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16th Annual Upstate New York Immunology Conference Major Corporate Sponsor BD Biosciences Grant Funding NIH National Institute of Allergy and Infectious Diseases Trainee Awards American Association of Immunologists October 20-23, 2013 The Sagamore Resort and Conference Center Bolton Landing, NY

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Page 1: 16th Annual Upstate New York Immunology Conference · immunology. Selection is based on career promise and presentation of an outstanding first-author abstract selected for oral presentation

16th Annual Upstate New York Immunology Conference

Major Corporate Sponsor

BD Biosciences

Grant Funding

NIH National Institute of Allergy and Infectious Diseases

Trainee Awards

American Association of Immunologists

October 20-23, 2013 The Sagamore Resort and Conference Center

Bolton Landing, NY

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UPSTATE NEW YORK IMMUNOLOGY CONFERENCE (NYIC)

We’ve come a long way from Garnet Hill! This meeting started in 1997 as a small retreat to facilitate interactions among young scientists, institutions, and renowned experts in the field of Immunology. In just a few short years, the number of attendees grew and a larger venue was needed to meet the future needs of the Conference.

We are happy to announce the American Association of Immunologists (AAI) is once again providing 20 Young Investigator Awards. Awards will be given to those chosen for Poster Oral Presentations. There will also be two new Workshops. Representa-tives from NIH/NIAID will present information on trainee fellowships and research awards. Dr. Pamela Fink will discuss the intricacies of publishing scientific papers.

As part of our leisure activities, there will be a cruise on Lake George, as well as a recreational night including a bonfire, miniature golf, WII, X-box, wiffle ball, and movies! Once again there will be an opportunity to win one of two iPads. The raffle is open to all trainees. You must be present at the drawing to win!

While all these elements lend to the atmosphere, one simple principle of this Conference re-mains: To provide an opportunity for young and senior scientists to gather in a setting that is di-verse enough to meet the needs of all attendees while remaining small enough to allow for per-sonal interactions. While always challenging, it is the goal of the NYIC Scientific Advisory Board and the NYIC Conference Organizers to give graduate students and postdoctoral fellows the oppor-tunity to present their research and engage in conversations that will stimulate further discus-sions, collaborations and interest in pursuing a new or different way of looking at their research.

We hope you share our enthusiasm and enjoy your time with us!

THE SAGAMORE RESORT

The Sagamore Resort and Conference Center celebrated it’s 125 year anniversary in 2008. Since that time, the resort has gone through some remarkable renovations. If this is your first visit to the Resort, take some time to enjoy the beauty that surrounds you. There are many breath-taking views to be seen.

The staff are friendly, courteous, and hard-working. If you require any information or have a special need, please see either Dawn Bellville, Administrative Coordinator for NYIC, or any of the Resort personnel. Many thanks to Lori Rehm (Director of Sales), Derrick Hammond (Conference Services Manager), Don Vilmar (Banquet Manager), Jerid McKinney (Banquet Captain), Joel Clark (Function Set-up Manager) and his amazing crew, Dave Reynolds (CMI Communications-Audio/Visual Manager) and his crew, Glen and Al, along with all of the associates who attend to our many needs. Thank you!

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Conference and Venue ......................................................................................... 3

Schedule of Events .............................................................................................. 6

Platinum Corporate Sponsor

BD Biosciences ................................................................................................ 16

Gold Corporate Sponsor

Life Technologies ............................................................................................ 18

Enhanced Silver Corporate Sponsor

EMD Millipore .................................................................................................. 20

Silver Corporate Sponsors

BioLegend, Inc. ................................................................................................ 23

eBioscience ..................................................................................................... 24

Krackeler Scientific .......................................................................................... 25

Lonza Pharma .................................................................................................. 26

Bronze Corporate Sponsors

DeNovo Software ............................................................................................. 27

Special Sponsor

American Association of Immunologists ....................................................... 28

NYIC Scientific Advisory Board ............................................................................ 30

Institutional Financial Supporters ....................................................................... 31

Grant Support ....................................................................................................... 32

Keynote Speaker

Pamela J. Fink, Ph.D. ...................................................................................... 33

Symposium I

Gut Immunity ................................................................................................... 34

Table of Contents

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Symposium II

Regulation of T cell Immunity I ....................................................................... 38

Workshop I - NIH Presentation

“NIH Training and Career Development Programs” ...................................... 41

Poster Talks

Macrophage/Monocyte Immunobiology ........................................................ 42

Adaptive Immunology ...................................................................................... 48

Cancer Immunobiology ................................................................................... 54

Immunity and Disease .................................................................................... 60

Platinum Sponsor Presentation

BD Biosciences ................................................................................................ 66

Poster Abstracts ................................................................................................... 67

Symposium III

Regulation of T cell Immunity II ....................................................................... 132

Symposium IV

Immune Dysfunction ....................................................................................... 138

Workshop - II

“Do’s and Don’ts When Writing and Publishing a Scientific Manuscript” .. 142

Keynote Speaker

Christopher A. Hunter, Ph.D. ........................................................................... 143

Symposium V

Immunomodulation ......................................................................................... 144

Symposium VI

Antigen Presenting Cell Biology ...................................................................... 148

Attendee Contact Information ............................................................................. 152

Author Index .......................................................................................................... 159

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Upstate New York Immunology Conference Schedule of Events

Sunday, October 20th

3:00-6:00 p.m. Hotel Check-in (Main Hotel Lobby) 4:00-5:30 p.m. Conference Check-in (Conference Center Lobby) 5:30-6:30 p.m. Free Time 6:30-7:00 p.m. Dinner (Bellvue) 7:00-8:00 p.m. Keynote Presentation (Bellvue) Introduction: Jim Drake, Ph.D. Pamela J. Fink, Ph.D. Professor of Immunology Department of Immunology University of Washington “Post-thymic T Cell Maturation” 8:00 p.m. AAI Presentations & Photos Award Presenters:

Dr. Jennifer Meyers (AAI) Dr. Christopher Hunter Dr. Edith Lord Dr. Jonathan Harton Welcome Reception (Cash Bar for mixed drinks) Immediately Following Awards Presentation (Conference Center Foyer)

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Monday, October 21st 8:00-9: 00 a.m. Continental Breakfast (Triuna) 9:00-10:30 a.m. Symposium I: Mucosal Immunity (Nirvana) Chair: Dr. Christopher A. Hunter 9:00-9:30 Markus Mohrs, Ph.D. (Trudeau Institute) “Immunity to Infection with Gastrointestinal Helminths” 9:30-10:00 Stela Celaj, B.S. (Dartmouth) “Gut Microbiota Modulates Susceptibility to Liver Injury” 10:00-10:30 Dennis W. Metzger, Ph.D. (Albany Medical College) “Infections at Mucosal Sites: Viral-Bacterial Co-Infections in the Respiratory Tract”

10:30-10:45 a.m. Break (Conference Center Foyer) 10:45-11:45 a.m. Symposium II: Regulation of T cell Immunity I (Nirvana) Chair: Dr. Margaret Bynoe 10:45-11:15 Vandana Kalia, Ph.D. (Pennsylvania State University) “MicroRNA Regulation of T cell Memory” 11:15-11:45 Dorina Avram, Ph.D. (Albany Medical College) “Increased Plasticity of Th17 cells by Expression of Th2 Cytokines Causes Diverted Trafficking and Amelioration of Autoimmune Encephalitis” 11:45-12:15 p.m. Lunch Buffet (Conference Center Foyer)

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12:15-2:00 p.m. NIH Presentation (Wapanak) Introduction by: Dr. Dennis W. Metzger Lawrence J. Prograis, Jr., M.D. Katrin Eichelberg, M.Sc., Ph.D. “NIH Training and Career Development Programs: Grantsmanship, Funding, and Peer Review” 2:00-2:15 p.m. Display Posters (Bellvue) 2:15-3:30 p.m. Poster Talks: Macrophage/Monocyte Immunobiology (Albenia) Chair: Drs. Girish Kirimanjeswara and Kate MacNamara 2:15-2:30 Sara B. Cohen, B.A. (Cornell University) “CXCR3-Dependent CD4+ Th1 cells Activate Inflammatory Monocytes for Defense against Toxoplasma gondii Infection” (#28) 2:30-2:45 Amanda McCabe, B.S. (Albany Medical College) “IFNγ Limits Hematopoietic Stem and Progenitor Cell Mobilization during an Intracellular Bacterial Infection by Maintaining BM-resident Mφs” (#27) 2:45-3:00 David Williamson, B.S. (Pennsylvania State University) “A Novel Macrophage Entry and Adhesion Protein (Meap) Contributes to Francisella tularensis Live Vaccine Strain Invasion of Host Macrophages” (#39) 3:00-3:15 Christopher S. Anderson, B.S. (University of Rochester) “Identification of a Core Molecular Pathway Required for Phagocyte Migration to Apoptotic Cells” (#52) 3:15-3:30 Colleen Netherby, B.S. (Roswell Park Cancer Institute) “Myeloid IRF-8 Levels Regulate Tumor-Induced Aberrant Myelopoiesis and Immune Surveillance in the Metastatic

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Microenvironment” (#17) 2:15-3:30 p.m. Poster Talks: Adaptive Immunity (Evelley) Chair: Drs. Jim Drake and Deborah Fowell 2:15-2:30 Heidi R. Tucker, B.S. (Albany Medical College) “The Pathway of Antigen Processing Regulates MHC Class II Signaling and Determines B cell Fate” (#47)

2:30-2:45 Alison Gaylo, M.S. (University of Rochester) “The Inflamed Microenvironment Influences CD4+ T cell Interstitial Motility” (#31) 2:45-3:00 Gonzalo de la Rosa*, Ph.D. (University of Rochester) “Essential Role of Elmo1 in T lymphocyte Chemotaxis” (#51) 3:00-3:15 Tara Capece, M.S. (University of Rochester) “Regulation of Integrin LFA-1 during T cell Migration and Activation” (#4)

3:15-3:30 Haley Spangler, B.S. (Roswell Park Cancer Institute) “Dendritic Cell Differentiation: Wyatt IRF and the Shootout at the PKC Corral” (#10)

*Presenting for Catherine Stevenson 3:30-3:45 p.m. Break (Conference Center Foyer) 3:45-5:00 p.m. Poster Talks: Cancer Immunobiology (Albenia) Chair: Dr. Yasmin Thanavala 3:45-4:00 Amy Ku, B.S. (Roswell Park Cancer Center) “Myeloid-Derived Suppressor Cells Negatively Impact Trafficking of CD8 Effector T cells in the Tumor Microenvironment” (#56)

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4:00-4:15 Weishan Huang, M.S. (Cornell University) “ITK Regulates the Dynamics and Anti-tumor Immunity Development during CD8+ T cell Homeostatic Proliferation” (#21) 4:15-4:30 Shawn Egan, B.S. (Roswell Park Cancer Institute)

“MicroRNA30e*—Novel Regulator of Prostate Cancer Growth” (#9)

4:30-4:45 Kathleen M. Kokolus, B.S. (Roswell Park Cancer Institute)

“How is Anti-Tumor Immunity Impacted by Metabolism in Mice Housed at Sub-Thermoneutral Temperature?” (#14)

4:45-5:00 Stephanie Sass, B.S. (Roswell Park Cancer Institute)

“Role of Myeloid Cells in Prostate Cancer Progression” (#8) 3:45-5:00 p.m. Poster Talks: Immunity and Disease (Evelley) Chair: Drs. Paige Lawrence and Chris Norbury 3:45-4:00 Michael Davies, Ph.D. (Penn State College of Medicine) “MyD88-Dependent Immunity to a Natural Model of Vaccinia

Virus Infection Does not Involve Toll-Like Receptor 2 (TLR2)” (#38)

4:00-4:15 Megan Peppenelli, B.S. (SUNY Upstate Medical University) “Monocytes Infected by HCMV are Reprogrammed from Short-

lived Monocytes to Long-lived Macrophages” (#2) 4:15-4:30 Justin B. Callaway, B.S. (University of North Carolina) “Dengue-viral Source and Purity Determine Differential

Infection Requirements for Secretion of IL-1β by Primary Monocytes” (#1)

4:30-4:45 Gregory Berry, B.S. (Penn State College of Medicine)

“The Type 1 Diabetes Resistance Locus B10 Idd9.3 Impairs the Development of Mature B cells in a Mouse Model of Human

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Diabetes” (#15) 4:45-5:00 Bethany Winans, M.S. (University of Rochester)

“Early life AhR Signaling Leads to Altered DNA Methylation in CD8+ T cells” (#3)

5:30-6:00 p.m. Dinner (Wapanak) 6:00-6:30 p.m. Platinum Sponsor Presentation (Wapanak) Introduction by: Kate MacNamara, Ph.D. Erik Puffer, Ph.D. BD Biosciences “New Tools for Multicolor Flow Cytometry from BD Biosciences” 6:30-9:30 p.m. Poster/Vendor Mixer** (Bellvue) 6:45-7:45 Poster Viewing (Odd Numbers) 8:00-9:00 Poster Viewing (Even Numbers) **iPad drawing during this event Tuesday, October 22nd 7:30-8:30 a.m. Continental Breakfast (Albenia/Evelley) 8:30-10:00 a.m. Symposium III: Regulation of T cell Immunity II (Nirvana) Chair: Dr. Pamela Fink 8:30-9:00 Jacques Robert, Ph.D. (University of Rochester) “Evolutionary Conserved Role of Non-classical MHC in Invariant

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T cell Biology” 9:00-9:30 Brian Rudd, Ph.D. (Cornell University) “Alterations to Memory CD8+T cell Formation in Early Life” 10:00-10:30 Deborah J. Fowell, Ph.D. (University of Rochester)

“Dynamic Regulation of T cell Motility at Sites of Inflamed Tissues”

10:00-10:15 a.m. Break (Conference Foyer) 10:15-11:45 a.m. Symposium IV: Immune Dysfunction (Wapanak) Chair: Dr. James Gorham 10:15-10:45 Kirsi Jarvinen-Seppo, M.D., Ph.D. (Albany Medical College) “Maternal Influence on Development of Allergies in the

Infant” 10:45-11:15 Edward Doherty, B.S. (SUNY Upstate Medical University)

“Mitochondrial Overdrive: The Metabolic Dysfunction of SLE PBL, and the Effect of NAC Treatment”

11:15-11:45 Pamela Hankey, Ph.D. (Pennsylvania State University)

“The Ron Receptor Tyrosine Kinase, Macrophage Heteroge-neity and Obesity Associated Disease”

11:45-12:15 p.m. Lunch Buffet (Wapanak) 12:15-1:15 p.m. Scientific Writing Presentation (Wapanak) Introduction by: Dr. James Drake Pamela J. Fink, Ph.D. “Do’s and Don’ts When Writing and Publishing a Scientific Manuscript”

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1:30 p.m. Meet at Dock 1:45-3:15 p.m. Cruise aboard The Morgan 3:15-4:30 p.m. Free Time 4:30-5:30 p.m. Keynote Presentation (Nirvana) Introduction: Dr. Dennis Metzger Christopher A. Hunter, Ph.D. Professor and Chairman Department of Pathobiology University of Pennsylvania “Imaging the Immune Response to Infection” 5:30-6:30 p.m. Vendor Demonstrations (Bellvue) Colleague Collaborations 6:30-7:15 p.m. Dinner (Wapanak) 7:30-10:00 p.m. Bonfire and Rec Center Activities (Rec Center) (Cash Bar – Refreshments Provided) Wednesday, October 23rd 8:00-9:00 a.m. Continental Breakfast (Triuna) 9:00-10:30 a.m. Symposium V: Immunomodulation

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(Nirvana) Chair: Dr. Nicholas Mantis 9:00-9:30 Yasmin Thanavala, Ph.D. (Roswell Park Cancer Institute)

“High Immunosuppressive Burden in Advanced Hepatocellular Carcinoma Patients: Can Effector Functions Be Restored?”

9:30-10:00 Zachary M. Parker, Ph.D. (Dartmouth College) “STING is Dispensible for Protection of Mice from HSV-1

Encephalitis Following Corneal Challenge” 10:00-10:30 Elizabeth A. Repasky, Ph.D. (Roswell Park Cancer Institute)

“Leaving Immunosuppression Out in the Cold: New Insights into the Role of Thermoregulatory Metabolism in the Anti-Tumor Immune Response”

10:30-11:00 a.m. Beverage Break & Hotel Check-out (Conference Center Foyer) 11:00-12:00 p.m. Symposium VI: Antigen Presenting Cell Biology (Nirvana) Chair: Dr. James Drake 11:00-11:30 Nicholas J. Mantis, Ph.D. (Wadsworth Center)

“Dynamics of Antigen Sampling by Peyer’s patch Dendritic Cells”

11:30-12:00 Chris C. Norbury, Ph.D. (Penn State College of Medicine)

“Sensing a REAL Virus - Trafficking of Poxviral DNA and the Innate Immune Sensor TLR9”

12:00-12:15 p.m. Closing Remarks** 12:30-2:00 p.m. Lunch & Scientific Advisory Board Meeting (Shelving Rock) **iPad drawing during this event

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NYIC 2012

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Platinum Corporate Sponsor

BD Biosciences

Page 17: 16th Annual Upstate New York Immunology Conference · immunology. Selection is based on career promise and presentation of an outstanding first-author abstract selected for oral presentation

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Gold Corporate Sponsor

Life Technologies

Page 19: 16th Annual Upstate New York Immunology Conference · immunology. Selection is based on career promise and presentation of an outstanding first-author abstract selected for oral presentation

Find out more at lifetechnologies.com

For Research Use Only. Not for use in diagnostic procedures. ©2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. SB10370913

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Page 20: 16th Annual Upstate New York Immunology Conference · immunology. Selection is based on career promise and presentation of an outstanding first-author abstract selected for oral presentation

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Enhanced Silver Corporate Sponsor

EMD Millipore

Page 21: 16th Annual Upstate New York Immunology Conference · immunology. Selection is based on career promise and presentation of an outstanding first-author abstract selected for oral presentation
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Silver Corporate Sponsor

BioLegend, Inc.

Affymetrix/eBioscience

Krackeler Scientific, Inc.

Lonza Pharma - Bioscience Solutions

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Page 24: 16th Annual Upstate New York Immunology Conference · immunology. Selection is based on career promise and presentation of an outstanding first-author abstract selected for oral presentation
Page 25: 16th Annual Upstate New York Immunology Conference · immunology. Selection is based on career promise and presentation of an outstanding first-author abstract selected for oral presentation

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Bronze Corporate Sponsor

DeNovo Software

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In recognition of the significance of this meeting

and work being done by Graduate Students

and Postdoctoral Fellows the

American Association Of Immunologists

has provided Young Investigator Awards

to twenty attendees. Each will receive a money award,

as well as the opportunity to present their research both in poster

format and in brief talks.

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The 2014 AAI Awards will be presented in conjunction with

IMMUNOLOGY 2014™ • May 2–6, 2014 • Pittsburgh, PennsylvaniaQuestions? Contact AAI at 301-634-7178 or [email protected]

For complete AAI Travel Award and Grant application details, visit www.AAI.org/Awards.

Call for 2014 Award ApplicationsDeadline: January 9, 2014

Lustgarten-eBioscience Memorial AwardEstablished to honor the memory of AAI member Dr. Joseph Lustgarten, this award is intended to advance the career of a mid-career scientist who attends the AAI annual meeting and presents an outstanding abstract specifically in the area of immune regulation. The award recipient will receive up to $1,250 travel reimbursement and a certificate during an awards presentation program at the AAI annual meeting. This award is generously supported through a grant from eBioscience, An Affymetrix Company.

AAI Minority Scientist Travel AwardThis award provides travel support to eligible AAI members to attend the AAI annual meeting. Two types of awards are available (trainee, junior faculty), providing support of up to $1,850 for registration and meeting-related travel expenses. This award is generously supported through the FASEB Minority Access to Research Careers (MARC) program and a grant from the National Institute of General Medical Sciences (NIGMS), NIH.

AAI Trainee Abstract AwardThis award provides up to $500–750 travel reimbursement to AAI trainee members (students and postdoctoral fellows) whose first-author abstracts submitted to the AAI annual meeting are selected for presentation in block symposia.

AAI Undergraduate Faculty Travel GrantThese grants assist undergraduate faculty in attending the AAI annual meeting. Each grant will also support travel costs for an undergraduate student of the recipient’s selection. A grant of up to $1,250 is awarded to the undergraduate faculty member, and a grant of up to $1,000 is awarded to the selected undergraduate student (registration for an undergraduate student is complimentary).

AAI Laboratory Travel GrantThese grants assist mid-career investigators in attending the AAI annual meeting. Applicants must hold an appointment of associate professor or equivalent, have limited support for travel (total funding not to exceed $300,000 per year), and be a first or last author on one or more abstracts submitted to the annual meeting. Each grant will provide two travel awards of up to $1,250 each: one to the PI or laboratory director and another to a member of his or her lab, chosen by the PI or laboratory di-rector. Recipients will be reimbursed for registration and travel expenses.

AAI Trainee Poster AwardThis award provides up to $300 travel reimbursement to AAI trainee members (students and postdoctoral fellows) whose first-author abstracts submitted to the AAI annual meeting are selected for poster sessions only and found to be exceptional by the AAI Abstract Programming Chairs. Selection is based on the originality and significance of the research being presented.

Pfizer-Showell Travel AwardThis award recognizes the professional promise of an early career investigator (assistant professor or equivalent) by assisting the award recipient with travel to the AAI annual meeting. Selection is based on career progress and submission of an outstanding abstract selected for oral presentation in a block symposium at the meeting. The award recipient will be recognized and presented with a certificate at an awards presentation program at the AAI annual meeting. Support of up to $1,500 will be provided for meeting registration and travel. This award is sup-ported through an endowment from Henry J. Showell and Pfizer, Inc.

AAI-Life Technologies Trainee Achievement AwardThis award recognizes up to 6 promising trainees in the field of immunology. Selection is based on career promise and presentation of an outstanding first-author abstract selected for oral presentation in a block symposium. Awardees will receive a $1,000 cash prize and reimbursement for meeting expenses. This award is generously supported through a grant from Life Technologies Corporation.

AAI Early Career Faculty Travel GrantThese grants assist young investigators (assistant professor or equivalent) in attending the AAI annual meeting. Recipients will be reimbursed up to $1,250 for registration and travel expenses.

Chambers-eBioscience Memorial AwardEstablished to honor the memory of AAI member Dr. Cynthia Chambers, this award is intended to advance the career of an early career scientist who attends the AAI annual meeting and presents an outstanding abstract specifically in the area of cancer biology. The award recipient will receive a $1,000 cash award and a certificate during an awards pre-sentation program at the AAI annual meeting. This award is generously supported through a grant from eBioscience, An Affymetrix Company.

Applications are invited for the following AAI Travel Awards and Grants, which annually foster the promise and professional development of early- and mid-career investigators, including underrepresented minority scientists and trainees.

Imm2014_CFAwardApp_2.indd 1 8/27/13 1:58 PM

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NYIC Scientific Advisory Board Institutional Representatives

Albany Medical College

Jim Drake and Kate MacNamara (NYIC Conference Organizers)

Cornell University Margaret Bynoe

Dartmouth College

Brent Berwin

Pennsylvania State University Vandana Kalia and Surojit Sarkar

Roswell Park Cancer Institute

Yasmin Thanavala

SUNY Upstate Medical University Michael Princiotta

Trudeau Institute

Andrea Cooper and Steve Smiley

University at Buffalo Nejat Egilmez

University of Rochester Medical Center

Edith Lord

Wadsworth Center Nicholas Mantis and William Lee

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Institutional Financial Supporters

Albany Medical College Alumni Association

Cornell University

Department of Microbiology & Immunology

Dartmouth College Department of Microbiology & Immunology

Pennsylvania State University

The Huck Institutes of the Life Sciences

Roswell Park Cancer Institute Department of Immunology

SUNY Upstate Medical University

Microbiology & Immunology Program

Trudeau Institute

University at Buffalo Department of Microbiology & Immunology

University of Rochester Medical Center

Department of Microbiology & Immunology

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Grant support provided to

Graduate Students

and

Postdoctoral Fellows

by the

National Institutes of Health

National Institute of Allergy and Infectious Diseases

R13AI051522

“Thank You”

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Keynote Speaker

Pamela J. Fink, Ph.D. Professor of Immunology

Department of Immunology University of Washington

Seattle, Washington

“Post-thymic T Cell Maturation”

Recent thymic emigrants (RTEs) comprise the subset of peripheral T cells that have most recently undergone thymic maturation and egress to the lymphoid periphery. RTEs are an important lymphocyte population to study because they represent the major-ity of T cells in neonates and in adults recovering from lymphoablation, and contribute to T cell diversity throughout life. Using a transgenic mouse model (RAG2p-GFP Tg) in which GFP expression is driven by the RAG2 promoter, RTEs can be readily identified within the lymphoid periphery by their residual GFP signal and isolated from their mature but still na-ïve (MN) T cell counterparts for phenotypic and functional analyses. Using this model, our lab has discovered that T cell development, once thought to be completed within the thymus, continues for several weeks after cells have entered the lymphoid periphery. OT-I TCR Tg CD8 RTEs that recognize an ovalbumin (OVA) peptide formed fewer memory precursor effector cells (MPECs) and secreted less IFN-γ and IL-2 than mature T cells following infection with OVA-expressing Listeria monocytogenes. To investigate the response of CD8 RTEs to ligands of varying affinity, we infected mice with Listeria engineered to express OVA containing altered peptide ligands (Lm-ovaAPL), fol-lowing transfer of sorted RTE and MN CD8 OT-I TCR Tg T cells. Surprisingly, RTEs had higher expression of the adhesion molecules VLA-4 and Ly6C and were more prone to invade inflamed tissue than their mature counterparts, particularly in response to lower affinity APLs. These data suggest that, compared to mature T cells, RTEs show dimin-ished function, but are less sensitive to reduced ligand affinity and efficiently invade tis-sues. We have used a diabetes model system to test the notion that RTEs are invasive but more prone to tolerance induction than mature T cells. OT-I Tg CD8 RTEs and MN T cells were co-transferred into mice expressing OVA in the pancreatic islets (RIP-mOVA Tg mice). RTEs proliferated less and secreted less IL-2 and IFN-g than their mature coun-terparts. RTEs also over-expressed some anergy-associated genes. Finally, OT-I RTEs were significantly less efficient at inducing diabetes than mature T cells when recipients were infected with Lm-OVA. These results further emphasize the functional differences between RTE and MN T cells, and support our hypothesis that post-thymic maturation represents a period of heightened susceptibility to tolerance induction.

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Symposium I

Mucosal Immunity

Chair Dr. Christopher Hunter

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Immunity to Infection with Gastrointestinal Helminths

Markus Mohrs Trudeau Institute, Saranac Lake, NY

Interleukin-10 (IL-10) is a regulatory cytokine involved in curtailing immune responses to pre-vent immune mediated tissue damage. Its importance is apparent during infection with the enteric helminth, Trichuris muris, which is spontaneously expelled in WT C57BL6 mice, but in IL-10-deficient mice leads to significant morbidity and mortality. To identify the cellular source(s) of IL-10 at the site of T. muris infection, the cecum, we used eGFP/IL-10 reporter mice.. After infection, we ob-served a significant increase in GFP expression in cecum intraepithelial lymphocytes (cIELs), but not in lamina propria lymphocytes. Surprisingly, γδ T cells comprised the largest portion of GFP+ IELs and were the most abundant GFP+ IELs. While the induction of IL-10in γδ T cells is IL-27Rα depend-ent, direct IL-27Rα signaling is dispensable. Instead, we demonstrate that infection-induced IFN-γ pro-duction by αβ T cells in the cecum is IL-27Rα dependent. Furthermore, neutralization of IFN-γ during infection prevented IL-10 expression in γδ T cells, indicating a novel role for γδ T cells in regulating intestinal Th1 responses.

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Gut microbiota modulates susceptibility to liver injury

Stela Celaj, Michael W. Gleeson, Jie Deng and James D. Gorham

Dept. of Microbiology and Immunology, Geisel School of Medicine at Dartmouth

Inflammatory liver disease (ILD), stemming from a range of etiologies such as viral, drug-induced, alcoholic, autoimmune etc., is a major health concern worldwide. The rate of progression of ILDs is highly variable and the factors dictating the outcome remain poorly identified. Although envi-ronmental triggers are known to play a major role in shaping disease course, the exact mechanisms have not been completely elucidated. Therefore, variability in disease progression remains one of the most challenging aspects of the effective management of patients with ILD and identifying factors that regulate liver inflammation and injury is critical to understanding how and why some patients progress rapidly.

We show that resident gut microbiota is a major regulator of hepatic injury during Concana-valin A and Fas-mediated liver damage. First, inflammation was induced in BALB/c mice through ad-ministration of Con A, and mice from different vendors (Jackson Labs (JAX); Taconic (TAC)) exhib-ited different levels of liver damage following Con A: JAX mice were highly sensitive and TAC mice were more resistant. Analysis of 1400 SNPs genome-wide showed that JAX and TAC BALB/c mice were genetically indistinguishable. Assessment of fecal microbiota using 16S rRNA deep sequencing showed distinct microbial populations in JAX mice and TAC mice. Importantly, sensitivity to Con A could be transferred between mice following co-housing.

Since Concanavalin A has been shown to require Fas/Fas ligand system we investigated the role of microbiota in regulating Fas activation. Interestingly, JAX mice were much more sensitive than TAC mice to liver damage induced by injection of the Fas activating mAb Jo-2, which induces apop-tosis directly by activating Fas on hepatocytes.

Treatment of JAX mice with oral antibiotics greatly reduced Jo-2 induced liver injury. Thus, the micro-biota by acting at the level of the hepatocyte, serves as a rheostat to modulate the hepatocellular re-sponse to cell death.

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Infections at Mucosal Sites: Viral-Bacterial Co-Infections in the Respiratory Tract

Dennis W. Metzger, Ph.D.

Center for Immunology and Microbial Disease

Albany Medical College

Secondary pulmonary infections by encapsulated bacteria including Streptococcus pneumoniae

and Staphylococcus aureus following influenza represent a common and challenging clinical problem. The reasons for this polymicrobial synergy are still not completely understood, hampering development of effective prophylactic and therapeutic interventions. While it has been commonly thought that viral-induced epithelial cell damage allows bacterial invasiveness, recent studies have now implicated dys-functional innate immune defenses following influenza as the primary culprit for enhanced susceptibil-ity to secondary bacterial infections. In particular, it appears that an elicited adaptive immune response against viral infection (an intracellular pathogen) impairs innate immune defenses against bacterial in-fection (an extracellular pathogen). This new paradigm should ultimately allow development of novel immune intervention strategies for the broad-spectrum prevention and management of secondary bacte-rial infections following influenza. .

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Symposium II

Regulation of T cell Immunity I

Chair

Dr. Margaret Bynoe

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MicroRNA Regulation of T cell Memory

Arif A. KhanF, Laura A. PennyF, Florian BaumannF, Yevgeniy YuzefpolskiyF, Surojit SarkarF,H and Vandana KaliaF,H

FCenter for Molecular Immunology and Infectious Diseases, Department of Veterinary and Biomedical Sciences and The Huck Institutes of Life Sciences, The Pennsylvania State University, University Park,

PA16802; HCorresponding authors

MicroRNAs (miRNAs) are small RNAs (~22 nucleotide, nt) that regulate gene expression by inhibiting protein translation or by degrading the mRNA transcript. This highly evolutionarily con-served miRNA pathway is emerging as a major player in gene regulation during immune cell develop-ment, differentiation and function, by controlling a diverse array of biological processes such as cell proliferation, apoptosis and signaling. T cell activation and proliferation, the very first steps in induc-tion of adaptive immune responses to pathogens, are dysregulated in the absence of the RNase-III en-zyme Dicer, which is required for mature miRNA production. However, the precise microRNAs and their target cellular processes involved in generation of durable T-cell immunity remain undefined. Our studies show a dynamic regulation of microRNAs as CD8 T-cells differentiate from naïve to effector and memory states, with short-lived effectors transiently expressing higher levels of oncogenic miR-17~92 compared to the relatively less proliferating memory-fated effectors. Conditional CD8 T-cell-intrinsic gain or loss of expression of miR-17~92 in mature cells following activation resulted in strik-ing reciprocal effects compared to wild-type counterparts in the same infection milieu – miR-17~92 deletion resulted in lesser proliferation of antigen-specific cells during primary expansion, while favor-ing enhanced IL-7Rα and Bcl-2 expression and multi-cytokine polyfunctionality; in contrast, constitu-tive expression of miR-17~92 promoted terminal effector differentiation, with decreased formation of polyfunctional lymphoid memory cells. Increased proliferation upon miR-17~92 overexpression corre-lated with decreased expression of tumor suppressor PTEN and increased PI3K-AKT-mTOR signaling. Thus, these studies identify miR17~92 as a critical regulator of CD8 T-cell expansion and effector and memory lineages in the physiologically relevant context of acute infection, and present miR-17~92 as a potential target for modulating immunological outcome following vaccination or immunotherapeutic treatments of cancer, chronic infections or autoimmune disorders.

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Increased plasticity of Th17 cells by expression of Th2 cytokines causes diverted trafficking and amelioration of autoimmune encephalitis

Danielle Califano and Dorina Avram

Center for Immunology and Microbial Disease, Albany Medical Center, 47 New Scotland Avenue, MC-151, Albany, New York 12208, USA

Recent evidence suggests that T-helper subsets, though differentiated, possess plasticity. Th17 cells have been shown to express Th1 cytokines in specific conditions, however nothing is known about their plasticity toward Th2 lineage. The transcription factor Bcl11b is expressed in mature T-helper cells, but its role in these cells has not been defined. We report that Bcl11b deficiency in Th17-mediated experimental autoimmune encephalomyelitis (EAE) caused increased plasticity through loss of Gata3 repression, concomitant with IL-4 production, with no impact on RORgt, IL-17 and GM-CSF expression. We further demonstrate that IL-4 signaling in concert with GM-CSF had a major impact on T-helper cell trafficking, diverting their migration from the draining lymph nodes-central nervous sys-tem (CNS) to the mesenteric lymph nodes-gut, thereby ameliorating EAE. Mechanistic studies demon-strate that IL-4 in concert with GM-CSF induced production of retinoic acid by dendritic cells, fol-lowed by gut imprinting on T-helper cells and their diverted migration from the draining lymph nodes-CNS to the mesenteric lymph nodes-gut. Similarly, treatment of EAE wild type mice in Th2 conditions ameliorated the disease, without causing alterations in Th17 cytokines, however resulting in diverted trafficking from the draining lymph nodes-CNS to the mesenteric lymph nodes-gut, which opens ave-nues for multiple sclerosis treatment through diverted migration. Thus increased IL-4 production dur-ing EAE does not strictly affect RORgt and Th17 lineage-specific cytokines, but rather trafficking, demonstrating that increased plasticity of T helper lineage affects trafficking, another critical compo-nent of the immune response.

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NIH Presentation

“NIH Training and Career Development Programs: Grantsmanship, Funding, and Peer Review”

Lawrence J. Prograis, Jr., M.D.

Katrin Eichelberg, M.Sc., Ph.D.

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Poster Talks (Albenia)

Macrophage/Monocyte Immunobiology

Chairs: Dr. Girish Kirimanjeswara

Dr. Kate MacNamara

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#28 CXCR3-Dependent CD4+ Th1 cells Activate Inflammatory Monocytes

for Defense Against Toxoplasma gondii Infection

Sara B. Cohen1, Kirk J. Maurer1, Charlotte E. Egan1, Steve Oghumu2, Abhay R. Satoskar2, and Eric Y. Denkers1

1Cornell University, Ithaca, NY, 2The Ohio State University, Columbus, OH

Chemokines and their receptors orchestrate immunity to microbial pathogens, including the orally acquired Th1-inducing protozoan parasite Toxoplasma gondii. Chemokine receptor CXCR3 is associated with Th1 responses, and we use bicistronic CXCR3-eGFP knock-in reporter mice to demon-strate upregulation of this chemokine receptor on T lymphocytes, but not natural killer (NK) cells, dur-ing Toxoplasma infection. We show a critical role for CXCR3 in resistance to the parasite in the intesti-nal mucosa. Absence of the receptor in Cxcr3-/- mice resulted in selective loss of ability to control T. gondii in the lamina propria compartment. CD4+ T cells and CD11b+Ly6C/G+ inflammatory mono-cytes, recently reported as major anti-Toxoplasma effectors in the intestine, were both recruited to the intestine during infection, but only CD4+ T cells were impaired in recruitment in the absence of CXCR3. Furthermore, intestinal Cxcr3-/- CD4+ T cells, but not CD8+ T cells or NK cells, were impaired in their ability to secrete IFN-g (gamma) upon stimulation. While local recruitment of inflammatory monocytes was not impacted by loss of CXCR3, inflammatory monocyte activation status, as measured by dual production of TNF-a (alpha) and IL-12, was severely impaired in Cxcr3-/- mice. Strikingly, adoptive transfer of wild-type but not Ifng(gamma)-/- CD4+ T lymphocytes into Cxcr3-/- animals prior to infection corrected the defect in inflammatory macrophage activation, simultaneously reversing the susceptibility phenotype of the knockout animals. Our results establish a central role for CXCR3 in co-ordinating innate and adaptive immunity, ensuring generation of Th1 effectors and their trafficking to the frontline of infection to program microbial killing by inflammatory monocytes.

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#27 IFNg limits hematopoietic stem and progenitor cell mobilization during an intracellular bacterial

infection by maintaining BM-resident Mφs

Amanda McCabe, Yubin Zhang, Maura Jones, Kenny Thai, Katherine MacNamara

Albany Medical College, Albany NY

The primary site of hematopoiesis is the bone marrow (BM), however hematopoietic stem and

progenitor cells (HSPCs) can be released from the BM and into the circulation in a process known as mobilization. While it is thought that mobilization may impact immunity and host defense, very little is known about how mobilization is regulated under infectious conditions. We observed that during infec-tion with an intracellular bacterial pathogen HSPC mobilization was impaired by interferon gamma (IFNg). In IFNgR-deficient mice, blood and spleen HSPCs were significantly increased relative to wildtype infected mice. As bone marrow (BM)-resident macrophages (Mfs) preserve stromal cell func-tion, and thus retain HSPCs in the BM, we next investigated Mf function within the BM. An IFNg-dependent increase in frequency and number of highly phagocytic BM-resident Mfs was observed dur-ing infection. The increase in BM-resident Mfs correlated with increased mesenchymal stem cells (MSC), key niche cells that produce HSPC retention factors. IFNg signaling specifically in Mfs was required for increased MSCs and the impairment of HSPC mobilization. These data demonstrate that infection-induced IFNg limits HSPC mobilization by supporting niche cell function and HSPC reten-tion in the BM, which may be critical for the appropriate immune response to infection. Moreover, the observation that IFNg maintains BM HSPC niches has direct implications for clinically relevant proce-dures including HSPC transplantation.

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#39 A novel macrophage entry and adhesion protein (Meap) contributes to Francisella tularensis Live

Vaccine Strain invasion of host macrophages

David Williamson1,2, Kalyan Dewan1, Rachel Markley1,2, David Place1,2, Vicky Avanzato, Bhuvana Katkere1, Girish Kirimanjeswara1

1The Dept. of Veterinary and Biomedical Science, 2Immunology and Infectious Disease Graduate Program, The Pennsylvania State University, University Park, PA 16802

The gram negative bacterium Francisella tularensis is one of the most infectious organisms known. In order to cause disease, the bacterium must enter host cells and gain access to the cytosol, where it replicates to high numbers, shielded from extracellular immune mechanisms and actively evading intracellular immunity. As few as 10 organisms are sufficient to cause severe disease, suggest-ing the bacterium possesses highly efficient mechanisms to adhere to and enter host cells.

Macrophages are one of the predominant cellular targets in the initial stages of infection, and F. tularensis has been shown to trigger engulfment via spacious, asymmetrical ‘pseudopod loops’. This morphologically distinct form of phagocytosis is considered unique to F. tularensis. Though a number of host receptors have been implicated in the entry process, bacterial factors responsible for inducing this unique form of uptake by macrophages have yet to be identified, and factors the bacterium utilizes to adhere to host cells have been incompletely characterized.

A transposon mutant library of Francisella tularensis Live Vaccine Strain (LVS) was gener-ated, with over 6,600 mutants representing approximately four-fold coverage of the genome. We screened mutants for deficiency in entry into RAW 264.7 macrophages, and discovered that mutants with a disruption in a particular gene, which we designate as macrophage entry and adhesion protein (Meap), enter macrophages at 10-fold lower efficiency, but enter the hepatocyte cell line HepG2 at nor-mal frequency. Compared to wild type, the mutants showed equivalent growth in metabolically mini-mal media and equal tolerance of H2O2, SDS, Triton-X100 and pH – suggesting the mutants are defi-cient in adhesion and entry rather than structurally or metabolically compromised. Co-infection with wild-type did not increase uptake of the mutant, arguing Meap is involved in a highly localized interac-tion with host cell membranes.

This work represents the first ever study of Meap, which may contribute to the extraordinary infec-tivity of the category A select bioterrorism agent F. tularensis.

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#52 Identification of a core molecular pathway required for phagocyte migration to apoptotic cells

Christopher S. Anderson, Taylor J. Moon & Michael R. Elliott

Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology,

University of Rochester Medical Center, Rochester New York 14642, USA

The rapid and non-inflammatory phagocytic clearance of apoptotic lymphocytes is essential to prevent aberrant immune activation and inflammation. Lymphocytes undergoing apoptosis release chemoattractant ‘find-me’ signals that recruit professional phagocytes such as monocytes, macrophages and dendritic cells. Neutrophils, by contrast, are not recruited to apoptotic cells. This selective recruit-ment of non-inflammatory phagocytes is an important feature of immunologically silent cell clearance, although the mechanisms that underlie this selectivity are not well understood. We recently identified ATP and UTP as the dominant find-me signal released by apoptotic lymphocytes through a novel apop-tosis-specific release mechanism. Hexameric pannexin 1 channels on the lymphocyte plasma mem-brane are activated by effector caspases to permit the regulated release of ATP/UTP during apoptosis. Migration of monocytes and macrophages to extracellular ATP/UTP from apoptotic cells is dependent on the G protein-coupled ATP/UTP receptor P2Y2. Interestingly, neutrophils also express high levels of P2Y2 yet fail to respond chemotactically to ATP/UTP and are not recruited to apoptotic cells. To understand the mechanisms responsible for the selective recruitment of macrophages by find-me sig-nals, we studied the activation of the Rho family GTPases by apoptotic cell find-me signals in mono-cytes. Rho proteins act as molecular switches to regulate cell morphology and motility in response to chemotactic stimuli. RhoA and Rac1 are the predominant GTPases expressed in monocytes, and we found that supernatants from apoptotic cells caused a rapid and transient activation of both RhoA and Rac1 in monocytes. Similarly ATP/UTP alone, at levels comparable to those found in apoptotic cell supernatants, was sufficient to induce similar levels of RhoA activation and cell migration. However, using RNAi depletion and pharmacological inhibition approaches we found that RhoA was essential, and Rac1 dispensable, for monocyte migration to apoptotic cell supernatants. Furthermore, we found that enzymatic depletion of nucleotides, overexpression of pannexin 1 dominant negative mutants in apoptotic cells, and inhibition of P2Y2 on monocytes ablated find-me signal-dependent RhoA activa-tion and cell migration. The identification of a core molecular pathway required for phagocyte migra-tion to apoptotic cells provides an opportunity to define key differences in P2Y2 signaling between monocyte/macrophages and neutrophils that determine selective recruitment of these cells to apoptotic cells.

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#17

Myeloid IRF-8 Levels Regulate Tumor-Induced Aberrant Myelopoiesis and Immune Surveillance in the Metastatic Microenvironment

Colleen Netherby*, Jeremy D. Waight*, Austin Miller%, Paul N. Bogner^, Kebin Liu# and Scott I.

Abrams* *Department of Immunology, %Department of Biostatistics and Bioinformatics and ^Department of Pa-thology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263; and #Department

of Biochemistry and Molecular Biology, Georgia Health Sciences University, Augusta, GA 30912

Tumor-induced dysregulation of myelopoiesis is recognized as an integral manifestation in ani-mal tumor models. Likewise, disruptions in myelopoiesis are also common in patients with diverse malignancies, including breast cancer. Because the myeloid compartment is essential for the induction of host immunity, alterations in myelopoiesis may help to explain host failure to effectively control neoplastic progression. Therefore, understanding the molecular basis by which the neoplastic process alters myelopoiesis may improve the efficacy of anticancer therapies that require a competent myeloid compartment. Given that interferon regulatory factor-8 (IRF-8) is indispensable for normal myeloid cell differentiation, we hypothesized that alterations in this ‘master’ regulator represent a novel mecha-nism for myeloproliferative phenotypes commonly found in malignancy, such as the expansion of mye-loid-derived suppressor cells (MDSCs). Consistent this hypothesis, we recently demonstrated a novel role for IRF-8 in regulating MDSC development. We found that tumors downregulate myeloid IRF-8 expression in a cytokine-driven STAT3- or STAT5-dependent manner. The finding that IRF-8 loss was causal to MDSC development raised the notion that IRF-8 is a key ‘rheostat’ regulating 1) the na-ture of the myeloid response and, consequently, 2) the efficacy of a myeloid-dependent antitumor re-sponse. To test this hypothesis, we examined the impact of IRF-8 enhancement on both experimental endpoints in two separate mouse mammary tumor models of spontaneous lung metastasis. To deter-mine the precise role for IRF-8, we used a newly developed genetic (transgenic) gain-of-function ap-proach. In both tumor models, we observed a significant diminution in the number of spontaneous lung metastases in IRF-8 transgenic mice compared to the wild-type controls, which was accompanied by a significant decline the frequency of CD11b+Gr-1+ MDSC populations. Altogether, these results demonstrate an unrecognized role for myeloid IRF-8 in the host response against metastatic mammary cancer, which may have important implications for the use of immune-related transcription factors as therapeutic targets.

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Poster Talks (Evelley)

Adaptive Immunity

Chairs: Dr. Jim Drake

Dr. Deborah Fowell

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#47 The pathway of antigen processing regulates MHC class II signaling and determines B cell fate

Heidi R. Tucker, Kathleen Nichol, Lei Jin and James R. Drake

Albany Medical College, Albany NY

The T-dependent B cell response to protein antigen requires that B cells receive a primary signal through the B cell receptor (BCR) and a secondary signal through peptide-MHC class II (p-MHCII). The B cells die if both signals are not received. Our lab reported that BCR-primed B cells that take up antigen through non-specific fluid phase (FP) uptake die when the resulting p-MHCII complexes are engaged, while those presenting BCR-derived peptide do not, confounding the simple two signal model. Our goal is to provide a mechanistic explanation of how the pathway of antigen uptake regu-lates the signal coming through the resulting p-MHCII complexes. We use the I-Ak mouse MHC class II model in which there is an antibody against total MHC class II and another against the lipid raft subset to seek our answer. Selectively engaging the lipid raft subset halts proliferation of a self-replicating B cell line and increases annexin V binding, while engaging to-tal I-Ak does not. STING, a protein that transmits the MHC class II-induced death signal, is associated with both raft and non-raft MHC class II. STING’s presence with all MHC class II, but its inability to initiate apoptosis outside a lipid raft, suggests that membrane microenvironment plays a key role in regulating MHC class II signaling. BCR-derived antigen is loaded selectively onto lipid raft-resident MHC class II, while FP uptake generates both raft and non-raft p-MHCII complexes. It was reported that CD79, the protein that transmits the calcium signal, associates with MHC class II following BCR stimulation. Our lab reported that engaging the lipid raft subset of I-Ak produces a calcium signal, while engaging total I-Ak does not. We show that BCR stimulation drives the formation of a peptide loading complex (PLC) between MHC class II and the BCR, providing a mechanism for the selective transfer of CD79 to lipid raft resident p-MHC class II complexes. We hypothesize that the pathway of antigen processing regulates B cell fate decisions by determining the lipid raft environment of the re-sulting p-MHCII complexes and the balance of associated signaling proteins.

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#31 The inflamed microenvironment influences CD4+ T cell interstitial motility

Alison Gaylo, Dillon Schrock and Deborah Fowell

David H. Smith Center for Vaccine Biology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester NY

In order to mount an effective immune response, CD4+ T cells must rapidly migrate to sites of infection, interact with resident antigen presenting cells, and carry out effector functions. Although homing to and entry into inflamed tissues is well studied, it remains unclear how CD4+ T cells migrate through interstitial spaces. It has been previously shown that T cells and other leukocytes employ actin/myosin based non-adhesive migratory mechanisms for migration through artificial collagen matrices in vitro and in non-inflamed skin. However, inflammation can result in extensive remodeling of the ex-tracellular matrix, which may necessitate alternative strategies for T cell migration. Through intravital multiphoton microscopy, we showed that Th1s require αv (alpha-v) β1 (beta-1)/β3 (beta-3) integrins for motility in the microbially inflamed dermis, which was accompanied by corresponding changes to the extracellular matrix. Preliminary data suggests that distinct modes of inflammation may remodel the ECM differently in terms of collagen density, which has been shown to be an important factor for the mode of T cell migration in vitro. In fact, Th1s show integrin-independent migration in a model of ster-ile inflammation characterized by dense ECM collagen, in contrast to findings in microbial inflamma-tion, where collagen density is greatly reduced. We therefore hypothesize that the mechanism used by T cells for interstitial motility is dictated by environmental factors. A better understanding of T cell mi-gratory requirements may provide insight into the pathogenesis of autoimmune disease or pathogen clearance. Exploiting potential differences in migration could provide for more specific therapies to inhibit autoimmune disease and pathology from chronic inflammation without disturbing effective re-sponses to pathogens.

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#51 Essential role of Elmo1 in T lymphocyte chemotaxis

Catherine Stevenson, Gonzalo de la Rosa, Patrick S. Murphy, Christopher S. Anderson,

Tara Capece, Minsoo Kim & Michael R. Elliott*

1Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology,

University of Rochester Medical Center, Rochester New York 14642, USA.

The small GTPase Rac critically regulates lymphocyte migration downstream of multiple chemokine receptors. The Rac-specific exchange factor Dock2 is essential for Rac-dependent lympho-cyte migration, but the signals that control Dock2 function during T cell migration are poorly under-stood. Elmo1 and Elmo2 are highly homologous cytoplasmic adapter proteins that can interact with SH3-containing Dock family members (e.g. Dock1 and Dock2). Interestingly, Dock2 is restricted to the hematopietic lineage and regulates lymphocyte migration. In immortalized cell lines, interaction with Elmo has been shown to enhance the Rac activation potential and membrane targeting of Dock as well as decrease Dock protein turnover. However, the role of Elmo proteins in regulating Dock2 function in primary T cells is not known. To address this, we measured mRNA and proteins levels of Elmo1, El-mo2 and Dock2 in splenic lymphocyte populations isolated from WT and Elmo1-/- mice. qPCR analysis showed comparable levels of mRNA for all three genes in WT versus Elmo1-/- cells. However, im-munoblot analyses revealed that Dock2 protein levels were reduced by five-fold in T and B cells from Elmo1-/- mice. Treatment with the proteasome inhibitor MG132 partially restored Dock2 levels in El-mo1-/- T cells. A similar reduction in Dock2 levels was observed upon acute RNAi-mediated depletion of Elmo1 in human Jurkat and THP-1 cell lines. Using transwell and time-lapse imaging approaches, we found that T cells from Elmo1-/- mice were profoundly impaired in CCR7- and CXCR4-dependent chemotaxis. Surface levels of these receptors were comparable between WT and Elmo1-/- T cells, as was p44/42 MAPK phosphorylation in response to chemokine stimulation. However, polarization and Rac activation upon chemokine stimulation were severely impaired in Elmo1-/- T cells, indicating that Elmo1 plays a specific and non-redundant role in regulating CCR signaling to Rac. In genetic rescue experiments, ectopic expression of Elmo1, but not the Dock-binding mutant Elmo1T629, restored CXCR4-dependent migration in Elmo1-/- T cells. Together these findings demonstrate that Elmo1 has an essential and non-redundant role in T cell migration through interaction-dependent regulation of Dock2 protein levels. Thus targeting Elmo1-Dock2 interaction could provide a novel and specific therapeutic target for regulating the trafficking of pathogenic lymphocytes in tissues.

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#4

Regulation of integrin LFA-1 during T cell migration and activation

Tara Capece, Anna Abid, Minsoo Kim University of Rochester Medical Center, Rochester, NY, USA.

T cells spend the majority of their time perusing lymphoid organs in search of cognate antigen presented by antigen presenting cells (APCs) and quickly recirculating through the bloodstream to an-other lymph node. Extravasation into a given lymph node occurs through high endothelial venules (HEV) and is dependent upon several key events, including activation of chemokine receptor CCR7 by chemokine CCL19/21 and adhesion of integrin LFA-1 (CD11a/CD18) to its ligand ICAM-1. T cells must become highly migratory to scan the densely packed organ for their cognate antigen on APCs, utilizing active LFA-1 at the leading edge of a migrating T cell. Upon antigen challenge, T cell forms a stable interaction with the APC, which is known as the immunological synapse (IS). Active LFA-1 is relocated to the IS, stabilizing the interaction between the T cell and APC. Although LFA-1 is a key adhesive force for both migration and IS formation, the outcome of chemokine and T cell receptor (TCR) signaling is quite different, as the former induce “go” and the latter mediate “stop” signals. We hypothesize that the magnitude of chemokine and TCR signals received by the T cell will deter-mine the outcome of LFA-1 mediated interactions and thus T cell activation.

To investigate the role of LFA-1 during T cell activation and migration, we generated fluores-cent knock-in (KI) mice that will allow us to visualize expression, distribution, and activation patterns of LFA-1. Having mated these KI mice with OTI TCR transgenic mice, we have begun studying LFA-1 redistribution and activation in T cells activated by varying TCR signal strength with altered peptide ligands (APL). Thus far, we have shown that LFA-1 clusters faster at the contact site between CD8+ T cells and dendritic cells (DCs) pulsed with cognate antigen compared to those pulsed with a weaker APL. Additionally, LFA-1 remains clustered at contact site longer with cognate antigen than the APL. We will repeat these experiments in our LFA-1 FRET mice, which allow us to visualize the activation status of LFA-1, to determine if LFA-1 activation induces the LFA-1 clustering seen in interactions with cognate antigen. We will also incorporate a photoactivatable chemokine receptor, PA-CCR7, to assess the effect of chemokine signal strength on LFA-1 activation.

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#10

Dendritic Cell Differentiation: Wyatt IRF and the shootout at the PKC corral Haley Spangler, Matthew Farren, Louise Carlson, Scott Abrams, Kelvin Lee

Roswell Park Cancer Institute, Buffalo NY

Dendritic cell (DC) differentiation is often targeted in pathological settings, resulting in a loss of multiple DC subsets and impaired immune responses. While the loss of Protein Kinase C βII(PKCβ(beta)II), and Interferon Regulatory Factors 4 and 8 (IRF4 and IRF8) results in diminished DC differentiation, we hypothesized that a relationship exists between these pathways which can be targeted to enhance DC differentiation. We found PKC activation upregulates IRF4 and IRF8 expres-sion in both human progenitor-like cell lines and murine bone marrow (BM) cells treated. This upregu-lation can in turn be prevented when these cells are pre-treated with classical PKC inhibitors. GM-CSF driven conventional DC differentiation requires IRF4, while FLT3-L driven plasmacytoid DC differen-tiation requires IRF8. Since we have already established that PKCβ(beta)II is the classical PKC required for conventional DC differentiation, we inspected whether it was also important for the FLT3-L driven process. Using FLT3 expressing THP-1 cells and ImageStream analysis we determined that PKCβ(beta)II and PKCα(alpha) are the two classical PKCs activated by FLT3-L. To verify the role of these PKCs in FLT3-L driven DC differentiation, we treated murine BMcells with FLT3-L in the pres-ence of a classical PKC inhibitor, and found it blocked differentiation. However, when we treated mur-ine PKCα(alpha) knockout BM cells with FLT3-L the cells were still able to differentiate, indicating that PKCβ(beta)II is the classical PKC regulating GM-CSF and FLT3-L driven DC differentiation. We then inspected the role of PKCβ(beta)II in regulating these IRFs over the course of a 7 day treatment with either cytokine. We found that inhibiting PKCβ(beta)II prevented human and murine progenitor cells from becoming fully differentiated and functional DCs, and that IRF4 and IRF8 expression was downregulated in conjunction with the loss of PKCβ(beta)II. This supports the idea that PKCβ(beta)II is regulating FLT3-L and GM-CSF mediated IRF8 and IRF4 (respectively) expression in DC progeni-tor cells. To examine which progenitor populations utilize this relationship, we used murine IRF8-GFP BM cells and treated them with PMA over several hours. The PMA treatment showed IRF8 upregula-tion as early as the multipotent progenitor cells (MPP2 and MPP3), and continuing through myeloid progenitor cells. Interestingly, while we have found that IRF8 knockout mice have diminished myeloid cell populations, we also found that their differentiated DCs have increased PKCβ(beta)II levels, suggesting a feedback mechanism by which these IRFs can be autoregulated. Thus, PKCβII regulating these IRFs may be the key to understanding and manipulating DC differentiation.

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Poster Talks (Albenia)

Cancer Immunobiology

Chair Dr. Yasmin Thanavala

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#56

Myeloid-Derived Suppressor Cells Negatively Impact Trafficking of CD8 Effector T Cells in the Tumor Microenvironment

Amy Ku1, Jason Muhitch1, Michael Diehl1, Scott I. Abrams1, and Sharon S. Evans1

1Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY

Immune based therapies offer compelling treatment options for cancer as they can generate site-specific and durable anti-tumor responses. The success of T cell-based immunotherapy depends on the trafficking of blood-borne cytotoxic T cells to tumor tissue in order to initiate contact-dependent killing of tumor targets. Our laboratory has previously demonstrated that deficits in effector T cell trafficking in multiple tumor models (e.g., CT26 colorectal carcinoma and B16 melanoma) is partially overcome by thermal preconditioning therapy (39.5 ± 0.5ºC for 6 h) which upregulates the vascular expression of the hallmark trafficking molecule intercellular adhesion molecule-1 (ICAM-1). There are a number of immunosuppressive cells (i.e., myeloid-derived suppressor cells, MDSC; regulatory T cells, Treg; and tumor-associated macrophages, TAM) that interfere with T cell function within tumor tissues, but whether these cells actively impede T cell entry into the tumor microenvironment remains an out-standing question. Here, we report on a newly discovered role of MDSC in limiting CD8 T cell traf-ficking within the tumor microenvironment. Analysis of a subset of thermally-resistant murine tumors (4T1 mammary, Polyoma Middle T-transgenic mammary, and Pan02 pancreatic tumors) revealed a strong correlation between high MDSC burden and the failure to boost CD8 T cell trafficking as deter-mined by intravital microscopic imaging. To investigate whether MDSC contribute to poor effector T cell trafficking, thermally-sensitive CT26 or B16 tumor cells were admixed with syngeneic CD11b+Gr-1+ MDSC isolated from spleens of tumor-bearing mice at a ratio of 2:1, thus mimicking the high MDSC burden detected in thermally-refractive tumors. Although the frequency of CD11b+Gr-1+ MDSC remained elevated within the tumor during tumor outgrowth, there was no impact on vessel density or baseline expression of ICAM-1 on tumor vessels. However, tumor vessels became refrac-tory to ICAM-1 induction by thermal preconditioning therapy in tumors admixed with MDSC. Taken together, these findings provide the first evidence that MDSC negatively regulate trafficking molecule expression in tumor vascular gateways and lay the foundation for future studies to identify the mecha-nisms underlying immunotherapy resistance in tumors with high MDSC burden. Supported by the NIH (CA79765, CA085183) and the Jennifer Liscott Tietgen Family Foundation.

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#21

ITK regulates the dynamics and anti-tumor immunity development during CD8+ T cell homeostatic proliferation

Weishan Huang1, Lu Huang1,2, Fei Huang1 and Avery August1,2

1Department of Microbiology & Immunology, Cornell University, Ithaca, NY 14853 2Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853

Tec family kinase ITK is an essential regulator of T cell development and function. The ab-sence of ITK leads to spontaneous acquisition of innate memory phenotype (IMP) in CD8+ T cells, due to bystander-derived IL-4. CD8+ T cells undergone homeostatic proliferation (HP) in lymphopenic con-ditions share similar memory markers with IMP CD8+ T cells. To examine the role of ITK during HP of CD8+ T cells, we compared naïve CD8+ T cells from WT and Itk-/- OTI-Rag-/- transgenic mice. We found that they had similar CD44loCD122- naïve phenotype. When transferred into lymphopenic Rag-/- recipients, Itk-/- naïve CD8+ T cells exhibited rapid early expansion, giving rise to > 10 fold expansion over that of WT CD8+ T cells by 10 days post-transfer, which was followed by massive retraction. The absence of ITK during HP resulted in defects in expression of cytokine receptors and Bcl-2, accompa-nied by enhanced apoptosis and Fas expression. These data indicate an important role of ITK in regu-lating the dynamics and maintenance of CD8+ T cells during HP, likely by regulating the expression of cytokine receptors required for sustained survival of these cells. Moreover, the hyperactive prolifera-tion in early stage in the absence of ITK is CD8+ T cell-intrinsic, independent of APC-MHCI and T cell-T cell interaction. Itk-/- HP CD8+ T cells exhibited a strong effector memory phenotype and had sig-nificantly enhanced anti-tumor activity compared to WT HP CD8+ T cells. This work suggests that tar-geting the ITK pathway may allow manipulation of CD8+ T cell number and function in lymphopenic conditions due to infection or irradiation, thus enhancing CD8+ T cell anti-tumor immunity following lymphopenia caused by chemo- or radio- therapies in cancer patients.

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#9 MicroRNA30e*—Novel regulator of prostate cancer growth

Egan, Shawn1 and Gollnick, Sandra2

Department of 1Immunology and 2Cell Stress Biology: Roswell Park Cancer Institute, Buffalo, NY 14263

Prostate cancer (CaP) has the highest incidence rate and second highest mortality rate in Ameri-can men, making it a prevalent and serious concern for the aging male population. NF-κB hyperactiva-tion is a hallmark of CaP; however mechanisms that perpetuate the uncontrolled NF- κB activation re-main unknown and thus a critical gap in the field. microRNA (miR) are known to regulate the expres-sion of a number of molecules required for NF-κB activation. One such miR, miR30e*, targets IκBα leading to increased NF-κB activation and has a previously defined role in cancer. The expression and oncological impact of miR30e* has never been characterized as a function of prostate cancer progres-sion.

Utilizing the autochthonous TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model we identified a significant increase in miR30e* expression throughout CaP progression that was most pronounced in late stage disease. The expression of miR30e* also correlated with the inflamma-tion present throughout disease progression. Functionally we have discovered that miR30e* signifi-cantly increases the proliferation of prostate cancer cells both in vitro and in vivo. We have validated that miR30e* targets IκBα allowing more NF-κB to translocate to the nucleus. Increased NF- κB acti-vation drives increased cyclin D1, Rb and p-Rb (ser 795), which we presume to be the dominant mechanism behind the increased proliferation. We have begun to investigate whether miR30e* inhibi-tion has clinical significance. Docetaxel or Taxotere© is usually the first chemotherapeutic drug given to CaP patients. We have discovered that the combination of Taxotere© treatment and miR30e* inhibi-tion is more effective at controlling prostate cancer cell proliferation than Taxotere© alone. We also discovered that miR30e* expression is high in CD11b+ populations within the tumor and that tumor cells themselves secrete a factor that leads to increased miR30e* expression in the CD11b+ population. When miR30e* function is reduced in macrophages their response to TLR4 stimulation is starkly re-duced. Surprisingly, inhibition of miR30e* prompts an increase in the iNOS/Arg1 ratio of macrophages which is an indication they have become more M1-like. M1 macrophages or classically activated macrophages are potent killers of tumor cells.

The proposed studies have the potential to significantly advance our understanding of the role of the miR30e*:NF-κB axis in CaP as well as the possibility of identifying a novel therapeutic target. Supported by: Student training grant

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#14 How is Anti-Tumor Immunity Impacted by Metabolism in Mice Housed at Sub-Thermoneutral

Temperature?

Kathleen M. Kokolus1, Maegan Capitano1, Jason Eng1, Bonnie Hylander1, Sandra Buitrago1, Chi-Chen Hong1, Christopher J. Gordon2, Scott I. Abrams1, Elizabeth A. Repasky1

1Roswell Park Cancer Institute; 2U.S. Environmental Protection Agency

An increased requirement of metabolic energy is necessary for laboratory animals, which are housed under standard conditions enlisting a chronic cold stress, simply to maintain body temperature. Since body temperature maintenance is a high priority for survival, we hypothesized that metabolic en-ergy may be allocated to body temperature maintenance at the expense of the anti-tumor immune re-sponse. Thus, we are working to better understand the impact of cold stress on energy demanding metabolic processes and the relationship to the anti-tumor immune response in mouse models.

At ambient temperatures of 30-31°C, which is considered the thermoneutral temperature (TT) for mice, resting metabolic rate is sufficient to generate enough heat to maintain body temperature. However, mice are kept at a cooler standard temperature (ST; 21-23°C). Our data reveals heat-seeking behavior in tumor-bearing mice housed at ST indicating a physiological drive for additional heat, likely needed to lessen the energetic burden of maintaining temperature. Additionally, we saw in increase in glucose uptake in the lymph nodes of tumor-bearing mice as well as an increase in Glut-1 expression on CD8+ T cells in the spleen of mice maintained at TT suggesting increased activity. We wondered if reducing the energy required for maintaining body temperature (by housing at TT), would allow more energy to be available for the anti-tumor immune response.

We compared tumor growth in mice housed at ST and TT and found significantly delayed tu-mor growth rates and a reduction in metastasis in animals housed at TT. Because of the energetic de-mands of activating CD8+ T cells we investigated immunodefficient mice and mice depleted for CD8+ T cells and found no differences in tumor growth between mice housed at ST and TT suggesting a role of the adaptive immune response. Our next studies revealed more antigen specific CD8+ T cells within the lymph node and tumor microenvironment of animals housed at TT compared to those housed at ST. Our data has also shown an increased number of immunosuppressive cell subsets including GR-1+CD11b+ myeloid derived suppressor cells and Foxp3+ cells in mice maintained at ST.

This data demonstrates that tumor growth and anti-tumor immune control in laboratory mice under standard conditions is highly dependent upon ambient temperature and the availability of meta-bolic energy. Since metabolism and its role in T cell activation is increasingly modeled in laboratory mice, it is essential to take into account the impact that housing temperature has on anti-tumor immune activity.

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#8 Role of Myeloid Cells in Prostate Cancer Progression

Stephanie Sass1 and Sandra O. Gollnick2

Departments of Immunology1 and Cell Stress Biology2: Roswell Park Cancer Institute, Buffalo, NY

Prostate cancer (CaP) is highly prevalent among the American male population and the current treatment strategies aren’t always effective. A contributing factor to the lack of efficacious treatments is the difficulty in differentiating between indolent and progressing disease. A more complete under-standing of the factors that influence prostate tumor progression is necessary to achieve more favorable clinical outcomes. Some factors that influence this progression include epithelial mesenchymal transi-tion (EMT), vasculature formation and the myriad tumor-promoting activities of tumor-associated myeloid cells (TAMC). The presence and role of TAMC in CaP has never been characterized, how-ever recent evidence in the literature suggests that TAMC may play a role in disease progression.

To study this, our lab utilized isogenic cell lines derived from the autochthonous TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model: TRAMP C2 and TRAMP C3. Although C2 and C3 were derived from the same 32 week TRAMP tumor, C2 is tumorigenic mimicking progressing disease while C3 is non-tumorigenic representing indolent disease. We hypothesized that TAMC pro-mote disease progression in prostate cancer. To test our hypothesis, TAMC isolated from an estab-lished C2 tumor were mixed with the non-tumorigenic C3 cells, implanted sub-cutaneously in C57BL/6 mice and tumor growth was monitored.

C3 tumors grew in the presence of TAMC, but not in the presence of myeloid cells isolated from C2 tumor-bearing spleens, naïve spleens, or bone marrow-derived macrophages. To our knowl-edge, this is the first time that C3 tumor growth was observed. Cells isolated from C3 tumors, termed C3X cells, were tumorigenic in the absence of TAMC, suggesting that TAMC induce stable changes in the C3 cells which lead to tumorigenesis. PCR microarray analysis showed that C3X cells expressed higher levels of cell cycle factors such as Cyclin D1 as well as various angiogenic, EMT and metabo-lism genes as compared to C3 cells.

This data suggests that TAMC are initiating numerous changes within the C3 cells which allow the cells to become tumorigenic. Future studies will determine which specific TAMC population is contributing the most to C3 tumorigenesis and will determine whether these changes are initiated by secreted factors or by contact-dependent mechanisms. The proposed studies have the potential to iden-tify factors critical in the conversion of indolent disease to progressing disease, which may lay the ground work for future treatment strategies. Supported by: NIH CA109480 and CA98156.

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Poster Talks (Evelley)

Immunity and Disease

Chairs Dr. B. Paige Lawrence

Dr. Chris Norbury

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#38 MyD88-Dependent Immunity to a Natural Model of Vaccinia Virus Infection

Does not Involve Toll-Like Receptor 2 (TLR2)

Michael Davies1, Janet Sei1, Nicholas Siciliano2, Laurence Eisenlohr2, Christopher Norbury1

1Dept of Microbiology and Immunology, Penn State College of Medicine, Hershey, PA 2Dept of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA

Vaccinia virus (VACV) is an orthopoxvirus used to immunize people against smallpox, and is also effective at immunizing mice against fatal ectromelia virus (mousepox) infection. VACV is used as a vector for other antigens in human and mouse immunization, as intradermal inoculation induces immu-nity against a wide variety of epitopes but the virus remains limited to the skin. TLR2 is one of several receptors that signal through adaptor protein MyD88, which activates proinflammatory and interferon responses. Many vaccine adjuvants are TLR agonists, making it important to study whether VACV it-self is a TLR agonist when inoculated intradermally. VACV contains an unknown TLR2 ligand that activates cells in vitro, but the role of TLR2 and other MyD88-dependent receptors during real VACV infection is unclear. In vitro VACV also acti-vates the AIM2 inflammasome, leading to secretion of IL-1beta which binds IL-1R, another MyD88-dependent receptor. In this study we investigate the importance of TLR2, MyD88, IL-1R, and AIM2 in in vivo immunity to VACV. Disseminated VACV infection (through i.v., i.p. or i.n. routes) can lead to severe illness in mice, and death in some knockout strains (MyD88-/-, IFNAR-/-). Some studies using i.v. and i.p. VACV in-fection have suggested that TLR2 is important for limiting VACV replication and spread. Instead of disseminated VACV, we use intradermal VACV infection, which mimics the natural route of orthopox-virus infection. Here we show that MyD88-/- mice are far more susceptible to both disseminated and intradermal infection than TLR2-/- mice, and in fact TLR2-/- mice are indistinguishable from wild-type in their response to intradermal VACV, whether measured by pathogenesis, viral titers, or T-cell and antibody responses. Likewise, IL-1R-/- mice mount a successful and timely response against intrader-mal VACV. Although monocyte/macrophages are important cells in detecting VACV early in infec-tion, we also observe no immune deficit in mice with conditional knockout of MyD88 in monocyte and neutrophil lineages (LysMcre x MyD88flox). In this study we show for the first time that MyD88 is important in the response to intradermal VACV. We also show that unlike in i.v. or i.p. VACV infection, intradermal VACV infection is unaf-fected by the absence of TLR2 or IL-1R. Evidently there is considerable redundancy in MyD88-dependent immunity to VACV – redundancy both between receptors and between cells. Although a single TLR is crucial for immunity to intradermal ectromelia virus (mousepox), we conclude that TLR2 is superfluous for immunity to intradermal VACV.

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#2 Monocytes infected by HCMV are reprogrammed from short-lived monocytes to long-lived

macrophages

Megan Peppenelli, Olesea Cojohari, and Gary Chan SUNY Upstate Medical University, Syracuse NY

Human Cytomegalovirus (HCMV), a betaherpes virus, has a high prevalence throughout the United States, with about 80% of the population being seropositive. HCMV causes deleterious affects mainly in immunocompromised individuals such as transplant recipients, AIDS patients, and neonates. HCMV infection in these immunocompromised individuals often leads to multi system organ failure. The development of multi system organ failure is dependent on the ability of HCMV to spread from the initial point of infection to peripheral organs, which is mediated by peripheral blood monocytes. In order for monocytes to mediate hematogenous spread, HCMV must extend the short 48hr lifespan of monocytes. We have previously shown that HCMV promotes the survival of infected monocytes; however, the mechanism employed by HCMV leading to survival is still unknown. To address this mechanism, we performed a transcriptome analysis on infected monocytes. Our data demonstrated that HCMV promotes the upregulation of myeloid leukemia sequence 1 (Mcl-1) and heat shock protein 27 (HSP27), which block the two proteolytic cleavages necessary for the formation of active caspase 3, an inducer of apoptosis. Using siRNA knockdown, we found that infected cells deficient in Mcl-1 had in-creased rates of apoptosis, while cells knocked down for HSP27 did not induce apoptosis. However, infected cells knocked down for both Mcl-1 and HSP27 had enhanced apoptosis rates compared to cells knocked down for only Mcl-1, suggesting that Mcl-1 is playing a predominate role in the inhibition of caspase 3 activation, while HSP27 plays a supportive role. Interestingly, when compared to other mye-loid stimulation factors known to induce monocyte survival, HCMV infection was found to more effi-ciently upregulate Mcl-1 and HSP27 suggesting a unique virus specific mechanism of induction. Thus, we examined the involvement of receptor: ligand interactions and downstream signaling cascades, since viral anti-apoptotic proteins are not expressed during the first 48h of infection. We determined that HCMV viral glycoprotein gB binding to cellular EGFR and gH binding to cellular αVβ3 integrin during viral entry are responsible for the upregulation of Mcl-1 and HSP27, respectively, during the initial 48h of infection. Overall, these data indicate a unique inhibition in the activation of caspase 3 by HCMV, through the manipulation of Mcl-1 and HSP27; ensuring short-lived monocyte survival past the normal 48hr lifespan, a key event necessary for viral dissemination.

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#1 Dengue-viral source and purity determine differential infection requirements for secretion of IL-

1beta by primary monocytes

Justin B. Callaway1, Douglas G. Widman2, Scott A. Smith4,5, Karen P. McKinnon1, Dirk P. Dittmer1,3, James E. Crowe, Jr4,6,7, Aravinda M. de Silva1, Jenny P.-Y. Ting1,3

1Department of Microbiology and Immunology, 2Department of Epidemiology, and 3Lineberger Com-prehensive Cancer Center, Institute of Inflammatory Diseases and Center for Translational Immunol-ogy, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 4The Vanderbilt Vaccine Center and the 5Departments of Medicine, 6Pathology, Microbiology and Immunology, and 7Pediatrics, Vanderbilt Medical Center, Nashville, TN, USA.

While most patients ill from dengue virus (DV) suffer from the mild disease dengue fever, a portion develop severe dengue, with hypovolemic shock due to vascular leakage. With four DV sero-types, severe dengue is typically associated with a secondary infection with a serotype differing from the primary infection, leading to theories that cross-reactive immunity enhances disease. Antibody-dependent enhancement (ADE) of DV infection is one theory thought to contribute to severe dengue, in which pre-existing antibodies cause antibody-mediated viral uptake into Fc-receptor-bearing cells. Though poorly understood, severe dengue pathogenesis is caused in part by a “cytokine storm” of many cytokines. Inflammatory cytokine IL-1beta, matured by the cleavage of immature pro-IL-1beta into active IL-1beta by caspase-1 and the multimolecular inflammasome complex, is elevated in many severe dengue patient profiles and has known in vitro effects on vasculature. With little known about the production and contribution of IL-1beta to severe dengue, we hypothesized that ADE of DV leads to enhanced IL-1beta secretion. Consistent with previous findings, we found primary human mono-cytes, which have high Fc-receptor levels and are known IL-1beta producers, were susceptible to ADE of DV infection. However, secretion of IL-1beta was equivalent regardless of the presence of antibod-ies, requiring only inoculation with DV supernatant derived from infected Vero cells, a common DV preparation for studies. Viral inactivation and Fc-receptor inhibition confirmed that viral replication and ADE were dispensable for IL-1beta secretion. Interestingly, purifying DV virions away from full supernatant rendered ADE necessary for induction of IL-1beta secretion, as purified DV only induced IL-1beta secretion with antibodies present. Full DV supernatant derived from C7/10 mosquito cells be-haved similarly to purified, Vero-derived virus. This suggests that in the absence of soluble moieties present in Vero-derived DV supernatant, ADE is necessary for DV-induced IL-1beta secretion by monocytes, while infected mosquito cells produce no additional moiety inflammatory to monocytes, as ADE of C7/10 supernatant was required for significant IL-1beta secretion. Inflammasome analysis showed that DV inoculation only induced pro-IL-1β generation, which a constitutively active NLRP3 inflammasome subsequently cleaved. We conclude that DV induces IL-1beta production by monocytes via two distinct factors: virions that are susceptible to antibody enhancement and soluble, inflammatory components produced upon infection of certain cells. ADE of DV in vivo could lead to enhanced pro-duction of both of these factors, with monocytes primed to secrete IL-1β quickly in response to expo-sure due to constitutive caspase-1 activity.

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#15

The type 1 diabetes resistance locus B10 Idd9.3 impairs the development of mature B cells in a mouse model of human diabetes

Gregory Berry and Hanspeter Waldner

Pennsylvania State University College of Medicine, Hershey, PA 17033, USA Department of Microbiology & Immunology, College of Medicine, Pennsylvania State University,

Hershey, PA 17033, USA

Type 1 diabetes (T1D) is a polygenic autoimmune disease, which is characterized by the T cell-

mediated destruction of the insulin-producing β-cells of the pancreas, resulting in insulitis and hyper-glycemia. B cells, functioning as antigen-presenting cells (APC) that activate self-reactive CD4+ T cells, are critical for T1D pathogenesis in the NOD mouse, a well-established model of human T1D. Several insulin-dependent diabetes (Idd) loci confer protection from T1D development in humans and NOD mice. NOD.B10 Idd9.3 mice are NOD mice in which the Idd9.3 locus, containing 18 genes, was replaced with the Idd9.3 from T1D resistant C57BL/10 mice, resulting in significant protection from diabetes development as compared to NOD mice.

To investigate whether B10 Idd9.3 mediates differences in the development or function of NOD B cells, we analyzed B cells from the bone marrow and spleen of NOD.B10 Idd9.3 mice. We demon-strate that B10 Idd9.3 mediates an intrinsic impairment in B cell lymphopoiesis in the bone marrow, resulting in a paucity of peripheral B cells in NOD.B10 Idd9.3 mice compared to NOD mice. We fur-ther show that the development from transitional to mature B cells in the spleen is impaired in NOD.B10 Idd9.3 mice. Consistent with an immature phenotype, splenic NOD.B10 Idd9.3 B cells were profoundly hyporesponsive to in vitro stimulation via the B cell receptor, toll-like receptors, and CD40 in contrast to NOD B cells. To investigate the mechanism responsible for impaired B cell development in NOD.B10 Idd9.3 mice, we analyzed B cell progenitors for the expression of forkhead box protein P1 (Foxp1), a critical transcription factor for B cell lymphopoiesis. Our analysis revealed markedly re-duced levels of Foxp1 expression in B cell progenitors of NOD.B10 Idd9.3 mice compared to NOD mice. Furthermore, we show that the expression of microRNA (miR)-34a, which is encoded by Idd9.3 and represses Foxp1 expression, is significantly increased in NOD.B10 Idd9.3 B cell progenitors com-pared to those from NOD mice.

In summary, we demonstrate that the Idd9.3 from T1D resistant C57BL/10 mice mediates im-paired development of mature NOD B cells, which appears to be regulated by the miR-34a-Foxp1 axis. Thus, our findings suggest that B10 Idd9.3 contributes to T1D protection by impairing the development of NOD B cells.

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#3 Early life AhR signaling leads to altered DNA methylation in CD8+ T cells

Bethany Winans, Anusha Nagari, Minho Chae, W. Lee Kraus and B. Paige Lawrence

University of Rochester Medical Center, Rochester, NY and University of Texas Southwestern Medical Center, Dallas, TX

The developing immune system is sensitive to early life environmental insults, which likely contribute to altered immune function later in life. However, how developmental exposures alter immune function later in life remains poorly understood. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that acts as an environmental sensor and binds many dioxins and polychlorinated biphenyls (PCBs), pollutants to which humans are constantly exposed. AhR also plays a role in the development and function of the immune system. Human and animal data demonstrate that early life exposure to AhR ligands correlates with persistent alterations in immune function, supporting the idea that AhR activation influences the developing immune sys-tem. In order to study immune function following developmental exposure to a potent AhR ligand, offspring of exposed dams are infected with influenza A virus at maturity. Mice with developmental AhR activation have alterations in both the adaptive and innate immune response to infection, including persistently reduced clonal expansion and differentiation of CD8+ T cells. This functional alteration of CD8+ T cells is due to direct AhR signaling in the offspring, and results from a direct effect on hematopoietic cells. Furthermore, altered function occurs without detectable changes in lymphoid organ cellularity, or the distribution of immune cell subpopula-tions in naïve animals. These findings suggest that inappropriate activation of AhR influences the epigenetic regulation of the developing immune system, leading to persistent changes in immune function. To test this idea directly we examined genome-wide DNA methylation patterns in naïve and activated CD8+ T cells isolated from mice with early life AhR activation. The DNA methylation profile of CD8+ T cells from developmentally ex-posed, virally infected mice was markedly different than in cells from mice developmentally exposed to vehicle control. Notably, changes in DNA methylation were observed in genes involved in T cell function and epige-netic regulation, suggesting that this altered DNA methylation may lead to the observed reduction in CD8+ T cell function. These novel data provide an important framework for understanding how altered epigenetic regulation, cued by developmental exposure, may affect gene expression and T cell function.

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Platinum Sponsor Presentation

New Tools for Multicolor Flow Cytometry from BD Biosciences

Erik Puffer, Ph.D. Technical Applications Specialist

BD Biosciences

Developed from pioneering dye technology, BD Horizon™ Brilliant Violet™ polymer conju-gates are brighter than conventional fluorochromes. Improved dye brightness enables detection of low density antigens, allowing for resolution of previously obscured cell populations. During this presenta-tion, we will discuss the new BD Horizon™ Brilliant Violet™ dyes and will describe how to incorpo-rate these dyes into multi-color panels. These new dyes are compatible with most existing violet-laser configurations. We will describe the violet-laser configuration recommendations in details. We will also discuss new instrument offerings from BD, including the BD FACSAria™ Fusion Cell Sorter and BD LSRFortessa™ X-20. The BD FACSAria™ Fusion improves on the solid foundation of patented technologies, exceptional multicolor performance, and ease-of-use that was first brought to the world of sorting with the launch of the BD FACSAria™ sorter in 2003. This sorting know-how, combined with best-in-class biosafety expertise, allowed us to create a fully integrated advanced cell sorter and biosafety solution for research laboratories: the BD FACSAria Fusion. The BD LSRFortessa™ X-20 cell analyzer delivers high performance multicolor analysis with the most compact footprint in its class. The BD LSRFortessa X-20 can be configured with up to 5 lasers, enabling detection of up to 20 pa-rameters simultaneously. Together, these new tools demonstrate BD’s commitment to the evolving ap-plication requirements of advanced multicolor flow cytometry.

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#1 Justin B. Callaway*

#2 Megan Peppenelli*

#3 Bethany Winans*

#4 Tara Capece*

#5 Lisbeth A. Boule

#6 Neva Watson

#7 Mohammad N. Uddin

#8 Stephanie Sass*

#9 Shawn Egan*

#10 Haley Spangler*

#11 Yuexin Xu

#12 Adam Utley

#13 Samira Mansouri

#14 Kathleen M. Kokolus*

#15 Gregory Berry*

#16 Open

#17 Colleen Netherby*

#18 Stela Celaj

#19 Anand Ravindran

#20 Debarati Banik

#21 Weishan Huang*

#23 Jocelyn Jie Wang

#24 Emma C. Reilly

#24 Rachel Markley

#25 Kathryn M. Pietrosimone

#26 Norah Smith

#27 Amanda McCabe*

#28 Sara B. Cohen*

#29 John C. Hu

#30 Christopher J. Greene

#31 Alison Gaylo*

#32 Dillon Schrock

#33 Alison C. Billroth-MacLurg

#34 Nikesha Haynes

#35 Bhuvana Katkere

#36 Open

#37 Katherine E. Herman

#38 Michael Davies*

#39 David Williamson*

#40 Lauren A. Weiler

#41 Sivakumar Periasamy

#42 Open

#43 Gaurav N. Joshi

#44 Ian Perry

#45 Nicholas Leigh

#46 James Rice

#47 Heidi R. Tucker*

#48 Patrick S. Murphy

#49 David A. Hoekstra, II

#50 Julie Sahler

#51 Catherine Stevenson*

#52 Christopher S. Anderson*

#53 Olesea Cohohari

#54 Yubin Zhang

#55 Margaret L. Barlow

#56 Amy Ku*

#57 Don Steiner

#58 Brian J. Franz

#59 Alan M. Sanfilippo

#60 Chelsey Reed

#61 Joanne Y.H. Lim

#62 Quang-Tam Nguyen

#63 Laura Bennett

#64 Kaitlin L. McDaniel

NYIC 2013 Poster Numbers

*Chosen to receive an AAI Young Investigator Award and given Poster Oral Presentation

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#1 Dengue-viral source and purity determine differential infection requirements for secretion of

IL-1beta by primary monocytes

Justin B. Callaway1, Douglas G. Widman2, Scott A. Smith4,5, Karen P. McKinnon1, Dirk P. Dittmer1,3, James E. Crowe, Jr4,6,7, Aravinda M. de Silva1, Jenny P.-Y. Ting1,3

1Department of Microbiology and Immunology, 2Department of Epidemiology, and 3Lineberger Com-prehensive Cancer Center, Institute of Inflammatory Diseases and Center for Translational Immunol-ogy, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 4The Vanderbilt Vaccine Center and the 5Departments of Medicine, 6Pathology, Microbiology and Immunology, and 7Pediatrics, Vanderbilt Medical Center, Nashville, TN, USA.

While most patients ill from dengue virus (DV) suffer from the mild disease dengue fever, a portion develop severe dengue, with hypovolemic shock due to vascular leakage. With four DV sero-types, severe dengue is typically associated with a secondary infection with a serotype differing from the primary infection, leading to theories that cross-reactive immunity enhances disease. Antibody-dependent enhancement (ADE) of DV infection is one theory thought to contribute to severe dengue, in which pre-existing antibodies cause antibody-mediated viral uptake into Fc-receptor-bearing cells. Though poorly understood, severe dengue pathogenesis is caused in part by a “cytokine storm” of many cytokines. Inflammatory cytokine IL-1beta, matured by the cleavage of immature pro-IL-1beta into active IL-1beta by caspase-1 and the multimolecular inflammasome complex, is elevated in many severe dengue patient profiles and has known in vitro effects on vasculature. With little known about the production and contribution of IL-1beta to severe dengue, we hypothesized that ADE of DV leads to enhanced IL-1beta secretion. Consistent with previous findings, we found primary human mono-cytes, which have high Fc-receptor levels and are known IL-1beta producers, were susceptible to ADE of DV infection. However, secretion of IL-1beta was equivalent regardless of the presence of antibod-ies, requiring only inoculation with DV supernatant derived from infected Vero cells, a common DV preparation for studies. Viral inactivation and Fc-receptor inhibition confirmed that viral replication and ADE were dispensable for IL-1beta secretion. Interestingly, purifying DV virions away from full supernatant rendered ADE necessary for induction of IL-1beta secretion, as purified DV only induced IL-1beta secretion with antibodies present. Full DV supernatant derived from C7/10 mosquito cells be-haved similarly to purified, Vero-derived virus. This suggests that in the absence of soluble moieties present in Vero-derived DV supernatant, ADE is necessary for DV-induced IL-1beta secretion by monocytes, while infected mosquito cells produce no additional moiety inflammatory to monocytes, as ADE of C7/10 supernatant was required for significant IL-1beta secretion. Inflammasome analysis showed that DV inoculation only induced pro-IL-1β generation, which a constitutively active NLRP3 inflammasome subsequently cleaved. We conclude that DV induces IL-1beta production by monocytes via two distinct factors: virions that are susceptible to antibody enhancement and soluble, inflammatory components produced upon infection of certain cells. ADE of DV in vivo could lead to enhanced pro-duction of both of these factors, with monocytes primed to secrete IL-1β quickly in response to expo-sure due to constitutive caspase-1 activity.

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#2

Monocytes infected by HCMV are reprogrammed from short-lived monocytes to long-lived macrophages

Megan Peppenelli, Olesea Cojohari, and Gary Chan.

SUNY Upstate Medical University, Syracuse NY Human Cytomegalovirus (HCMV), a betaherpes virus, has a high prevalence throughout the United States, with about 80% of the population being seropositive. HCMV causes deleterious affects mainly in immunocompromised individuals such as transplant recipients, AIDS patients, and neonates. HCMV infection in these immunocompromised individuals often leads to multi system organ failure. The development of multi system organ failure is dependent on the ability of HCMV to spread from the initial point of infection to peripheral organs, which is mediated by peripheral blood monocytes. In order for monocytes to mediate hematogenous spread, HCMV must extend the short 48hr lifespan of monocytes. We have previously shown that HCMV promotes the survival of infected monocytes; however, the mechanism employed by HCMV leading to survival is still unknown. To address this mechanism, we performed a transcriptome analysis on infected monocytes. Our data demonstrated that HCMV promotes the upregulation of myeloid leukemia sequence 1 (Mcl-1) and heat shock protein 27 (HSP27), which block the two proteolytic cleavages necessary for the formation of active caspase 3, an inducer of apoptosis. Using siRNA knockdown, we found that infected cells deficient in Mcl-1 had in-creased rates of apoptosis, while cells knocked down for HSP27 did not induce apoptosis. However, infected cells knocked down for both Mcl-1 and HSP27 had enhanced apoptosis rates compared to cells knocked down for only Mcl-1, suggesting that Mcl-1 is playing a predominate role in the inhibition of caspase 3 activation, while HSP27 plays a supportive role. Interestingly, when compared to other mye-loid stimulation factors known to induce monocyte survival, HCMV infection was found to more effi-ciently upregulate Mcl-1 and HSP27 suggesting a unique virus specific mechanism of induction. Thus, we examined the involvement of receptor: ligand interactions and downstream signaling cascades, since viral anti-apoptotic proteins are not expressed during the first 48h of infection. We determined that HCMV viral glycoprotein gB binding to cellular EGFR and gH binding to cellular aVb3 integrin during viral entry are responsible for the upregulation of Mcl-1 and HSP27, respectively, during the initial 48h of infection. Overall, these data indicate a unique inhibition in the activation of caspase 3 by HCMV, through the manipulation of Mcl-1 and HSP27; ensuring short-lived monocyte survival past the normal 48hr lifespan, a key event necessary for viral dissemination.

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#3

Early life AhR signaling leads to altered DNA methylation in CD8+ T cells

Bethany Winans, Anusha Nagari, Minho Chae, W. Lee Kraus and B. Paige Lawrence University of Rochester Medical Center, Rochester, NY and

University of Texas Southwestern Medical Center, Dallas, TX

The developing immune system is sensitive to early life environmental insults, which likely contribute to altered immune function later in life. However, how developmental exposures alter im-mune function later in life remains poorly understood. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that acts as an environmental sensor and binds many dioxins and poly-chlorinated biphenyls (PCBs), pollutants to which humans are constantly exposed. AhR also plays a role in the development and function of the immune system. Human and animal data demonstrate that early life exposure to AhR ligands correlates with persistent alterations in immune function, supporting the idea that AhR activation influences the developing immune system. In order to study immune func-tion following developmental exposure to a potent AhR ligand, offspring of exposed dams are infected with influenza A virus at maturity. Mice with developmental AhR activation have alterations in both the adaptive and innate immune response to infection, including persistently reduced clonal expansion and differentiation of CD8+ T cells. This functional alteration of CD8+ T cells is due to direct AhR sig-naling in the offspring, and results from a direct effect on hematopoietic cells. Furthermore, altered function occurs without detectable changes in lymphoid organ cellularity, or the distribution of immune cell subpopulations in naïve animals. These findings suggest that inappropriate activation of AhR influ-ences the epigenetic regulation of the developing immune system, leading to persistent changes in im-mune function. To test this idea directly we examined genome-wide DNA methylation patterns in naïve and activated CD8+ T cells isolated from mice with early life AhR activation. The DNA methylation profile of CD8+ T cells from developmentally exposed, virally infected mice was markedly different than in cells from mice developmentally exposed to vehicle control. Notably, changes in DNA methy-lation were observed in genes involved in T cell function and epigenetic regulation, suggesting that this altered DNA methylation may lead to the observed reduction in CD8+ T cell function. These novel data provide an important framework for understanding how altered epigenetic regulation, cued by develop-mental exposure, may affect gene expression and T cell function.

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#4

Regulation of integrin LFA-1 during T cell migration and activation

Tara Capece, Anna Abid, Minsoo Kim University of Rochester Medical Center, Rochester, NY, USA.

T cells spend the majority of their time perusing lymphoid organs in search of cognate antigen presented by antigen presenting cells (APCs) and quickly recirculating through the bloodstream to an-other lymph node. Extravasation into a given lymph node occurs through high endothelial venules (HEV) and is dependent upon several key events, including activation of chemokine receptor CCR7 by chemokine CCL19/21 and adhesion of integrin LFA-1 (CD11a/CD18) to its ligand ICAM-1. T cells must become highly migratory to scan the densely packed organ for their cognate antigen on APCs, utilizing active LFA-1 at the leading edge of a migrating T cell. Upon antigen challenge, T cell forms a stable interaction with the APC, which is known as the immunological synapse (IS). Active LFA-1 is relocated to the IS, stabilizing the interaction between the T cell and APC. Although LFA-1 is a key adhesive force for both migration and IS formation, the outcome of chemokine and T cell receptor (TCR) signaling is quite different, as the former induce “go” and the latter mediate “stop” signals. We hypothesize that the magnitude of chemokine and TCR signals received by the T cell will deter-mine the outcome of LFA-1 mediated interactions and thus T cell activation.

To investigate the role of LFA-1 during T cell activation and migration, we generated fluores-cent knock-in (KI) mice that will allow us to visualize expression, distribution, and activation patterns of LFA-1. Having mated these KI mice with OTI TCR transgenic mice, we have begun studying LFA-1 redistribution and activation in T cells activated by varying TCR signal strength with altered peptide ligands (APL). Thus far, we have shown that LFA-1 clusters faster at the contact site between CD8+ T cells and dendritic cells (DCs) pulsed with cognate antigen compared to those pulsed with a weaker APL. Additionally, LFA-1 remains clustered at contact site longer with cognate antigen than the APL. We will repeat these experiments in our LFA-1 FRET mice, which allow us to visualize the activation status of LFA-1, to determine if LFA-1 activation induces the LFA-1 clustering seen in interactions with cognate antigen. We will also incorporate a photoactivatable chemokine receptor, PA-CCR7, to assess the effect of chemokine signal strength on LFA-1 activation.

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#5

Activation of the aryl hydrocarbon receptor during development leads to an altered CD4+ T cell response to influenza A virus infection

Lisbeth A. Boule1, Bethany Winans2, and B. Paige Lawrence1,2 1Departments of Microbiology and Immunology and 2Environmental Medicine,

University of Rochester Medical Center, Rochester, NY

Recent reports suggest developmental exposures to certain pollutants lead to lower antibody responses to childhood immunizations, but the mechanism by which this occurs is unknown. The tran-scription factor aryl hydrocarbon receptor (AhR) is an intracellular receptor activated by a variety of chemicals. It is expressed by many cell types, including immune cells, and can alter their function upon activation. Previously, we have shown that developmental triggering of the AhR by one of its most po-tent ligands, the pollutant 2,3,7,8-tetrochlorodibenzo-p-dioxin (TCDD), results in a decrease in the CD8+ T cell response to influenza virus infection. However, the possible effect of maternal exposure to AhR ligands on the function of CD4+ T cells has never been directly examined, yet establishment of robust antibody responses to infection requires CD4+ T cells. We report here the initial characterization of the consequences of developmental triggering of AhR on the activation and differentiation of CD4+ T cells during infection with a common respiratory viral infection. Similar to the CD8+ T cell compart-ment, in the absence of infection there are no discernable effects of developmental exposure on the fre-quency of CD4+ T cells. However, upon influenza virus infection there are fewer activated and virus-specific CD4+ T cells in the draining lymph nodes of infected adult mice that were developmentally exposed to TCDD, compared to offspring of control dams. In addition, there are fewer Th1 cells in the lymph nodes of these mice, which is significant given that these are critical effector cells in the re-sponse to influenza virus. CD4+ T follicular helper (Tfh) cells are key facilitators of the class-switched, virus-specific antibody response. Similar to Th1 cells, the number of Tfh cells is decreased after devel-opmental exposure to TCDD; this finding is consistent with our observation that levels of virus-specific IgG isotypes are diminished in developmentally exposed mice. Conversely, the percentage of CD4+CD25+Foxp3+ regulatory T cells (Tregs), responsible for suppressing immune responses, is in-creased in infected mice that had been developmentally exposed. By using reciprocal adoptive transfers of naïve CD4+ T cells, we demonstrate that the effects of developmental activation of the AhR on CD4+

T cells occur through both direct and indirect pathways. These findings suggest that maternal exposure to AhR ligands may lead to decreased antibody responses to infections and immunizations in children by causing long lasting changes in the functional capacity of CD4+ T cells.

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#6 The role of SHP-1 in virus-induced myositis

Neva Watson and Paul Massa

SUNY Upstate Medical University

Microbial infection of tissues results in considerable inflammation mediated by myeloid and lymphoid immune cells which eradicate pathogens and promote subsequent tissue repair. Tight regula-tion of the immune response is critical as excessive immunity may cause substantial tissue destruction in the process of pathogen clearance, while a suppressed immune response may be insufficient to eradi-cate microbes and infected cells. The key immune regulatory phosphatase SHP-1 (Src homology re-gion 2 domain-containing phosphatase-1) has been described as an essential modulator of both innate and adaptive immune responses to pathogens. Consistent with this role, mice with either a null or hypo-morphic mutation in SHP-1 succumb to excessive tissue inflammation induced by environmental trig-gers (e.g. pathogens) associated with prominent tissue destruction by immune cells. We have shown a similar destruction of white matter in the central nervous system (CNS) following peripheral infection with an attenuated strain of Theiler’s murine encephalomyelitis virus (TMEV) mediated primarily by inflammatory monocytes/macrophages in SHP-1-deficient mice. Remarkably, we have now discovered that wild type animals, although free of CNS disease, develop a severe debilitating skeletal muscle dis-ease not seen in SHP-1-/- mice. While it has been documented that TMEV readily infects skeletal myo-fibers and induces myositis following peripheral inoculation of various strains of wild type mice, the lack of disease pathology in SHP-1-/- mice indicates a critical role for inflammatory cells in mitigating muscle pathology. Although extensive infection of myofibers elicits a massive inflammatory response of monocytes/macrophages within skeletal muscle of both SHP-1-/- mice and wild type littermates, an accumulation of calcified myofibers has only been observed in wild type mice. As SHP-1 functions as a key negative regulator of macrophage phagocytic activity we predict that hyper-responsive SHP-1-/- macrophages restrict myofiber calcification through rapid clearance of virus-infected and degenerating myofibers. This hypothesis is in accord with the preponderance of arginase-1-positive macrophages (i.e. alternatively-activated) in TMEV-infected muscles which are known to possess high phagocytic function. Experiments utilizing targeted deletion of SHP-1 in either skeletal muscle fibers or mono-cytes/macrophages are in progress to determine the site of SHP-1 function in virus-induced muscle pa-thology. We anticipate that TMEV-induced muscle disease will be controlled at the level of monocytes/macrophages. These findings demonstrate a unique role of SHP-1 in promoting disease pathology by restricting beneficial functions of macrophages, suggesting that in the case of skeletal muscle, a tightly regulated immune response to an attenuated virus is detrimental to the host.

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#7 TNF α(alpha)-dependent NK hematopoiesis following Bcl11b deletion in T cells restricts

metastatic melanoma

Mohammad N. Uddin, Yubin Zhang, Jonathan A. Harton, Katherine C. MacNamara, Dorina Avram Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY 12208

TNFα (alpha) is currently used for the treatment of advanced soft tissue sarcomas and metastatic mela-nomas. TNFα (alpha) is known to inhibit tumor-associated vasculature, however its role in anti-tumor immune response is poorly understood. Using several tumor models we demonstrate that mice deficient in Bcl11b in T cells, though having reduced numbers of T cells in the peripheral lymphoid organs, developed significantly less tumors compared to wild type mice. We demonstrate that Bcl11b-/- CD4+ T cells, producing elevated TNFα (alpha) levels, but not Bcl11b-/- CD8+ T cells, were required for the reduced tumor burden, as were NK1.1+ cells, found in increased numbers in Bcl11bF/F/CD4-Cre mice. Among NK1.1+ cells, the NK cell population was predominant in number and displayed elevated Granzyme B levels and increased degranulation. Although the NK1.1+ population expressing intracellular CD3 was also increased, it only represented a small fraction of the total NK1.1+ cells and changes in Granzyme B and degranulation were insignificant compared to wild type mice. The increase in NK cell numbers was associated with increased bone marrow and splenic hematopoiesis, along with elevated frequencies and numbers of NK precursors. Finally, the reduced tumor burden, increased numbers of NK cells and precursors, and increased hematopoiesis, were all dependent on TNFα (alpha). Moreover, TNFα(alpha) treatment of wild type mice bearing metastatic lung tumors also reduced the tumor burden and increased NK numbers and progenitors. These studies reveal a novel role for TNFα (alpha) in the anti-tumor immune re-sponse, specifically in stimulating hematopoiesis and increasing production of NK cells.

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#8 Role of Myeloid Cells in Prostate Cancer Progression

Stephanie Sass1 and Sandra O. Gollnick2

Departments of Immunology1 and Cell Stress Biology2: Roswell Park Cancer Institute, Buffalo, NY

Prostate cancer (CaP) is highly prevalent among the American male population and the current treatment strategies aren’t always effective. A contributing factor to the lack of efficacious treatments is the difficulty in differentiating between indolent and progressing disease. A more complete under-standing of the factors that influence prostate tumor progression is necessary to achieve more favorable clinical outcomes. Some factors that influence this progression include epithelial mesenchymal transi-tion (EMT), vasculature formation and the myriad tumor-promoting activities of tumor-associated myeloid cells (TAMC). The presence and role of TAMC in CaP has never been characterized, how-ever recent evidence in the literature suggests that TAMC may play a role in disease progression.

To study this, our lab utilized isogenic cell lines derived from the autochthonous TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model: TRAMP C2 and TRAMP C3. Although C2 and C3 were derived from the same 32 week TRAMP tumor, C2 is tumorigenic mimicking progressing disease while C3 is non-tumorigenic representing indolent disease. We hypothesized that TAMC pro-mote disease progression in prostate cancer. To test our hypothesis, TAMC isolated from an estab-lished C2 tumor were mixed with the non-tumorigenic C3 cells, implanted sub-cutaneously in C57BL/6 mice and tumor growth was monitored.

C3 tumors grew in the presence of TAMC, but not in the presence of myeloid cells isolated from C2 tumor-bearing spleens, naïve spleens, or bone marrow-derived macrophages. To our knowl-edge, this is the first time that C3 tumor growth was observed. Cells isolated from C3 tumors, termed C3X cells, were tumorigenic in the absence of TAMC, suggesting that TAMC induce stable changes in the C3 cells which lead to tumorigenesis. PCR microarray analysis showed that C3X cells expressed higher levels of cell cycle factors such as Cyclin D1 as well as various angiogenic, EMT and metabo-lism genes as compared to C3 cells.

This data suggests that TAMC are initiating numerous changes within the C3 cells which allow the cells to become tumorigenic. Future studies will determine which specific TAMC population is contributing the most to C3 tumorigenesis and will determine whether these changes are initiated by secreted factors or by contact-dependent mechanisms. The proposed studies have the potential to iden-tify factors critical in the conversion of indolent disease to progressing disease, which may lay the ground work for future treatment strategies.

Supported by: NIH CA109480 and CA98156.

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#9 MicroRNA30e*—Novel regulator of prostate cancer growth

Egan, Shawn1 and Gollnick, Sandra2

Department of 1Immunology and 2Cell Stress Biology: Roswell Park Cancer Institute, Buffalo, NY 14263

Prostate cancer (CaP) has the highest incidence rate and second highest mortality rate in Ameri-can men, making it a prevalent and serious concern for the aging male population. NF-κB hyperactiva-tion is a hallmark of CaP; however mechanisms that perpetuate the uncontrolled NF- κB activation re-main unknown and thus a critical gap in the field. microRNA (miR) are known to regulate the expres-sion of a number of molecules required for NF-κB activation. One such miR, miR30e*, targets IκBα leading to increased NF-κB activation and has a previously defined role in cancer. The expression and oncological impact of miR30e* has never been characterized as a function of prostate cancer progres-sion.

Utilizing the autochthonous TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model we identified a significant increase in miR30e* expression throughout CaP progression that was most pronounced in late stage disease. The expression of miR30e* also correlated with the inflamma-tion present throughout disease progression. Functionally we have discovered that miR30e* signifi-cantly increases the proliferation of prostate cancer cells both in vitro and in vivo. We have validated that miR30e* targets IκBα allowing more NF-κB to translocate to the nucleus. Increased NF- κB acti-vation drives increased cyclin D1, Rb and p-Rb (ser 795), which we presume to be the dominant mechanism behind the increased proliferation. We have begun to investigate whether miR30e* inhibi-tion has clinical significance. Docetaxel or Taxotere© is usually the first chemotherapeutic drug given to CaP patients. We have discovered that the combination of Taxotere© treatment and miR30e* inhibi-tion is more effective at controlling prostate cancer cell proliferation than Taxotere© alone. We also discovered that miR30e* expression is high in CD11b+ populations within the tumor and that tumor cells themselves secrete a factor that leads to increased miR30e* expression in the CD11b+ population. When miR30e* function is reduced in macrophages their response to TLR4 stimulation is starkly re-duced. Surprisingly, inhibition of miR30e* prompts an increase in the iNOS/Arg1 ratio of macrophages which is an indication they have become more M1-like. M1 macrophages or classically activated macrophages are potent killers of tumor cells.

The proposed studies have the potential to significantly advance our understanding of the role of the miR30e*:NF-κB axis in CaP as well as the possibility of identifying a novel therapeutic target. Supported by: Student training grant

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#10 Dendritic Cell Differentiation: Wyatt IRF and the shootout at the PKC corral

Haley Spangler, Matthew Farren, Louise Carlson, Scott Abrams, Kelvin Lee

Roswell Park Cancer Institute, Buffalo NY

Dendritic cell (DC) differentiation is often targeted in pathological settings, resulting in a loss of multiple DC subsets and impaired immune responses. While the loss of Protein Kinase C βII(PKCβ(beta)II), and Interferon Regulatory Factors 4 and 8 (IRF4 and IRF8) results in diminished DC differentiation, we hypothesized that a relationship exists between these pathways which can be targeted to enhance DC differentiation. We found PKC activation upregulates IRF4 and IRF8 expres-sion in both human progenitor-like cell lines and murine bone marrow (BM) cells treated. This upregu-lation can in turn be prevented when these cells are pre-treated with classical PKC inhibitors. GM-CSF driven conventional DC differentiation requires IRF4, while FLT3-L driven plasmacytoid DC differen-tiation requires IRF8. Since we have already established that PKCβ(beta)II is the classical PKC required for conventional DC differentiation, we inspected whether it was also important for the FLT3-L driven process. Using FLT3 expressing THP-1 cells and ImageStream analysis we determined that PKCβ(beta)II and PKCα(alpha) are the two classical PKCs activated by FLT3-L. To verify the role of these PKCs in FLT3-L driven DC differentiation, we treated murine BMcells with FLT3-L in the pres-ence of a classical PKC inhibitor, and found it blocked differentiation. However, when we treated mur-ine PKCα(alpha) knockout BM cells with FLT3-L the cells were still able to differentiate, indicating that PKCβ(beta)II is the classical PKC regulating GM-CSF and FLT3-L driven DC differentiation. We then inspected the role of PKCβ(beta)II in regulating these IRFs over the course of a 7 day treatment with either cytokine. We found that inhibiting PKCβ(beta)II prevented human and murine progenitor cells from becoming fully differentiated and functional DCs, and that IRF4 and IRF8 expression was downregulated in conjunction with the loss of PKCβ(beta)II. This supports the idea that PKCβ(beta)II is regulating FLT3-L and GM-CSF mediated IRF8 and IRF4 (respectively) expression in DC progeni-tor cells. To examine which progenitor populations utilize this relationship, we used murine IRF8-GFP BM cells and treated them with PMA over several hours. The PMA treatment showed IRF8 upregula-tion as early as the multipotent progenitor cells (MPP2 and MPP3), and continuing through myeloid progenitor cells. Interestingly, while we have found that IRF8 knockout mice have diminished myeloid cell populations, we also found that their differentiated DCs have increased PKCβ(beta)II levels, suggesting a feedback mechanism by which these IRFs can be autoregulated. Thus, PKCβII regulating these IRFs may be the key to understanding and manipulating DC differentiation.

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#11 Photoactivatable Chemokine Receptor

Yuexin Xu, Young-min Hyun, Christina Baker, Minsoo Kim

David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immu-nology, University of Rochester, Rochester, NY, USA

One of the challenges in immunology experimental design is the need to control one type of cells in the organ while leaving others unaltered, which is hard to be achieved by conventional meth-ods, such as knockdown, overexpression, mutation. A possible solution to this challenge may be a light (laser) mediated control because it could be delivered to a very small and defined area in precisely timed pulses. The use of light to probe/control immune function avoids the need for direct physical contact with the tissue thus won’t interfere with immune functions. . Therefore we propose to develop photoactivatable chemokine receptors that leverage common structure–function relationships between two different GPCR families (rhodopsin receptor and chemokine receptor) to recruit and control T cell migration. We fused the bovine rodopsin extracellular loops and transmembrane domains with CXCR4 intracellular loops to generate a photoactivatable CXCR4 (PA-CXCR4). PA-CXCR4 successfully transmitted intracellular CXCR4 signals in response to 505-nm light. Localized activation of PA-CXCR4 induced T cell polarization and directional migration (“phototaxis”) both in vitro and in vivo. Direct light exposure onto the melanoma was sufficient to recruit PA-CXCR4-expressing tumor-targeting cytotoxic T cells and furthermore improved the efficacy of adoptive T cell transfer immuno-therapy with a significant reduction in tumor growth in mice. These findings suggest that the strategy of photoactivatable chemokine receptors allows remotely controlled leukocyte trafficking with out-standing spatial resolution in tissues and may be feasible in other cell transfer therapies.

This project was supported by National Institute of Health (Grants HL087088 and HL018208, M. K.).

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#12 CD28 signaling in long-lived plasma cells regulates oxidative phosphorylation for survival and

antibody production

Adam Utley, Cheryl Rozanski, Louise Carlson, Megan Murray and Kelvin Lee

Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY

Recent evidence suggests that sustained humoral immunity is dependent upon bone marrow resident long-lived antibody secreting plasma cells (LLPCs). However, the signaling pathways that de-fine LLPC survival and function are not completely understood. We have shown that CD28, the proto-typic T cell co-stimulatory molecule, is necessary for LLPC survival and sustained antibody produc-tion. In T cells, CD28 is known to regulate the induction of glycolysis during activation at the expense of oxidative phosphorylation. What is unclear is how CD28 regulates metabolism in LLPCs. Surpris-ingly, CD28 signaling in LLPCs induces reactive oxygen species (ROS) production, a major byproduct of oxidative phosphorylation. To understand how CD28 induces ROS, we compared wild type mice with mice that contain a mutated CD28 cytoplasmic tail at the Grb2-Vav binding domain. Here we demonstrate that the Grb2-Vav domain is required for sustained presence of ROS producing LLPCs in vivo. Furthermore, the Grb2-Vav deficient mice cannot mount sustained long-term antibody production in vivo. Moreover, Grb2-Vav deficient LLPCs fail to survive with CD28 activation in vitro. Taken to-gether these data suggest a model wherein CD28-Grb2-Vav signaling regulates oxidative phosphoryla-tion in LLPCs for survival and sustained antibody production. By understanding the metabolic regula-tion of plasma cell induction, maintenance, and function, we may find insight into how targeting me-tabolism may alter long-term humoral immunity as well as autoimmune disorders characterized by autoantibody production.

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#13

The Role of HectD3 in Th17 Immune Responses

Samira Mansouri and Dorina Avram

Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY

E3 ubiquitin ligases are good therapeutic targets due to their substrate specificity as well as their critical roles in many biological processes. HectD3 is a novel E3 ubiquitin ligase that is expressed in T cells, however its role remains unknown. Recent work in cancer cells has found that HectD3 interacts and stabilizes the NF-kB regulator, mucosa-associated lymphoid tissue 1 (Malt1), through K-63 linked polyubiquitin chains. Malt1 was found to play a critical role in Th17 differentiation and effector function in experimental autoimmune encephalo-myelitis (EAE) through the activation of the NF-kB pathway. The absence of Malt1 resulted in the reduction of IL-17 and GM-CSF production while levels of the Th17 master regulator, Rorgt, remained unchanged. Our re-sults show that HectD3 deficient mice have reduced EAE severity and produce reduced levels of the Th17 cyto-kines, IL-17 and GM-CSF, in the central nervous system (CNS), suggesting that HectD3 is an important regula-tor of Th17 immune responses. Additionally, levels of the Th17 transcription factor Rorgt were also reduced in T cells deficient in HectD3. Based on these observations, we hypothesize that HectD3 modulates Th17 immune responses by (1) regulating NF-kB signaling through the stabilization of Malt1 and (2) regulating levels of Rorgt through a Malt1 independent pathway.

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#14 How is Anti-Tumor Immunity Impacted by Metabolism in Mice Housed at Sub-Thermoneutral

Temperature?

Kathleen M. Kokolus1, Maegan Capitano1, Jason Eng1, Bonnie Hylander1, Sandra Buitrago1, Chi-Chen Hong1, Christopher J. Gordon2, Scott I. Abrams1, Elizabeth A. Repasky1

1Roswell Park Cancer Institute; 2U.S. Environmental Protection Agency An increased requirement of metabolic energy is necessary for laboratory animals, which are

housed under standard conditions enlisting a chronic cold stress, simply to maintain body temperature. Since body temperature maintenance is a high priority for survival, we hypothesized that metabolic en-ergy may be allocated to body temperature maintenance at the expense of the anti-tumor immune re-sponse. Thus, we are working to better understand the impact of cold stress on energy demanding metabolic processes and the relationship to the anti-tumor immune response in mouse models.

At ambient temperatures of 30-31°C, which is considered the thermoneutral temperature (TT) for mice, resting metabolic rate is sufficient to generate enough heat to maintain body temperature. However, mice are kept at a cooler standard temperature (ST; 21-23°C). Our data reveals heat-seeking behavior in tumor-bearing mice housed at ST indicating a physiological drive for additional heat, likely needed to lessen the energetic burden of maintaining temperature. Additionally, we saw in increase in glucose uptake in the lymph nodes of tumor-bearing mice as well as an increase in Glut-1 expression on CD8+ T cells in the spleen of mice maintained at TT suggesting increased activity. We wondered if reducing the energy required for maintaining body temperature (by housing at TT), would allow more energy to be available for the anti-tumor immune response.

We compared tumor growth in mice housed at ST and TT and found significantly delayed tu-mor growth rates and a reduction in metastasis in animals housed at TT. Because of the energetic de-mands of activating CD8+ T cells we investigated immunodefficient mice and mice depleted for CD8+ T cells and found no differences in tumor growth between mice housed at ST and TT suggesting a role of the adaptive immune response. Our next studies revealed more antigen specific CD8+ T cells within the lymph node and tumor microenvironment of animals housed at TT compared to those housed at ST. Our data has also shown an increased number of immunosuppressive cell subsets including GR-1+CD11b+ myeloid derived suppressor cells and Foxp3+ cells in mice maintained at ST.

This data demonstrates that tumor growth and anti-tumor immune control in laboratory mice under standard conditions is highly dependent upon ambient temperature and the availability of meta-bolic energy. Since metabolism and its role in T cell activation is increasingly modeled in laboratory mice, it is essential to take into account the impact that housing temperature has on anti-tumor immune activity.

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15# The type 1 diabetes resistance locus B10 Idd9.3 impairs the development of mature B cells in a

mouse model of human diabetes

Gregory Berry and Hanspeter Waldner Pennsylvania State University College of Medicine, Hershey, PA 17033, USA

Department of Microbiology & Immunology, College of Medicine, Pennsylvania State University, Hershey, PA 17033, USA

Type 1 diabetes (T1D) is a polygenic autoimmune disease, which is characterized by the T cell-mediated destruction of the insulin-producing β-cells of the pancreas, resulting in insulitis and hyper-glycemia. B cells, functioning as antigen-presenting cells (APC) that activate self-reactive CD4+ T cells, are critical for T1D pathogenesis in the NOD mouse, a well-established model of human T1D. Several insulin-dependent diabetes (Idd) loci confer protection from T1D development in humans and NOD mice. NOD.B10 Idd9.3 mice are NOD mice in which the Idd9.3 locus, containing 18 genes, was replaced with the Idd9.3 from T1D resistant C57BL/10 mice, resulting in significant protection from diabetes development as compared to NOD mice.

To investigate whether B10 Idd9.3 mediates differences in the development or function of NOD B cells, we analyzed B cells from the bone marrow and spleen of NOD.B10 Idd9.3 mice. We demon-strate that B10 Idd9.3 mediates an intrinsic impairment in B cell lymphopoiesis in the bone marrow, resulting in a paucity of peripheral B cells in NOD.B10 Idd9.3 mice compared to NOD mice. We fur-ther show that the development from transitional to mature B cells in the spleen is impaired in NOD.B10 Idd9.3 mice. Consistent with an immature phenotype, splenic NOD.B10 Idd9.3 B cells were profoundly hyporesponsive to in vitro stimulation via the B cell receptor, toll-like receptors, and CD40 in contrast to NOD B cells. To investigate the mechanism responsible for impaired B cell development in NOD.B10 Idd9.3 mice, we analyzed B cell progenitors for the expression of forkhead box protein P1 (Foxp1), a critical transcription factor for B cell lymphopoiesis. Our analysis revealed markedly re-duced levels of Foxp1 expression in B cell progenitors of NOD.B10 Idd9.3 mice compared to NOD mice. Furthermore, we show that the expression of microRNA (miR)-34a, which is encoded by Idd9.3 and represses Foxp1 expression, is significantly increased in NOD.B10 Idd9.3 B cell progenitors com-pared to those from NOD mice.

In summary, we demonstrate that the Idd9.3 from T1D resistant C57BL/10 mice mediates im-paired development of mature NOD B cells, which appears to be regulated by the miR-34a-Foxp1 axis. Thus, our findings suggest that B10 Idd9.3 contributes to T1D protection by impairing the development of NOD B cells.

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#16

OPEN DUE TO LATE WITHDRAWAL

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#17 Myeloid IRF-8 Levels Regulate Tumor-Induced Aberrant Myelopoiesis and Immune Surveillance

in the Metastatic Microenvironment

Colleen Netherby*, Jeremy D. Waight*, Austin Miller%, Paul N. Bogner^, Kebin Liu# and Scott I. Abrams*

*Department of Immunology, %Department of Biostatistics and Bioinformatics and ^Department of Pa-thology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263; and #Department

of Biochemistry and Molecular Biology, Georgia Health Sciences University, Augusta, GA 30912

Tumor-induced dysregulation of myelopoiesis is recognized as an integral manifestation in ani-mal tumor models. Likewise, disruptions in myelopoiesis are also common in patients with diverse malignancies, including breast cancer. Because the myeloid compartment is essential for the induction of host immunity, alterations in myelopoiesis may help to explain host failure to effectively control neoplastic progression. Therefore, understanding the molecular basis by which the neoplastic process alters myelopoiesis may improve the efficacy of anticancer therapies that require a competent myeloid compartment. Given that interferon regulatory factor-8 (IRF-8) is indispensable for normal myeloid cell differentiation, we hypothesized that alterations in this ‘master’ regulator represent a novel mecha-nism for myeloproliferative phenotypes commonly found in malignancy, such as the expansion of mye-loid-derived suppressor cells (MDSCs). Consistent this hypothesis, we recently demonstrated a novel role for IRF-8 in regulating MDSC development. We found that tumors downregulate myeloid IRF-8 expression in a cytokine-driven STAT3- or STAT5-dependent manner. The finding that IRF-8 loss was causal to MDSC development raised the notion that IRF-8 is a key ‘rheostat’ regulating 1) the na-ture of the myeloid response and, consequently, 2) the efficacy of a myeloid-dependent antitumor re-sponse. To test this hypothesis, we examined the impact of IRF-8 enhancement on both experimental endpoints in two separate mouse mammary tumor models of spontaneous lung metastasis. To deter-mine the precise role for IRF-8, we used a newly developed genetic (transgenic) gain-of-function ap-proach. In both tumor models, we observed a significant diminution in the number of spontaneous lung metastases in IRF-8 transgenic mice compared to the wild-type controls, which was accompanied by a significant decline the frequency of CD11b+Gr-1+ MDSC populations. Altogether, these results demonstrate an unrecognized role for myeloid IRF-8 in the host response against metastatic mammary cancer, which may have important implications for the use of immune-related transcription factors as therapeutic targets.

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#18 Gut microbiota modulates susceptibility to liver injury

Stela Celaj, Michael W. Gleeson, Jie Deng and James D. Gorham

Dept. of Microbiology and Immunology, Geisel School of Medicine at Dartmouth

Variability in disease progression is common to all liver diseases and is one of the most chal-lenging aspects of the effective management of patients with inflammatory liver disease. Identifying factors that regulate liver inflammation and injury is critical to understanding how and why some pa-tients progress rapidly. We show that resident gut microbiota is a major regulator of hepatic injury dur-ing Concanavalin A and Fas-mediated liver damage. First, inflammation was induced in BALB/c mice through administration of Con A, and mice from different vendors (Jackson Labs (JAX); Taconic (TAC)) exhibited different levels of liver damage following Con A: JAX mice were highly sensitive and TAC mice were more resistant. Analysis of 1400 SNPs genome-wide showed that JAX and TAC BALB/c mice were genetically indistinguishable. Assessment of fecal microbiota using 16S deep se-quencing showed distinct microbial populations in JAX mice and TAC mice. Importantly, sensitivity to Con A could be transferred between mice following co-housing.

Since Concanavalin A has been shown to require Fas/Fas ligand system we investigated the role of microbiota in regulating Fas activation. Interestingly, JAX mice were much more sensitive than TAC mice to liver damage induced by injection of the Fas activating mAb Jo-2, which induces apop-tosis directly by activating Fas on hepatocytes. Treatment of JAX mice with oral antibiotics greatly reduced Jo-2 induced liver injury. Thus, the micro-biota by acting at the level of the hepatocyte, serves as a rheostat to modulate the hepatocellular re-sponse to cell death.

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#19 Ultraviolet Radiation (UVB)-Induced migration of Skin Dendritic Cell subsets is mediated

through TGF-β Signaling

Anand Ravindran*, Javed Mohammed, Andrew Gunderson and Adam B. Glick The Pennsylvania State University, State College, PA 16802

Ultraviolet radiation (UVB) is the leading cause of skin cancer worldwide. UVB also modulates certain inflammation driven cutaneous pathologies such as delayed type hypersensitivity and contact hypersensitivity through actions on skin resident dendritic cell (DC) subsets. Transforming Growth Factor-b1 (TGF-b1) is a potent immunoregulatory cytokine in the skin microenvironment and we found that UVB at erythema doses (MED) activates the TGF-b signaling pathway measured in terms of phos-phorylation of Smad2 and Smad3. Here, we show that this pathway activation is required for UVB in-duced activation and migration of dendritic cells to the skin draining lymph nodes. We irradiated skin of Skin Hairless (SKH1) mice with UVB in the presence or absence of SB431542, a small molecule inhibitor of the TGF-b type I receptor and measured lymph node migration of skin DC subsets at acute time points. Topical inhibition of TGF-b1 pathway with SB431542 suppressed the migration of skin DC subsets, primarily CD103+ CD207+ and CD207- DC populations to the lymph nodes in response to UVB irradiation. In addition, in an ex vivo skin explant assay for the migration of dendritic cells, UVB-induced DC migration into culture media was suppressed with topical inhibition with SB431542. Treatment with SB431542 also suppressed UVB-induced Interferon γ (IFNγ) secretion as well as the effector differentiation of T lymphocytes within the lymph nodes. Consistent with decreased activation within the lymph nodes, SB431542 decreased UVB activation of the skin infiltrating CD4+ and CD8+ lymphocytes after acute treatments and in UVB-induced skin tumors. In mice expressing a dominant negative receptor for TGF-b in CD11c+ dendritic cells (CD11c-TbRII DNR), UVB induced migration of DC subsets- CD103+ CD207+ and CD207- was suppressed directly linking TGF-b signaling in DCs to UVB-induced migration of DCs. This suppression of UVB-induced DC migration was in turn cou-pled to decreased T cell activation and decreased ear thickness in a contact hypersensitivity assay (CHS). Together, these data show that the TGF-b1 signaling pathway is important for the initiation of the inflammatory response to UVB irradiation of the skin, mediated primarily through the dendritic cells.

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#20 Regulation of Tumor Progression via a Novel IRF-8/MMP3 Interaction

Debarati Banik and Scott I. Abrams

Roswell Park Cancer Institute

Recently, we identified novel roles for interferon regulatory factor (IRF-8) in both myeloid and solid tumor cell biology. IRF-8 was originally characterized as a transcription factor essential for nor-mal myelopoiesis. In fact, IRF-8-loss within the myeloid compartment results in myeloproliferative disorders due, in part, to a decrease in apoptotic sensitivity. In solid tumor models, IRF-8-loss also en-hances tumor growth in vivo. Interestingly, the enhanced tumor growth rate is not readily explained solely by differences in apoptotic phenotype, suggesting that additional IRF-8-regulated events influ-enced tumor growth. Gene expression profiling revealed an intriguing inverse relationship between IRF-8 and MMP3 expression, later confirmed at the protein level. While tumor-derived MMPs, namely MMP2 and MMP9 are not unique to neoplasia, much less is known about the role of MMP3. Thus far, MMP3 expression was reported to play important roles during tumor initiation. Here, we tested the hypothesis that MMP3 also acts at later stages of tumor progression including metastasis. To do so, we made use of MMP3 loss- or gain-of-function approaches. Loss-of-function studies were per-formed using the 4T1 mammary tumor model of spontaneous lung metastasis, which expressed high levels of MMP3, while gain-of-function studies were performed using a sarcoma model (CMS4) which expressed low levels of MMP3. Altogether, we found that: 1) overexpressing MMP3 in CMS4 cells increased primary tumor growth rate; 2) silencing MMP3 in 4T1 cells reduced primary tumor growth, as well as spontaneous lung metastasis; 3) changes in MMP3 expression influenced tumorigenic behav-ior at multiple levels, including invasive ability, generation of active MMP9 and intra-tumoral accu-mulation of pro-tumor myeloid populations; 4) IRF-8 downregulated MMP3 expression at both mes-sage and promoter levels; and 5) inhibiting MMP3 expression rendered IRF-8-deficient tumors less ag-gressive, suggesting that IRF-8 impacts tumor behavior in an MMP3-dependent manner. Collectively, these data identify previously undescribed roles for an IRF-8/MMP3 axis in solid tumor biology.

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#21 ITK regulates the dynamics and anti-tumor immunity development during CD8+ T cell

homeostatic proliferation

Weishan Huang1, Lu Huang1,2, Fei Huang1 and Avery August1,2 1Department of Microbiology & Immunology, Cornell University, Ithaca, NY 14853

2Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853

Tec family kinase ITK is an essential regulator of T cell development and function. The ab-sence of ITK leads to spontaneous acquisition of innate memory phenotype (IMP) in CD8+ T cells, due to bystander-derived IL-4. CD8+ T cells undergone homeostatic proliferation (HP) in lymphopenic con-ditions share similar memory markers with IMP CD8+ T cells. To examine the role of ITK during HP of CD8+ T cells, we compared naïve CD8+ T cells from WT and Itk-/- OTI-Rag-/- transgenic mice. We found that they had similar CD44loCD122- naïve phenotype. When transferred into lymphopenic Rag-/- recipients, Itk-/- naïve CD8+ T cells exhibited rapid early expansion, giving rise to > 10 fold expansion over that of WT CD8+ T cells by 10 days post-transfer, which was followed by massive retraction. The absence of ITK during HP resulted in defects in expression of cytokine receptors and Bcl-2, accompa-nied by enhanced apoptosis and Fas expression. These data indicate an important role of ITK in regu-lating the dynamics and maintenance of CD8+ T cells during HP, likely by regulating the expression of cytokine receptors required for sustained survival of these cells. Moreover, the hyperactive prolifera-tion in early stage in the absence of ITK is CD8+ T cell-intrinsic, independent of APC-MHCI and T cell-T cell interaction. Itk-/- HP CD8+ T cells exhibited a strong effector memory phenotype and had sig-nificantly enhanced anti-tumor activity compared to WT HP CD8+ T cells. This work suggests that tar-geting the ITK pathway may allow manipulation of CD8+ T cell number and function in lymphopenic conditions due to infection or irradiation, thus enhancing CD8+ T cell anti-tumor immunity following lymphopenia caused by chemo- or radio- therapies in cancer patients.

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#22 Regulating mTOR to fine-tune memory CD8+ T cell formation in early life

Jocelyn Jie Wang, Norah L. Smith and Brian D. Rudd

Cornell University, Ithaca NY

Neonatal infection is a major cause of morbidity and mortality worldwide. While adults gener-ate robust immunity to most intracellular pathogens, neonates can be repeatedly infected with the same pathogen, indicating an impaired ability to develop long-lasting immunity. CD8+ T cells are important in clearing intracellular pathogens from the host, which is largely dependent on memory T cells to launch a rapid and robust immune response against subsequent infections. It is clear that neonates and young children have difficulty developing effective CD8+ T cell memory. However, the mechanistic details underlying defective formation of memory CD8+ T cells in young children and neonates are presently unclear. To examine this more closely, we compared the development of neonatal and adult memory CD8+ T cells and found that neonatal CD8+ T cells more rapidly become terminally differen-tiated compared to adult CD8+ T cells. We hypothesized that excessive proliferation and terminal dif-ferentiation impaired the ability of neonatal CD8+ T cells to transition into the long-lived memory pool. To test our hypothesis, we reduced neonatal CD8+ T cell proliferation by targeting mTOR, a ma-jor pathway for cellular growth and metabolism. Interestingly, limiting mTOR activity significantly reduced terminal differentiation and increased the numbers of neonatal memory CD8+ T cells to levels observed in adults. These results indicate that excessive mTOR activity contributes to impaired neona-tal memory CD8+ T cell formation and suggest that metabolic inhibitors may be useful strategy to en-hance immunity in early life.

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#23 Neonatal oxygen supplementation leads to persistent alterations in NK cell production of IL-22

during influenza A infection

Emma C. Reilly1, Kyle C. Martin1, Guangbi B. Jin1, Michael A. O’Reilly1,2, and B. Paige Lawrence1, 3 Departments of Environmental Medicine1, Pediatrics2, and Microbiology & Immunology3

University of Rochester, Rochester, NY

Supplemental oxygen treatment is critical for increasing survival in preterm infants. However, this treatment often has damaging and long-lasting effects, including more severe respiratory infec-tions. A mouse model of neonatal hyperoxia, in which pups are exposed to 100% oxygen between post-natal days 0-4, results in alveolar simplification, poorer survival, and exacerbated pathology when mice are challenged with influenza A virus later in life. For instance, mice exposed to hyperoxia as neonates exhibit excess leukocytes in the infected lung, and the mice that survive infection develop hallmarks of pulmonary fibrosis, suggesting an atypical repair response to infection. Natural Killer (NK) cells are well defined as key players in antiviral immunity; identifying and killing infected cells. However, re-cent evidence suggests NK cells also help maintain a homeostatic environment in peripheral tissues, including the lung, by promoting epithelial cell regeneration via the release of IL-22. In the studies pre-sented here, we tested the hypothesis that adult mice previously exposed to hyperoxia as neonates have altered NK cell responses to infection, including altered IL-22 production. We find that mice exposed to 100% oxygen as neonates had as much as a 50% reduction in IL-22 producing interstitial NK cells on days 5, 7, and 9 after influenza virus infection. In an effort to further define whether these effects were a result of intrinsic changes to the hematopoietic compartment, reciprocal bone marrow chimera mice were created. Irrespective of whether the host was exposed to oxygen or room air, mice that re-ceived bone marrow from an oxygen exposed donor displayed reduced numbers of IL-22 producing NK cells 3 days after influenza virus infection. These results indicate the possible contributions from both non-hematopoietic cells and immune cells. Overall, our findings demonstrate that neonatal hyper-oxia leads to long-term changes in IL-22 production by NK cells, which in turn, may contribute to al-tered host responses to infection.

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#24 Modulation of Tularemia Pathogenesis Through Selenium Supplementation

Rachel Markley 1,2, Kalyan Dewan 1, Bhuvana Katkere 1, David Place 1,2, David Williamson 1,2, K. San-

deep Prabhu 1 and Girish Kirimanjeswara 1; 1The Department of Veterinary and Biomedical Science, 2Immunology and Infectious Disease Graduate Program, The Pennsylvania State University, University

Park, PA, USA

The micronutrient Selenium (Se) is known to be critical in maintaining the Red-Ox status of

eukaryotic cells. Additionally, previous work has shown that selenium supplementation is able to dampen NF-êB induced nitric oxide synthase (iNOS) expression after LPS stimulation. Reactive Oxy-gen Species (ROS) are important intracellular signal transduction molecules, and have been reported to play a role in autophagy initiation and maturation. However, the ability of selenium to directly or indi-rectly affect autophagy has not been clearly elucidated. Here, we utilize the ability of Se to modulate the Red-Ox status of macrophages to understand its influence on autophagy. Macrophages maintained in media containing 200 nM sodium selenite had a 12 – 16 fold increase in the selenoprotein, glu-tathione peroxidase 1 (GPx-1) compared to the expression in macrophages maintained under Se defi-cient conditions. Using GPx-1 expression as an indication of selenium status we then measured expres-sion of autophagy protein markers LC3-I and LC3-II in parallel. Our preliminary data indicated that the ratio of LC3-I to LC3-II expression is greater in selenium-supplemented macrophages as compared to untreated macrophages, which suggests that Se decreases autophagy below the basal level.

Francisella tularensis, the causative agent of tularemia, is a gram-negative intracellular zoono-tic bacterium. Upon entry into a host cell, F. tularensis is able to escape the phagosomal compartment, and initiate replication, multiplying on average around 100 fold with in 24 hours. This replicative burst requires a large amount of metabolic substrates, which the bacteria must procure from its host. One of the mechanisms for a host cell to generate copious amounts of nutrients during starvation is through induction of autophagy. Since Se is able to directly or indirectly repress autophagy, we hypothesized that Se supplementation creates an intracellular environment where bacterial replication is restricted. Indeed, our preliminary data indicates that Se supplemented macrophages support only limited bacte-rial replication. We are currently in the process of further establishing the relationship between Se, autophagy and bacterial replication. Dietary supplementation as a means to decrease intracellular bacte-rial burden may provide insight into the influence of nutrients on infectious disease pathogenesis.

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#25 The Immunomodulatory and protective Effects of a Pseudmonas aeruginosa Metallothionein

Kathryn M. Pietrosimone1, Stephanie Davis1, Debby Laukens2, Michael A. Lynes1

1University of Connecticut, Storrs CT 2University of Ghent, Belgium Small, cysteine-rich proteins called metallotheins (MT’s) bind essential divalent heavy metal cations, such as zinc and copper, as well as toxic heavy metals, such as cadmium and mercury. Expres-sion of MT’s in eukaryotic cells increase under stressful conditions, such as exposure to these cations, reactive oxygen species (ROS), or reactive nitrogen species (RNS). The cysteine residues in MT al-low the protein to bind these heavy metals and to neutralize the toxic effects of ROS. Eukaryotic MT’s also share sequence similarities with chemokines and can act as a chemoattractant for human leuko-cytes. A bacterial MT (SmtA) expressed in Pseudomonas aeruginosa shares some molecular features with eukaryotic MT’s, including size and cysteine motifs. These structural similarities suggest that SmtA may also share immunomodulatory functions with mammalian MT. The expression of SmtA P. aeruginosa influences the host immune response by detoxifying ROS produced by inflammatory cells and by influencing immune cell trafficking, each of which may aid in the survival of the bacteria in the host. SmtA, with a GST tag, was purified from Escherichia coli MC1061 carrying the pGEX-6p-smtA plasmid. Jurkat T cells pre-incubated with SmtA and subsequently exposed to a gradient of SDF-1a, lost the ability to move in a persistent direction, while Jurkat T cells pre-incubated with GST did re-spond to the SDF-1a gradient. Incubation with SmtA also decreased SDF-1a-induced internalization of the receptor CXCR4 on Jurkat T cells. SmtA may also increase survival in the host by influencing im-mune cell movement and combating ROS-mediated immune cell defenses against pathogens. The SmtA mutant P. aeruginosa strain, PW4670, is more sensitive to oxidant exposure than the wildtype PA01 strain. SmtA’s ability to disrupt immune cell trafficking and detoxify ROS suggests it plays a role in pathogenesis and may be a novel target for therapeutic intervention.

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#26 Development of memory CD8+ T cells is intrinsically defective in early life

Norah Smith, Erin Wissink, Andrew Grimson and Brian Rudd

Cornell University, Ithaca, NY

CD8+ T cell memory formation contributes to the success of adaptive immunity and immu-nological memory. While adults efficiently generate memory CD8+ T cells following infection with intracellular pathogens, the development of memory CD8+ T cells in neonates is less robust and repeat infections with the same pathogen are common. We set out to determine whether the defect in neona-tal memory CD8+ T formation was due to an inability of naïve CD8+ T cells to respond to infection or an impaired ability of effector cells to transition into the long-lived memory pool. First, we compared different aged CD8+ T cells in vitro, and found that neonatal CD8+ T cells actually divided more than their adult counterparts in response to peptide stimulation. This data argues against the notion that na-ïve CD8+ T cells in neonates are inherently defective in responding to stimulation. Next, we examined cell intrinsic differences in memory cell differentiation in vivo. For these experiments, we set up an in vivo co-transfer model using Listeria monocytogenes, which elicits strong CD8+ mediated immunity, and tracked CD8+ T cells from neonatal and adult donor mice in the same adult recipient. During the primary infection, we saw that both neonatal and adult cell populations underwent antigen specific ex-pansion and differentiation. However, at the peak of the primary response, most neonatal CD8+ T cells were KLRG1hi CD127low, a phenotype typical of short-lived effector cells (SLECs). In contrast, adult CD8+ T cells expressed a memory precursor phenotype (KLRG1lowCD127hi) and exhibited an altered gene expression profile. At late stages of infection, the memory pool was almost entirely comprised of adult donor cells, which dominated the recall response following secondary challenge. Given that we have also observed these trends following infection with vaccinia virus, we believe this increase in ter-minal differentiation and reduced memory potential is likely a common feature among neonatal effec-tor CD8+ T cells. These results broaden our fundamental understanding of neonatal immunity and pro-vide critical insight into enhancing immunity in early life.

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#27 IFNg limits hematopoietic stem and progenitor cell mobilization during an intracellular bacterial

infection by maintaining BM-resident Mfs

Amanda McCabe, Yubin Zhang, Maura Jones, Kenny Thai, Katherine MacNamara Albany Medical College, Albany NY

The primary site of hematopoiesis is the bone marrow (BM), however hematopoietic stem and progenitor cells (HSPCs) can be released from the BM and into the circulation in a process known as mobilization. While it is thought that mobilization may impact immunity and host defense, very little is known about how mobilization is regulated under infectious conditions. We observed that during infec-tion with an intracellular bacterial pathogen HSPC mobilization was impaired by interferon gamma (IFNg). In IFNgR-deficient mice, blood and spleen HSPCs were significantly increased relative to wildtype infected mice. As bone marrow (BM)-resident macrophages (Mfs) preserve stromal cell func-tion, and thus retain HSPCs in the BM, we next investigated Mf function within the BM. An IFNg-dependent increase in frequency and number of highly phagocytic BM-resident Mfs was observed dur-ing infection. The increase in BM-resident Mfs correlated with increased mesenchymal stem cells (MSC), key niche cells that produce HSPC retention factors. IFNg signaling specifically in Mfs was required for increased MSCs and the impairment of HSPC mobilization. These data demonstrate that infection-induced IFNg limits HSPC mobilization by supporting niche cell function and HSPC reten-tion in the BM, which may be critical for the appropriate immune response to infection. Moreover, the observation that IFNg maintains BM HSPC niches has direct implications for clinically relevant proce-dures including HSPC transplantation.

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#28 CXCR3-Dependent CD4+ Th1 cells Activate Inflammatory Monocytes

for Defense Against Toxoplasma gondii Infection

Sara B. Cohen1, Kirk J. Maurer1, Charlotte E. Egan1, Steve Oghumu2, Abhay R. Satoskar2, Eric Y. Denkers1

1Cornell University, Ithaca, NY, 2The Ohio State University, Columbus, OH

Chemokines and their receptors orchestrate immunity to microbial pathogens, including the orally acquired Th1-inducing protozoan parasite Toxoplasma gondii. Chemokine receptor CXCR3 is associated with Th1 responses, and we use bicistronic CXCR3-eGFP knock-in reporter mice to demon-strate upregulation of this chemokine receptor on T lymphocytes, but not natural killer (NK) cells, dur-ing Toxoplasma infection. We show a critical role for CXCR3 in resistance to the parasite in the intesti-nal mucosa. Absence of the receptor in Cxcr3-/- mice resulted in selective loss of ability to control T. gondii in the lamina propria compartment. CD4+ T cells and CD11b+Ly6C/G+ inflammatory mono-cytes, recently reported as major anti-Toxoplasma effectors in the intestine, were both recruited to the intestine during infection, but only CD4+ T cells were impaired in recruitment in the absence of CXCR3. Furthermore, intestinal Cxcr3-/- CD4+ T cells, but not CD8+ T cells or NK cells, were impaired in their ability to secrete IFN-γ (gamma) upon stimulation. While local recruitment of inflammatory monocytes was not impacted by loss of CXCR3, inflammatory monocyte activation status, as measured by dual production of TNF-α (alpha) and IL-12, was severely impaired in Cxcr3-/- mice. Strikingly, adoptive transfer of wild-type but not Ifnγ(gamma)-/- CD4+ T lymphocytes into Cxcr3-/- animals prior to infection corrected the defect in inflammatory macrophage activation, simultaneously reversing the susceptibility phenotype of the knockout animals. Our results establish a central role for CXCR3 in co-ordinating innate and adaptive immunity, ensuring generation of Th1 effectors and their trafficking to the frontline of infection to program microbial killing by inflammatory monocytes.

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#29 Intradermal administration of type II heat-labile enterotoxins LT-IIb and LT-IIc of

Escherichia coli enhances both humoral and cellular immunity

John C. Hu1, Camila Mathias-Santos2, Christopher J. Greene1, Natalie D. King-Lyons1, Luís C. S. Ferreira2, Terry D. Connell1

1Department of Microbiology and Immunology and Witebsky Center for Microbial Pathogenesis and Immunology, the University at Buffalo, Buffalo, New York

2Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, Sao Paulo, Brazil

Vaccinations are extremely effective at combating infectious diseases. Many conserved antigen tar-gets, however, are poorly immunogenic. Additionally, protein subunit vaccines frequently only elicit humoral responses and are unable to protect against complex intracellular pathogens such as Mycobac-terium tuberculosis and HIV. These inadequacies in current vaccine development can be overcome by the use of appropriate adjuvants. Heat-labile enterotoxins (HLT) from Escherichia coli have potent ad-juvant properties. In this study, type-II heat-labile enterotoxins LT-IIb and LT-IIc were employed in an intradermal immunization model. Both adjuvants enhanced antigen-specific antibody levels and CD8+ T cell responses to ovalbumin, a poorly immunogenic model antigen. While LT-IIb and LT-IIc en-hanced similar types of humoral responses, LT-IIc induced a more rapid and sustained expansion of CD8+ T cells. This difference in expansion kinetics, however, did not improve cytotoxic function of LT-IIc adjuvanted mice when compared to LT-IIb. Furthermore, both LT-IIb and LT-IIc enhanced the expression of costimulatory molecule CD80 on dendritic cells (DC) in the draining lymph node at a point 24 hours after administration. However, only LT-IIc enhanced CD80 expression in the CD8α+ DC subpopulation. Finally, both LT-IIb and LT-IIc induced minimal inflammation as analyzed by both swelling and cellular infiltrate at the site of injection. Therefore, LT-IIb and LT-IIc are attractive com-prehensive adjuvants that enhance both humoral and cellular immunity when intradermally adminis-tered.

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#30 PspA is a critical virulence factor expressed by biofilm-dispersed Streptococcus pneumoniae fol-lowing influenza infection and confers protection against disease without affecting colonization

using an intradermal immunization regimen

Christopher J. Greene1,2*, Laura R. Marks1,2*, John C. Hu1,2, Lorrie Mandell1,2, Hazeline Hakansson1,2, Terry D. Connell1,2, Anders P. Hakansson1,2,3

1 The Witebsky Center for Microbial Pathogenesis and Immunology, 2 The Department of Microbiology & Immunology, and 3The New York State Center of Excellence in

Bioinformatics and Life Sciences, The University at Buffalo, Buffalo, NY 14214 * Indicates equal contribution to the work

Influenza A virus (IAV) infection is often complicated by bacterial super-infection, most fre-quently by Streptococcus pneumoniae, converting benign bacterial colonization into a severe or even fatal infection. Pneumococcal surface protein A (PspA) has a major role during invasive disease caused by planktonic, broth grown bacteria. Yet, the effect of PspA expression on host colonization and invasive disease of physiologically relevant, biofilm-derived bacteria has not been evaluated. To date, vaccine development has been aimed at eliminating colonization of S. pneumoniae; this strategy, however, opens niches in the oropharynx to colonization by new pathogens (e.g., Staphylcoccus aureus). Therefore, development of new vaccines for S. pneumoniae that do not alter oropharygeal colonization while protecting against invasive disease is highly desirable. Herein, we demonstrate that PspA is a critical virulence factor for pneumococci that have been dispersed from biofilms in response to IAV infection. Furthermore, PspA is required for development of secondary pneumococcal infec-tions from biofilm-dispersed bacteria following IAV infection. Intradermal immunization with recom-binant PspA and LT-IIb(T13I), a non-toxic type II heat-labile enterotoxin that possesses potent i.d. ad-juvant properties, induced strong antigen (Ag)-specific serum IgG antibodies (Ab), but no detectible salivary IgA Ab. This immunization strategy effectively prevented development of pneumonia, otitis media, and sepsis following IAV infection without inhibiting nasopharyngeal colonization and biofilm formation by pneumococci. Our results indicate that targeting PspA provides an effective strategy to prevent dissemination and disease of otherwise sterile sites, such as the middle ear, lung and blood, without affecting nasopharyngeal colonization burden.

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#31 The inflamed microenvironment influences CD4+ T cell interstitial motility

Alison Gaylo, Dillon Schrock and Deborah Fowell

David H. Smith Center for Vaccine Biology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester NY

In order to mount an effective immune response, CD4+ T cells must rapidly migrate to sites of infection, interact with resident antigen presenting cells, and carry out effector functions. Although homing to and entry into inflamed tissues is well studied, it remains unclear how CD4+ T cells migrate through interstitial spaces. It has been previously shown that T cells and other leukocytes employ actin/myosin based non-adhesive migratory mechanisms for migration through artificial collagen matrices in vitro and in non-inflamed skin. However, inflammation can result in extensive remodeling of the ex-tracellular matrix, which may necessitate alternative strategies for T cell migration. Through intravital multiphoton microscopy, we showed that Th1s require av (alpha-v) b1 (beta-1)/b3 (beta-3) integrins for motility in the microbially inflamed dermis, which was accompanied by corresponding changes to the extracellular matrix. Preliminary data suggests that distinct modes of inflammation may remodel the ECM differently in terms of collagen density, which has been shown to be an important factor for the mode of T cell migration in vitro. In fact, Th1s show integrin-independent migration in a model of ster-ile inflammation characterized by dense ECM collagen, in contrast to findings in microbial inflamma-tion, where collagen density is greatly reduced. We therefore hypothesize that the mechanism used by T cells for interstitial motility is dictated by environmental factors. A better understanding of T cell mi-gratory requirements may provide insight into the pathogenesis of autoimmune disease or pathogen clearance. Exploiting potential differences in migration could provide for more specific therapies to inhibit autoimmune disease and pathology from chronic inflammation without disturbing effective re-sponses to pathogens.

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#32

CD4 T Cell Interactions with the Extracellular Matrix

Dillon Schrock, Jill Ford PhD, Alison Gaylo, Michael Overstreet PhD and Deborah Fowell PhD

David H. Smith Center for Vaccine Biology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester NY

The ability of CD4 T cells to carry out their effector function at sites of inflammation in periph-

eral tissues is indispensible for the efficient clearance of a variety of pathogens as well as for mainte-nance of normal immune homeostasis. Much attention has been paid to the processes by which CD4 T cell extravasate from the peripheral vasculature and contribute to the inflammatory response (in terms of cytokines, chemokines, and surface co-stimulatory factors) at the site of insult. However, the mecha-nisms of CD4 T cell migration through the intervening tissue and the role that the complex and dy-namic extracellular matrix (ECM) plays in this process are poorly understood. Our lab recently dem-onstrated that CD4 T cells differentially require integrin-mediated adhesion to the ECM for efficient migration through the dermis, depending on the mechanism of inflammation. We have also observed that the ECM composition and density can vary greatly between tissues and amongst different inflam-matory conditions. Experiments to date have revealed the ECM to be an important facilitator of the CD4 T cell response to inflammatory stimuli and have hinted at a possible role for ECM proteins in CD4 T cell activation and differentiation. Using second harmonic generation (SHG) via two-photon microscopy, we have devised a robust metric for quantifying the density of dermal fibrillar collagen. This tool, in combination with conditional fluorescent and/or knockout CD4 T cells will allow us to further study the particulars of these processes. We anticipate that future experiments will reveal a piv-otal role for ECM components in many stages of the CD4 T cell response to inflammatory stimuli.

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#33 FoxP3+ Regulatory T Cells in Inflamed Dermis

Alison C. Billroth-MacLurg, Alison Gaylo, Jill Ford, Ph.D., Deborah J. Fowell, Ph.D.

David H. Smith Center for Vaccine Biology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester NY

Maintaining immune homeostasis is imperative for controlling deleterious responses against self-antigens and damage from inflammation. Regulatory CD4+ T cells (Tregs), classically defined by expression of CD25 and the transcription factor FoxP3, are necessary suppressor cells that curb the im-mune response. Tregs are implicated in suppressing within diverse tissues in response to pathogen clearance, maintenance of homeostasis against commensal bacteria, or allergy. Furthermore, it is hy-pothesized that Treg properties can be modulated in these specialized inflamed locales. However, Treg recruitment kinetics, duration of retention, and suppressive function during an immune response at environmental interfaces, such as the dermis, remains unclear. Studying how inflammation may in-duce unique Treg accumulation and function will guide development of therapies to limit or promote Tregs in disease, as well as considerably enhance our understanding of basic Treg biology. Our re-search focuses on the modulation of both endogenous and antigen-specific Tregs within the dermis dur-ing different types of inflammation. We established inflammation to a protein antigen OVA(Ovalbumin) within mouse ears using different types of Th1 or Th2-inducing adjuvants. ALUM (Aluminum Salts) (only widely used adjuvant in humans) and CFA (Complete Freund’s Adjuvant) led to strikingly high frequencies of polyclonal and OVA-specific TCR Tg+ Treg accumulation in the der-mis. In contrast, low numbers of Tregs accumulated with the adjuvant SEA (Schistosoma mansoni egg antigen). This increase in Treg accumulation was sustained for weeks post-immunization. Differences in IFNg (Interferon-gamma) and IL-4 (Interleukin 4) cytokine expression in the tissue do not appear to be responsible for the differential Treg accumulation. However, we did find a correlation between Treg accumulation in the tissue and their efficiency of interstitial migration by multiphoton micros-copy. Further investigations exploring local Treg expansion and Treg retention in the tissue under dif-ferent inflammatory settings are ongoing. Defining tissue-specific inflammatory signals responsible for modulating Treg function would aid in improving the design of therapeutics that enhance or inhibit Treg accumulation and activity at the inflammatory site.

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#34 A Critical Role of Nonclassical MHC in Tumor Immune Evasion Revealed in vivo in the

Amphibian Xenopus Model

Nikesha Haynes1,2 , Eva Stina Edholm1,PhD, Maureen Banach1, Jacques Robert1,PhD University of Rochester Medical Center, Rochester, NY, 1Department of Microbiology and Immunol-

ogy, 2Department of Pathology & Laboratory Medicine

Nonclassical class Ib (class Ib) genes are found in all jawed vertebrates, including the amphib-ian Xenopus, which possesses at least 20 Xenopus nonclassical class Ib genes (XNCs). As an immune evasion strategy, tumors often down-regulate surface expression of classical MHC class Ia (class Ia) molecules. In contrast, cancers commonly express class Ib molecules, presenting an alternative for tu-mor immune recognition. Certain class Ib’s are also critically involved in the differentiation and func-tion of invariant (i)T-cells, especially when class Ia expression is suboptimal. We have previously char-acterized a novel XNC gene, XNC10, functionally similar to CD1d from a class Ia-deficient thymic lymphoid tumor (15/0), which grows aggressively in Xenopus LG15 cloned animals and is necessary for the differentiation of a distinct iT-cell subset. We hypothesized that XNC10 provides inhibitory sig-nals these iT cells, preventing 15/0-tumor cell killing. To investigate the roles of XNC10 in antitumor immunity, we generated stable 15/0-transfectants with modulated XNC10 expression. Notably, silenc-ing of XNC10 (but not XNC6 nor scramble shRNA) in the 15/0-tumor resulted in acute tumor rejection with a concomitant increase of tumor infiltrating lymphocytes in syngenic tadpoles, with the greatest effect in tumors with more efficient XNC10 knockdown. Our optimized in vivo killing assay shows that tumor rejection is due to a cell-mediated cytotoxic immune response elicited by the tadpole host, which can be further enhanced by priming. Flow cytometry reveals that XNC10-deficient tumor rejec-tion is associated with an accumulation of CD8+ T cells, presumably invariant, as well as other leuko-cytes. Strikingly, when XNC10 on the 15/0-tumor is overexpressed, even though the tumor continues to grow, it causes a robust infiltration of the recently identified XNC10-restricted invariant T cells (CD8neg/XNC10 tetramer+) into the tadpole peritoneum. These data suggest a complex and dynamic role of class Ib molecules in tumor immunity where the types and expression levels of various class Ib molecules may stimulate different subsets of cells with varying and distinct effector functions, thus de-termining the tumor outcome. To further investigate class Ib’s in tumor immunity, we have developed a novel intra-vital semi-solid tumor model in transparent tadpoles to visualize infiltration of CD8 T cells, MHC Class II+ leukocytes, tumor vascularization and dying tumor cells with apoptotic nuclei in real time and determine the distinct differences of XNC10-deficient tumors. Our study shows for the first time cell-mediated cytotoxic responses in the Xenopus tadpole and provides insight into the critical but complex roles of class Ib’s in tumor immunity in vivo.

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#35 Role of Selenium in the Modulation of B Lymphocyte Functions.

Bhuvana Katkere1,2, Justin P. Delgado1, Kalyan Dewan1, David Place1, Rachel Markley1, David Williamson1, K. Sandeep Prabhu1 and Girish S. Kirimanjeswara1, 1Veterinary and Biomedical

Sciences Department, The Pennsylvania State University, University Park, PA-16802

Selenium (Se), a micronutrient, has been shown to exert several beneficial effects such as pre-venting cancer, supporting immune functions, delaying aging and onset of AIDs. Several pathological conditions such as myopathy, cardiomyopathy and immune dysfunction are positively associated with with Se deficiency. While there have been reports that dietary Se modulates B cell maturation and B cell –mediated immunity, the underlying mechanisms are poorly understood. We sought to elucidate the role of Se and selenoproteins in B cell functions. Preliminary studies show that B cell receptor (BCR) endocytosis is accelerated in the presence of Se. Interestingly, surface expression of BCR is also upregulated in Se-supplemented B cells. These data suggested that selenoproteins are involved in antigen uptake and processing. We are currently investigating the role of selenoproteins in BCR-mediaed antigen presentation.

2e-mail: [email protected]

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#36

OPEN DUE TO LATE WITHDRAWAL

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#37 Susceptibility of Human Placental Trophoblast-like Cells to Viral Infection is Associated with

Hypo-responsiveness to Interferons

Katherine E. Herman, Catherine G. Burke, Benson Y.H. Cheng, Luis Martinez-Sobrido, and Shawn P. Murphy

University of Rochester School of Medicine and Dentistry, Rochester, NY Pregnant women are preferentially susceptible to infection by a variety of intracellular patho-gens, such as viruses. These infections are associated with severe complications, including miscarriage, congenital infection, preterm birth, in utero growth restriction, and maternal death. However, the fac-tors accounting for the increased susceptibility of pregnant women to infection are incompletely under-stood. Previous studies demonstrated that viruses can infect the placenta, but little is known about the pathogenesis of viral infection in this organ. Placental trophoblast cells (TBCs) are the only blastocyst-derived cells that directly contact maternal blood and immune cells and play critical roles in successful pregnancy. Here, we show that vesicular stomatitis virus (VSV) and lymphocytic choriomeningitis vi-rus (LCMV) readily infected and replicated more rapidly in human TBCs compared to human cervical epithelial cells. We subsequently investigated whether this was due to aberrant antiviral responses by TBCs. Following viral infection, TBCs expressed mRNAs encoding both type I and III interferons (IFNs), which promote an antiviral state in a variety of cell types. Furthermore, TBCs secreted antiviral cytokines in response to infection. However, compared to epithelial cells, pretreatment of TBCs with type I IFN protected weakly against subsequent VSV infection. Despite expressing all of the JAK-STAT pathway components necessary for IFN signaling, TBCs were hypo-responsive to both type I and type III IFNs. Specifically, IFN-induced STAT activation and IFN-responsive gene expression were compromised in TBCs relative to epithelial cells. We propose that decreased TBC responses to IFNs may play an important role in the susceptibility of pregnant women to viruses that infect the pla-centa.

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#38 MyD88-Dependent Immunity to a Natural Model of Vaccinia Virus Infection

Does not Involve Toll-Like Receptor 2 (TLR2).

Michael Davies1, Janet Sei1, Nicholas Siciliano2, Laurence Eisenlohr2, Christopher Norbury1

1Dept of Microbiology and Immunology, Penn State College of Medicine, Hershey, PA 2Dept of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA

Vaccinia virus (VACV) is an orthopoxvirus used to immunize people against smallpox, and is also effective at immunizing mice against fatal ectromelia virus (mousepox) infection. VACV is used as a vector for other antigens in human and mouse immunization, as intradermal inoculation induces immu-nity against a wide variety of epitopes but the virus remains limited to the skin. TLR2 is one of several receptors that signal through adaptor protein MyD88, which activates proinflammatory and interferon responses. Many vaccine adjuvants are TLR agonists, making it important to study whether VACV it-self is a TLR agonist when inoculated intradermally. VACV contains an unknown TLR2 ligand that activates cells in vitro, but the role of TLR2 and other MyD88-dependent receptors during real VACV infection is unclear. In vitro VACV also acti-vates the AIM2 inflammasome, leading to secretion of IL-1beta which binds IL-1R, another MyD88-dependent receptor. In this study we investigate the importance of TLR2, MyD88, IL-1R, and AIM2 in in vivo immunity to VACV. Disseminated VACV infection (through i.v., i.p. or i.n. routes) can lead to severe illness in mice, and death in some knockout strains (MyD88-/-, IFNAR-/-). Some studies using i.v. and i.p. VACV in-fection have suggested that TLR2 is important for limiting VACV replication and spread. Instead of disseminated VACV, we use intradermal VACV infection, which mimics the natural route of orthopox-virus infection. Here we show that MyD88-/- mice are far more susceptible to both disseminated and intradermal infection than TLR2-/- mice, and in fact TLR2-/- mice are indistinguishable from wild-type in their response to intradermal VACV, whether measured by pathogenesis, viral titers, or T-cell and antibody responses. Likewise, IL-1R-/- mice mount a successful and timely response against intrader-mal VACV. Although monocyte/macrophages are important cells in detecting VACV early in infec-tion, we also observe no immune deficit in mice with conditional knockout of MyD88 in monocyte and neutrophil lineages (LysMcre x MyD88flox). In this study we show for the first time that MyD88 is important in the response to intradermal VACV. We also show that unlike in i.v. or i.p. VACV infection, intradermal VACV infection is unaf-fected by the absence of TLR2 or IL-1R. Evidently there is considerable redundancy in MyD88-dependent immunity to VACV – redundancy both between receptors and between cells. Although a single TLR is crucial for immunity to intradermal ectromelia virus (mousepox), we conclude that TLR2 is superfluous for immunity to intradermal VACV.

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#39 A novel macrophage entry and adhesion protein (Meap) contributes to Francisella tularensis Live

Vaccine Strain invasion of host macrophages.

David Williamson1,2, Kalyan Dewan1, Rachel Markley1,2, David Place1,2, Vicky Avanzato, Bhuvana Katkere1, Girish Kirimanjeswara1.

1The Dept. of Veterinary and Biomedical Science, 2Immunology and Infectious Disease Graduate Pro-gram, The Pennsylvania State University, University Park, PA 16802

The gram negative bacterium Francisella tularensis is one of the most infectious organisms known. In order to cause disease, the bacterium must enter host cells and gain access to the cytosol, where it replicates to high numbers, shielded from extracellular immune mechanisms and actively evading intracellular immunity. As few as 10 organisms are sufficient to cause severe disease, suggest-ing the bacterium possesses highly efficient mechanisms to adhere to and enter host cells.

Macrophages are one of the predominant cellular targets in the initial stages of infection, and F. tularensis has been shown to trigger engulfment via spacious, asymmetrical ‘pseudopod loops’. This morphologically distinct form of phagocytosis is considered unique to F. tularensis. Though a number of host receptors have been implicated in the entry process, bacterial factors responsible for inducing this unique form of uptake by macrophages have yet to be identified, and factors the bacterium utilizes to adhere to host cells have been incompletely characterized.

A transposon mutant library of Francisella tularensis Live Vaccine Strain (LVS) was gener-ated, with over 6,600 mutants representing approximately four-fold coverage of the genome. We screened mutants for deficiency in entry into RAW 264.7 macrophages, and discovered that mutants with a disruption in a particular gene, which we designate as macrophage entry and adhesion protein (Meap), enter macrophages at 10-fold lower efficiency, but enter the hepatocyte cell line HepG2 at nor-mal frequency. Compared to wild type, the mutants showed equivalent growth in metabolically mini-mal media and equal tolerance of H2O2, SDS, Triton-X100 and pH – suggesting the mutants are defi-cient in adhesion and entry rather than structurally or metabolically compromised. Co-infection with wild-type did not increase uptake of the mutant, arguing Meap is involved in a highly localized interac-tion with host cell membranes.

This work represents the first ever study of Meap, which may contribute to the extraordinary infec-tivity of the category A select bioterrorism agent F. tularensis.

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#40 Role of myeloid cell subsets in the immune response to a peripheral poxvirus infection

Lauren A. Weiler, Janet J. Sei, Christopher C. Norbury

Department of Microbiology and Immunology, Penn State University College of Medicine, Hershey, PA 17033, USA.

Upon peripheral infection with a virus, a race ensues between the virus and the immune system. The virus attempts to counteract the immune response in order to replicate and spread from the site of infection, while the immune system deploys various cells and molecules to restrict spread of the virus and mediate survival. Infection with ectromelia virus (ECTV), the causative agent of mousepox, is characterized by spread of the virus from the peripheral site of infection and death from systemic dis-ease in susceptible mice. Following peripheral ECTV infection in the footpad, there is an early expan-sion of myeloid cells after infection in resistant mice. Resistant mice injected with clodronate lipo-somes die, indicating a requisite role for phagocytic innate immune cells in protective immunity. How-ever, the complexity of the myeloid lineage, combined with the plasticity of the lineage during infec-tion have meant that the phagocytic immune cell subset(s) responsible for protective immunity to ECTV, the effector mechanisms deployed by these immune cells, and the mechanisms responsible for activation and recruitment of these cells are currently unknown.

To isolate the requisite role(s) of different myeloid cell subsets, we characterized both the effec-tiveness of depletion and the immune response to ECTV in myeloid cell depletion models challenged with ECTV. We used a combination of depletion mechanisms, including injection of clodronate lipo-somes, Macrophage Fas-Induced Apoptosis (MaFIA) mice, inducible diphtheria toxin receptor trans-genic mice, mice lacking specific myeloid cell populations, and injection of antibodies directed against myeloid cell populations to ascertain the cell populations that mediate resistance to ECTV. Addition-ally, we have also used adoptive transfer of specific innate immune cell subsets into depleted mice challenged with ECTV. Our results indicate that cells derived from the monocytic lineage and den-dritic cell subpopulations cooperate to mediate a required protective innate immune response following ECTV infection. These results represent a unique study in which the role of myeloid cell subsets in mediating protective immunity and preventing mortality following a peripheral poxvirus infection has been carefully delineated.

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#41 Positive and negative impact of the inflammasome pathway on myeloid cell function during

Francisella tularensis infection

Sivakumar Periasamy, Ellen Duffy, HongNga T. Le, and Jonathan A. Harton Center for Immunology and Microbial Disease, Albany Medical College, Albany NY

Pneumonic tularemia is an acute bacterial disease caused by Francisella tularensis, which is classified as a category ‘A’ biothreat agent by the U.S. Centers for Disease Control. Aerosol infection with a few as 10 bacteria can cause a severe necrotizing pneumonia and septicemic death. Being a fac-ultative intracellular bacterium, F. tularensis adopts several immune evasive strategies (e.g. escaping the phagolysosome compartment and inhibiting oxidative burst in phagocytes) for its survival. In addi-tion to bacterial evasive strategies, the development of regulatory or immunosuppressive cells limits the host ability to defend against F. tularensis infection. It has recently been appreciated that myeloid lineage cells also acquire regulatory phenotypes and immunosuppressive capabilities during infections. In addition, large numbers of neutrophils and macrophages are shown to undergo apoptosis and necro-sis causing collateral lung damage. Inflammasome activation is an important mechanism in inflamma-tion and has, in addition, been linked to myeloid cell death and necrosis. In our mouse model of tularemia, IL-1β production is delayed until the late phase (> 3 dpi) of disease, but recruitment of myeloid cells occurs as early as 2 dpi, yet these cells undergo cell death by 3 dpi in lungs. This sug-gests that activation of the inflammasome may mediate both cytokine production and cell death in lungs. Il1r-/- mice show increased susceptibility to Francisella infection. consistent with a protective role for IL-1β. Surprisingly, Asc-/- mice exhibit some protection, albeit limited, suggesting a more complex interplay between the inflammasome and IL-1β in the pathophysiology of Francisella infection. Further, this protection appears enhanced in the absence of Nlrp3. Our results suggest that Casp1-mediated IL-1β plays a protective role, while Nlrp3-mediated cell death could be deterimental for resistance to F. tularensis infection and may influence the phenotype, function, and/or survival of myeloid cells generated in tularemia.

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#42

OPEN DUE TO LATE WITHDRAWAL

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#43 Investigation of the role of ROS in silica induced phago-lysosomal leakage and cell death in

alveolar macrophages

Gaurav N. Joshi, Christopher Cheng, David A. Knecht University of Connecticut, Storrs, CT

Silicosis is a chronic lung disease induced by inhalation of crystalline silica dust resulting in

fibrotic scarring of lung tissue. Phagocytosis of silica particles by macrophages results in phago-lysosomal leakage leading to NALP3 inflammasome activation and the apoptosis. We are interested in understanding the role of intra-phagosomal ROS species in phago-lysosome leakage. Previous studies have shown that treatment with anti-oxidants reduces inflammation and cell death. We hypothesize that H2O2 generated as part of the oxidative burst is converted to a more membrane damaging ROS (HO) by the silica surface leading to phago-lysosomal leakage. In order to test this hypothesis, we have used several schemes to quantify ROS generation in the phagosome and cytoplasm using time-lapse micros-copy.

ROS generation was detected in macrophages treated with either silica particles or non-toxic latex particles. Particles were labeled with dihydro-hexafluorofluorescein-BSA (H2HFF-BSA) as a gen-eral ROS sensor. Phagosomal H2O2 was detected using Peroxyfluor6-AM (PF6-AM). To detect phago-lysosomal leakage cells were loaded with FITC or TRITC dextran. Cytoplasmic ROS was detected by loading cells with CM-H2DCFDA or roGFP2-Orp1, a hydrogen peroxide sensor.

Latex particle associated H2HFF fluorescence increased within 10 minutes of uptake and con-tinued to increase for 40 minutes. No phago-lysosomal leakage occurred with latex particles. We were not able to measure H2HFF fluorescence in silica phagosomes because H2HFF became oxidized in the presence of silica during particle coating. Using PF6-AM, an increase in fluorescence due to H2O2 was detected in silica phagosomes and phago-lysosomal leakage began approximately 2 minutes later. In cells treated with DPI, a NADPH oxidase inhibitor, the start of leakage was delayed about 55 minutes. When tested for cytoplasmic ROS using CM-H2DCFDA only 20% of cells showed an increase in CM-H2DCFDA fluorescence when exposed to either latex or silica particles. Cytoplasmic H2O2 could not be detected using the roGFP2-Orp1 sensor consistent with the idea that minimal phagosomal ROS can be detected in the cytoplasm. During cell blebbing associated with apoptosis, which occurs many hours after particle uptake, both probes showed a dramatic increase in fluorescence.

Although phagocytosis of both latex and silica particles result in an oxidative burst, the inability of latex particles to cause phago-lysosomal leakage points to a complex chemistry between ROS and silica within the phago-lysosomal microenvironment.

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#44 Investigating the susceptibility of the human placenta to Salmonella typhimurium infection

Ian Perry, Michelle Dziejman Ph.D. and Shawn Murphy Ph.D.

University of Rochester School of Medicine and Dentistry, Rochester NY

Infections by intracellular pathogens pose a special risk during pregnancy, contributing to fetal and maternal morbidity and mortality. Salmonella infections are prevalent worldwide, and Salmonella enterica serovar Typhimurium (ST) is both the leading cause of Salmonella infection in Sub-Saharan Africa and North America, and the leading cause of death from food-borne illness in the United States. Pregnant females from a number of mammalian species are particularly susceptible to Salmonella, and infection is associated with mis-carriage and maternal death. Studies of pregnant mice demonstrate that ST replicates rapidly within the mur-ine placenta, which results in destruction of the placenta and subsequent maternal death. However, there is a gap in our knowledge regarding the potential role of the placenta in the susceptibility of pregnant women to Salmonella infection.

To investigate the extent to which the human placenta is susceptible to ST infection, we developed an in vitro model using floating placental explants from 1st trimester and term tissues. Placental infection was assessed by staining with antibodies to ST lipopolysaccharide and whole mount immunofluorescence micros-copy. We detected Salmonella-induced filament formation on the outer layer of the placenta in both 1st trimes-ter and term tissues. In addition, the level of infection appeared higher in 1st trimester compared to term sam-ples. These results suggest that ST has the capacity to infect the human placenta. Thus, the placenta may play a role in the susceptibility of pregnant women to infection by ST.

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#45 Perforin is important for both CD4+ and CD8+ T cell-mediated graft-versus-tumor effect but plays differential roles in CD4+ and CD8+ T cell expansion after allogeneic transplantation

Nicholas Leigh1, Guanglin Bian1, Wei Du1, George Chen2, Hong Liu2, Philip McCarthy2, and

Xuefang Cao1

1Department of Immunology, 2Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263, USA

Graft versus tumor (GVT) effect is dependent on T cell mediated recognition and elimination of residual host tumor present after allogeneic bone marrow transplantation (allo-BMT). Recent work from our lab has found a detrimental role for granzyme B (GzmB) in GVT effect due to its role in fa-cilitating activation induced cell death (AICD) in CD8+ T cells. As a result, GzmB-/- CD8+ T cells exhibited higher expansion after allo-BMT and subsequently provided better tumor control.

This current study sought to determine the role of perforin (Prf1) in GVT effect mediated by both CD4+ and CD8+ T cells. Using the MHC-mismatched C57BL/6 (H-2b) to BALB/c (H-2d) allo-BMT model, we first confirmed previous findings that CD8+ T cells require Prf1 to mediate GVT ef-fect against allogeneic A20 lymphoma. In addition, our data suggest that Prf1 is also required for CD4+ T cells to effectively mediate GVT effect against A20.

Our previous findings show that lack of GzmB allows for less AICD and subsequently more CD8+ T cell accumulation. Here, we show a similar effect for Prf1 in terms of accumulation, as Prf1-/- CD8+ T cells outcompete WT CD45.1+ CD8+ T cells when CD8+ T cells from these two genotypes are mixed in equal ratio and transplanted into tumor bearing BALB/c mice. This competitive advan-tage was due to less AICD in the Prf1-/- CD8+ T cells. We next tested the effect of Prf1 in AICD in CD4+CD25- T cells, and co-transplanted WT CD45.1+ and Prf1-/- CD4+CD25- T cells into tumor bearing mice. Unexpectedly, WT CD4+CD25- T cells accumulate to significantly higher numbers when in direct competition with Prf1-/- CD4+CD25- T cells. When we measured apoptotic cells, we found that WT CD4+CD25- T cells still had significantly more AICD. This result suggests that while Prf1 has an important role in AICD; it may also play a role in another feature of CD4+ T cell biology. Preliminary results suggest that Prf1 may enhance T cell proliferation, as Prf1-/- CD4+ T cells have less actively dividing cells post allo-BMT.

In summary, our results suggest that Prf1 plays an important role in GVT responses mediated by both CD8+ and CD4+ T cells. In addition, Prf1 can cause AICD to both CD4+ and CD8+ T cells after allo-BMT. However, while Prf1-induced AICD reduces CD8+ T cell expansion, Prf1 appears to play a previously unrecognized role enhancing CD4+ T cell proliferation via an unidentified mechanism.

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#46 Metallothionein affects the regulation of intracellular labile zinc during T cell activation

and differentiation

James Rice1, Katherine Han1, David Lawrence2, and Michael Lynes1 1University of Connecticut, Storrs CT, 2Wadsworth Center, Albany NY

Metallothioneins (MTs) are a family of cysteine rich, small molecular weight (~7 kD) proteins that are induced in response to oxidative stress, glucocorticoids, inflammatory cytokines, and biologi-cally relevant metals, most notably zinc (Zn2+). Recent studies suggest that the reduced (TR), oxidized (TO) and metal bound (MT) forms of the protein are all present in vivo. In both adaptive and innate im-mune cells, the intracellular [Zn2+] is modulated following activation through receptors on the surface. These Zn signals, in turn, differentially active MAPK signaling cascades and are a requirement for sig-nal propagation. To determine if MT affects regulation of intracellular [Zn2+] in lymphocytes we meas-ured intracellular Zn in the context of different CD4+ activation conditions in both MT knockout mice (MT-KO) and congenic WT controls (MT-WT). We observed lower baseline levels of intracellular [Zn2+] in MT-KO CD4+ T cells compared with MT-WT controls, indicating a role for MT in intracel-lular Zn homeostasis in lymphocytes. During exposure to ROS, both the amplitude and rate of the in-crease in intracellular [Zn2+] are greater in cells expressing MT, demonstrating the redox sensitivity of MT and concomitant release of Zn2+ following oxidation. Activation of PKC by PMA also releases Zn in CD4+ T cells and activates the p38 MAPK pathway. The release of Zn from intracellular stores fol-lowing exposure to PMA occurs in both MT-WT and MT-KO mice, but the significantly weaker Zn signal in MT-KO cells correlates with a smaller increase in p38 activation. Regulation of MAPK sig-naling cascades via modulation of intracellular [Zn2+] demonstrates a novel mechanism of MT medi-ated immunomodulation and may provide a new target for the therapeutic manipulation of T cell acti-vation.

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#47 The pathway of antigen processing regulates MHC class II signaling and determines B cell fate

Heidi R. Tucker, Kathleen Nichol, Lei Jin and James R. Drake

Albany Medical College, Albany NY

The T-dependent B cell response to protein antigen requires that B cells receive a primary signal through the B cell receptor (BCR) and a secondary signal through peptide-MHC class II (p-MHCII). The B cells die if both signals are not received. Our lab reported that BCR-primed B cells that take up antigen through non-specific fluid phase (FP) uptake die when the resulting p-MHCII complexes are engaged, while those presenting BCR-derived peptide do not, confounding the simple two signal model. Our goal is to provide a mechanistic explanation of how the pathway of antigen uptake regu-lates the signal coming through the resulting p-MHCII complexes. We use the I-Ak mouse MHC class II model in which there is an antibody against total MHC class II and another against the lipid raft subset to seek our answer. Selectively engaging the lipid raft subset halts proliferation of a self-replicating B cell line and increases annexin V binding, while engaging to-tal I-Ak does not. STING, a protein that transmits the MHC class II-induced death signal, is associated with both raft and non-raft MHC class II. STING’s presence with all MHC class II, but its inability to initiate apoptosis outside a lipid raft, suggests that membrane microenvironment plays a key role in regulating MHC class II signaling. BCR-derived antigen is loaded selectively onto lipid raft-resident MHC class II, while FP uptake generates both raft and non-raft p-MHCII complexes. It was reported that CD79, the protein that transmits the calcium signal, associates with MHC class II following BCR stimulation. Our lab reported that engaging the lipid raft subset of I-Ak produces a calcium signal, while engaging total I-Ak does not. We show that BCR stimulation drives the formation of a peptide loading complex (PLC) between MHC class II and the BCR, providing a mechanism for the selective transfer of CD79 to lipid raft resident p-MHC class II complexes. We hypothesize that the pathway of antigen processing regulates B cell fate decisions by determining the lipid raft environment of the re-sulting p-MHCII complexes and the balance of associated signaling proteins.

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#48 Defining age-related changes in peritoneal macrophage inflammatory responses

Patrick S. Murphy & Michael R. Elliott

Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology,

University of Rochester Medical Center, Rochester, New York 14642, USA.

Aging in humans is associated with elevated levels of inflammatory cytokines both in circula-tion and specific tissues. Chronic low-level inflammation in the elderly is an important component of multiple age-related diseases, but the underlying causes remain poorly defined. Macrophages are key mediators of inflammation based on their ability to produce a wide range of cytokines in response to varied environmental stimuli. However, the contribution of macrophages to age-related chronic inflam-mation is poorly understood. In this study we consider how age-related alterations in macrophage func-tions might contribute to chronic inflammation in aged mice by studying cytokine responses of perito-neal macrophages to i.p. LPS challenge. Using aged (20mo) and young (4mo) C57BL/6 mice we first establish baseline values of peritoneal immune populations as well as the cytokines TNF, IL-6, IL-10 and IL-12/23p40 in serum and lavage fluid of these mice. We have identified significant changes in the steady-state peritoneal cavity of aged mice including increased leukocyte cellularity and elevated levels of IL-12/23p40 in the peritoneal lavage. Next, young and aged mice were challenged i.p. with LPS and macrophage cytokine production was measured by intracellular cytokine staining, as well as cytokine ELISAs on serum and lavage fluid. Finally, we stimulated purified macrophages ex vivo with toll-like receptor (TLR) agonists to assess if macrophage response differences seen were specific to TLR4 or encompassed multiple TLR family members. Future studies will be aimed at defining specific defects in molecular pathways that contribute to age-related changes in inflammatory responses of macro-phages. Also, we will study effects of aging on inflammation-related macrophage functions including phagocytosis and pro-resolution responses. Together, these data will further our understanding of macrophage-specific defects that contribute to the chronic inflammatory state in aged individuals and may identify novel targets for clinical intervention.

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#49 Hijacking oncogenic kinases to activate apoptotic machinery: A novel rewiring of Bcr-Abl

and PKC βII

David A Hoekstra II, Louise Carlson, Kelvin P. Lee Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY, 14263

Despite recent success using aberrantly expressed viral kinases to selectively eliminate infected cells, this approach has not been successful in an oncogenic setting. Bcr-Abl is the result of a chromo-somal translocation of the abl and bcr genes, and is exclusively expressed in a variety of leukemias, predominantly in chronic myeloid leukemia (CML). Since CML is dependent upon Bcr-Abl for sur-vival and is exclusively expressed in transformed cells, we targeted Bcr-Abl to activate a ‘suicide’ pro-drug that is not normally activated by Bcr-Abl. We have previously shown that direct activation of pro-tein kinase C isoform βII (PKCβII) drives dendritic cell differentiation and apoptosis in leukemic cells. Using this as a pro-drug, we have rewired its signaling such that it is activated by Bcr/Abl. Using mo-lecular modeling techniques, the Abl kinase target motif was inserted into a specific regulatory se-quence in PKCβII to generate a mutated PKCβII (A25Y-PKCβII). To determine whether these mutated constructs were activated by Bcr-Abl, we transfected them into both Bcr-Abl+ and Bcr-Abl- cells. Upon transfection of WT-PKCβII in both Bcr-Abl+ and Bcr-Abl- cells, WT-PKCβII was inactive; however, A25Y-PKCβII was active in Bcr-Abl+ cells, but not in Bcr-Abl- cells. Cells treated with the Bcr-Abl inhibitor imatinib resulted in a significant decrease in activation of A25Y-PKCβII mutant constructs, suggesting that this activation is dependent upon Bcr-Abl activity. When Bcr-Abl was stably expressed in cells that are normally Bcr-Abl-, we found that A25Y-PKCβII was activated. PKCβII activation re-sults in its rapid degradation and an upregulation of phospho-ERK 1/2. Consistent with this, expression of WT-PKCβII decreased by 20% while expression of A25Y-PKCβII decreased by 90%. Moreover, relative to WT-PKCβII, expression of A25Y-PKCβII in Bcr-Abl+ cells increased phosphorylation of Erk 1/2, while no increase was observed in Bcr-Abl- cells. A25Y-PKCβII induced a 4-fold increase in apoptosis when compared to WT PKCβII in Bcr-Abl+ cells. However, there was no increase in apop-tosis in Bcr-Abl- cells as evidenced by AnnexinV and cleaved caspase-3 staining. Lastly, relative to WT-PKCβII, we found a significant decrease in the growth of Bcr-Abl+ cells transfected with A25Y-PKCβII. These findings demonstrate that Bcr-Abl mediated activation of PKCβII is feasible and in-duces death in leukemic cells, and provides a novel mechanism by which oncogenic kinases can be re-wired to activate pro-death agents and can have broad therapeutic applications.

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#50 Microparticle composition impacts inflammatory responses of target cells

Julie Sahler, Collynn Woeller, Sherry Spinelli, Neil Blumberg, Richard Phipps

University of Rochester School of Medicine and Dentistry, Rochester NY

Microparticles are submicron plasma membrane-derived vesicles that circulate in blood and other bodily fluids. Our lab has previously shown that the internal composition of microparticles can be transferred into the cytoplasm of target cells and directly influence cell physiology. Therefore, the composition of circulating blood microparticles may have a profound impact on the homeostatic in-flammatory state within the vasculature. Type 2 Diabetes (T2D) is a disease affecting over 300 million people worldwide and is associated with vascular inflammation. Our initial results show that circulat-ing microparticles from T2D have a proinflammatory composition compared to non-diabetic subjects. In our previous studies, we demonstrate that the anti-inflammatory transcription factor, peroxisome proliferator activated receptor-gamma (PPARγ), was transferred from microparticles into target cells, where it regulated the transcription of downstream target genes. We wanted to further investigate whether inflammatory responses of target cells are affected by microparticle composition. We set out to address this question using two microparticle model systems: 1) comparing engineered megakaryo-cyte cell line culture-derived control microparticles versus microparticles that overexpress PPARγ, and 2) comparing non-diabetic plasma microparticles versus T2D microparticles. Human target cells used to deliver microparticles included the monocytic cell line THP-1 and freshly isolated human peripheral blood mononuclear cells (PBMCs). Our results show, for the first time, that among the various PBMC populations, monocytes are the predominant target cells of microparticles. After activation of target cells, we found that control culture-derived microparticles induced more proinflammatory cytokine production compared to microparticles overexpressing PPARγ. Additionally, primary circulating mi-croparticles from T2D patients induced more proinflammatory cytokine production from target cells than microparticles derived from healthy human donors. These results reveal that microparticle com-position impacts inflammatory responses of human PBMCs, particularly monocytes. Identifying mi-croparticle composition could then, act as a biomarker to provide insight into vascular inflammation of T2D or other inflammatory diseases.

This work is supported by T90-DE021985, T32-HL066988, HL095467, UL-1RR024160 and ES01247.

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#51 Essential role of Elmo1 in T lymphocyte chemotaxis

Catherine Stevenson, Gonzalo de la Rosa, Patrick S. Murphy, Christopher S. Anderson,

Tara Capece, Minsoo Kim & Michael R. Elliott* 1Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology,

University of Rochester Medical Center, Rochester New York 14642, USA.

The small GTPase Rac critically regulates lymphocyte migration downstream of multiple chemokine receptors. The Rac-specific exchange factor Dock2 is essential for Rac-dependent lympho-cyte migration, but the signals that control Dock2 function during T cell migration are poorly under-stood. Elmo1 and Elmo2 are highly homologous cytoplasmic adapter proteins that can interact with SH3-containing Dock family members (e.g. Dock1 and Dock2). Interestingly, Dock2 is restricted to the hematopietic lineage and regulates lymphocyte migration. In immortalized cell lines, interaction with Elmo has been shown to enhance the Rac activation potential and membrane targeting of Dock as well as decrease Dock protein turnover. However, the role of Elmo proteins in regulating Dock2 function in primary T cells is not known. To address this, we measured mRNA and proteins levels of Elmo1, El-mo2 and Dock2 in splenic lymphocyte populations isolated from WT and Elmo1-/- mice. qPCR analysis showed comparable levels of mRNA for all three genes in WT versus Elmo1-/- cells. However, im-munoblot analyses revealed that Dock2 protein levels were reduced by five-fold in T and B cells from Elmo1-/- mice. Treatment with the proteasome inhibitor MG132 partially restored Dock2 levels in El-mo1-/- T cells. A similar reduction in Dock2 levels was observed upon acute RNAi-mediated depletion of Elmo1 in human Jurkat and THP-1 cell lines. Using transwell and time-lapse imaging approaches, we found that T cells from Elmo1-/- mice were profoundly impaired in CCR7- and CXCR4-dependent chemotaxis. Surface levels of these receptors were comparable between WT and Elmo1-/- T cells, as was p44/42 MAPK phosphorylation in response to chemokine stimulation. However, polarization and Rac activation upon chemokine stimulation were severely impaired in Elmo1-/- T cells, indicating that Elmo1 plays a specific and non-redundant role in regulating CCR signaling to Rac. In genetic rescue experiments, ectopic expression of Elmo1, but not the Dock-binding mutant Elmo1T629, restored CXCR4-dependent migration in Elmo1-/- T cells. Together these findings demonstrate that Elmo1 has an essential and non-redundant role in T cell migration through interaction-dependent regulation of Dock2 protein levels. Thus targeting Elmo1-Dock2 interaction could provide a novel and specific therapeutic target for regulating the trafficking of pathogenic lymphocytes in tissues.

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#52 Identification of a core molecular pathway required for phagocyte migration to apoptotic cells

Christopher S. Anderson, Taylor J. Moon & Michael R. Elliott

Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester New York 14642, USA

The rapid and non-inflammatory phagocytic clearance of apoptotic lymphocytes is essential to prevent aberrant immune activation and inflammation. Lymphocytes undergoing apoptosis release chemoattractant ‘find-me’ signals that recruit professional phagocytes such as monocytes, macrophages and dendritic cells. Neutrophils, by contrast, are not recruited to apoptotic cells. This selective recruit-ment of non-inflammatory phagocytes is an important feature of immunologically silent cell clearance, although the mechanisms that underlie this selectivity are not well understood. We recently identified ATP and UTP as the dominant find-me signal released by apoptotic lymphocytes through a novel apop-tosis-specific release mechanism. Hexameric pannexin 1 channels on the lymphocyte plasma mem-brane are activated by effector caspases to permit the regulated release of ATP/UTP during apoptosis. Migration of monocytes and macrophages to extracellular ATP/UTP from apoptotic cells is dependent on the G protein-coupled ATP/UTP receptor P2Y2. Interestingly, neutrophils also express high levels of P2Y2 yet fail to respond chemotactically to ATP/UTP and are not recruited to apoptotic cells. To understand the mechanisms responsible for the selective recruitment of macrophages by find-me sig-nals, we studied the activation of the Rho family GTPases by apoptotic cell find-me signals in mono-cytes. Rho proteins act as molecular switches to regulate cell morphology and motility in response to chemotactic stimuli. RhoA and Rac1 are the predominant GTPases expressed in monocytes, and we found that supernatants from apoptotic cells caused a rapid and transient activation of both RhoA and Rac1 in monocytes. Similarly ATP/UTP alone, at levels comparable to those found in apoptotic cell supernatants, was sufficient to induce similar levels of RhoA activation and cell migration. However, using RNAi depletion and pharmacological inhibition approaches we found that RhoA was essential, and Rac1 dispensable, for monocyte migration to apoptotic cell supernatants. Furthermore, we found that enzymatic depletion of nucleotides, overexpression of pannexin 1 dominant negative mutants in apoptotic cells, and inhibition of P2Y2 on monocytes ablated find-me signal-dependent RhoA activa-tion and cell migration. The identification of a core molecular pathway required for phagocyte migra-tion to apoptotic cells provides an opportunity to define key differences in P2Y2 signaling between monocyte/macrophages and neutrophils that determine selective recruitment of these cells to apoptotic cells.

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#53 Human Cytomegalovirus Manipulates Monocyte PI3K Signaling to Induce Survival and

M1 Differentiation

Olesea Cojohari, Megan Peppenelli, and Gary Chan SUNY Upstate Medical University, Syracuse, NY

Monocytes are considered to be responsible for disseminating human cytomegalovirus (HCMV) to various organ sites, which often leads to multiorgan failure and death in immunocompro-mised individuals. However, monocytes are short-lived cells and are naturally programmed to undergo apoptosis about 48h after release into the circulation. Moreover, monocytes are non-permissive for HCMV replication, and they sustain no viral lytic gene expression, which creates a conundrum as to how monocytes mediate viral spread. We have shown that upon infection in monocytes, HCMV pro-motes their survival and polarization into an M1 (pro-inflammatory) phenotype, which exhibits en-hanced motility and ability to extravasate into tissues. In order to promote monocyte survival, HCMV rapidly upregulates the activity of cellular phosphoinositide 3-kinase (PI3K), a pro-survival protein. However, chronically high PI3K activity in monocytes also induces their differentiation into an M2 (anti-inflammatory) phenotype with decreased motility. This raised a dilemma as to how the virus is able to do both: induce survival via high PI3K activity, but also polarize monocytes towards an M1 phenotype, where low PI3K activity is critically needed. Through a time-course analysis, we have shown that although initially the virus upregulates PI3K activity in monocytes, in later time-points HCMV reduces PI3K activity in order to achieve an M1 macrophage polarization profile. The virus accomplishes this by inducing an apoptotic protein specific to hematopoietic cells, SHIP-1, which is also a known negative regulator of PI3K. Moreover, we have evidence that in compliment with SHIP-1, HCMV may be using a viral microRNA, hcmv-miR-US25-2-5p, to target p110δ, a PI3K subunit pre-dominantly found in leukocytes, in order to further decrease PI3K output and promote M1 differentia-tion. Overall, our data suggest that the virus usurps monocyte PI3K signaling in order to concurrently promote monocyte survival and M1 differentiation, two key processes required for viral dissemination and persistence.

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#54

Type I Interferons Drive Pathogenesis during Lethal Ehrlichial Infection

Yubin Zhang, Vinh Thai, Amanda McCabe, Maura Jones and Katherine C. MacNamara Center for Immunology and Microbial Disease, Albany Medical College, 47 New Scotland Avenue,

Albany, NY 12208, USA

The impact of type I interferons (IFNs) on pathogenesis during bacterial infections is complex, and type I IFNs (alpha (α) and beta (β)) can be protective or pathogenic depending upon the organism in question. Here we investigated the role of type I IFNs on pathogenesis in a model of lethal rickettsial infection caused by the pathogen Ixodes ovatus ehrlichia (IOE). In contrast to infection with a related and more attenuated pathogen, Ehrlichia muris, infection with IOE elicited production of type I IFNs that contributed to host death. Using intracellular IFNα staining and IFNβ reporter mice, we identified the major sources of type I IFNs during IOE infection to be plasmacytoid dendritic cells and monocytes. In the absence of IFNαβR-mediated signaling, we observed increased pathogen-specific antibody responses, increased circulating IFN-gamma (IFNγ), increased myelopoiesis, and a significant increase in survival. Relative to wildtype mice, mice that lacked IFNγhad higher bacterial burden dur-ing IOE infection. Using mixed bone marrow chimeric mice, we found intrinsic type I IFN signaling on CD4 T cells inhibited IFNγ production. While increased IFNγ was essential for increased ehrlichia-specific IgM and monopoiesis in IFNαβR-deficient mice, IFNγ was not essential for increased survival, as administration of neutralizing antibodies to IFNγ did not impair host resistance in IFNαβR-deficient mice. We identify a previously unknown pathogenic role for type I IFNs during severe ehrlichiosis, and our preliminary data suggest that type I IFN-mediated signaling in non-hematopoietic cells is responsi-ble for IOE-induced lethality.

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#55 Radiation-induced depletion of cutaneous dendritic cells:

Functional consequences of a breakdown in the cutaneous immunological barrier

Margaret L. Barlow, Ryan J. Cummings, Julie L. Ryan, Constantine G. Haidaris, Edith M. Lord, and Scott A. Gerber

University of Rochester, Rochester NY

The United States continues to be a prime target for attack by terrorist organizations in which nuclear detonation or dispersal of radiological material are legitimate threats. These types of attacks could result in devastating consequences to a multitude of individuals in the form of radiation injury to particular organ systems. One of these organs, the cutaneous system, which provides the first line of defense against invading pathogens, is particularly sensitive to ionizing radiation (IR) leaving the host vulnerable to cutaneous pathogen invasion. Our laboratory demonstrated that exposure to IR depleted murine cutaneous dendritic cells (cDCs); a network of cells responsible for forming a cutaneous immu-nological barrier that is critical in protecting the host against microorganism infection. This IR-induced depletion was not the result of IR-induced apoptosis, but rather a migration of cDCs out of the skin re-sponding to abnormal upregulation of cDC migratory factors, CCR7 and CCL21. As a result, this breakdown in the immunological barrier left the host susceptible to a cutaneous Candida albicans chal-lenge where the pathogen was not contained locally but rather disseminated systemically to the kid-neys; a serious form of disease that is described clinically and is often lethal. Interestingly, an intrader-mal treatment with IL-12 prevented the IR-induced depletion of cDCs offering a potential radiation countermeasure to preserve the cutaneous immunological barrier and perhaps prevent the lethal dis-semination of opportunistic pathogens following radiation exposure.

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#56 Myeloid-Derived Suppressor Cells Negatively Impact Trafficking of CD8 Effector T Cells in the

Tumor Microenvironment

Amy Ku1, Jason Muhitch1, Michael Diehl1, Scott I. Abrams1, and Sharon S. Evans1 1Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY

Immune based therapies offer compelling treatment options for cancer as they can generate site-specific and durable anti-tumor responses. The success of T cell-based immunotherapy depends on the trafficking of blood-borne cytotoxic T cells to tumor tissue in order to initiate contact-dependent killing of tumor targets. Our laboratory has previously demonstrated that deficits in effector T cell trafficking in multiple tumor models (e.g., CT26 colorectal carcinoma and B16 melanoma) is partially overcome by thermal preconditioning therapy (39.5 ± 0.5ºC for 6 h) which upregulates the vascular expression of the hallmark trafficking molecule intercellular adhesion molecule-1 (ICAM-1). There are a number of immunosuppressive cells (i.e., myeloid-derived suppressor cells, MDSC; regulatory T cells, Treg; and tumor-associated macrophages, TAM) that interfere with T cell function within tumor tissues, but whether these cells actively impede T cell entry into the tumor microenvironment remains an out-standing question. Here, we report on a newly discovered role of MDSC in limiting CD8 T cell traf-ficking within the tumor microenvironment. Analysis of a subset of thermally-resistant murine tumors (4T1 mammary, Polyoma Middle T-transgenic mammary, and Pan02 pancreatic tumors) revealed a strong correlation between high MDSC burden and the failure to boost CD8 T cell trafficking as deter-mined by intravital microscopic imaging. To investigate whether MDSC contribute to poor effector T cell trafficking, thermally-sensitive CT26 or B16 tumor cells were admixed with syngeneic CD11b+Gr-1+ MDSC isolated from spleens of tumor-bearing mice at a ratio of 2:1, thus mimicking the high MDSC burden detected in thermally-refractive tumors. Although the frequency of CD11b+Gr-1+ MDSC remained elevated within the tumor during tumor outgrowth, there was no impact on vessel density or baseline expression of ICAM-1 on tumor vessels. However, tumor vessels became refrac-tory to ICAM-1 induction by thermal preconditioning therapy in tumors admixed with MDSC. Taken together, these findings provide the first evidence that MDSC negatively regulate trafficking molecule expression in tumor vascular gateways and lay the foundation for future studies to identify the mecha-nisms underlying immunotherapy resistance in tumors with high MDSC burden. Supported by the NIH (CA79765, CA085183) and the Jennifer Liscott Tietgen Family Foundation.

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#57 A protective role for macrophages in pneumonic Francisella tularensis infection

Don Steiner, Yoichi Furuya, and Dennis W. Metzger

Albany Medical College, Albany, NY

Francisella tularensis is a highly infectious pathogen and a potential agent of biological terror-ism. F. tularensis is believed to replicate intracellularly and to target macrophages for infection; conse-quently, we hypothesized that depletion of the alveolar macrophage population would inhibit bacterial replication and protect mice from pneumonic F. tularensis infection. To the contrary, we found that mice treated intranasally with the phagocyte-depleting drug clodronate showed increased sensitivity to even low infectious doses of F. tularensis. Additional experiments showed that mice with a macro-phage-specific insensitivity to IFN-γ (interferon gamma; MIIG mice) exhibited similar sensitivity to intranasal F. tularensis infection, as well as increased bacterial burden in the lungs, indicating that sur-vival of infection requires macrophages to be present in the lungs and receptive to IFN-γ stimulation. MIIG mice also exhibited reduced neutrophil recruitment to the lungs, and systemic depletion of neu-trophils rendered wild-type mice extremely sensitive to infection. These results indicate that macro-phages are critical for protection against pneumonic F. tularensis infection and are likely to protect by recruiting neutrophils to the lungs.

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#58 Fcγ(gamma)RIIB limits protection against F. tularensis LVS challenge induced by

inactivated F. tularensis immunogen

Brian J. Franz1 and Ying Li2, Constantine Bitsaktsis3, Bibiana V. Iglesias4, Giang Pham1, Sarah Rosa1, and Edmund J. Gosselin1

1Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY 12208 2Regeneron Pharmaceuticals, 81 Columbia Turnpike, Rensselaer, NY 12144

3Department of Biological Sciences, Seton Hall University, South Orange, NJ 07079 4Regeneron Pharmaceuticals, 777 Old Saw Mill River Road, Tarrytown, NY, 10591

We have previously shown that intranasal immunization with inactivated Francisella tularensis live vaccine strain (LVS) targeted to Fc receptors (FcR) induces partial protection against a mucosal challenge with F. tularensis SchuS4 and that this protection is antibody (Ab) and IFN-γ(gamma) de-pendent. Fcγ(gamma)RIIB, the only inhibitory Fcγ(gamma)R, has been reported to regulate IgG pro-duction, which plays a major role in protection against F. tularensis LVS and SchuS4 challenge. Thus, we sought to determine the impact of Fcγ(gamma)RIIB on immune protection against F. tularensis in-fection. We utilized inactivated F. tularensis as an immunogen. Naïve or inactivated F. tularensis-immunized Fcγ(gamma)RIIB knockout versus wildtype C57BL/6 mice were challenged with varying doses of F. tularensis LVS. While we observed no difference in survival between naïve knockout ver-sus wildtype mice, knockout mice immunized with inactivated F. tularensis showed significantly in-creased survival as compared to similarly immunized wildtype mice. Also, both F. tularensis-specific IgG and IgA production were elevated in serum and bronchoalveolar lavage fluid from inactivated F. tularensis-immunized knockout versus wildtype mice, as was IFN-γ(gamma) production. In summary, our studies show that Fcγ(gamma)RIIB limits the level of protection against F. tularensis challenge generated by inactivated F. tularensis immunization, by limiting the production of F. tularensis-specific IgG, IgA, and IFN-γ(gamma), which have been shown to be important for protection against F. tularensis infection. (Supported by NIH grant numbers P01 AI056320 and R01 AI076408.)

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#59 Resistance to invasive pneumococcal infection in mice with acute allergic lung inflammation

Alan M. Sanfilippo, Yoichi Furuya and Dennis W. Metzger

Albany Medical College, Albany, NY

Streptococcus pneumoniae is the most common cause of bacterial pneumonia which results in 175,000 hospitalizations as well as 40,000 deaths in the United States annually. Recent work has iden-tified a link between asthma and invasive pneumococcal disease (IPD) prompting the Advisory Com-mittee on Immunization Practices to recommend that all adults with asthma receive vaccination. To investigate the effects asthma has on the immune response to a pneumococcal infection in the lung, we used an acute model of ovalbumin (OVA)-induced allergic lung inflammation (ALI) and hypothesized that mice with acute ALI would be more susceptible than naïve mice. Surprisingly, mice with acute ALI were more resistant to infection than naïve controls and this increased resistance was found to be greatest 10 days following the final OVA challenge. While inflammatory cell infiltration and cytokine levels in the bronchoalveolar lavage (BAL) fluid had returned to levels similar to naïve controls by day 10, this time point also correlated with increased IgA expression in the BAL fluid. Indeed, protection against IPD was lost in IgA KO mice with ALI as well as in naïve IgA KO mice, suggesting a critical role for non-specific IgA in protection against IPD. Accordingly, future experiments will address the role non-specific IgA plays in defense against pulmonary pneumococcal infections. (Supported by NIH grant R01 AI41715.)

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#60 Efficacy of gemcitabine in a mouse model of pancreatic cancer is lost at thermoneutrality: role of

cold-stress in preclinical models

Chelsey Reed, Jason Eng, Kathleen Kokolus, Elizabeth Repasky, Bonnie Hylander Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY

Cancer researchers rely on mouse models to test therapies and select promising ones for further evaluation in clinical trials. However, therapies which appeared promising often fail to achieve the same efficacy in the clinic. Gemcitabine is one such example which appeared efficacious in animal models but, in the clinic, resulted in minimal increased survival and became the first line treatment for pancreatic ductal adenocarcinoma largely due to better quality of life. Recent studies have shown that laboratory mice are metabolically stressed due to housing conditions and suggested that this could compromise the predictive power of these models. One underappreciated factor which causes signifi-cant stress in mice is ambient housing temperature (22-23 °C) which is several degrees below the ani-mals’ thermoneutral temperature (~30°C) and results in systemic & metabolic adaptations to maintain normal body temperature. Our lab has focused on how this cold-stress might affect outcomes of experi-ments using tumor models. We have found extensive differences in many aspects of tumor initiation, growth, response to therapy and critical elements of the anti-tumor immune response when comparing tumor models in mice housed at standard temperature vs. thermoneutrality.

Here, we investigated the effect of cold-stress and ambient housing temperature on the growth of the murine pancreatic tumor Pan02 and its response to gemcitabine. Our data shows that when Pan02 is grown in syngeneic C57BL/6 mice, the tumors grow vigorously at 22 ° C and this growth is significantly inhibited by gemcitabine. However, tumor growth itself is significantly suppressed by housing at 30 ° C and gemcitabine provided no additional benefit in controlling tumor growth. Inter-estingly, we found this difference is lost when tumors are grown in SCID mice, suggesting a significant role for the adaptive immune system. Recent literature has reported that, in addition to its reported kill-ing of tumor cells directly through apoptosis, gemcitabine specifically deletes immunosuppressive cells. From our initial findings, we hypothesize that the primary mechanism of action of gemcitabine in tumor-bearing mice maintained at 22°C is through targeting immunosuppressive cell populations and that if this population of cells is diminished (through housing mice at thermoneutrality) then the apparent efficacy of this drug is significantly diminished. These results point out the importance of conducting experiments under more than one set of conditions in order to understand the range of pos-sible responses and to be better prepared for what may occur in the clinic. Funded by NIH R01 CA135368 and Roswell Park Alliance Foundation.

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#61 Use of Listeria monocytogenes-based Vaccine to Enhance Cancer Radiation Therapy

Joanne Y.H. Lim, Scott A. Gerber, and Edith M. Lord

University of Rochester, Rochester, NY

Radiation therapy (RT) has been used for the treatment of cancer for over a century. It was

thought that the therapeutic capacity of radiation is solely dependent on direct cancer cell death by dou-ble-stranded DNA breaks. More recently, our lab and others have demonstrated that anti-tumor im-mune responses induced following RT play a critical role in the efficacy of the treatment. Despite the generation of an immune response, radiation therapy alone is often insufficient to overcome the immu-nosupressive microenvironment of tumors. Consequently, initial reduction in tumor sizes and growth delays occur, but tumors eventually continue to grow. Therefore, combining immunotherapy and RT to further enhance radiation-induced anti-tumor immunity represents a promising cancer treatment strat-egy.

Listeria monocytogenes (Lm) is an intracellular bacteria that elicits strong innate and adaptive immune responses in the infected host. This attribute led to the development of attenuated Lm as a vec-tor for cancer immunotherapy. Deletion of two virulence factors, ActA (ΔactA) and internalin B (ΔinlB) enables rapid clearance of the bacteria by hosts yet maintains immunopotency. ΔactA/ΔinlB Lm-expressing tumor-associated antigens has been demonstrated to generate effective anti-tumor re-sponses and is currently being tested in phase II clinical trials. In this study, we propose that combining OVA-expressing ΔactA/ΔinlB Lm with RT will further delay growth of B16-OVA melanoma, com-pared to either of the treatments alone. Indeed, although both Lm vaccine and RT delayed tumor growth when used as monotherapies, tumors in mice given the combination treatment (CT) grew even slower. Tumor control resulting from CT, as well as Lm vaccine alone, was abolished in mice depleted of CD8+ T cells, suggesting the induction of cytolytic T cells by Lm but not RT. To gain further insight into mechanisms of action, we examined tumor-infiltrating immune cells using flow cytometry. Our data indicated that CT promotes several aspects of anti-tumor immunity including an increase in CD69 expression on both CD4+ and CD8+ T cells, suggesting that these cells acquired a more activated phe-notype in response to CT. Moreover, we observed an increase in the number of NK cells, OVA-specific CD8+ T cells, and a decrease in the number of granulocytic MDSCs that are known to be immunosup-pressive.

Taken together, our data suggest that Lm vaccine when combined with RT, can result in better tumor control, and this is at least partly through enhancing immune responses generated against the tu-mor.

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#62 Intranasal Administration of Interleukin-12 Generates a Protective Immune Response against

Lung Methicillin-Resistant Staphylococcus aureus Infection

Quang-Tam Nguyen, Yoichi Furuya, Sean Roberts and Dennis W. Metzger Center for Immunology & Microbial Disease, Albany Medical College, Albany New York 12208

Methicillin-resistant Staphylococcus aureus (MRSA) represents the most common pathogen associated with nosocomial pneumonia and is an increasing cause of severe community-acquired pneu-monia. In the present study, the role of interleukin-12 (IL-12) in inducing protective immunity in the lungs against intranasal infection of mice with MRSA was investigated. It was found that IL-12 defi-cient mice showed a lower survival rate, a higher bacterial burden in both the lung and spleen, and a lower level of interferon gamma (IFN-ã) in the lung than wild-type (WT) mice. Furthermore, exoge-nous IL-12 treatment of WT mice 24 h before intranasal infection with a lethal dose of MRSA signifi-cantly improved bacterial clearance and resulted in 100% protection from death. These IL-12 treated mice had higher levels of IFN-ã in the lung and serum, and had a greater number of the lung neutro-phils and macrophages compared to control mice. We also examined the influence of linezolid, a potent antibiotic that inhibits bacterial protein synthesis. A combination therapy with linezolid and IL-12 fol-lowing intranasal infection with MRSA significantly increased survival compared to mice receiving linezolid or IL-12 alone. This protection was dependent upon expression of IFN-ã. Our results indicate that IL-12 can play an important role in protection against MRSA infection.

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#63 Stress erythropoiesis plays a key role in the initial response to inflammation.

Laura Bennett1, Robert F. Paulson1,2

1Intercollege Graduate Program in Genetics, 2 Department of Veterinary and Biomedical Sciences, and Center for Molecular Immunology and Infectious Disease, Pennsylvania State University,

University Park, PA 16802 Acute anemia results in a severe drop in hematocrit that cannot be compensated for by homeo-static bone marrow erythropoiesis. There is a dedicated stress response that is BMP4 dependent and results in extramedullary erythropoiesis to rapidly produce large numbers of red blood cells. Chronic inflammation has been shown to inhibit steady state bone marrow erythropoiesis, but very little is known about the relationship between chronic inflammation and stress erythropoiesis. Flexed-tail (f/f) mice have defects in stress erythropoiesis and show a delayed recovery from phenylhydrazine-induced anemia. When inflammation is induced in f/f mice, they exhibit a significantly higher mortality rate compared to wild-type C57BL/6 mice (56.6% vs 7.2%), suggesting an important role for stress erythro-poiesis in the initial response to inflammation. Inducing inflammation results in stabilization of Hif-2 alpha in the kidney followed by a rise in serum Epo despite the absence of anemia. We also observe an up-regulation of BMP4 and an increase in stress BFU-Es in the spleen at 24 hours after inducing in-flammation with the number of stress BFU-Es peaking at 36 hours. These data suggest that the stress erythropoiesis pathway is activated early in the response to inflammatory stimuli and play a key role in survival. Our data also demonstrate that the BMP4 dependent stress erythropoiesis pathway is activated in the absence of anemia and accompanying tissue hypoxia. These results suggest that novel signals induced by inflammation activate stress erythropoiesis. We are continuing to examine the role stress erythropoiesis plays in this initial response to inflammation and the mechanism through which the pathway is activated in this situation.

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#64 Retinoic acid supplementation of vitamin A deficient mice promotes clearance of an infection

with Citrobacter rodentium

Kaitlin L. McDaniel1,4, Katherine H. Restori2, A. Catharine Ross3 and Margherita T. Cantorna 2,4 1 Pathobiology Graduate Program; 2 Center for Immunology and Infectious Disease; 3 Department of

Nutritional Sciences; 4 Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, PA 16802

Vitamin A is critical for normal immune function and vitamin A supplementation has been shown to decrease mortality from infectious respiratory and diarrheal diseases in children. Here we determined the role of vitamin A on mucosal T cells and resistance to Citrobacter rodentium infection in mice. Vitamin A sufficient (A+) and RA supplemented vitamin A deficient mice cleared a C. roden-tium infection within 25 days of infection. Conversely, vitamin A deficient (A-) mice continued to shed over 10,000 C. rodentium up to 37 days following infection, indicating that C. rodentium is a chronic infection in A- mice. Supplementation of A- mice with RA after infection (d 14 post-infection) was also effective for clearance of C. rodentium. A- mice had fewer immune cells in the small intes-tine and colon compared to RA supplemented and A+ mice. In the small intestine the frequency of γδ T cells and CCR9+ γδ T cells were reduced in A- mice compared to A+ and RA treated mice. In the colon of A- mice, there were fewer CD8αα+ Tcrβ+ T cells and an increased frequency of CD4+ Tcrβ+ T cells. The expression of IL-22, an essential cytokine for barrier defenses against bacterial infections, was lower in the colon of uninfected A- mice compared to A+ mice. This work indicates that vitamin A is important for clearance of C. rodentium. Alterations of mucosal T cell populations and responses in A- mice that correlated with the inability of the A- mice to resolve the C. rodentium infection, sug-gesting that vitamin A is an important factor for gastrointestinal homeostasis and protection from infec-tion.

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Symposium III

Regulation of T cell Immunity

II

Chair Dr. Pamela Fink

Page 133: 16th Annual Upstate New York Immunology Conference · immunology. Selection is based on career promise and presentation of an outstanding first-author abstract selected for oral presentation

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Evolutionary conserved role of nonclassical MHC in invariant-T cell biology

Jacques Robert, Leon Grayfer, Nikesha Haynes, Maureen Banach, and Eva-Stina Edholm

Department of Microbiology & Immunology, University of Rochester Medical Center

In jawed vertebrates, classical MHC class Ia (class Ia) genes encode highly polymorphic and ubiqui-tously expressed molecules essential for the differentiation and function of conventional CD8 T cells. In addition, jawed vertebrates have variable numbers of heterogeneous nonclassical MHC class Ib (class Ib) genes, which encode molecules structurally similar to class Ia, but typically, with a more lim-ited tissue distribution and lower polymorphism. Increasing evidence suggests that some class Ib mole-cules play important roles in the development and function of unconventional invariant T cell popula-tions such as mammalian invariant natural killer T cells (iNKT) and mucosal-associated invariant T cells (MAIT). However, despite recent progress many aspects of class Ib involvement in bacterial, viral and tumor immunity are still poorly understood.

Using the amphibian Xenopus as an advantageous multifaceted in vivo model, we have demonstrated the evolutionary conservation and essential role of a class Ib gene in differentiation and function of a distinct iT cell subset. By means of Xenopus class Ib tetramers and RNAi loss-of-function by trans-genesis, we identified a prominent class Ib (XNC10)-dependent CD8/CD4 double negative iT cell sub-set in healthy frogs and tadpoles. Most notably, our findings indicate that this XNC10 class Ib molecule is critical to immune responses against viral infections and lymphoid tumor in young tadpoles when class Ia protein expression is suboptimal. To further investigate XNC10 in tumor immunity, we have developed a novel intra-vital semi-solid tumor model in transparent tadpoles in order to visualize leu-kocytes infiltration and tumor neo-vascularization in real time.

The restriction and requirement of class Ib molecules for development as well as antiviral and tumor immunity of a mammalian iNKT counterpart in the amphibian Xenopus show the importance of iT cells in the in the emergence, evolution and regulation of the vertebrate adaptive immune system.

Research Support: 2R24-AI-059830-06, Kesel Fund Award 20115123 (E.-S.E.).

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Alterations to memory CD8+ T cell formation in early life

Brian D. Rudd Assistant Professor

Department of Microbiology and Immunology Cornell University

Neonates are highly susceptible to infection and often experience more severe disease than adults. Recurrent infections with the same intracellular pathogen (e.g. RSV, Streptococcus) are com-mon, indicating an impaired ability to develop long-lasting protective CD8+ T cell immunity. How-ever, the basic mechanisms underlying the generation of poor memory CD8+ T cell responses in neo-nates are unknown, making it impossible to develop treatments and vaccines to promote effective CD8+ T cell immunity in early life. A key question is whether neonatal memory CD8+ T cell forma-tion is altered because of cell-intrinsic differences that are present prior to infection or environmental factors acting during the response. To differentiate these possibilities, we have used a basic approach of transferring neonatal and adult CD8+ T cells into adult recipient mice combined with modeling of T cell dynamics to understand the most important mechanisms contributing to poor CD8+ T cell immu-nity in early life. Unexpectedly, our data indicated that neonatal CD8+ T cells may form poor memory, not because of an inability to respond, but rather because they more quickly become terminally differ-entiated. Data illustrating these findings will be presented and discussed.

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Dynamics of tissue surveillance by CD4+ T cells at sites of inflammation

Michael Overstreet, Alison Gaylo, Alison Billroth-MacLurg, Angie Hughson, Dillon Schrock and Deborah J Fowell

David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester NY

Fundamental to immune-mediated protection is the ability of leukocytes to survey barrier sur-faces and quickly respond to infection or tissue damage. Signals from cytokines, chemokines and cells within the tissue provide critical cues for T cell migration into and within sites of inflammation, but the mechanics of T cell motility in response to these signals is poorly understood. Elegant studies in the lymph node have defined the structures and mechanochemical processes for T cell migration. T cells move along an extracellular matrix (ECM)-based reticular network interacting, not with the matrix it-self but, with fibroblastic reticular cells that ensheath the ECM. Migration is guided by chemokines and driven by the actinomyosin cytoskeleton rather than integrin adhesion. These conduits appear to pro-vide a scaffold that promotes T cell-DC interactions. Far less is known about how T cells scan and lo-cate APCs in the inflamed tissue.

We find that the ability to effectively move through inflamed tissue is dependent on the integrin αv (alpha v) binding to RGD-containing ECM components. The expression of αv is upregulated in ef-fector T cells in the lymph node and ‘marks’ effector T cells that are destined to exit the LN. Such upregulation of αv occurs in response to a number of disparate inflammatory signals (Th1 and Th2 in-ducing pathogens) suggesting αv expression is linked to the fundamental programming of effector T cell functions, similar to the upregulation of CD44. The extravasation of effector T cells from the blood into the inflamed tissue is independent of this matrix-binding integrin. However, once within the tissue, effector T cells utilize αv to survey the inflamed site. Modulation of αv-expression, by Ab blockade or shRNA knock-down strategies, significantly impairs effector T cell motility and attenuates T cell production of effector cytokines. Thus, matrix-binding integrins are key regulators of immune function and can be used to modulate cytokine responses in the inflamed/infected tissue. Defining key parameters for efficient T cell surveillance and effector activity in the inflamed dermis will inform new inflammation-specific therapies.

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Alterations to memory CD8+ T cell formation in early life

Brian D. Rudd Assistant Professor

Department of Microbiology and Immunology Cornell University

Neonates are highly susceptible to infection and often experience more severe disease than

adults. Recurrent infections with the same intracellular pathogen (e.g. RSV, Streptococcus) are com-mon, indicating an impaired ability to develop long-lasting protective CD8+ T cell immunity. How-ever, the basic mechanisms underlying the generation of poor memory CD8+ T cell responses in neo-nates are unknown, making it impossible to develop treatments and vaccines to promote effective CD8+ T cell immunity in early life. A key question is whether neonatal memory CD8+ T cell forma-tion is altered because of cell-intrinsic differences that are present prior to infection or environmental factors acting during the response. To differentiate these possibilities, we have used a basic approach of transferring neonatal and adult CD8+ T cells into adult recipient mice combined with modeling of T cell dynamics to understand the most important mechanisms contributing to poor CD8+ T cell immu-nity in early life. Unexpectedly, our data indicated that neonatal CD8+ T cells may form poor memory, not because of an inability to respond, but rather because they more quickly become terminally differ-entiated. Data illustrating these findings will be presented and discussed.

Page 137: 16th Annual Upstate New York Immunology Conference · immunology. Selection is based on career promise and presentation of an outstanding first-author abstract selected for oral presentation

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Dynamics of tissue surveillance by CD4+ T cells at sites of inflammation

Michael Overstreet, Alison Gaylo, Alison Billroth-MacLurg, Angie Hughson, Dillon Schrock and Deborah J Fowell

David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester School of Medicine

and Dentistry, Rochester NY

Fundamental to immune-mediated protection is the ability of leukocytes to survey barrier sur-faces and quickly respond to infection or tissue damage. Signals from cytokines, chemokines and cells within the tissue provide critical cues for T cell migration into and within sites of inflammation, but the mechanics of T cell motility in response to these signals is poorly understood. Elegant studies in the lymph node have defined the structures and mechanochemical processes for T cell migration. T cells move along an extracellular matrix (ECM)-based reticular network interacting, not with the matrix it-self but, with fibroblastic reticular cells that ensheath the ECM. Migration is guided by chemokines and driven by the actinomyosin cytoskeleton rather than integrin adhesion. These conduits appear to pro-vide a scaffold that promotes T cell-DC interactions. Far less is known about how T cells scan and lo-cate APCs in the inflamed tissue.

We find that the ability to effectively move through inflamed tissue is dependent on the integrin αv (alpha v) binding to RGD-containing ECM components. The expression of αv is upregulated in ef-fector T cells in the lymph node and ‘marks’ effector T cells that are destined to exit the LN. Such upregulation of αv occurs in response to a number of disparate inflammatory signals (Th1 and Th2 in-ducing pathogens) suggesting αv expression is linked to the fundamental programming of effector T cell functions, similar to the upregulation of CD44. The extravasation of effector T cells from the blood into the inflamed tissue is independent of this matrix-binding integrin. However, once within the tissue, effector T cells utilize αv to survey the inflamed site. Modulation of αv-expression, by Ab blockade or shRNA knock-down strategies, significantly impairs effector T cell motility and attenuates T cell production of effector cytokines. Thus, matrix-binding integrins are key regulators of immune function and can be used to modulate cytokine responses in the inflamed/infected tissue. Defining key parameters for efficient T cell surveillance and effector activity in the inflamed dermis will inform new inflammation-specific therapies.

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Symposium IV

Immune Dysfunction

Chair

Dr. Jim Gorham

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Maternal Influence on Development of Food Allergy in the Infant

Kirsi M Järvinen, M.D., Ph.D.1,2 , Jennifer E. Westfall Ph.D.1, Magdia De Jesus, Ph.D.3, Nick Mantis, Ph.D.3, Dennis W. Metzger, Ph.D.1, Hugh A. Sampson, M.D.2 and Cecilia Berin, Ph.D.2

1 Department of Medicine & Center for Immunology and Microbial Diseases, Albany Medical Col-lege, Albany, NY; 2 Division of Pediatric Allergy & Immunology and Jaffe Institute for Food Allergy, Icahn

School of Medicine at Mt. Sinai, New York, NY; 3 Division of Infectious Diseases, Wadsworth Center, New York State Department of Health, Albany, NY

Background. The role of maternal diets restricting allergenic foods during pregnancy / lactation in preven-tion of food allergy is under debate. Little is known regarding the effects of such diets on breast milk com-position, antigen uptake or induction of humoral responses in the offspring. Objective. 1) To assess the association between maternal diatery antigen avoidance during pregnancy / breastfeeding and specific IgA levels in breast milk, and development of antibodies and food allergy in off-spring. 2) To determine the role of breast milk in food antigen uptake. Methods. We utilized breast milk, maternal and infant sera from a prospective birth cohort of 145 dyads. Maternal sera and milk were assessed for casein and beta-lactoglobulin (BLG)-specific IgA and IgG; 37 mothers initiated cow’s milk (CM) avoidance diet due to a family history or infant’s symptoms. Infants’ sera were assessed for CM antibodies; CM allergy was confirmed by an oral food challenge. The impact of breast milk on BLG uptake was assessed in transcytosis assays utilizing a Caco-2 human intestinal epithe-lial cell line. In mice, maternal IgG antibodies are actively taken up by the neonatal γ receptor (FcRn). Util-izing a murine model of peanut (PN) allergy, PN tolerant females were either fed or not fed PN during preg-nancy and lactation; serum and milk PN antibodies were measured. After weaning, offspring were subjected to a PN sensitization or oral tolerance protocol and antibodies assessed. To investigate the impact of murine milk on antigen uptake, mice were gavaged with FITC-labeled PN +/- immunized murine milk; at 24 hrs mice were sacrificed and Peyer’s patches harvested for immunostaining. Results. In human, mothers avoiding CM had lower casein- and BLG-specific IgA in milk (p=0.019 and p=0.047), their infants had lower serum casein- and BLG-specific IgG1 (p=0.025 and p<0.001) and BLG-specific IgG4 levels (p=0.037) and their casein- and BLG-specific IgA was less often detectable (p=0.003 and p=0.007) than in those with no CM elimination diet. Lower CM-specific IgG4 and IgA levels in turn were associated with infant allergy. Transcytosis of BLG was impaired by milk with high, but not with low levels of specific IgA. In mice, maternal serum PN-IgG1 and IgG2a levels correlated with antibody levels seen in milk and in offspring at weaning (p<0.001 and p=0.002), but maternal PN exposure during preg-nancy & lactation had no impact on antibody levels in milk, offspring serum at weaning or sensitization / tolerance development. In vivo studies to assess the impact of milk on PN uptake are ongoing. Conclusions. In humans, maternal CM ingestion was associated with higher levels of specific IgA in breast milk and specific IgG in serum, and prevention of allergy in infants. Transcytosis studies suggested that specific IgA in breast milk may play a role in preventing excessive, uncontrolled food antigen uptake in the gut lumen. In mice, maternal PN exposure had no impact on antibody levels in milk or offspring nor sensiti-zation / tolerance development. These differences between human and mouse may be due to differences in maternal transfer of antibodies or antigen used. Randomized controlled clinical studies are needed to address the impact of maternal exposure to PN on allergy risk in the infant.

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Mitochondrial Overdrive: The metabolic dysfunction of SLE PBL, and the effect of NAC treatment

Edward Doherty and Andras Perl

Department of Microbiology and Immunology SUNY Upstate Medical University, Syracuse, NY 13210

Systemic lupus erythematosus (SLE) peripheral blood lymphocytes (PBL) show mitochondrial

dysfunction that has been characterized by elevated mitochondrial transmembrane potential (Δψm) and mass and attributed to increased production of nitric oxide (NO) and depletion of GSH. To understand the molecular bases of mitochondrial dysfunction we measured the activity of the electron transport chain (ETC) and its regulation by N-acetylcysteine (NAC) that reversed GSH depletion and improved disease activity in SLE. PBL from 65 SLE patients and 30 healthy controls, matched for patients’ age within ten years, gender, and ethnicity were studied. Compared to controls (2.137nmol/ml/min±0.153; p=0.027), O2 consumption by lupus PBL was increased at baseline (2.492nmol/ml/min±0.196) and with complex IV substrates (SLE: 7.722nmol/ml/min ±0.419, control: 7.006nmol/ml/min ±0.505; p=0.028). SLE PBL also consumed more O2 upon in-chamber T-cell activation (SLE: 4.157nmol/ml/min ±0.186, control: 3.655nmol/ml/min ±0.167; p=0.012). After overnight CD3/CD28 pre-stimulation SLE PBL consumed less O2 (SLE: 2.535nmol/ml/min±0.271. control: 3.208nmol/ml/min±0.206; p=0.016), however, their respiration was increased by complex I (SLE: 1.606nmol/ml/min±0.273, con-trol: 0.709nmol/ml/min±0.169; p= 0.001) and complex IV substrates (SLE: 7.212nmol/ml/min±0.572, control: 6.341nmol/ml/min±0.517; p= 0.040). Respiration through complex I remained elevated after compensating for increased mitochondrial mass of lupus PBL. SLE PBL resisted NO-mediated inhibi-tion of respiration (SLE: 1.405nmol/ml/min ±0.206; control: 1.277nmol/ml/min ±0.150 one-way ANOVA p=0.026). NAC diminished the Δψm /mitochondrial mass ratio and respiration through com-plex I both in control (untreated: 2.433 nmol/ml/min ±0.283, NAC-treated: 1.019 nmol/ml/min ±0.161; p=0.005) and SLE PBL (untreated: 2.820 nmol/ml/min ±0.360, NAC-treated: 1.231 nmol/ml/min ±0.163; p=0.0001) and normalized H2O2 levels in lupus PBL. The results of this study suggest that SLE PBL have defective ETC activity at complex I. While the drug NAC helps maintain a reducing envi-ronment, possibly by directly blocking respiration at complex I.

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The Ron receptor tyrosine kinase, macrophage heterogeneity and obesity associated disease

1,2,3Pamela Hankey, 1,2Shan Yu and 1,3Joselyn Allen.

The Pennsylvania State University, 1Department of Veterinary and Biomedical Sciences, 2Graduate Program in Physiology, 3Graduate Program in Immunology and Infectious Disease.

Macrophages play a central role in the progression of obesity associated disease. Macrophages are generally classified into two distinct programs, termed M1 and M2 macrophages, polar ends of a spectrum of potential macrophage activation states. The ability to tip the balance of macrophage activa-tion away from inflammatory M1 cells and toward a reparative M2 phenotype could be beneficial in the treatment of chronic inflammation associated with obesity. The Ron receptor is expressed on tissue resident macrophages and plays a critical role in suppressing inflammation while promoting hallmarks of M2 macrophage activation. Here we show that Ron is expressed in a subset of macrophages in the atherosclerotic lesions and fatty liver of mice on a high fat diet. The Ron positive macrophages are characterized by high levels of arginase expression and low levels of iNOS, indicating that Ron expres-sion is tightly associated with a reparative macrophage phenotype in diseased tissues in vivo. Further-more, the percentage of Ron positive cells in the lesions and liver decreases with increasing obesity, suggesting these cells are either lost or that Ron expression is actively suppressed in response to chronic inflammation. Using Ron-deficient mice, we demonstrate that Ron plays a protective role in the progression of atherosclerosis and hepatic steatosis in obese animals, associated with decreased in-flammatory cytokine expression and decreased lipid deposition in these tissues. In lean animals, Ron is highly expressed on resident macrophages in adipose tissue. Upon weight gain, the expression of Ron is decreased and there is shift in the relative percentages of M1 (CDllc+) vs. M2 (CD11c-) cells in Ron deficient mice, favoring an inflammatory environment. With time, the Ron-deficient mice exhibit in-creased glucose tolerance and insulin resistance. Taken together, these results indicate that Ron plays a critical role in regulating macrophage heterogeneity and chronic inflammation in obese animals.

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Scientific Writing Presentation

“Do’s and Don’ts When Writing and Publishing a Scientific Manuscript”

Pamela J. Fink, Ph.D.

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Keynote Speaker

Christopher A. Hunter, Ph.D. Professor and Chairman

Department of Pathobiology University of Pennsylvania Philadelphia, Pennsylvania

“Imaging the Immune System to Infection”

The development of genetically encoded fluorescent reporters to study immune function combined with advances in intravital imaging has afforded many opportunities to visualize the function of the immune system in situ, in real time. Using the genetically trac-table pathogen Toxoplasma gondii, several model systems have been developed that al-low us to visualize the motility of effector CD8 T cell responses and Treg populations dur-ing this infection. Combined with mathematical approaches to describe these populations it becomes apparent that the behavior of these populations are fundamentally different and may be linked to their distinct functions in the immune system.

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Symposium V

Immunomodulation

Chair Dr. Nick Mantis

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High immunosuppressive burden in advanced hepatocellular carcinoma patients: Can effector functions be restored?

Suresh Kalathil1, Amit A. Lugade1, Austin Miller2, Renuka Iyer3, and Yasmin Thanavala1

Departments of 1Immunology, 2Biostatistics, and 3Medicine, Roswell Park Cancer Institute, Buffalo, New York

The extent to which T-cell–mediated immune surveillance is impaired in human cancer remains a question of major importance. Hepatocellular carcinoma (HCC) is the fifth-most common cancer in the world and the third highest cause of cancer related mortality globally. HCC develops in patients with chronic hepatitis, either due to chronic hepatitis B or C virus infection, due to inflammation fol-lowing aflatoxin ingestion or excessive alcohol consumption. We have recently completed the first global analysis of immune dysfunction in patients with advanced hepatocellular carcinoma (HCC). The immune dysfunction elicited by developing neoplasms occurs at several checkpoints during tumor progression and overlapping immunosuppressive mecha-nisms act in concert for malignant cells to evade antitumor immunity. Using multi-parameter fluores-cence-activated cell sorting analysis, we quantified the cumulative frequency of regulatory T cells (Treg), exhausted CD4+ helper T cells, and myeloid-derived suppressor cells (MDSC) to gain concur-rent views on the overall level of immune dysfunction in these inoperable patients. We found aug-mented numbers of Tregs, MDSC, PD-1+–exhausted T cells, and increased levels of immunosuppres-sive cytokines in patients with HCC, compared with normal controls, revealing a network of potential mechanisms of immune dysregulation in patients with HCC. We reasoned that these processes by dampening T-cell–mediated antitumor immunity, may facilitate HCC progression and thwart the effi-cacy of immunotherapeutic interventions. In testing this hypothesis, we showed that combined regi-mens to deplete Tregs, MDSC, and PD-1+ T cells in patients with advanced HCC restored production of granzyme B by CD8+ T cells, reaching levels observed in normal controls and also modestly in-creased the number of IFN-g producing CD4+ T cells. Our findings suggest that in the clinical setting further enhancement of endogenous antitumor responses will have to rely on the "science of combina-tion." Thus, immunosuppressive cell depletion along with concomitant expansion of effector T cells in conjunction with immunotherapeutic strategies may be effective for patients with HCC.

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STING is dispensable for protection of mice from HSV-1 encephalitis following corneal challenge

Zachary M. Parker, Aisling A. Murphy, David A. Leib

Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon, NH

Rapid recognition of pathogen associated molecular patterns (PAMPs) is critical for host sur-vival from infection. TLR-independent responses to foreign DNA mediate a swift interferon response to invading DNA containing microbes. One such TLR- independent pathway hinges on the stimulator of interferon genes (STING) protein which responds to both DNA as well as cyclic dinuclotides in the cytoplasm, generating a robust antiviral response to herpes simplex virus 1 (HSV-1) and other patho-gens. While STING-deficient mice succumb rapidly to HSV-1 infection following intracerebral and intravenous infection, corneally infected mice are resistant to HSV-1 challenge. To further elucidate the effect of STING on viral pathogenesis and tropism, we used a luciferase- expressing HSV-1 strain in conjunction with bioluminescence imaging to monitor real-time infection in vivo. This approach re-vealed increased bioluminescence, correlating with increased viral replication in the oculofacial skin of corneally infected STING-/- mice, but surprisingly, these mice eventually cleared the virus. Our data are consistent with the hypothesis that STING is required in a tissue-specific fashion for an efficient re-sponse to viral infection.

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Leaving Immunosuppression Out in the Cold: New Insights into the Role of Thermoregulatory Metabolism in the Anti-Tumor Immune Response

Elizabeth A. Repasky, Ph.D.

Dept. of Immunology Roswell Park Cancer Institute, Buffalo, NY 14263

[email protected]

This presentation will highlight new research in our laboratory demonstrating that tumor growth control by the immune response is significantly impaired by the sub-thermoneutral ambient tempera-ture (TA) standard in all research mouse colonies, a temperature which is substantially cooler than the TA mice would select in nature and which results in an increase in adaptive thermogenesis. Moreover, our data reveals that the ratio between regulatory cells (Treg and MDSC) and anti-tumor effectors (Ag-specific CD8+ T cells) is significantly increased by chronic cold stress typically experienced by our tumor bearing mice. We have found that tumor bearing mice actually prefer a TA much higher than non-tumor-bearing mice and overall, our findings suggest the hypothesis that the immune response against tumors in mice housed at sub-thermoneutrality is actively suppressed when metabolic resources are required to sustain adaptive thermogenesis. Our data also points to a major role for the stress hor-mone norepinephrine in regulation of immunosuppression. A better understanding of the metabolic interrelationships between overall energy allocation and the immune system could lead to improved immunotherapeutic strategies.

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Symposium VI

Antigen Presenting Cell Biology

Chair

Dr. Jim Drake

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Dynamics of Antigen Sampling by Peyer’s Patch Dendritic Cells

Magdia de Jesus, Maria O. Hernandez, Sarita Ahlawat and Nicholas J Mantis* Division of Infectious Diseases, Wadsworth Center, NY State Department of Health,

Albany, NY 12208

Peyer’s patches (PPs) are macroscopic aggregates of lymphoid follicles present throughout the small intestines of humans and mice and are the primary sites where luminal antigens, commensal bac-terial, and pathogenic microbes are sampled by the mucosal immune system. PPs are also where im-munological “decisions” are made between the onset of oral tolerance and the onset of mucosal immu-nity, which characterized in large by the production of secretory IgA (SIgA) antibodies. We are inter-ested in the dynamics of antigen sampling by PP dendritic cells (DCs) and factors that influence SIgA responses to oral vaccines. Glucan particles (GPs) are 2-4 μm hollow, porous particles composed of 1,3-β-D-glucan that have been effectively used for oral targeted-delivery of a wide range payloads, including small mole-cules, siRNA, DNA and protein antigens. Here we report that, following transepithelial transport, fluo-rescently labeled-GPs accumulate in CD11c+ DCs situated in PP sub-epithelial dome (SED) regions. Phenotypic immunoanalysis identified the GP-containing DCs as CD11b-, CD8α- , and lysozyme M- , indicating that they are the so-called “double negative” (DN) population of PP DCs. We found that the GP-bearing CD11c+ DN population of DCs do not express measurable levels of Dectin-1 or the man-nose receptor (MR), but were invariably positive for Langerin (CD207), a C-type lectin receptor (CLR) with known specificity for β-D-glucan and highly mannosylated glycans. GP-bearing Langerin+ DCs were capable of sampling additional GPs, as well as fluorescent poly(lactic-co-glycolic acid) nanoparti-cles, and remained in the SED >72 h. However, GPs were also detected in mesenteric lymph nodes as early as 15 min following oral gavage, suggesting that a subset of the Peyer’s patch DCs migrate to draining lymph nodes immediately following particle uptake. These data highlight the complex dy-namics of particle sampling by Peyer’s patch DCs and have important implications for the future ef-forts aimed at mucosal delivery of molecular payloads, including vaccines and therapeutics In an effort to capture the complexity and ever-changing nature of PPs in response to antigens and vaccines, we have developed a novel macro for Fiji, the open source image-processing package based on ImageJ, that enabled the stacking and linear blending of serial cryosections and the three di-mensional (3D) reconstruction of immunofluorescent-labeled mouse Peyer’s patches. By simultane-ously labeling cyrosections for surface markers CD45R, CD3 and CD11c, we provide a 3D image as well as quantitative measures of B cells, T cells and DCs populations at steady state and following ex-posure to the mucosal adjuvant cholera toxin. This technology will facilitate the tracking of GP-containing DCs as they migrate within the PP and as they make their way to the MLN.

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Sensing a REAL virus – Trafficking of Poxviral DNA and the Innate Immune Sensor TLR9

Yayoi Izu1, Tracy E. Krouse1, Mary Ellen Truckenmiller1, Lauren A. Weiler1, William A. Rose III2, Luis J. Sigal3, Cynthia A. Leifer2, Chris C. Norbury1

Department: 1Immunology & Microbiology, 2 Cornell University College of Veterinary Medicine, Ithaca, NY, 3 Fox Chase Cancer Center, Philadelphia, PA

Double-stranded DNA is recognized by Toll-like receptor 9 (TLR9) in endosomes and ly-sosomes within antigen presenting cells, leading to the initiation of innate and adaptive immune re-sponse. The majority of studies examining recognition of dsDNA have used exogenous DNA, despite the proposed importance of this pathway in recognition of virus-encapsulated DNA. Ectromelia virus (ECTV) is a member of the orthopoxvirus family that causes mousepox, a lethal infection in suscepti-ble mouse strains. Wild-type C57BL/6 mice are resistant to ECTV infection, but mice lacking TLR9 (but not any other TLR) die following challenge, indicating a requirement for recognition of the double stranded DNA genome of ECTV by TLR9 in protective immunity. Here, we track the internalization of DNA within ECTV virions and demonstrate a novel pathway for the activation of TLR9 in dendritic cells (DC) in response to mousepox infection. ECTV was internalized along with a fluid phase marker in an actin-dependent entry pathway that resembles “spacious phagocytosis”. In response to ECTV en-try, TLR9 translocates rapidly to the primary phagosomes where it colocalizes with viral DNA. Sur-prisingly, in contrast to the published results using CpG DNA, the viral DNA and TLR9 colocalize with a lysosomal marker, Lamp1, but not early endosome markers, Rab5 and EEA1, suggesting that the ECTV-containing primary phagosomes fuse directly with lysosomes. Following exposure to the proto-typic TLR9 ligands CPGA and CPGB Inhibition of lysosomal acidification blocks the nuclear localiza-tion of both NF-KB and IRF7, essential steps in activation of the dendritic cells. However, ECTV-triggered nuclear localization of NF-KB and IRF7 does not require lysosomal acidification, indicating that signaling occurs from a distinct intracellular compartment. Taken together, these results indicate BMDC engulf ECTV through formation of “macropinocytosis-like” spacious phagosomes that deliver their contents directly to lysosomal compartments, altering the potential functional outcomes of TLR9 ligation following exposure to ECTV.

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Please join us next year for the

17th Annual Upstate New York Immunology Conference

October 19-22, 2014

at

The Sagamore Resort and Conference Center

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Amobi, Adaobi Roswell Park Cancer Institute 716-583-0875 [email protected] Anderson, Christopher University of Rochester 585-729-6838 [email protected] Avram, Dorina Albany Medical College 518-262-6731 [email protected] Balandaram, Gaythri Pennsylvania State University 269-873-2039 [email protected] Banik, Debarati Roswell Park Cancer Institute 716-845-3352 [email protected] Barlow, Margaret University of Rochester 216-410-5140 [email protected] Bellville, Dawn Albany Medical College 518-262-5365 [email protected] Bennett, Laura Pennsylvania State University 814-863-5613 [email protected] Berry, Gregory Pennsylvania State University 610-739-5074 [email protected]

Bevan, Michael University of Washington 206-802-8476 [email protected] Bhandari, Sadikshya University of Connecticut 580-647-0228 [email protected] Billroth-MacLurg, Alison University of Rochester 585-273-2902 [email protected] Boule, Lisbeth University of Rochester 585-275-2013 [email protected] Burkard, Lauren Roswell Park Cancer Institute 716-207-5269 [email protected] Bucsek, Mark Roswell Park Cancer Institute 814-573-6974 [email protected] Bynoe, Margaret Cornell University 607-253-4023 [email protected] Callaway, Justin University of North Carolina 919-966-2662 [email protected] Capece, Tara University of Rochester 585-273-1435 [email protected]

NYIC 2013 Participant Contact Information

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Celaj, Stela Dartmouth Medical College 508-873-0306 [email protected] Cohen, Sara Cornell University 607-273-3270 [email protected] Cojohari, Olesea SUNY Upstate Medical University 315-944-5434 [email protected] Connell, Terry University at Buffalo 716-829-3364 [email protected] Connelly, Kelli University of Rochester 585-275-6746 [email protected] Consiglio, Camila Roswell Park Cancer Institute 716-845-4106 [email protected] Davies, Michael Pennsylvania State University 717-531-0624 [email protected] de la Rosa, Gonzalo University of Rochester 585-273-1437 [email protected] Denkers, Eric Cornell University 607-253-4022 [email protected] Dhume, Shaffina Baxter BioTherapeutics 203-272-1638 [email protected]

Doherty, Edward SUNY Upstate Medical University 315-464-5486 [email protected] Drake, James Albany Medical College 518-262-9337 [email protected] Egan, Shawn Roswell Park Cancer Institute 716-845-8167 [email protected] Egilmez, Nejat University at Buffalo 716-829-6059 [email protected] Elliott, Michael University of Rochester 585-273-4793 [email protected] Fink, Pamela University of Washington 206-685-3608 [email protected] Fowell, Deborah University of Rochester 585-273-3680 [email protected] Franz, Brian 518-262-1650 Albany Medical College [email protected] Gaylo, Alison University of Rochester 585-273-2902 [email protected] Gerber, Scott University of Rochester 585-275-6747 [email protected]

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Gilmore, Nikesha University of Rochester 585-275-5359 [email protected] Gomez, Audry Fernandez Roswell Park Cancer Institute 716-845-4106 [email protected] Gorham, James Dartmouth College 603-650-8373 [email protected] Greene, Christopher University at Buffalo 716-829-5426 [email protected] Hankey, Pamela Pennsylvania State University 814-692-4978 [email protected] Harton, Jonathan Albany Medical College 518-262-4445 [email protected] Herman, Katherine University of Rochester 513-225-5766 [email protected] Herrera, Cristina Wadsworth Center 518-402-4081 [email protected] Hoekstra, David Roswell Park Cancer Institute 716-845-8231 [email protected] Hu, John University at Buffalo 585-991-8047 [email protected]

Huang, Weishan Cornell University 607-253-4014 [email protected] Hunter, Christopher University of Pennsylvania 215-573-7772 [email protected] Jarvinen-Seppo, Kirsi Albany Medical College 518-262-9824 [email protected] Joshi, Gaurav University of Connecticut 732-476-7402 [email protected] Kalia, Vandana Pennsylvania State University 814-863-8533 [email protected] Katkere, Bhuvana Pennsylvania State University 518-339-8133 [email protected] Kennedy, Jeff Wadsworth Center 518-486-4395 [email protected] Khan, Irfan Albany Medical College 518-262-9039 [email protected] Kirimanjeswara, Girish University of Rochester 814-863-5350 [email protected] Kokolus, Kathleen Roswell Park Cancer Institute 716-845-3288 [email protected]

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Kosanovich, Jessica University at Buffalo 724-831-6731 [email protected] Ku, Amy Roswell Park Cancer Institute 716-845-3157 [email protected] Lawrence, B. Paige University of Rochester 585-275-1974 [email protected] Le, Thi Hong Nga Albany Medical College 518-262-4447 [email protected] Leifer, Cynthia Cornell University 607-253-4258 [email protected] Levinson, Kara Wadsworth Center 518-402-4081 [email protected] Lim, Joanne University of Rochester 585-275-6747 [email protected] Leigh, Nicholas Roswell Park Cancer Institute 716-845-3553 [email protected] Liu, Kun Albany Medical College 518-262-6220 [email protected] Lord, Edith University of Rochester 585-275-5855 [email protected]

Ludewick, Herbert Albany Medical College 518-262-0096 [email protected] Lynes, Michael University of Connecticut 860-486-4350 [email protected] MacNamara, Kate Albany Medical College 518-262-0921 [email protected] Mansouri, Samira Albany Medical College 518-262-6690 [email protected] Mantis, Nicholas Wadsworth Center, NYSDOH 518-402-2750 [email protected] Markley, Rachel Pennsylvania State University 610-613-1406 [email protected] McCabe, Amanda Albany Medical College 518-262-0922 [email protected] McDaniel, Kaitlin Pennsylvania State University 307-202-1734 [email protected] Metzger, Dennis Albany Medical College 518-262-6750 [email protected] Meyers, Jennifer American Association of Immunologists 301-634-7146 [email protected]

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Meyers, Jessica University of Rochester 585-275-2013 [email protected] Mohrs, Markus Trudeau Institute 518-891-3080 [email protected] Moon, Taylor University of Rochester 315-796-7728 [email protected] Murphy, Patrick University of Rochester 585-520-2194 [email protected] Murthy, Aditi University of Rochester 508-813-3363 [email protected] Netherby, Colleen Roswell Park Cancer Institute 716-845-3352 [email protected] Ngyuen, Quang-Tam Albany Medical College 518-262-6220 [email protected] Norbury, Christopher Pennsylvania State University 717-531-7204 [email protected] O’Connor, William Albany Medical College 518-262-6548 [email protected] O’Hara, Joanne Wadsworth Center 518-402-4081 [email protected]

Parker, Zachary Dartmouth College 919-619-0097 [email protected] Peppenelli, Megan SUNY Upstate Medical University 315-723-2260 [email protected] Periasamy, Sivakumar Albany Medical College 518-262-4447 [email protected] Perry, Ian University of Rochester 585-697-4798 [email protected] Pietrosimone, Kathryn University of Connecticut 860-486-3648 [email protected] Podolsky, Michael Pennsylvania State University 215-378-3215 [email protected] Ravindran, Anand Pennsylvania State University 814-777-2851 [email protected] Reed, Chelsey Roswell Park Cancer Institute 716-946-4571 [email protected] Reilly, Emma University of Rochester 585-275-2013 [email protected] Repasky, Elizabeth Roswell Park Cancer Institute 716-845-3133 [email protected]

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Rice, James University of Connecticut 860-486-3648 [email protected] Robert, Jacques University of Rochester 585-275-1722 [email protected] Rudd, Brian Cornell University 607-253-3486 [email protected] Sahler, Julie University of Rochester 585-752-4825 [email protected] Sanfilippo, Alan Albany Medical College 518-462-6220 [email protected] Sass, Stephanie Roswell Park Cancer Institute 716-845-8167 [email protected] Schrock, Dillon University of Rochester 585-273-2909 [email protected] Simmons, Katrina Wadsworth Center, 518-210-6077 [email protected] Smith, Kayla Pennsylvania State University 717-781-5885 [email protected] Smith, Norah Cornell University 607-227-0721 [email protected]

Soucy, Alicia Albany Medical College 518-262-6220 [email protected] Spangler, Haley Roswell Park Cancer Institute 716-845-4348 [email protected] Steiner, Don Albany Medical College 518-262-6220 [email protected] Taffet, Steven SUNY Upstate Medical University 315-464-5419 [email protected] Thanavala, Yasmin Roswell Park Cancer Institute 716-845-8536 [email protected] Twum, Danielle Roswell Park Cancer Institute 413-230-0984 [email protected] Tucker, Heidi Albany Medical College 518-262-5395 [email protected] Uddin, Mohammad Albany Medical College 518-262-7499 [email protected] Utley, Adam Roswell Park Cancer Institute 336-847-4725 [email protected] Vance, Dave Wadsworth Center 518-402-4081 [email protected]

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Wang, Jocelyn Cornell University 607-379-3219 [email protected] Watson, Neva SUNY Upstate Medical University 203-858-5889 [email protected] Weiler, Lauren Pennsylvania State University 717-531-0624 [email protected] Williamson, David Pennsylvania State University 814-769-9805 [email protected] Winans, Bethany University of Rochester 585-275-2013 [email protected] Xu, Yuexin University of Rochester 585-305-9405 [email protected] Yermakova, Anastasiya Wadsworth Center 518-402-4081 [email protected] Zhang, Yubin Albany Medical College 518-262-0922 [email protected]

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Authors Index

KS - Keynote Speaker

O - Oral Poster Presentation

P# - Poster Number

S – Speaker Page (s)

***********************************************************************************

Abid, Anna ................................................................................................... 52, 71

Abrams, Scott I. ........................................................................... 47, 53, 55, 58, 77, 81, 84, 87, 123

Ahlawat, Sarita ............................................................................................. 149

Allen, Joselyn ............................................................................................... 141

Anderson, Christopher S. (P52, O) .............................................................. 46, 51, 118, 119

August, Avery .............................................................................................. 56, 88

Avanzato, Vicky .......................................................................................... 45, 106

Avram, Dorina (S) ....................................................................................... 40, 74, 80

Baker, Christina ........................................................................................... 78

Banach, Maureen ......................................................................................... 101, 133

Banik, Debarati (P20) .................................................................................. 87

Barlow, Margaret L. (P55) ........................................................................... 122

Baumann, Florian ......................................................................................... 39

Bennett, Laura (P63) .................................................................................... 130

Berin Cecilia ................................................................................................ 139

Berry, Gregory (P15, O) .............................................................................. 64, 82

Bian, Guanglin ............................................................................................. 112

Billroth-MacLurg, Alison C. (P33) .............................................................. 100, 137

Bitsaktsis, Constantine ................................................................................. 125

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Blumberg, Neil ............................................................................................. 117

Bogner, Paul N. ............................................................................................ 97, 84

Boule, Lisbeth A. (P5) ................................................................................. 72

Buitrago, Sandra .......................................................................................... 58, 81

Burke, Catherine G. ..................................................................................... 104

Califano, Danielle ........................................................................................ 40

Callaway, Justin B. (P1, O) .......................................................................... 63, 68

Cantorna, Margherita T. ............................................................................... 131

Cao, Xuefang ............................................................................................... 112

Capece, Tara (P4, O) .................................................................................... 51, 52, 71, 118

Capitano, Maegan ........................................................................................ 58, 81

Carlson, Louise ............................................................................................ 53, 77, 79, 116

Celaj, Stela (P18, S) ..................................................................................... 36, 85

Chae, Minho ................................................................................................. 65, 70

Chan, Gary ................................................................................................... 62, 69, 120

Chen, George ............................................................................................... 112

Cheng, Benson Y.H. .................................................................................... 104

Cheng, Christopher ...................................................................................... 110

Cohen, Sara B. (P28, O) ............................................................................... 43, 95

Cojohari, Olesea (P53) ................................................................................. 62, 69, 120

Connell, Terry D. ......................................................................................... 96, 97

Crowe, James E., Jr. ..................................................................................... 63, 68

Cummings, Ryan J. ...................................................................................... 122

Davies, Michael (P38, O) ............................................................................. 61, 105

Davis, Stephanie .......................................................................................... 92

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De Jesus, Magdia ......................................................................................... 139, 149

de la Rosa, Gonzalo ..................................................................................... 51, 118

de Silva, Aravinda M. .................................................................................. 63, 68

Delgado, Justin P. ........................................................................................ 102

Deng, Jie ...................................................................................................... 36, 85

Denkers, Eric Y. ........................................................................................... 43, 95

Dewan, Kalyan ............................................................................................. 45, 91, 102, 106

Diehl, Michael .............................................................................................. 55, 123

Dittmer, Dirk P. ............................................................................................ 63, 68

Doherty, Edward (S) .................................................................................... 140

Drake, James R. ........................................................................................... 49, 114

Du, Wei ........................................................................................................ 112

Duffy, Ellen .................................................................................................. 108

Dziejman, Michelle ...................................................................................... 111

Edholm, Eva-Stina ....................................................................................... 101, 133

Egan, Charlotte E. ........................................................................................ 43, 95

Egan, Shawn (P9, O) .................................................................................... 57, 76

Egilmez, Nejat K. ......................................................................................... 109

Eichelberg, Katrin (S) .................................................................................. 41

Eisenlohr, Laurence ..................................................................................... 61, 105

Elliott, Michael R. ........................................................................................ 46, 51, 115, 118, 119

Eng, Jason .................................................................................................... 58, 81, 127

Evans, Sharon S. .......................................................................................... 55, 123

Farren, Matthew ........................................................................................... 53, 77

Ferreira, Luis C. S. ....................................................................................... 96

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Fink, Pamela J. (KS) .................................................................................... 33

Ford, Jill ....................................................................................................... 99, 100

Fowell, Deborah (S) ..................................................................................... 50, 98, 99, 100, 137

Franz, Brian J. (P58) .................................................................................... 125

Furuya, Yoichi ............................................................................................. 124, 126, 129

Gaylo, Alison (P31, O) ................................................................................ 50, 98, 99, 100, 137

Gerber, Scott A. ........................................................................................... 122, 128

Gleeson, Michael W. .................................................................................... 36, 85

Glick, Adam B. ............................................................................................ 86

Gollnick, Sandra O. ...................................................................................... 57, 59, 75, 76,

Gordon, Christopher J. ................................................................................. 58, 81

Gorham, James D. ........................................................................................ 36, 85

Gosselin, Edmund J. .................................................................................... 125

Grayfer, Leon ............................................................................................... 133

Greene, Christopher J. (P30) ........................................................................ 96, 97

Grimson, Andrew ......................................................................................... 93

Gunderson, Andrew ..................................................................................... 86

Haidaris, Constatine G. ................................................................................ 122

Hakansson, Anders P. .................................................................................. 97

Hakansson, Hazeline .................................................................................... 97

Han, Katherine ............................................................................................. 113

Hankey, Pamela (S) ..................................................................................... 141

Harton, Jonathan A. ..................................................................................... 74, 108

Haynes, Nikesha (P34) ................................................................................. 101, 133

Herman, Katherine E. (P37) ......................................................................... 104

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Hernandez, Maria O. .................................................................................... 149

Hoekstra II, David A. (P49) ......................................................................... 116

Hong, Chi-Chen ........................................................................................... 58, 81

Hu, John C. (P29) ......................................................................................... 96, 97

Huang, Fei .................................................................................................... 56, 88

Huang, Lu .................................................................................................... 56, 88

Huang, Weishan (P21, O) ............................................................................ 56, 88

Hughson, Angie ........................................................................................... 137

Hunter, Christopher A. (KS) ........................................................................ 143

Hylander, Bonnie ......................................................................................... 58, 81, 127

Hyun, Young-min ........................................................................................ 78

Inglesias, Bibiana V. .................................................................................... 125

Iyer, Renuka ................................................................................................. 145

Izu, Yayoi ..................................................................................................... 150

Jarvinen-Seppo, Kirsi M. (S) ....................................................................... 139

Jin, Guangi B. .............................................................................................. 90

Jin, Lei .......................................................................................................... 49, 114

Jones, Maura ................................................................................................ 44, 94, 121

Joshi, Gaurav N. (P43) ................................................................................. 110

Kalathil, Suresh ............................................................................................ 145

Kalia, Vandana (S) ....................................................................................... 39

Katkere, Bhuvana (P35) ............................................................................... 45, 91, 102, 106

Khan, Arif A. ............................................................................................... 39

Kim, Minsoo ................................................................................................ 51, 52, 71, 78, 118

King-Lyons, Natalie D. ................................................................................ 96

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Kirimanjeswara, Girish ................................................................................ 45, 90, 102, 106

Knecht, David A. ......................................................................................... 110

Kokolus, Kathleen M. (P14, O) ................................................................... 58, 81, 127

Kraus, W. Lee .............................................................................................. 65

Krouse, Tracy E. .......................................................................................... 150

Ku, Amy (P56, O) ........................................................................................ 55, 123

Laukens, Debby ........................................................................................... 92

Lawrence, B. Paige ...................................................................................... 65, 70, 72, 90

Lawrence, David .......................................................................................... 113

Le, HongNga T. ........................................................................................... 108

Lee, Kelvin P. .............................................................................................. 53, 77, 79, 116

Leib, David A. .............................................................................................. 146

Leifer, Cynthia A. ........................................................................................ 150

Leigh, Nicholas (P45) .................................................................................. 112

Li, Ying ........................................................................................................ 125

Lim, Joanne Y.H. (P61) .............................................................................. 128

Liu, Hong ..................................................................................................... 112

Liu, Kebin .................................................................................................... 47, 84

Lord, Edith M. .............................................................................................. 122, 128

Lugade, Amit A. .......................................................................................... 145

Lynes, Michael A. ........................................................................................ 92, 113

MacNamara, Katherine C. ........................................................................... 44, 74, 94, 121

Mandell, Lorrie ............................................................................................ 97

Mansouri, Samira (P13) ............................................................................... 80

Mantis, Nicholas J. (S) ................................................................................. 139, 149

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Markley, Rachel (P24) ................................................................................. 45, 91, 102, 106

Marks, Laura R. ........................................................................................... 97

Martin, Kyle C. ............................................................................................ 90

Martinez-Sobrido, Luis ................................................................................ 104

Massa, Paul .................................................................................................. 73

Mathias-Santos, Camila ............................................................................... 96

Maurer, Kirk J. ............................................................................................. 43, 95

McCabe, Amanda (P27, O) .......................................................................... 44, 94, 121

McCarthy, Philip .......................................................................................... 112

McDaniel, Kaitlin L. (P64) .......................................................................... 131

McKinnon, Karen P. .................................................................................... 63, 68

Metzger, Dennis W. ..................................................................................... 37, 124, 126, 129, 139

Miller, Austin ............................................................................................... 47, 84, 145

Mohammad, Uddin N. (P7) ......................................................................... 74

Mohammed, Javed ....................................................................................... 86

Mohrs, Markus (S) ....................................................................................... 35

Moon, Taylor J. ............................................................................................ 46, 119

Muhitch, Jason ............................................................................................. 55, 123

Murphy, Aisling A. ...................................................................................... 146

Murphy, Patrick S. (P48) ............................................................................. 51, 115, 118

Murphy, Shawn P. ........................................................................................ 104, 111

Murray, Megan ............................................................................................ 79

Nagari, Anusha ............................................................................................ 65, 70

Netherby, Colleen (P17, O) ......................................................................... 47, 84

Nguyen, Quang-Tam (P62) .......................................................................... 129

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Nichol, Kathleen .......................................................................................... 49, 114

Norbury, Christopher C. (S) ......................................................................... 61, 105, 107, 150

Oghumu, Steve ............................................................................................. 43, 95

O’Reilly, Michael A ..................................................................................... 90

Overstreet, Michael ..................................................................................... 99, 137

Parker, Zachary M. (S) ................................................................................. 146

Paulson, Robert F. ........................................................................................ 130

Penny, Laura A. ........................................................................................... 39

Peppenelli, Megan (P2, O) ........................................................................... 62, 69, 120

Periasamy, Sivakumar (P41) ........................................................................ 108

Perl, Andras .................................................................................................. 140

Perry, Ian (P44) ............................................................................................ 111

Pham, Giang ................................................................................................. 125

Phipps, Richard ............................................................................................ 117

Pietrosimone, Kathryn M. (P25) .................................................................. 92

Place, David ................................................................................................. 45, 91, 102, 106

Prabhu, K. Sandeep ...................................................................................... 90, 102

Prograis, Lawrence J. ................................................................................... 41

Puffer, Erik (S) ............................................................................................. 66

Ravindran, Anand (P19) .............................................................................. 86

Reed, Chelsey (P60) ..................................................................................... 127

Reilly, Emma C. (P23) ................................................................................. 90

Repasky, Elizabeth A. (S) ............................................................................ 58, 81, 127, 147

Restori, Katherine H. ................................................................................... 131

Rice, James (P46) .......................................................................................... 113

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Robert, Jacques (S) ...................................................................................... 103, 133

Roberts, Sean ............................................................................................... 129

Rosa, Sarah .................................................................................................. 125

Rose III, William A. ..................................................................................... 150

Ross, Catharine ............................................................................................ 131

Rozanski, Cheryl .......................................................................................... 79

Rudd, Brian D. (S) ....................................................................................... 89, 93, 134

Ryan, Julie L. ............................................................................................... 122

Sahler, Julie (P50) ........................................................................................ 117

Sampson, Hugh A ........................................................................................ 139

Sanfilippo, Alan M. (P59) ............................................................................ 126

Sarkar, Surojit .............................................................................................. 39

Sass, Stephanie (P8, O) ................................................................................ 59, 75

Satoskar, Abhay R. ...................................................................................... 43, 95

Schrock, Dillon (P32) .................................................................................. 50, 98, 99, 137

Sei, Janet ...................................................................................................... 61, 105, 107

Siciliano, Nicholas ....................................................................................... 61, 105

Sigal, Luis J. ................................................................................................. 150

Smith, Norah L. (P26) .................................................................................. 89, 93

Smith, Scott A. ............................................................................................. 63, 68

Spangler, Haley (P10, O) .............................................................................. 53, 77

Spinelli, Sherry ............................................................................................ 117

Steiner, Don (P57) ....................................................................................... 124

Stevenson, Catherine (P51, O) ..................................................................... 51, 118

Thai, Vinh Kenny ......................................................................................... 44, 94, 121

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Thanavala, Yasmin (S) ................................................................................. 145

Ting, Jenny P.-Y. ......................................................................................... 63, 68

Truckenmiller, Mary Ellen ........................................................................... 150

Tucker, Heidi R. (P47, O) ............................................................................ 49, 114

Utley, Adam (P12) ....................................................................................... 79

Waight, Jeremy D. ....................................................................................... 47, 84

Waldner, Hanspeter ...................................................................................... 64, 82

Wang, Jocelyn Jie (P22) .............................................................................. 89

Watson, Neva (P6) ....................................................................................... 73

Weiler, Lauren A. (P40) ............................................................................... 107, 150

Westfall, Jennifer E. ..................................................................................... 139

Widman, Douglas G. .................................................................................... 63, 68

Williamson, David (P39, O) ........................................................................ 45, 90, 102, 106

Winans, Bethany (P3, O) ............................................................................. 65, 70, 72

Wissink, Erin ................................................................................................ 93

Woeller, Collynn .......................................................................................... 117

Xu, Yuexin (P11) .......................................................................................... 78

Yu, Shan ....................................................................................................... 141

Yuzefpolsky, Yevgeniy ................................................................................ 39

Zhang, Yubin (P54) ..................................................................................... 44, 74, 94, 121