL-22 production in activation assays with CD1a+ K562 cells +/- CD1a-blocking antibody. By IL-22 ELISPOT, CD1a-dependent responses across donors (n=13) were variable, with 7/13 exhibiting CD1a-dose-dependent and blockable numbers of CD1a-dependent IL-22-producing cells. A second group (3/13) had an elevated frequency in IL-22-producing T cells at baseline that was CD1a-dose dependent but only partially reduced by CD1a-blocking, suggesting that CD1a-reactive T cells make up a variable fraction of IL-22+ skin T cells by donor. Using a newly developed IL-22 capture reagent, we are positively selecting CD1a-dependent IL-22-secreting cells for further phenotyping and high throughput sequencing of TCRα/δ and TCRβ genes. By adapting a described 3D culture method for skin, we are capturing CD1a-reactive T cells from their tissue microenvironment.

185

MAIT cells in the human oral mucosa exhibit a CD69+/CD103+ tissue resident memory phenotype and lowered cytolytic potential

Michal J Sobkowiak1, Haleh Davanian2, Annelie Tjernlund3, Anna Gibbs3, Sushrusha Nayak1, Joana Dias1, Soo Aleman4,

Carina Krüger-Weiner2, Markus Moll1, Robert Heymann2, Edwin Leeansyah1, 5, Margaret Sällberg Chen2, Johan K Sandberg1 1. Center for Infectious Medicine, Karolinska Institutet, Stockholm, Sweden

2. Department of Dental Medicine, Karolinska Institutet, Stockholm, Sweden


3. Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden

4. Department of Medicine, Huddinge, Karolinska Institutet, Stockholm, Sweden

5. Duke-NUS Medical School, Singapore

Mucosa-associated invariant T (MAIT) cells are a class of non-classical T lymphocytes defined by their invariant Vα7.2-containing MR1-restricted T cell receptor, as well as high expression of CD161. At present, the characteristics of MAIT cells in the oral mucosa are poorly defined. In this study, we have analyzed buccal biopsies and matched blood samples from a cohort of healthy volunteers. MAIT cells were present in the buccal mucosa, with a tendency to cluster around the basal layer, a region also found to contain a higher frequency of MR1-expressing antigen-presenting cells. The frequency of MAIT cells was similar in the buccal mucosa compared to matched peripheral blood. However, buccal mucosal MAIT cells displayed a tissue resident activated profile, with high CD69, HLA-DR and PD-1 expression, as well as skewed subset distribution with higher representation of CD4/CD8 double-negative cells and of CD8αα+ cells within the CD8+ subset. The Jα chain usage in the MAIT cell TCR was more variable in oral mucosal MAIT cells than in peripheral blood MAIT cells. Functionally, oral mucosal MAIT cells had lower perforin levels, indicative of a lower cytolytic potential. Up to three quarters of oral MAIT cells had a CD69+/CD103+ tissue resident memory phenotype, characterized by an increased CD8 and CD38 expression, and further lowered perforin levels. Interestingly, aspects of the oral MAIT cell population were associated with gender and life-style factors such as consumption of bacterially fermented milk products and alcohol intake. Together, these data show that MAIT cells form a part of the oral mucosal T cell compartment, where they exhibit a tissue-resident activated profile. Thus, MAIT cells are positioned in the oral cavity and equipped to handle resident microbiota as well as invading pathogens.

186

Natural killer T cells promote cholestatic liver disease in bile duct ligated mice

Laura Valestrand1, 2, 3, Natalie L. Berntsen1, 2, 3, Fei Zheng1, 2, 3, Bjarte Fosby4, Tom H. Karlsen1, 2, 3, 5, Pål Dag Line3, 4, Xiaojun

Jiang1, 2, Espen Melum1, 2, 5 1. Norwegian PSC Research Center, Division of Surgery, Inflammatory Medicine and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway

2. Research Institute of Internal Medicine, Division of Surgery, Inflammatory Medicine and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway

3. Institute of Clinical Medicine, University of Oslo, Oslo, Norway

4. Department of Transplantation Medicine, Oslo University Hospital, Rikshospitalet, Oslo, Norway

5. Section of Gastroenterology, Division of Surgery, Inflammatory Medicine and Transplantation, Surgery, and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway

Background:

Natural Killer T (NKT) cells are abundant in the mouse liver and we have previously demonstratedthat CD1d on cholangiocytes can present lipid antigens to NKT cells. We hypothesised that during cholestasis, i.e., when bile builds up in the liver, NKT cells play a regulatory role.

Methods:

To induce cholestasis we ligated the common bile duct of wild type (WT) and CD1d knockout (Cd1d-/-) mice. The mice were weighed daily and after three days we sampled livers for histological assessment and lymphocyte isolation for flow cytometry. Serum was sampled and analysed for alkaline phosphatase (AP), bilirubin and alanine transferase (ALT).

Results:

After bile duct ligation (BDL) (n=5) or sham operation (n=7) of WT mice there was a clear increase of the activation marker CD69 on the intrahepatic NKT cells at day 3 (1458 median fluorescense intensity (MFI) vs. 709 MFI, P<0.0001). There was no change in CD69 expression for the remaining hepatic T-cell population. We then investigated whether Cd1d -/- mice were protected against cholestasis. Mice lacking NKT cells had less weight loss following BDL than WT animals (weight loss day 3; 7% vs. 15%, P=0.0003). In line with this, the serum levels of the cholestatic parameters AP and bilirubin were significantly lower in Cd1d-/-mice; (AP; Cd1d -/-: 345 U/L vs. WT: 511 U/L, P=0.036) and (bilirubin; CD1d -/- :115 μmol/L vs. WT: 224 μmol/L, P=0.003).

Conclusion:

The intrahepatic NKT cell population is activated following BDL and Cd1d-/- mice are protected from cholestatic liver damage. This contrast previous reports in Jα18-/- mice. A possible explanation may be that the non-invariant NKT cells that the Cd1d -/- mouse also lacks, play the detrimental role during cholestasis.

1. Schrumpf E, Tan C, Karlsen TH, et al. The biliary epithelium presents antigens to and activates natural killer T cells. Hepatology. April 2015. doi:10.1002/hep.27840.

2. Wintermeyer P, Cheng C-W, Gehring S, et al. Invariant natural killer T cells suppress the neutrophil inflammatory response in a mouse model of cholestatic liver damage. Gastroenterology. 2009;136(3):1048-1059. doi:10.1053/j.gastro.2008.10.027.

3. Duwaerts CC, Sun EP, Cheng C-W, van Rooijen N, Gregory SH. Cross-activating invariant NKT cells and kupffer cells suppress cholestatic liver injury in a mouse model of biliary obstruction. PLoS One. 2013;8(11):e79702. doi:10.1371/journal.pone.0079702.

187


Gamma delta T cells producing IL-17A regulate adipose Treg homeostasis and thermogenesis

Ayano C Kohlgruber2, 1, Shani Gal-Oz3, Nelson LaMarche2, 1, Danielle Duquette4, Hung Nguyen2, Amir Mina4, Moto Shimazaki2, Derek Sant'Angelo5, Ali Tavakkoli6, Ulrich von Andrian1, 7, Alexander S Banks4, Tal Shay3, Michael B Brenner2, Lydia Lynch4,

8 1. Division of Medical Sciences, Harvard Medical School, Boston, MA, USA

2. Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital , Boston, MA, USA

3. Department of Life Sciences, Ben-Gurion University of the Negev, Israel

4. Endocrinology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

5. Graduate School of Biomedical Sciences, Rutgers Robert Wood Johnson Medical School, Brunswick, NJ, USA

6. Department of General and Gastrointestinal Surgery, BWH, Boston, MA, USA

7. Department of Microbiology and Immunology, Harvard Medical School, Boston, MA, USA

8. School of Biochemistry and Immunology, Trinity College, Dublin, Ireland

γδ T cells are guardians at barrier sites and rapidly respond to the presence of extracellular fungi, bacterial infections, and cellular stress by producing chemokines and cytokines. However, little is known about their steady state role in non-barrier tissues. Here, we characterize a highly enriched, tissue-resident population of γδ T cells in adipose tissue that regulates age-dependent adipose Treg expansion and controls core body temperature in response to environmental fluctuations. These adipose γδ T cells express and rely on the transcription factor PLZF and transcriptional profiling of PLZF+ γδ T cells reveals their innate-phenotype and functional capacity to produce TNF and IL-17A. Mechanistically, TNF and IL-17A synergize to enhance IL-33 production by adipose stromal cells and augment Treg numbers. As such, mice genetically deficient in PLZF+ γδ T cells or IL-17A in vivo, exhibit striking reductions in both ST2+ Tregs and IL-33 levels in visceral adipose tissue. Surprisingly, we find that mice deficient in PLZF+ γδ T cells also lack the ability to regulate core body temperature at thermoneutrality and cold challenge due to their inability to support UCP-1 dependent adaptive thermogenesis. γδ T cell regulation of body temperature thus opens a new dimension in adipose biology, whereby the dynamic cross talk between innate lymphocytes and tissue-specific stromal cells dictates local immune homeostasis and systemic energy expenditure. Together, our studies underscore the emerging roles of innate γδ T cells as critical modulators of both immune and tissue homeostatic biology.

188

CD1a presentation of endogenous antigens by group 2 innate lymphoid cells

Clare S Hardman1, Yi-Ling Chen1, Maryam Salimi1, Rachael Jarrett1, David Johnson2, Valtteri Jarvinen3, Raymond Owens3,

Emmanouela Repapi4, David Cousins5, Jillian Barlow6, Andrew McKenzie6, Graham Ogg1 1. Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, Oxfordshire, UK

2. Department of Plastic and Reconstructive Surgery, John Radcliffe Hospital, Oxford University Hospitals NHS Trust, Oxford, Oxfordshire, UK

3. OPPF – UK, Harwell and Wellcome Trust Centre for Human Genetics , University of Oxford, Oxford, Oxfordshire, UK

4. CBRG, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, Oxfordshire, UK

5. Department of Infection, Immunity and Inflammation, NIHR Leicester Respiratory Biomedical Research Unit , University of Leicester, Leicester, Leicestershire, UK

6. MRC Laboratory of Molecular Biology, Laboratory of Molecular Biology, Cambridge, Cambridgeshire, UK


189

Lipid antigens in bile from patients with liver diseases activate NKT cells

Laura Valestrand1, 2, 3, Natalie L. Berntsen1, 2, 3, Elisabeth Schrumpf1, 2, 3, Tom H. Karlsen1, 2, 3, 4, Richard S. Blumberg5, Espen

Melum1, 2, 4 1. Norwegian PSC Research Center, Division of Surgery, Inflammatory Medicine and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway

2. Research Institute of Internal Medicine, Division of Surgery, Inflammatory Medicine and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway

3. Institute of Clinical Medicine, University of Oslo, Oslo, Norway

4. Section of Gastroenterology, Division of Surgery, Inflammatory Medicine and Transplantation, Surgery, and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway

5. Division of Gastroenterology, Hepatology and Endoscopy, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA


Background

Natural Killer T (NKT) cells are abundant in the liver of mice and humans. We have previously demonstrated that CD1d on the biliary epithelium can present antigens to NKT cells1. We hypothesized that lipids dissolved in bile could act as antigens and activate NKT cells.

Methods

Bile was collected from ten patients undergoing liver transplantation due to primary sclerosing cholangitis (PSC) (n=5), cryptogenic cirrhosis (n=3), autoimmune hepatitis (n=1), or hepatocellular carcinoma (n=1). Murine invariant (24.7, 24.8 and DN32.D3) and non-invariant (14S.6, 14S.7, 14S.10 and 14S.15) NKT cell hybridomas covering a range of different antigen specificities were used to test bile samples for reactivity. α-Galactosylceramide was used as a positive antigen control. Cell-culture plates were coated with a murine CD1d-monomer and incubated with the different bile samples over night. NKT hybridomas were added to the plates and IL-2 was analyzed in the culture supernatants by ELISA.

Results

Eight out of ten bile samples activated at least one of the NKT hybridomas. The NKT hybridoma that demonstrated the clearest activation by the bile samples was 14S.10, which is a non-invariant NKT hybridoma14S.10 was further used in the subsequent experiments along with four bile samples that demonstrated distinct activation. The bile samples were then tested in dilutions from 1:100 to 1:1.000.000. For all of the samples there was an evident dose-response relationship. Of note there was clear activation even at the highest dilutions.

Conclusions

Bile from patients with a diverse set of liver diseases contains antigens that activate both invariant and non-invariant NKT hybridomas. The biliary antigens that activate these cells demonstrate a dose-response relationship and do not seem to be present in bile from all patients. These findings indicate that activation of NKT cells by lipid antigens in bile can be of relevance in the disease development of inflammatory biliary diseases.

1. Schrumpf E, Tan C, Karlsen TH, et al. The biliary epithelium presents antigens to and activates natural killer T cells. Hepatology. April 2015. doi:10.1002/hep.27840.

190

Function of MR1-Restricted Mucosal-Associated Invariant T Cells during Citrobacter rodentium infection

Elodie Macho-Fernandez1, Nathaniel Barton1, Mahantappa Halimani1, Claudio Nunes-Alves2, Mary H Young3, Natacha

Veerapen4, Samuel M Behar2, Gurdyal Besra4, Laurent Gapin3, Manfred Brigl1 1. Brigham and Women's Hospital-Harvard Medical School, Brookline, MA, United States

2. Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, United States

3. Department of Immunology and Microbiology, University of Colorado Denver, Aurora, CO, United States

4. School of Bioscience, University of Birmingham, Edgbaston, Birmingham , United Kingdom

Mucosal-Associated Invariant T (MAIT) cells are prominently localized in gastro-intestinal mucosal tissues and recognize MR1-presented microbial vitamin metabolites that are produced by both intestinal commensals and pathogens. However, very little is known about the role and function of MAIT cells during intestinal infection, which is in large part due to the low numbers of MAIT cells in commonly used laboratory mouse strains. Here, we used several mouse strains, including novel traceable MAIT T cell receptor (TCR) retrogenic (Rg) mice, iVa19 TCR transgenic mice, and wild-type mice, together with antigen-loaded MR1 tetramers to investigate the function of MAIT cells during Citrobacter rodentium infection. At steady state, intestinal MAIT cells displayed a CD44highCD62Lneg activated/memory phenotype and expressed PLZF and T-bet. However, two populations remained distinct based on the expression of RORgt. Following in vitro C. rodentium infection, MAIT cells secreted large amounts of IFN-g, TNF-a, IL-17A and IL-22. Secretion of IFN-g and IL-17A required both activating cytokines and TCR stimulation mediated by C. rodentium antigen-loaded MR1 molecules. In contrast, efficient IL-22 release required secretion of IL-1b and IL-23 by infected antigen presenting cells and occurred in the absence of concomitant TCR ligation. In vivo, C. rodentium infection led to the rapid activation of MAIT cells and their accumulation in the colon. Importantly, early during C. rodentium infection, mice with a high frequency of MAIT cells showed significantly reduced bacterial burden. Furthermore, MAIT cell activation enhanced expression of antimicrobial peptides by intestinal epithelial cells, a key function of the intestinal barrier contributing to protective immunity. Taken together, our data suggest that MAIT cell effector functions are differentially regulated by TCR- and cytokine-mediated signals, and reveal an important contribution of MAIT cells in the early immune response to bacterial infection of the intestinal tract.

192

Characterizing MAIT cells in human mucosal cancers

Jason Kelly1, 2, Brandan Toohey1, Daniel Gray3, George Kannourakis2, Stuart P Berzins1, 2


1. Fiona Elsey Cancer Research Institute, Ballarat, VIC, Australia

2. Federation University Australia, Redan, VIC, Australia

3. The Walter and Eliza Hall Institute of Medical Research , Melbourne, VIC, Australia

Mucosal-associated invariant T (MAIT) cells are an evolutionary conserved subset of innate-like T cells that are enriched in mucosal tissues within the human body. Upon activation, MAIT cells rapidly produce cytokines that can regulate inflammatory responses in and around mucosal tissues. Mucosal cancers of the lung and large bowel are among the most commonly diagnosed human cancers in Australia and despite advances in conventional treatments, still maintain very high mortality rates. As mucosal cancers and pre-cancerous lesions involve a breach of the mucosal barrier, T cells within these areas may interact with resident microbiota. We are investigating MAIT cell activity in these tissues and whether it impacts cancer evolution and progression. Using flow cytometry and an array of microscopy techniques, we have analysed MAIT cells in human tissues containing both mucosal tissue and mucosal cancer. We are also investigating the function of MAIT cells within the tumour microenvironment, and whether there are changes in MR1 antigen presentation within this environment that influence MAIT cell responses.

193

6-FP Pretreatment Demonstrates an MR1 Recycling Pathway

David Lewinsohn1, Elly Karamooz1 1. OHSU / PVAMC, Portland, OREGON, United States

Introduction

MR1 is a unique non-classical class I molecule in that 1) it is monomorphic, 2) very little MR1 is on the cell surface and 3) it presents small molecule, non-peptidic antigens, some of which are derived from the riboflavin biosynthesis pathway. In airway epithelial cells (BEAS-2Bs) expressing MR1GFP, MR1 resides in the endoplasmic reticulum (ER) and in discrete intracellular vesicles. Currently, little is known about the precise trafficking of native MR1 or about MR1 recycling.

Methods

We used BEAS-2Bs and A549 MR1 knockout cells as our antigen presenting cells. Our exogenous antigen was filtered Mycobacterium smegmatis (Msmeg) supernatant. Antigen presentation was measured via IFN-γ release by MR1-restricted T cells (MR1Ts). For experiments utilizing MR1GFP, we used the plasmid TET-MR1GFP, which has a doxycycline inducible promoter to control MR1GFP synthesis.

Results

BEAS-2Bs treated overnight with the MR1 antagonist 6-formylpterin (6-FP) demonstrated an augmented response to Msmeg supernatant compared to controls. To understand the mechanism of this “boosting” effect, we transfected A549 MR1 knockout cel ls with TET-MR1GFP to see if new protein synthesis accounted for the increased response. We found that 6-FP pretreatment, even in the absence of new protein synthesis, resulted in more IFN-γ release by MR1Ts in response to Msmeg supernatant. Using flow cytometry we found that 6-FP treated cells have more MR1, indicating that the possible mechanism of 6-FP “boosting” is that MR1 is rescued from degradation and is then recycled for re-use.

Conclusions

Our data suggest that 6-FP rescues MR1 from degradation and that 6-FP loaded MR1 can be reloaded with exogenous mycobacterial antigen. This indicates the existence of a recycling pathway for MR1 and shows that MR1 can be loaded with antigen even when MR1 is outside of the ER.


SPONSORS AND EXHIBITORS

Gold Plus Sponsor Kyowa Kirin Pharmaceutical Research, Inc. Kyowa Kirin Pharmaceutical Research, Inc. (KKR) is a wholly owned U.S. subsidiary of Kyowa Hakko Kirin Co., Ltd. dedicated to discovering first-in- class therapeutics that address unmet medical needs and improve the health of patients. KKR serves as Kyowa Hakko Kirin’s primary drug discovery center in the United States with more than 30 research staff members working each day to discover biologics, including antibodies, in the therapeutic areas of immunology, allergy and fibrotic disease. Since its inception, KKR has focused on open innovation through several industry–academia collaborative programs including supporting the research of La Jolla Institute for Allergy and Immunology (LJI) for more than 20 years. Industry–academia partnerships are one of KKR’s principal missions to enhance its network-based drug discovery activities.

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Silver Sponsors BioLegend BioLegend develops and manufactures highly recognized, world-class antibodies and reagents at an outstanding value to customers for biomedical research. Our broad product portfolio includes flow cytometry, cell biology, and biofunctional molecules for research in immunology, neuroscience, cancer, cell biology, and stem cells. Our product development program, accomplished through technology licensing, collaborations, and internal research and development, has produced over 17,000 products, which have been collectively cited in over 25,000 peer-reviewed journals. BioLegend’s reagents are supported by superior customer service and a quality management system dedicated to continuous improvement that is certified for ISO 9001:2008 and ISO 13485:2003. Cell Medica


Cell Medica is committed to transforming patients’ lives through developing cellular immunotherapies to treat cancer. In collaboration with our strategic partners, Cell Medica is developing products using three proprietary technologies: activated T cells, chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs). Our lead product CMD-003 is being tested in two Phase II trials for treating Epstein Barr virus-associated cancers. We are working with Baylor College of Medicine and the University of North Carolina to develop next generation CAR-modified NKT cells, including off-the- shelf products. We are collaborating with University College London to develop the Dominant TCR technology platform.

Bronze Sponsors BD Biosciences BD Biosciences is a world leader in bringing innovative diagnostic and research tools to life scientists, clinical researchers, laboratory professionals and clinicians who are involved in basic research, drug discovery and development, biopharmaceutical production and disease management. The BD Biosciences segment is focused on continually advancing the science and applications associated with cellular analysis and products that help grow living cells and tissue. ProImmune Ltd BD Biosciences is a world leader in bringing innovative diagnostic and research tools to life scientists, clinical researchers, laboratory professionals and clinicians who are involved in basic research, drug discovery and development, biopharmaceutical production and disease management. The BD Biosciences segment is focused on continually advancing the science and applications associated with cellular analysis and products that help grow living cells and tissue. GE Healthcare The Life Sciences business of GE Healthcare provides bioprocessing solutions for the development and manufacture of high-quality biotherapeutics and vaccines. We help customers increase speed to market, while reducing costs and improving drug manufacturing performance. With high-quality products and services, we support the biopharmaceutical industry in making health visions a reality.

Supporters American Association of Immunologists The American Association of Immunologists (AAI) is an association of professionally trained scientists from all over the world dedicated to advancing the knowledge of immunology and its related disciplines, fostering the interchange of ideas and information among investigators, and addressing the potential integration of immunologic principles into clinical practice. www.aai.org Bio X Cell Bio X Cell is a biotechnology company that manufactures high quality, bulk quantity (milligram to gram) monoclonal antibodies for biomedical research. All of our antibodies are of high purity and low endotoxin levels, well suited for in-vivo preclinical studies. University of Melbourne The Department of Microbiology and Immunology at the University of Melbourne is recognised internationally for scientific excellence and leadership across the fields of microbiology and immunology and is home to a number of Australia's most eminent biomedical scientists including Nobel Laureate, Professor Peter Doherty AC. The Department is located in the Peter Doherty Institute for Infection and Immunity, a new state-of-the-art facility that combines research and training in infectious diseases and immunology with laboratory diagnostic services, clinical services and infectious diseases surveillance and epidemiology.


Exhibitors IMMUDEX Based in Copenhagen, Denmark, with North American operations based in Fairfax, Virginia, Immudex provides MHC Dextramers for the monitoring of antigen-specific T cells. Under an agreement with the US Cancer Immunotherapy Consortium (CIC) and the European Cancer Immunotherapy Consortium (CIMT), Immudex also provides MHC Multimer and Elispot proficiency panel services worldwide. o date we have achieved quantitation of over 1000 CD8 T cell specificities in a single, small blood sample. To find out more about how Immudex is improving immune monitoring and enabling better treatment decisions please visit us at www.immudex.com. To learn more about proficiency panel participation email [email protected]. Miltenyi Biotec Inc Miltenyi Biotec’s mission is to improve scientific understanding and medical progress. We innovate products that address sample preparation, separation of cells and their analysis, and that advance the concept of cellular therapy. We provide products and services that advance biomedical research and cellular therapy.


ATTENDEES Erin Adams University of Chicago, United States Cole Anderson University of Utah, United States Holger Babbe Janssen R&D, United States Li Bai University of Science & Technology of China, China Maureen Banach University of Rochester Medical Center, United States Patricia Barral King's College London, United Kingdom Melissa Bedard University of Oxford, United Kingdom Yasmine Belkaid National Institute of Health, United States Albert Bendelac The University of Chicago, United States Julia Berkson University of Washington, United States Jay Berzofsky National Cancer Institute, NIH, United States Alessandra Bigi San Raffaele Scientific Institute, Italy Niklas Bjorkstrom Karolinska Institutet, Sweden Jordan Bloom The Scripps Research Institute, United States Richard Blumberg Brigham and Women's Hospital, United States Bradley Bolling University of Wisconsin-Madison, United States Dominique Bollino University of Maryland Baltimore, United States Caroline Boulouis Karolinska Institutet, Sweden Michael Bowman Pfizer Research Inc, United States Patrick Brennan Brigham and Women's Hospital / Harvard Medical School, United States

Michael Brenner Harvard Medical School/Brigham and Women's Hospital, United States Manfred Brigl Brigham and Women's Hospital, Harvard Medical School, United States Randy Brutkiewicz Indiana University School of Medicine, United States Liang Cao Northwestern University, United States Susanna Cardell University of Gothenburg, Sweden Giulia Casorati San Raffaele Scientific Institute, Italy A. Raul Castano Universidad Autonoma de Barcelona, Spain Chiara Ceriotti Center for Regenerative Therapies Dresden, Germany Andrew Chancellor University of Southampton, United Kingdom Shilpi Chandra La Jolla Institute for Allergy and Immunology, United States Zhenjun Chen Peter Doherty Institute,University of Melbourne, Australia Tan-Yun Cheng Brigham and Women's Hospital, Harvard Medical School, United States Shian-Huey Chiang Pfizer, United States Asako Chiba Juntendo University School of Medicine, Japan Hyunwoo Cho MD Anderson Cancer Center, United States Michela Consonni San Raffaele Scientific Institute, Italy Alexandra Corbett University of Melbourne, Australia Rachel Cotton Harvard University, United States Amy Courtney Baylor College of Medicine, United States

Catherine Crosby La Jolla Institute for Allergy and Immunology, United States Patrick Darcy The Child Health Insitiute of New Jersey, Rutgers University, United States Nyambayar Dashtsoodol RIKEN, Japan Louise D'Cruz University of Pittsburgh, United States Annemieke de Jong Columbia University, United States Gennaro De Libero University of Basel, Switzerland Gloria Delfanti San Raffaele Scientific Institute, Italy Paolo Dellabona San Raffaele Scientific Institute, Italy Kavita Dhodapkar Yale University, United States Madhav Dhodapkar Yale University, United States Derek Doherty Trinity College Dublin, Ireland Carlos Donado Harvard Medical School / Brigham and Women's Hospital, United States Catarina Filipa dos Santos Sa e Almeida The University of Melbourne, Australia Sidonia Eckle Peter Doherty Institute for Infection and Immunity, Australia Eva-Stina Edholm University of Rochester Medical Center, United States Dirk Elewaut Ghent University Hospital and Flanders Institute of Biotechnology, Belgium Amy Ellis University of Wisconsin-Madison, United States Isaac Engel La Jolla Institute for Allergy & Immunology, United States Mark Exley University of Manchester, United Kingdom Martin Flajnik University of Maryland Baltimore, United States


Stephan Gadola F.Hoffmann-La Roche & University of Southampton/UK, Switzerland Gloria Galvan MD Anderson Graduate School of Biomedical Sciences, United States Laurent Gapin University of Colorado, United States Lucy Garner University of Oxford, United Kingdom Olivier Gasser Malaghan Institute of Medical Research, New Zealand Sabine Geiselhart Medical University of Vienna, Austria Thomas Gensollen Brigham and Women's Hospital, United States Yasmeen Ghnewa King's College London, United Kingdom Martine Gilleron CNRS, France Dale Godfrey The University of Melbourne, Australia Katharine Goodall Monash University, Australia Jean-Baptiste Gorin Karolinska Institutet, Sweden Srinath Govindarajan VIB-UGENT, Center for Inflammation Research, Belgium Jenny Gumperz University of Wisconsin School of Medicine and Public Health, United States Thomas Hägglöf University of Texas Health Science Center at San Antonio, United States Clare Hardman University of Oxford, United Kingdom Melanie Harriff Oregon Health & Sciences University, United States Nadine Hartmann La Jolla Institute For Allergy And Immunology, United States André Herbelin INSERM, France Ian Hermans Malaghan Institute of Medical Research, New Zealand Nicholas Hess University of Wisconsin-Madison, United States

Andrew Hogan Maynooth University, Ireland Kristin Hogquist University of Minnesota, United States Shouxiong Huang University of Cincinnati College of Medicine, United States Piotr Humeniuk Medical University of Vienna, Austria Fumie Ihara Chiba University, Japan Jin Im MDACC, United States Naoki Ishimori Hokkaido University Hospital, Japan Kazuya Iwabuchi Kitasato University School of Medicine, Japan Charlotte James University of Washington, United States Ellie-May Jarvis Malaghan Institute of Medical Research, New Zealand Rebeca Jimeno KINGS COLLEGE LONDON, United Kingdom Anna Helena Jonsson Brigham and Women's Hospital, United States Jennifer Juno University of Melbourne, Australia Harry Kane Trinity College Dublin, Ireland Mikael Karlsson Karolinska Institutet, Sweden Jim Kaufman University of Cambridge, United Kingdom Edy Kim Brigham & Women's Hospital, United States Ji Hyung Kim Boston Children's Hospital and Harvard Medical School, United States Paul Klenerman University Of Oxford, United Kingdom Jonas Klingström Karolinska Institutet, Sweden Hui-Fern Koay The University of Melbourne, Australia Ayano Kohlgruber Harvard Medical School, United States

Mitchell Kronenberg La Jolla Institute for Allergy & Immunology, United States Sai Harsha Krovi University of Colorado Denver, United States Paul Kubes University of Calgary, Canada Corinna Kulicke University of Oxford, United Kingdom Vipin Kumar University of California San Diego, United States Kerri Lal Military HIV Research Program, United States Nelson LaMarche Brigham and Women's Hospital / Harvard Medical School, United States Roeland Lameris VU University medical center – Cancer Center Amsterdam, Netherlands Rajesh Lamichhane University of Otago, New Zealand Joshua Lange Malaghan Institute of Medical Research, New Zealand Olivier Lantz Institut curie, France Erik Layton University of Washington, United States Elizabeth Leadbetter UTHSCSA, United States Michael Lee University of Maryland, Baltimore, United States You Jeong Lee IBS / POSTECH, South Korea Edwin Leeansyah Karolinska Institutet, Sweden Francois Legoux Institut Curie, France Agnes Lehuen Institut Cochin, France Marco Lepore Immunocore Ltd, United Kingdom Daniel Leung University of Utah, United States David Lewinsohn OHSU / PVAMC, United States Deborah Lewinsohn Oregon Health & Science University, United States


Joel Linden La Jolla Institute for Allergy and Immunology, United States Bin Liu Baylor college of Medicine, United States Jianyun Liu Indiana University, School of Medicine, United States Amadeu Llebaria IQAC-CSIC, Spain Lydia Lynch Harvard Medical School, United States Fatima Macedo i3S - Instituto de Investigacao e Inovacao em Saude, Porto University, Portugal Elodie Macho-Fernandez Brigham and Women's Hospital-Harvard Medical School, United States Jeffrey Mak University of Queensland, Australia Kimia Maleki Karolinska Institutet, Sweden Thierry Mallevaey University of Toronto, Canada Claudia Mauri Univeristy College London, United Kingdom Jim McCluskey University of Melbourne, Australia Hamish McWilliam The University of Melbourne, Australia Erin Meermeier Oregon Health & Science University, United States Ashanty Melo Trinity College Dublin, Ireland Espen Melum Oslo Universitetssykehus - Rikshospitalet, Norway Leonid Metelitsa Baylor College of Medicine, United States Sachiko Miyake Juntendo University, Japan Gwen Monnot Columbia University Medical Center, United States Branch Moody Brigham and Women's Hospital, Harvard Medical School, United States Lucia Mori University of Basel, Switzerland

Shinichiro Motohashi Chiba University, Japan Goh Murayama JUNTENDO UNIVERSITY SCHOOL OF MEDICINE, TOKYO, JAPAN, Japan Gitanjali Narayanan Oregon Health and Science University, United States Ho Ngai Baylor College of Medicine, United States Angela Nguyen Monash University, Australia Thao Nguyen Baylor College of Medicine, United States Catriona Nguyen-Robertson The Doherty Institute, The University of Melbourne, Australia Yoshikuni Obata Hokkaido University Graduate School of Medicine, Japan Aisling O'Brien National Childrens Research Centre, Ireland Shelby O'Connor University of Wisconsin-Madison, United States Graham Ogg MRC Human Immunology Unit, University of Oxford, United Kingdom Gavin Painter Victoria University of Wellington, New Zealand Tiphaine Parrot Karonlinska Institutet, Sweden Lise Pasquet NCI/NIH, United States Mallory Paynich La Jolla Institute for Allergy and Immunology, United States Daniel Pellicci University of Melbourne, Australia Johanne Pentier Immunocore Ltd, United Kingdom Avi Perna BioLegend, United States Jeanne Perroteau Université de Nantes, France Prabh Phalora University of Oxford, United Kingdom Nicholas Provine University of Oxford, United Kingdom Krystle Quan University of Washington, United States

Josephine Reijneveld Brigham and Women's Hospital, United States Peter Reinink Utrecht University, Netherlands Xavier Rios Texas Children's Hospital, United States Jacques Robert University of Rochester, United States Jamie Rossjohn Monash University, Australia Akimichi Saito Hokkaido University, Japan Mariolina Salio University of Oxford, United Kingdom Marion Salou Institut Curie, France Johan Sandberg Karolinska Institutet, Sweden Derek Sant'Angelo Child Health Institute, Rutgers Robert Wood Johnson Medical School, United States Charles Serhan Brigham and Women's Hospital, Harvard University, United States Chetan Seshadri University of Washington, United States Adam Shahine Monash University, Australia Akshat Sharma University of Wisconsin Madison, United States Siddhartha Sharma The Scripps Research Institute, United States Susannah Shissler University of Maryland, Baltimore, United States Dilip Shrestha Weatherall Institute of Molecular Medicine, University of Oxford, United Kingdom Daimon Simmons Brigham and Women's Hospital, United States Michal Sobkowiak Karolinska Institutet, Sweden Michael Souter Peter Doherty Institute, Australia Nick Stucky OHSU, United States


Sara Suliman Brigham and Women's Hospital, United States Mariko Takami Chiba University, Japan, Japan Masaru Taniguchi RIKEN IMS, Japan Masaki Terabe NIH/NCI, United States Luc Teyton The Scripps Research Institute, United States Ajitha Thanabalasuriar University of Calgary, Canada Maren Thomaier Universität Dresden, Germany Amine Toubal Institut Cochin, France Emmanuel Treiner Inserm, France Abel Trujillo-Ocampo The University of Texas M.D. Anderson Cancer Center, United States Ageliki Tsagaratou La Jolla Institute, United States Juan Umana University of Toronto, Canada James Ussher University of Otago, New Zealand Laura Valestrand Norwegian PSC research center, Norway Peter van den Elzen University of British Columbia, Canada

Hans van der Vliet VU University medical center, Netherlands Ildiko Van Rhijn Brigham and Women's Hospital-Harvard Medical School, United States Koen Venken University Ghent, Belgium Joshua Vieth The Child Health Institute of NJ, Rutgers University, United States Jose Villadangos University of Melbourne, Australia rik von Seth Karolinska Institutet, Sweden Chyung-Ru Wang Northwestern University, United States Haiguang Wang University of Minnesota, United States Huimeng Wang University of Melbourne, Australia Yuting Wang Center for Regenerative Therapies Dresden, Germany Hiroshi Watarai University of Tokyo, Japan Tonya Webb University of Maryland, United States Andreas Weigert Goethe-University Frankfurt, Germany Corinne Williams Journal of Clinical Investigation | JCI Insight, United States Florian Winau Harvard Medical School, United States

Gerhard Wingender Izmir Biomedicine and Genome Center, Turkey Jacinta Wubben Monash University, Australia Takashi Yamamura National Institute of Neuroscience, NCNP, Japan Lili Yang University of California, Los Angeles, United States Seung Hee Yang Seoul National University, South Korea Xuefen Yang Institute of Animal Science, Guangdong Academy of Agricultural Sciences, China Lin-Kin Yong Baylor College of Medicine, United States Weiming Yuan University of Southern California, United States Dirk Zajonc La Jolla Institute for Allergy and Immunology, United States Meng Zhao La Jolla Institute, United States Fei Zheng Norwegian PSC Research Center, Norway Christine Zimmer Karolinska Institutet, Sweden Nicholas Zumwalde University of Wisconsin - Madison, United States


NOTES

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