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Abstract Book

Tuesday, 22 September 2020

(Singapore & China Time/GMT+8)

08:30 - 08:45 Welcome speech by Assoc Prof Kevin Tan and Prof Wang Qingqing

Session I: Microbial pathogenesis and Innate immunity | Chairperson: Kevin Tan

08:45 - 09:30 Keynote 1 The development of fully human antibodies as

candidate medicines for infectious disease.

Speaker: Paul MACARY

NUS

09:30 - 10:00 T1 Action and mechanism for ongoing MCR family of

colistin resistance.

Speaker: FENG Youjun

ZJU

10:00 - 10:30 T2 Relative contribution of non-structural protein 1

(NS1) in dengue pathogenesis.

Speaker: Sylvie ALONSO

NUS

10:30 - 11:00 T3 FXYD3 in goblet cells is required for intestine

immune homeostasis.

Speaker: WANG Qingqing

ZJU

Break

Session I: Microbial pathogenesis and Innate immunity | Chairperson: Sylvie Alonso

11:15 - 11:45 T4 Experimental infections of DSS colitis mouse model

with a common Blastocystis subtype reveals

protective effects from the microbiome and

immunological perspectives.

Speaker: Kevin TAN

NUS

11:45 - 12:15 T5 Roles of eosinophils in airway inflammation &

carcinogenesis.

Speaker: YING Songmin

ZJU

12:15 - 12:45 T6 High-frequency transfer of chromosomal virulence

genes by pathogenicity islands.

Speaker: John CHEN

NUS

Lunch Break

Session II: Inflammation | Chairperson: Veronique Angeli

14:00 - 14:45 Keynote 2 Metabolic control of IL-1β production.

Speaker: WANG Di

ZJU

14:45 - 15:15 T7 IL-1alpha secretion in the tumor microenvironment.

Speaker: LIU Haiyan

NUS

15:15 - 15:45 T8 Exploration of MtrE-based vaccines as therapeutics

for multidrug-resistant gonococcal infections.

Speaker: Stijn van der VEEN

ZJU

15:45 - 16:15 T9 Harnessing lymphocyte trafficking for treatment of

inflammatory diseases.

Speaker: NGUYEN Nam Long

NUS

Break

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Session II: Inflammation | Chairperson: Liu Haiyan

16:30 - 17:00 T10 Unexpected function of LYVE-1 + macrophage in

skin.

Speaker: Veronique ANGELI

NUS

17:00 - 17:30 T11 Mechano-chemical regulation of TCR antigen

recognition.

Speaker: CHEN Wei Jack

ZJU

17:30 - 18:00 T12 Gasdermin-mediated cell death during bacterial

infection.

Speaker: Kaiwen CHEN

NUS

Wednesday, 23 September 2020

(Singapore & China Time/GMT+8)

Session III: T cell biology and Therapy | Chairperson: Zhang Yongliang

08:30 - 09:15 Keynote 3 T cell signal transduction regulation in T cells and

CAR-T cells.

Speaker: Nicholas GASCOIGNE

NUS

09:15 - 09:45 T13 Regulation of T-cell development and homeostasis

by NPAT.

Speaker: LU Linrong

ZJU

09:45 - 10:15 T14 A trans-splicing-based suicide gene therapy

approach targeting virus infection and cancer.

Speaker: Volker PATZEL

NUS

10:15 - 10:45 T15 The function of histone methyltransferase MLL4 in

ESC and T cell differentiation.

Speaker: WANG Chaochen

ZJU

Break

Session III: T cell biology and Therapy | Chairperson: Nicholas Gascoigne

11:00 - 11:30 T16 Epigenetic initiation of Th17 differentiation program

is promoted by Cxxc finger protein 1.

Speaker: WANG Lie

ZJU

11:30 - 12:00 T17 Altered gastric bacterial composition associated with

Gastric cancer progression.

Speaker: ZHANG Yongliang

NUS

12:00 - 12:30 T18 P2X7 is a high affinity receptor for ATP in antigen

presenting cells and promotes sterile inflammation.

Speaker: SHEN Xiao

ZJU

End

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Keynote 1 Transcending the Boundaries of Infection and Immunity Research in the Post-COVID-19 Era

The development of fully human antibodies as candidate medicines for infectious disease

Paul MacAry

Antibodies are multi-functional glycoproteins that encode hypervariable paratopes that

recognize and bind to target motifs (termed epitopes) on targets molecules with a high degree

of specificity. This activity underlies the wide employment of these molecules in research and

diagnostics where biochemical assays aimed at protein detection and/or quantitation form

essential components of the analyses. Traditional methodologies for the generation of

antibody reagents have relied upon the immunization of rodents to generate either polyclonal

sera or monoclonal mouse/rat antibody hybridomas. The advent of recombinant Human

antibody technology has resulted in their broader utilization for therapeutic applications. In my

talk, I will describe our efforts to develop candidate therapeutic Human antibodies for Dengue

virus, Hepatitis B virus and M.Tuberculosis.

T1 Transcending the Boundaries of Infection and Immunity Research in the Post-COVID-19 Era

Action and mechanism for ongoing MCR family of colistin resistance

Feng Youjun

Antimicrobial resistance is a prevalent health concern. Colistin is a last-resort defense against

carbapenem-resistant bacterial pathogens. A rainbow coalition of mobile colistin resistance

(MCR) genes raises significant global health concerns. This talk concentrates on research

work in Dr. Feng’s lab in the past four years, esp., mechanistic insights into MCR-like colistin

resistance: i) Complexity in global dissemination of transferable mcr-1; ii) a “ping-pong” trade-

off model for MCR-like enzymes, whose activity relies on an evolutionarily-conserved PE lipid

substrate-recognizable cavity; iii) phylogenetic and functional definition of

progenitor/ancestors of MCR-like members; and iv) functional dissection of a linker rending

PEA lipid A transferases to gain the resistance to polymyxin, an ultimate line of defense

antibiotic. Our long-term research plan aimed at providing molecular basis for the rational

development of inhibitors of small molecules that reverse the MCR resistance to colistin, a

last-resort antibiotic against multi-drug resistant pathogens.


Relative contribution of non-structural protein 1 in dengue pathogenesis

Pei Xuan Lee1,2, Donald Heng Rong Ting1,2, Clement Peng Hee Boey1,2, Eunice Tze Xin Tan1,2, Janice Zuo Hui Chia1,2, Li Ching Ong1,2, Yen Leong Chua1, Chanditha Hapuarachchi3, Lee Ching Ng3, Sylvie Alonso1,2

1 NUHS Infectious Diseases Translational Research Programme; Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. 2Immunology Programme, Life Sciences Institute, National University of Singapore, Singapore. 3Environmental Health Institute at National Environment Agency, Singapore. Dengue is an arthropod-borne viral disease that is endemic in the sub-tropical and tropical

world, and ranges from mild to life-threatening conditions, including haemorrhage and

shock, with the latter resulting from increased vascular permeability. There are no effective

treatments against dengue and the only vaccine available on the market (Dengvaxia), a live

attenuated vaccine, has been facing a serious setback due to the recent discovery that it could

increase the risk of severe dengue in serologically dengue naïve vaccinees. As such, sub-unit

dengue vaccine approaches have re-gained some traction, and the non-structural 1 (NS1)

protein of dengue virus (DENV) has surfaced as a potential candidate. Recent studies have

reported that NS1 plays a significant role in dengue pathogenesis, whereby secreted NS1

(sNS1) was shown to interact with the endothelium and induce vascular leakage both in vivo

and in vitro. Consistently, NS1 immunity was found to protect against dengue in symptomatic

mouse models. Those studies were conducted using either mouse-adapted DENV strains or

DENV strains of poor in vivo fitness that necessitate a very high infectious dose to establish

infection. However, we found that NS1 immunization failed to confer protection in symptomatic

dengue mouse models using two non-mouse-adapted DENV2 strains that are highly virulent.

Furthermore, exogenously administered NS1 did not enhance vascular leakage and disease

severity in sub-lethally infected mice. Virus chimerization approaches supported that the prME

structural region, but not NS1, plays a critical role in driving in vivo fitness and virulence of the

virus, through induction of key pro-inflammatory cytokines. This work highlights that the role

of NS1 in dengue pathogenesis is DENV strain-dependent and warrants re-evaluation of NS1

as a dengue vaccine candidate.


FXYD3 in goblet cells is required for intestine immune homeostasis

Wang Qingqing

Goblet cells (GCs) are specialized secretory cells that produce mucins, especially mucin 2

(MUC2) to form the mucous layer which is the first line of innate defense for intestine, which is

critical for the maintenance of intestine immune hemostasis. The mechanisms for regulating

the function of Goblet cells for intestine immune hemostasis remain largely unknown. FXYD3

belongs to a small family of FXYD-domain containing regulators of Na+/K+ ATPases, encoding

a cell membrane protein that may regulate the function of ion-pumps and ion-channels, the

role of FXYD3 in regulating immune response has been not reported. To investigate the

function of FXYD3 in regulating intestine immune hemostasis, we generated mice with

conditional knockout of FXYD3 gene in intestine epithelial cells and established the DSS

induced inflammatory bowel disease (IBD) model. We found that FXYD3 selectively expressed

in colon GCs, is a critical orchestrator of colon goblet cell mucin secretion. Deficiency of FXYD3

in intestinal epithelial cells (IECs) lead to the severer colitis induced by DSS. C. rodentium

infection in FXYD3 conditional knockout mice exhibited higher permeability of colon tissues.

FXYD3 conditional knockout mice showed impaired secretion of mucin 2, and FXYD3

knockdown inhibits autophagy in goblet cells. In terms of the regulation of FXYD3 expression,

we found that the expression of FXYD3 in goblet cells could be significantly induced by

interleukin 22 (IL-22) and decreased by ER stress-p53 axis during colitis. In clinical samples

of intestine tissues, the expression level of FXYD3 in IBD patients was significantly decreased

when compared to healthy controls. Our study identifies FXYD3 as an important regulator for

governing goblet cell mucin 2 secretion, which is required for the maintenance of intestine

immune homeostasis.


Experimental Infections of DSS Colitis Mouse Model with a Common

Blastocystis Subtype Reveals Protective Effects from the Microbiome and

Immunological Perspectives

Kevin S.W. Tan1, Lei Deng1,2, Chin Wen Png3, Lukasz W. Wojciech3, Eileen Y.L. Koh1,

Guangneng Peng2, Yongliang Zhang3, Nicholas Gascoigne3

1Laboratory of Molecular and Cellular Parasitology, Healthy Longevity Programme and Department of

Microbiology and Immunology, National University of Singapore, 5 Science Drive 2, Singapore 117545.

2The Key Laboratory of Animal Disease and Human Health of Sichuan Province, College of Veterinary

Medicine, Sichuan Agricultural University, Chengdu, Sichuan, 611130, China.

3Immunology Programme and Department of Microbiology and Immunology, Yong Loo Lin School of

Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117545.

Background: Blastocystis is a common gut protistan parasite in humans and animals

worldwide, but its causal role on the host gut microbiota and mucosal immune responses

remain poorly studied. By using a murine model of infections, this study aimed to examine the

interactions between a common Blastocystis subtype (ST4), host gut microbial community and

adaptive immune system.

Results: We observed that Blastocystis ST4 colonization in a steady-state condition leads to

the microbiome's remodeling, exerting a favorable effect on the Firmicutes phylum, particularly

on Clostridiales vadinBB60 group. Analysis of the LN and gut-derived T cells revealed that

simultaneously to the changes in microbial consortia, Blastocystis ST4 positively affects the T

helper 2 (Th2) response defined by IL-4, IL-5 and IL-13 cytokine makeup, and T regulatory

(Treg) subsets from colonic lamina propria (LP). In the DSS-induced colitis mice model,

Blastocystis ST4 colonization was associated with improved recovery. Importantly, this

Blastocystis-driven phenotype was accompanied by the increased diversity of the microbiota

communities and the expansion of the colonic Th2 and Treg T cells.

Conclusions: Our findings demonstrated for the first time that Blastocystis ST4 may be a

beneficial commensal and can alter the composition of the gut microbiota and immune

responses to promote host health and homeostasis.


Roles of Eosinophils in Airway Inflammation & Carcinogenesis

Ying Songmin

Eosinophils are a population of terminally differentiated white blood cells from hematopoietic

stem cells (HSCs) in the bone marrow. However, they display multi-regulatory roles in various

inflammatory and immunological responses. Using gene-edited mice with altered eosinophil

numbers, we found previously undetected functions of eosinophil in ovalbumin (OVA)-induced

airway inflammation and a tumor metastasis murine model. Briefly, eosinophil released C-C

chemokine ligand 6 (CCL6), disrupts HSC homeostasis by impairing HSC quiescence under

OVA challenge, promoting committed eosinophil differentiation and TH2 response to exert a

positive feedback regulation on airway inflammation; besides, in an experimental metastatic

model, airway eosinophilia tends to promote cancer cells migrating to the inflammatory locus,

and in an eosinophil-enriched Cd3δ-Il-5 transgenic (Il-5 Tg) mice, extensive number of

eosinophil contributes to increased bone metastasis sties. Taken together, our studies propose

unexpected and potential roles of eosinophil in airway inflammation and cancer development.


High-frequency transfer of chromosomal virulence genes by pathogenicity islands

John Chen

The Staphylococcus aureus genome contains highly variable regions that are enriched in

pathogenicity islands and virulence genes. Some of these regions are located immediately

adjacent to S. aureus pathogenicity islands (SaPIs) or their attachment sites. The SaPIs are

a family of highly mobile molecular parasites that exploit bacteriophages for their propagation

and dissemination. Here we show that SaPIs package and transfer large segmented spans of

the bacterial genome (adjacent to their attachment sites) at extremely high frequencies, in a

manner similar to the recently discovered phage-mediated lateral transduction. This work

provides a plausible mechanism to explain some of the highly variable regions in the S. aureus

genome. Furthermore, it shows that genetic transduction is not the exclusive domain of

phages, and that non-canonical modes of transduction play a much bigger role in pathogen

evolution than we previously understood.


Metabolic control of IL-1β production

Wang Di

Immune cell function depends on specific metabolic programs dictated by mitochondria,

including nutrient oxidation, macromolecule synthesis, and post-translational modifications.

Mitochondrial adaptations have been linked to acute and chronic inflammation, but the

metabolic cues and precise mechanisms remain unclear. Here we reveal that histone

deacetylase 3 (HDAC3) is essential for shaping mitochondrial adaptations for IL-1β production

in macrophages through non-histone deacetylation. In vivo, HDAC3 promoted

lipopolysaccharide-induced acute inflammation and highfat diet-induced chronic inflammation

by enhancing NLRP3-dependent caspase-1 activation. HDAC3 configured the lipid profile in

stimulated macrophages and restricted fatty acid oxidation (FAO) supported by exogenous

fatty acids for mitochondria to acquire their adaptations and depolarization. Rather than

affecting nuclear gene expression, HDAC3 translocated to mitochondria to deacetylate and

inactivate an FAO enzyme, mitochondrial trifunctional enzyme subunit α. HDAC3 may serve

as a controlling node that balances between acquiring mitochondrial adaptations and

sustaining their fitness for IL-1β-dependent inflammation.


IL-1alpha secretion in the tumor microenvironment

Liu Haiyan Interleukin-1α (IL-1α) is a pleiotropic cytokine and plays an important role in inflammation and

hematopoiesis. Many tumors have increased IL-1α expression. However, the immune

regulatory role of secreted IL-1α in tumor development and whether it can be targeted for

cancer therapy are still not clear. Here we investigated the functions of secreted IL-1α in

hepatocellular carcinoma. We constructed tumor cells stably expressing secreted IL-1α and

found that tumoral-secreted IL-1α had no significant effect on tumor cell growth in vitro.

However, it significantly promoted tumor development in vivo. Further studies showed that

tumoral-released IL-1α inhibited T and NK cell activation, as well as the killing capacity of

CD8+ T cells. Tumoral released IL-1α also decreased the number of DCs in the tumor

microenvironment. Moreover, MDSCs were dramatically increased by tumoral-released IL-1α

in both spleens and tumor-bearing livers, and depletion of MDSCs could block the tumor-

promoting effect of tumoral-secreted IL-1α. On the contrary, systemic administration of IL-1α

protein significantly inhibited the tumor development by promoting T cell activation. In fact, IL-

1α protein could promote T cell activation and enhance the cytotoxicity of CD8+ T cells in vitro.

Thus, our study demonstrated that tumoral-released IL-1α promoted tumor development

through recruiting MDSCs and inhibiting T cell activation, while systemic IL-1α promoted anti-

tumor T cell responses. We further identified calpain 1 as the major intracellular protease

mediating tumoral IL-1α secretion. Calpain 1 KO tumors had diminished IL-1α release and

reduced tumor development. Thus, our findings provide new insights of the functions of

secreted IL-1α in tumor immunity and its implications for tumor immunotherapy.


Exploration of MtrE-based vaccines as therapeutics for multidrug-resistant

gonococcal infections

Stijn van der Veen Neisseria gonorrhoeae is a human-specific bacterial pathogen that has proven to be highly

adaptable to our treatment protocols and it has developed resistance to nearly every antibiotic

introduced for treatment. Therefore, N. gonorrhoeae has been prioritized as an urgent public

health threat for which immediate action and development of alternative treatments are

needed. Gonococcal vaccines are commonly recognized as a major attribute to our future

ability to control this infectious disease. However, the development of an effective vaccine has

remained elusive due to antigenic and phase variability of surface-expressed proteins and the

lack of clear correlates of protection. Recently it was shown that specific enhancement of a

Th1 response during a primary infection allowed for the establishment of immune memory,

generation of specific antibodies, clearance of the infection, and protection against reinfection.

This suggests that a Th1-inducing vaccine might be suitable as a prophylactic as well as a

therapeutic vaccine to clear an already established infection, which is particularly important

for multidrug resistant infections for which antibiotic treatment options are no longer available.

Our studies identified the surface-expressed Loop2 of the outer-membrane protein MtrE as

the most highly conserved vaccine antigen that was able to elicit strong immune responses in

mice with antibodies displaying bactericidal activity against a collection of multidrug resistant

clinical isolates. Furthermore, CpG1826 was identified as an optimal Th1-polarizing adjuvant

for Loop2-based vaccines and these vaccines elicited high protein-specific antibody titers, with

antibodies displaying bactericidal and opsonophagocytosis activity. Furthermore, the

Loop2/CpG1826 vaccines accelerated clearance of N. gonorrhoeae from infected mice when

provided as a therapeutic vaccine, indicating that Loop2-based vaccines might be a suitable

alternative for anti-gonococcal therapy.


Harnessing lymphocyte trafficking for treatment of inflammatory diseases

Nguyen Nam Long

The role of sphingosine-1-phosphate (S1P) has been well appreciated in regulation of

lymphocyte trafficking. S1P exerts this function via stimulating its S1P receptors (S1P1-5) on

lymphocytes. Our lab has interests in determining the sources of extracellular S1P that are

required for signalling. We cloned out Mfsd2b from erythrocytes and platelets and determined

that Mfsd2b provides a major source of S1P. Endothelial cells also provide a small amount of

plasma S1P via Spns2. Interestingly, deletion of Mfsd2b only results in a mild reduction of

circulating lymphocytes, while deletion of Spns2 in mice significantly reduces circulating

lymphocytes. In this presentation, I will summarize the roles of Mfsd2b and Spns2 in regulation

of lymphocyte trafficking. Our results highlight that Spns2 may be a good target for

development of anti-inflammatory therapeutics.


Unexpected function of LYVE-1 + macrophage in skin

Veronique Angeli

The skin plays a critical role in protecting the body from external insults. Macrophages are strategically

situated in the skin, serving as sentinels against any evading pathogens and ensuring the restoration

of skin integrity upon injury. Despite largely known as a marker for lymphatic endothelial cells,

lymphatic vessel endothelial hyaluronic acid receptor-1 (LYVE-1) is also expressed by macrophages.

Several groups have demonstrated the presence of LYVE-1+ macrophages during wound and tissue

repair in mice models. However, little is known about the presence and functions of LYVE-1+

macrophages in the skin under non-inflammatory conditions. We demonstrated that LYVE-1+

macrophages are the most abundant subpopulation of macrophages in mouse and human skin. Mice

depleted specifically of LYVE-1+ macrophages exhibit marked changes in the content and organization

of the skin extracellular matrix (ECM) at steady state. Having shown that this subpopulation of

macrophages is particularly abundant in the fascia of the skin and its loss affects collagen in this layer,

we assessed the potential involvement of LYVE-1+ macrophages in the pathogenesis of fasciitis in a

mouse model induced by Group A Streptococcus infection. We showed that lesions and bacterial load

in skin of mice lacking LYVE-1+ macrophages were reduced compared to wild-type mice. Altogether,

our work reveals the unexpected function of skin LYVE-1+ macrophage in controlling ECM homeostasis.

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Mechanical Regulation of TCR Antigen Recognition

Chen Wei

TCRs recognize cognate pMHCs to initiate T cell signaling and adaptive immunity. Mechanical

force strengthens TCR-pMHC interactions to elicit agonist specific catch bonds to trigger TCR

signaling, but the underlying dynamic structural mechanism is unclear. We combined steered

molecular dynamics (SMD) simulation, single-molecule biophysical approaches, and in vitro

and in vivo functional assays to collectively demonstrate that mechanical force induces

conformational changes in pMHC-Is to enhance pre-existing contacts and activates new

interactions at the TCR-pMHC binding interface to resist bond dissociation under force,

resulting in TCR-pMHC catch bonds and T cell activation. Intriguingly, cancer-associated

somatic mutations in HLA-A2 that may restrict these conformational changes suppressed

TCR-pMHC catch bonds. Our findings not only reveal critical roles of force-induced

conformational changes in pMHC-Is for activating TCR-pMHC catch bonds but also have

implications for T cell-based immunotherapy.


Gasdermin-mediated cell death during bacterial infection

Kaiwen Chen

The extrinsic apoptosis pathway is traditionally regarded as an immunologically silent form of

cell death; however, emerging studies reveal that under specific conditions, apoptotic

caspase-8 and -3 cleave the pore-forming proteins gasdermin D (GSDMD) and GSDME to

trigger a form of lytic cell death called ‘pyroptosis’. Although the mechanisms of pyroptosis

induction is well characterised in vitro, the physiological function of gasdermin D (GSDMD)

and GSDME-dependent pyroptosis is unclear. Here, we demonstrate two opposing functions

for gasdermin activation in vivo: while GSDMD and GSDME activation in specific myeloid

cells subsets are required for anti-bacterial defence, GSDMD activation confers susceptibility

to TNF-induced lethality. These findings reveal gasdermins as important mediators of host

defence and potential therapeutic targets to reduce inflammation associated with mutations in

the death receptor signalling machinery.


T cell signal transduction regulation in T cells and CAR-T cells

Nicholas Gascoigne

We have recently compared signaling through T cell receptor with that through chimeric

antigen receptors (CAR) in T cells. Using a TCR and a CAR with the same specificity for

peptide-MHC, we compared their signal strength, kinetics, and specificity, as well as their

requirements for particular signal-transduction molecules. Signaling induced by CAR is

thought to be reliant on the activity of the SRC family kinase LCK, as with the TCR. However,

we found a non-canonical CAR signaling that is LCK-independent. A CD28 intracellular

domain within the CAR enables signaling dependent on a different kinase, FYN. We found

that LCK-deficient CAR-T cells have improved specificity, reduced exhaustion phenotype, and

were induced to be more memory-like. This results in better in vivo performance and efficacy

and should reduce the risk of graft-versus-host disease or autoimmunity by quenching

endogenous TCR, but not CAR signaling.

We have found that LCK itself is more active and more mobile when it is not bound to its co-

receptors CD4 or CD8, and as such may be more able to initiate TCR signaling.

In addition, we have identified an important role for the T-cell lineage-specific protein Themis

in peripheral T cells. Themis is known to be important in thymocyte positive selection, but its

role (if any) in mature T cells was unknown. We have identified Themis as an important

regulator of T cell metabolic upregulation in response to antigen, and in integrating signals

between TCR and cytokines for T cell homeostasis.


Regulation of T-cell development and homeostasis by NPAT

Lu Linrong T cell development process is instructed by microenvironmental stimulus in the thymus and

dependent on the precise regulation of lineage choice, cell proliferation and apoptosis. Our

previous research has revealed the essential role of protein phosphatase PP2A in ensuring

the survival of double positive (DP) thymocytes. In this study, we focus on the proliferation

capacity of developing thymocytes at different developmental stages and studied the role of

proliferation regulation during the development process through analyzing cell cycling related

protein Nuclear protein ataxia-telangiectasia (NPAT).

NPAT is a substrate of cyclin E-Cdk2 kinase and plays a critical role in coordinated activation

of histone genes during the G1/S-phase transition and in S-phase entry in mammalian cells.

We found that ablation of NPAT in mouse thymocytes results in severe thymus development

arrest in ISP stage reflected by substantial reduction of DP and total thymocyte cell numbers.

NPAT deficient thymocytes showed decreased proliferation in vivo. We also constructed a

mouse strain which conditionally delete NPAT expression in peripheral mature T cells. NPAT

deficiency in peripheral T cells causes decreased number of CD4 and CD8 T cells as well as

impaired proliferative capacity of naïve CD4 or CD8 cells following anti-CD3 and anti-CD28

stimulation in vitro, resulted from decreased expression of Histone genes.

Taken together, our data indicated that NPAT is essential regulator in both T cell development

and activation, through promoting cell proliferation.


A trans-splicing-based suicide gene therapy approach targeting virus infection and cancer

Sushmita Poddar1, Pei She Loh1, Carin Ingemarsdotter2, Zi Hao Ooi1, Farhana Osman1, Joachim Eul2, Xiaoou Jiang1, Andrew Lever3 and Volker Patzel1,2*

1Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 2Department of Medicine, Division of Infectious Diseases, University of Cambridge, Addenbrooke’s Hospital, Cambridge, UK 3INEIDFO GmbH, Berlin, Germany Spliceosome-mediated RNA trans-splicing enables correction or labelling of pre-mRNA

targets but therapeutic applications are hampered by issues related to delivery, activity and

target specificity of trans-splicing RNA (tsRNA). We employed computational RNA structure

design to improve both on-target activity and specificity of tsRNA in a Herpes simplex virus

thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene therapy approach. As molecular

targets we selected various pre-mRNA biomarkers of (i) hepatocellular carcinoma (HCC), (ii)

human papillomavirus type 16 (HPV-16) or (iii) human immunodeficiency virus type 1 (HIV-1).

HPV-16 and HIV-1 are integrating viruses and killing the infected cells could represent one

option to get rid of these viruses. Compared with the previous state-of-the-art, our new suicide

RNAs which were designed to target multiple biomarkers of the same disease, exhibited about

1000-fold improved cell death activity and about 10-fold higher on-target specificity. These

suicide RNAs efficiently triggered death of HPV-16-transduced, HIV-1-infected or

hepatoblastoma-derived human tissue culture or primary cells in vitro and ex vivo without any

evidence for off-target cell killing. Efficient target cell killing was achieved at non-toxic GCV

concentrations as low as 0.3 M, indicating the Michaelis-Menten constant of the HSVtk

enzyme but not the trans-splicing activity is the rate limiting factor. For delivery, we developed

non-viral, non-integrating dumbbell-shaped DNA minimal vectors which are not silenced in

primary cells and which are being featured with triantennary N-acetyl galactosamine ligands

to facilitate targeting of hepatocytes. Such designed suicide vectors were tailored for targeting

distinct patient-derived tumor cells which will be tested as xenografts in nude and humanized

mouse models. Our observations suggest trans-splicing represents a promising approach to

suicide gene therapy.


The function of histone methyltransferase MLL4 in ESC and T cell differentiation

Wang Chaochen Mixed-lineage leukemia4 (MLL4/KMT2D) is a major enhancer H3K4me1/2 methyltransferase with functional redundancy with MLL3 (KMT2C). We show that in mouse embryonic stem cells (ESCs) MLL4 associates with, but is surprisingly dispensable for the maintenance of, active enhancers of cell-identity genes. In contrast, MLL4 is required for enhancer-binding of H3K27 acetyltransferasep300, enhancer activation, and induction of embryonic developmental genes during ESC differentiation. In adult animals, we found that MLL4 deficiency compromised the development of regulatory T cells (Treg cells). And we found that MLL4 catalyzed methylation of H3K4 at distant unbound enhancers via chromatin looping. Taken together, we identified the essential role of MLL4 in controlling cell fate transition by orchestrating enhancer activation.


Epigenetic initiation of Th17 differentiation program is promoted by Cxxc finger protein 1

Wang Lie

IL-6/STAT3 signaling is known to initiate the TH17 differentiation program, but the upstream

regulatory mechanisms remain minimally explored. Here, we show that Cxxc finger protein 1

(Cxxc1) promoted the generation of TH17 cells as an epigenetic regulator and prevented their

differentiation into Treg cells. Mice with a T cell-specific deletion of Cxxc1 were protected from

experimental autoimmune encephalomyelitis and were more susceptible to Citrobacter

rodentium infection. Cxxc1 deficiency decreased IL-6Rα expression and impeded IL-6/STAT3

signaling, whereas the overexpression of IL-6Rα could partially reverse the defects in Cxxc1-

deficient TH17 cells in vitro and in vivo. Genome-wide occupancy analysis revealed that Cxxc1

bound to Il6rα gene loci by maintaining the appropriate H3K4me3 modification of its promoter.

Therefore, these data highlight that Cxxc1 as a key regulator governs the balance between

TH17 and Treg cells by controlling the expression of IL-6Rα, which affects IL-6/STAT3

signaling and has an impact on TH17-related autoimmune diseases.


Altered gastric bacterial composition in intestinal metaplasia and its association in gastric cancer progression

Chin Wen Png1,2, Wei Jie Lee Jonathan3,4, Shijia Chua Joy3, Feng Zhu3, Khay Guan Yeoh3,4,5, and Yongliang Zhang1,2 1Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, 2Immunology Programme, Life Science Institute, National University of Singapore, Singapore 117597, Singapore. 3Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228, Singapore. 4Division of Gastroenterology and Hepatology, University Medical Cluster, National University Health System, Singapore 119074, Singapore 5Singapore Gastric Cancer Consoritum BACKGROUND: Dysbiotic gastric microbiota was shown to be associated to the development

of gastric cancer (GC). Abundance and diversity of various bacterial taxa were identified to be

different particular between normal or gastritis compared to intestinal metaplasia (IM) and

gastric cancer. However, comparison with different IM OLGIM subgroups has not been shown.

In addition, investigation to determine bacterial signature in IM patients that could influence

the risk of GC progression have not been reported. Hence, our study aimed to characterise

the changes in bacterial diversity in non-IM, OLGIM1-2, OLGIM3-4 and early gastric neoplasia

(EGN)/cancer patient groups. In addition, bacterial profile in patients with IM that progressed

to cancer will also be determined for comparison with patients that did not show disease

progression. METHODS: Gastric mucosal-associated bacterial profile in IM, OLGIM1-2,

OLGIM3-4 and EGN/cancer patients was assessed by next generation sequencing of bacterial

16S rRNA gene before analysis using Dada2 and phyloseq R packages. Evaluation of gene

functions was carried out using Picrust2 and gene ontology analysis RESULTS: Differences

in bacterial diversity were not detected between non-IM, OLGIM1-2, OLGIM3-4 and

EGN/cancer group. Interestingly, the bacterial diversity were significantly different between

patients with baseline IM that progressed to EGN/cancer compared to non-progression group.

This resulted in the generation of a panel of bacterial taxa and gene functions that could

discriminate between the two patient groups. CONCLUSIONS: IM patients that progressed

to EGN/cancer harboured different bacterial profile which was predicative for cancer

progression risk. Further validation will be required to determine the level of accuracy of these

biomarkers.


P2X7 is a high affinity receptor for ATP in antigen presenting cells and triggers the inflammation associated with hypertension

Shen Xiao

The P2X7 receptor is a nucleotide-gated ion channel. It is generally thought that the P2X7

receptor has low affinity for ATP so that millimolar ATP is widely used to study P2X7-mediated

actions in immune cells. However, the studies of ATP-P2X7 affinity were mostly performed in

P2X7 transfected cell lines, which may not reflect the biological activity of P2X7 in antigen

presentation cells (APCs). Hypertension is accompanied with low-grade systemic

inflammation and T cells appear critical in disease progression. We found that hypertensive

mice have increased antigen specific T cell responses. A root cause for this is plasma ATP

levels, which rise to about 3 μM after the onset of hypertension and stimulate APC upregulation

of CD86. Three micromolar ATP could efficiently upregulate CD86 expression and IL-1β

secretion in APCs and these effects could be abrogated by P2X7 antagonists but not

antagonists against other purinergic receptors. However, unlike the effect of millimolar ATP on

P2X7, prolonged exposure to 3 μM ATP did not make APCs permeable to larger molecules

nor leads to cell death. In vitro, 3 μM ATP triggers Ca2+ influx in dendritic cells and

macrophages but not T cells nor APCs derived from P2X7-deficient mice. Mechanistic

analyses unveil that APCs contain high amount of lysophosphatidylcholine which increases

the sensitivity of P2X7. Pharmacological or genetic ablation of P2X7 activity suppressed the

T cell overactivity and blood pressure increase associated with hypertension. Thus, P2X7 is a

high affinity receptor for ATP in APCs and mediates the inflammation associated with

hypertension.

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