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Immunology The Journal of cells, molecules, systems and technologies Volume 140, Suppl. 1, December 2013

Abstracts of the Annual Congress of the British Society for Immunology 2–5 December 2013 Liverpool, UK

Editor Daniel Altmann

Disclaimer: This abstract book has been produced using author-supplied copy. Editing has been restricted to some corrections of spelling and style where appropriate. No responsibility is assumed for any claims, instructions, methods or drug dosages contained in the abstracts; it is recommended that these are verified independently.



Invited Abstracts BSI Congress 2013 Parallel Session: Cancer Therapy Through Immune Modulation 744 Breaking down barriers to effective anti-tumour immunity A. Gallimore, M. Scurr, M. Besneux, A. Bloom, E. Jones, E. Colbeck, D. Costa-Bento & A. Godkin Cardiff University, Cardiff, UK Effective anti-tumour immune responses are often limited through suppression by Foxp3+ regulatory T cells (Tregs). In mice, we have used a model of carcinogen-induced tumours to define the correlates of successful immunity following manipulation of Treg numbers. These studies indicate that immune activation as a result of Treg depletion is not sufficient for enabling tumour destruction; this process requires the presence of specialised blood vessels termed high endothelial venules which facilitate entry of lymphocytes into the tumour mass. In humans, our work is focussed on defining the role of the immune system in progression of colorectal cancer (CRC). The results of these investigations have revealed that both Foxp3+ and Foxp3 tumour-infiltrating CD4+ T cells are highly enriched within tumours. Both populations suppress conventional T cell responses, and may serve to promote tumour progression. The phenotypic and functional characteristics of these tumour-infiltrating Foxp3+ and Foxp3 Tregs will be described as well as clinical studies currently underway to examine whether manipulation of Treg activity boosts anti-tumour immune responses in patients with CRC.

746 Developing immunostimulatory monoclonal antibodies to promote anti-cancer immunity M. J. Glennie University of Southampton, Southampton, UK The ability of monoclonal antibodies (mAb) to block key immune regulators, such as CTLA-4 and PD-1, and promote anti-cancer responses in ‘difficult to treat’ cancers has generated considerable excitement and reinvigorated the belief that the immune system can provide durable control of malignancy. In addition, agonistic mAb, such as anti-CD40, which boost anti-cancer immunity by stimulating critical co-receptors on lymphocytes or APC are also showing promise in certain cancers. Despite such advances we still know relatively little about what properties constitute the optimal antagonistic or

agonistic mAb for delivering immunostimulatory activity. This has been further complicated by recent data suggesting that mAb which were designed to work by blocking CTLA-4, may work better when they are also able to recruit activatory FcR needed for cytotoxic activity. Such findings are consistent with the need to delete certain cells, perhaps Tregs. In this presentation we will explore the mechanisms of action of immunostimulatory mAb, particularly focusing on agonistic mAb such as anti-CD40 and -4-1BB. We will show that FcRIIb, rather than being inhibitory, is often essential for delivering the hyper-crosslinking that makes mAb agonistic. We will also demonstrate that different FcR may be needed for hyper-crosslinking function in different anatomical locations, supporting the idea of engineering different immunostimulatory mAb for use as systemic or local drugs.

757 Immunotherapy of pancreatic cancer: the need for powerful approaches R. Offringa German Cancer Research Center, Heidelberg, Germany Although pancreatic ductal adenocarcinoma (PDA), the predominant type of pancreatic cancer, has been branded poorly immunogenic, it is important to realize that the deadly characteristics of this disease are primarily due to the late detection and sheer malignancy of these tumors. Accordingly, we are able to establish in vitro TIL cultures from PDA tumor biopsies with efficiencies comparable to those for melanoma. We are currently analyzing the tumor reactivity of these TIL cultures by means of TCR repertoire analysis and by evaluating their in vitro and in vivo reactivity against autologous, patientderived xenograft tumors, with the aim of developing TIL therapy for recurrent PDA. Tumor-immune interaction in PDA also involves CD40-positive macrophages and dendritic cells that can be activated through CD40-ligation. Based on promising pre-clinical data, we are starting a neo-adjuvant PD-biomarker trial in resectable PDA that involves administration of agonist anti-CD40 Abs in conjunction with Gemcitabine chemotherapy. This study, which focuses on induction/monitoring of intratumoral immunity, is part of the EU FP7 program IACT (Immunostimulatory Agonist antibodies for Cancer Therapy). We will make use of the clinical anti-CD40 IS-Ab ChiLob7/4, which has been successfully tested in a single-agent phase I study (M. Glennie, P. Johnson, C. Ottensmeier, Southampton, UK).

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2 Abstracts

Parallel Session: Complement in Pathogenesis of Disease 749 Complement in the pathogenesis of inflammatory arthritis M. Holers University of Colorado Denver, Aurora, CO, USA The complement system is a major component of innate immunity, sits at the interface with adaptive immunity, and plays a central role in the mechanisms of autoimmunity that drive the pathogenesis of rheumatoid arthritis (RA). In this process, inappropriate complement recognition that is directed not in a beneficial manner to foreign pathogens but rather to self-tissues drives both the initial development of autoimmunity as well as subsequent cellular influx, synovial inflammation and bone erosions. Following upon informative human biomarker studies, we have used animal models of RA to understand the molecular basis for the injurious roles of complement. Our initial observations included that Complement Receptor Type 2 (CR2/CD21) engagement is necessary to develop pathogenic autoimmunity. Subsequently, while the complement classical and lectin pathways can initiate local activation following pathogenic autoantibody deposition, the alternative pathway is the only one of the three that is both necessary and sufficient to develop arthritis. In addition, each of the major complement effector mechanisms, C3a, C5a and the membrane attack complex, play important roles in promoting damage. Current studies are focused on determining how the normal regulatory mechanisms of complement are overcome in this environment, and elucidating additional ‘hard-wired’ amplification mechanisms that further increase damage.

765 Complement in Alzheimer’s disease A. J. Tenner University of California, Irvine, Irvine, CA, USA The classical and the alternative pathways of complement are activated by beta sheet fibrillar b amyloid, the major component of disease defining amyloid plaques in Alzheimer’s disease (AD). Our laboratory previously demonstrated that a C5a receptor (CD88) receptor antagonist reduced neuropathology and improved a memory task in mouse models of AD (Fonseca et al. 2009), suggesting that generation of the proinflammatory peptide C5a, can be detrimental in this disease. To further test the potential of C5a and/or C5aR as a therapeutic target, C5aR (CD88) knockout mice or mice containing an transgene for C5a under the control of the astrocyte specific GFAP promoter were crossed to mouse models of AD. While the AD mice exhibited a defect in the hippocampal dependent object location memory task (OLM) at 10 month old of age, the deficits were not apparent in the absence of C5aR, even in the presence of equal amyloid plaque deposits. In contrast, behavioral deficits were accelerated in GFAP-C5a transgene AD models. These data suggest a detrimental role of C5a through its interaction with C5aR, and supports the therapeutic targeting of this activation fragment or its proinflammatory receptor as a strategy to slow progression of AD in humans.

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Parallel Session: Molecular Basis and Prevention of Alloreactivity 748 Risks and benefits of alloreactivity M. H. M. Heemskerk Leiden University Medical Center, Leiden, The Netherlands TCRs can intrinsically be cross-reactive, binding and responding to multiple peptide-HLA complexes, enabling the adaptive immune system to protect the individual against a wide range of pathogens. This cross-reactivity of T cells is manifested clinically as graft versus host

or graft/organ rejection after HLA-mismatched transplantation. We have recently demonstrated that virus specific T cells often exhibit allo-HLA reactivity, and that the allo-HLA reactivity of T cells inducing graft versus host disease (GVHD) in vivo are single peptide specific. Besides detrimental GVHD and graft rejection, allo-HLA reactive T cells may also have beneficial anti-tumor specificities, demonstrated by the identification of high affinity PRAME specific allo-HLA restricted T cells. In this presentation I will present recent data demonstrating that the immunogenicity of foreign HLA can be exploited to generate strong immune response towards tumor-associated self-antigens useful for adoptive immunotherapeutic strategies.

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4 Abstracts

Parallel Session: Regulation of T Cell Function 750 Epigenetic control of stable Foxp3 expression in regulatory T cells J. Huehn Hemholtz Centre for Infection Research, Braunschweig, Germany Regulatory T cells (Tregs) are specialized immunosuppressive cells that are essential for the maintenance of self-tolerance and for the modulation of inflammatory immune responses. Tregs have been shown to either develop within the thymus or to be converted from naive T cells (Tnaive) in the periphery. In my talk, I will present recent data on the thymic and peripheral generation of Tregs, will discuss recent results on the epigenetic imprinting of the Treg phenotype and will discuss how microenvironmental signals influence decision-making steps during T cell differentiation.

751 TCR signal strength and differentiation C. G. King*, S. Keck†, M. Schmaler‡, D. Zehn§ & E. Palmer* *University of Basel, Basel, Switzerland, †Department of Transplantation Immunology and Nephrology, University of Basel, Basel, Switzerland, ‡University Hospital Basel, University of Basel, Basel, Switzerland, §Swiss Vaccine Research Institute, Lausanne, Switzerland An effective immune response relies on T cell activation, differentiation and development into memory T cells that contribute to protective recall responses. One way that heterogeneous T cell differentiation can be achieved is through asymmetric cell division, whereby one parent cell gives rise to two daughter cells with differential cell fates. Recent data from our laboratory have revealed asymmetric division as an important mechanistic link between TCR affinity and CD8+ effector T cell differentiation. Although TCR affinity also modulates T cell fitness and clonal dominance within the CD4+ T cell compartment, the influence of TCR affinity on the priming and differentiation of specific Th subsets is not well understood. Here we examined the impact of TCR affinity on the generation of Th1 and follicular helper (TFH) CD4+ T cell subsets. We found that T cells activated by low affinity antigen

are selectively defective in Th1 differentiation while maintaining their ability to differentiate into TFH effectors. Surprisingly, memory T cells generated after low affinity activation exhibited distinct recall effector responses compared to memory cells generated after high affinity activation. These data challenge the view that only strongly stimulated CD4+ T cells are capable of differentiating into the TFH and memory T cell compartments and suggest that differential strength of stimulation during primary T cell activation imprints unique and long lasting T cell differentiation programs. A potential role for asymmetric division in this process is discussed.

769 The influence of PTPN22 on the development of autoimmunity R. Zamoyska, R. Salmond, R. Brownlie & S. Sood University of Edinburgh, Edinburgh, UK Genome-wide association studies suggest that perturbations in T lymphocyte activation underpin disease pathogenesis for several autoimmune diseases. Good examples are allelic variants in PTPN22, encoding a cytoplasmic phosphatase, which negatively regulates T cell receptor signaling. Despite intense effort, it is unclear how PTPN22 mutations contribute to disease, however, loss of function PTPN22 mutations are associated with elevated T effector cell expansion and autoreactive B cells in human and mouse. Surprisingly, the complete absence of Ptpn22 in mice does not result in spontaneous autoimmunity. We have shown that Ptpn22 is also a key regulator of regulatory T cell (Treg) function by fine tuning TCR and integrin signals making Ptpn22 / Tregs more potent suppressors, able to restrain Ptpn22 / effector T cells under homeostatic conditions and maintain tolerance. Recent data from humans indicate that an expansion of Tregs is associated with expression of the PTPN22 disease-associated variants, as we see in mice. Ongoing studies are examining what tips the balance in favour of self-reactivity in T cells, which can lead to autoimmunity, and how PTPN22 mutations influence this process. References: Brownlie RJ, Miosge LA, Vassilakos D, Svensson LM, Cope A, Zamoyska R. Lack of the phosphatase PTPN22 increases adhesion of murine regulatory T cells to improve their immunosuppressive function. Sci Signal 2012; 5:ra87. doi: 10.1126/scisignal.2003365.

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Parallel Session: RNA Regulation of Development and Activation in the Immune System 752 The two faces of the RNA-binding protein HuR in inflammation and autoimmunity D. L. Kontoyiannis BSRC Fleming, Vari, Greece The phenotypes of immune cells are guided by flexible gene and RNA regulation circuits. Biochemical and genetic platforms demonstrate the importance of negative RNA regulation in cytokine gene expression by members of the so-called ‘ARE-binding proteins and suggest that blocking positive RNA regulators may be of clear clinical benefit against inflammatory pathologies’. The RBP Elavl1/HuR has been ascribed as such a positive RNA regulator. Using

conditional genetic platforms to silence or overexpress this RBP in the mouse, we unraveled a series of unprecedented, cell and signal specific responses requiring its function. In innate immune cells, HuR appears to act – not a as a positive but rather – as a regulatory RBP capable of suppressing systemic and organ specific inflammatory occurrences and cancer through changes in migration, polarization and plasticity programs. In adaptive immune cells, however, HuR has a pro-active role capable of regulating the thresholds of Tcell receptor activation, peripheral tolerance and autoimmunity. A systems view of the HuR:RNA and HuR:protein interactome in immune cells indicate that these seemingly opposing effects of HuR arise due to signal specific synergies and ribonucleoprotein assemblies controlling cellular phenotypes as a whole and not only restricted RNA subsets. Our current data highlight the importance of post-transcriptional nodes in the epigenetic coordination of cellular states and the management of immunopathologies.

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6 Abstracts

Plenary Session 1: Migration Tolerance versus Immunity

microenvironment can be exploited to create a therapeutic window to boost T cell-mediated antitumor immunity and immunotherapy. Supported the NIH (CA79765, CA094045 and AI082039) and Joanna M. Nicolay Melanoma Foundation.

743 IL-6 – friend or foe? Targeting the immune microenvironment for adoptive cell transfer cancer immunotherapy S. S. Evans Roswell Park Cancer Institute, Buffalo, NY, USA Proinflammatory cytokines including TNF, IL-1b, and IL-6 have emerged as major orchestrators of tumor progression through effects on the proliferation and survival of cancer cells as well as proangiogenic activities within the tumor microenvironment. Here, we report that IL-6 functions as a double-edged sword in murine tumor models. One the hand, IL-6 accelerates tumor growth by acting coordinately with an agonistic soluble form of the ligand-binding IL-6 receptor alpha subunit (sIL-6R) via a mechanism termed trans-signaling. Paradoxically, IL-6 produced by non-hematopoietic cells can be co-opted by systemic thermal therapy to stimulate CD8 T cellmediated antitumor immunity during adoptive transfer therapy. Live-imaging microscopy revealed that IL-6 trans-signaling enhances cytolysis of cognate tumor targets and tumor growth delay by augmenting E-P-selectin ligand, CXCR3, and LFA-1-dependent trafficking of tumor-specific CD8 T cells across tumor vascular endothelial barriers. Notably, homeostatic trafficking in the absence of thermal therapy was below the threshold necessary for sustained tumor control by adoptive T cell transfer therapy. Similar IL-6-dependent increases in LFA-1-dependent na€ıve CD8 T cell homing were observed in high endothelial venules in tumor-draining lymph nodes during systemic thermal therapy. A concomitant IL-6-dependent decrease in tumor infiltration by CD4+Foxp3+CD25+ regulatory T cells (Treg) during systemic thermal therapy resulted in substantial enhancement of CD8Teffector/Treg ratios (i.e., from ~5:1 to >35:1). Taken together, these findings suggest that the IL-6-enriched tumor

756 Neutrophil transmigration in vivo: mechanisms, dynamics and contribution to dissemination of systemic inflammation S. Nourshargh Queen Mary, University of London, London, UK It has long been known that inappropriate, excessive or prolonged leukocyte transmigration is associated with the pathogenesis of inflammatory disorders. However despite much research there has been a disappointingly slow progress in fruitful targeting of leukocyte trafficking for development of anti-inflammatory drugs, indicating a need for better understanding of the intricacies of targeted pathways. Using an advanced confocal intravital microscopy platform we have analysed details of neutrophil-vessel wall interactions in real-time in 3D and have noted previously unreported physiological responses (e.g sub-endothelial cell crawling1) and also potential pathological events such as neutrophil reverse transendothelial cell migration, a response that we have associated with dissemination of systemic inflammation2. Such studies have demonstrated that detailed analysis of leukocyte-vessel wall dynamics are likely to identify novel and disease-specific phenomena that could promote a change in thinking towards development of new therapeutic strategies. 1. Proebstl et al. J Exp Med 2012; 209:1219–34. 2. Woodfin A et al. Nat Immunol 2011; 12:761–9. This work was funded by The Wellcome Trust.

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Parallel Session: Antigen Presentation of MHC Class I and MHC Class I-like Molecules 739 Molecular aspects of MHC class I-restricted antigen processing P. Cresswell Department of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA The recognition of virus-infected cells or tumor cells by CD8-positive T lymphocytes relies on the formation of complexes of MHC class I molecules with virus-derived or tumor specific peptides, respectively. The peptides are generated in the cytosol and are translocated into the endoplasmic reticulum (ER), where peptide binding occurs, by the dimeric ATP-dependent Transporter associated with

Antigen Processing (TAP). TAP is a component of the multi-subunit Peptide Loading Complex (PLC). The PLC also incorporates the glycoprotein tapasin, a product of an MHC-linked gene, which physically links MHC class I-α2-microglobulin dimers to the PLC. The preferential association of MHC class I molecules with high affinity peptides is mediated by tapasin and is facilitated by an adaptation of the calnexin/calreticulin quality control cycle that normally facilitates the proper folding of a variety of glycoproteins in the ER prior to transport. The lectin chaperone calreticulin and the thiol oxidoreductase ERp57, two components of this folding pathway, are also associated with the PLC and play a critical role in the peptide binding process. The mechanism of action of the PLC and the nature of the quality control mechanisms underlying the formation of MHC class I-peptide complexes will be discussed.

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8 Abstracts

Parallel Session: B Cells; Bridging the Innate and Adaptive Immune Systems

766 The BAFF receptor transduces survival signals by co-opting the B cell receptor signaling pathway

753 INKT cells direct a unique B cell response against cognate glycolipid antigens

V. L. J. Tybulewicz*, E. Schweighoffer*, L. Vanes*, J. Nys*, D. Cantrell†, S. McCleary‡ & N. Smithers‡ *MRC National Institute for Medical Research, London, UK, † University of Dundee, Dundee, UK, ‡GlaxoSmithKline, Stevenage, UK

E. Leadbetter*, I. L. King*, E. Amielu*, M. Tighe*, K. Mohrs*, N. Veerapen†, G. Besra† & M. Mohrs* *Trudeau Institute, Saranac Lake, NY, USA, †University of Birmingham, Birmingham, UK Invariant Natural Killer T (iNKT) cells are glycolipid-specific innate lymphocytes emerging as critical players in the immune response to diverse infections and disease. iNKT cells are activated either through cognate interactions with lipid-loaded antigen-presenting cells, by antigen-independent cytokine-mediated signaling pathways, or a combination of both. Although iNKT cells directly influence humoral and cell-mediated immune responses, the spatio-temporal nature of these interactions and the cellular requirements for activation are largely undefined. Combining novel in situ confocal imaging of CD1d tetramer labeling to localize the endogenous iNKT cell population with cytokine reporter mice, we reveal the choreography of early murine splenic iNKT cell activation across diverse settings of glycolipid immunization and systemic infection with Streptococcus pneumoniae. We find that iNKT cells consolidate in the marginal zone and require dendritic cells lining the splenic marginal zone for activation following administration of cognate glycolipids and during systemic infection but not following exogenous cytokine administration. We also show that non-cognate iNKT-dependent mechanisms are sufficient to mediate effector outcomes throughout the entire splenic parenchyma. Collectively, these data provide new insight into how iNKT cells may serve as a natural adjuvant in facilitating adaptive immune responses irrespective of their tissue localization.

The survival of follicular B cells requires BAFFR, a receptor for BAFF, and the B cell antigen receptor (BCR). The requirement for the BCR is postulated to be due to a ligand-independent tonic signal from the receptor, which would be distinct from signals delivered following antigen binding. To investigate the nature of this tonic signal, we inducibly inactivated the Syk gene, coding for the Syk tyrosine kinase, a key signal transducer from the BCR following antigen binding. We show that loss of Syk results in death of most follicular B cells, because Syk-deficient B cells are unable to survive in response to BAFF. Genetic rescue studies demonstrate that Syk transduces key BAFFR survival signals via the ERK and PI3 kinase pathways. Surprisingly, we show that BAFFR signaling directly induces phosphorylation of both Syk and the BCR-associated Iga signaling subunit, and that BAFF-induced phosphorylation of Syk requires the BCR. We conclude that rather than delivering a tonic signal, the BCR and Iga are required for B cell survival because they function as adapter proteins in a BAFFR signaling pathway leading to the activation of Syk and hence to activation of ERK and PI3 kinase.

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Parallel Session: Conservation and Divergence of Mammalian Immune Systems 735 The role of a new pattern recognition receptor family (WC1) in activation of gamma delta T cells and promotion of vaccine efficacy C. Baldwin Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA, USA Effective vaccines will reduce the global health burden in both humans and food animals. To date, successful vaccines largely include those to diseases that only require antibody responses for protection. However activation of a particular type of white blood cell or leukocyte known as a gamma delta T lymphocyte may help advance the generation of vaccines against more difficult diseases such as tuberculosis that require a cellular Th1 type immune response. To date, these lymphocytes have received little attention for this purpose. The discovery of families of molecules known as pathogen recognition receptors (PRR) expressed on leukocytes and which recognize of a diverse array of bacteria and viruses has been an important advance for the study of immune responses and may be particularly relevant to engaging gamma delta T lymphocytes into protective immune responses. Such receptors do not display the diversity of other types of leukocyte receptors but they can effectively discriminate between self and non-self in an immediate rapid fashion. Thus, we have determined the specificity of a family of PRR molecules known as WC1 for their ability to interact with bacterial components and the role they play in stimulating protective cd T lymphocyte responses to the bacteria. Both of these bacteria cause serious infections of humans and livestock and T cells are critical to immune surveillance and protection. They are found as resident cells in many organs and tissues, including in humans and ruminants, and circulate at substantial numbers in the blood. We have shown that IFN-producing bovine T cells bearing the WC1 co-receptor are the major cell population responding in recall responses to Leptospira during the first month following priming by vaccination against serovar Hardjo. Activation of nonconventional T cells such as cd T cells that direct adaptive T cell responses has received little attention for improving vaccines because it is not clear how best to prime T cells for recall responses. Using RNA silencing we have shown that the WC1 co-receptor contributes to the ability of cd T cells to respond. Annotation of the bovine genome showed there were13 WC1 molecules which are distributed among cells to form a number of cd T cell subsets and that the response to bacteria is correlated with the WC1 molecules expressed. For example, the leptospiraresponsive T cells are found within a subset of the WC1.1+ T cell subpopulation. Data presented will thow that the WC1 molecules expressed act as pattern recognition receptors interacting directly with bacteria and point mutations in the WC1 molecule can disrupt the binding.

Consequently, different groups have diversified alternative NK cell receptor gene families that recognise MHC class I. This is a remarkable example of convergent evolution, which includes three rapidly evolving gene families from two structurally unrelated superfamilies, often making orthologs impossible to identify even between closely related species. Cattle and higher primates are the only mammals with an expanded killer-cell immunoglobulin-like (KIR) receptor gene complex. Cattle also have an expanded killer-cell lectin-like receptor (KLR) region and remain the only species known to have significantly expanded more than one receptor family. We have characterised the cattle KIR complex and compared it to KIR in another ruminant species, sheep. These immune genes have been under strong diversifying selection pressure for over 30 million years and in cattle, have not been significantly altered during domestication. Therefore this immunogenetic variation within cattle has evolved under natural selection and holds the potential to improve disease resistance through selective breeding. Moreover, a detailed understanding NK cell function in cattle will inform future vaccine studies and increase the utility of cattle as a model species.

758 Natural killer receptor diversity and function in humans and primates P. Parham Stanford University School of Medicine, Stanford, CA, USA In humans the killer-cell immunoglobulin-like receptors (KIR) are expressed by subpopulations of NK and T cells and recognize polymorphic epitopes of HLA class I molecules. Counterparts to the human system are found only in the catarrhine primates – Old world monkeys, apes and human – species in which the co-evolution of KIR with MHC class I epitopes can readily be discerned. A major event was the emergence of HLA-C from an HLA-B like ancestor, a transition observed in the orangutan, and the evolution of HLA-C to become the dominant source of KIR ligands in chimpanzees and humans. Driving the rapid evolution of KIR and MHC class I are the distinctive and competing functions that these ligand-receptor systems play in reproduction and immunity, as well as in NK-cell and T-cell immunity. Chimpanzees have a functionally more robust system that appears primarily driven by selection from pathogens, whereas the human system appears to be a compromise between the demands of reproduction and immune defense. The genetic basis for this compromise are the two distinctive forms of the KIR gene family, of which the A KIR haplotypes are more similar to chimpanzee KIR haplotypes and the B KIR haplotypes are uniquely human.

747 Differential NK receptor gene usage in mammals J. Hammond Pirbright Institute, Woking, UK Natural killer (NK) cells have fundamental roles in both the immune and reproductive systems. These essential functions are in part controlled by variable NK cell receptors that recognise MHC class I molecules. This receptor ligand system has therefore been subject to multiple and strong selection pressures during mammalian radiation.

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10 Abstracts

Parallel Session: Innate Lymphoid Cells 738 Lymphoid tissue inducer cells and their interactions with stromal cells in vivo ˜o J. Caaman University of Birmingham, Birmingham, UK Lymphoid tissue inducer (LTi) cells are dependent on the expression of the transcription factor Retinoid orphan receptor gamma t (Rorgt) for their development and belong to the group 3 of the recently characterized family of innate lymphoid cells (ILC 3). LTi cells have been described several years ago for their essential role on the development of secondary lymphoid tissues in the embryo. Interactions between these cells and stromal cells at the sites of secondary lymphoid tissue formation are essential for the development of these organs. Expression of the TNF family ligand Lymphotoxin alfa/beta on the surface of LTi cells binds to its receptor on stromal cells triggers (or induces) the maturation of the latter through a gene expression program that includes chemokines and cell adhesion molecules. These cell-cell interactions create a positive feedback loop to increase the recruitment of LTi cells and the maturation of stromal cells to create the lymph node anlagen. Importantly, adult LTi cells express OX40 and CD30L that are required for the generation of CD4+ T cell memory. Thus LTi cells have distinct roles in vivo during embryogensis as well as in adults. Our recent work shows that LTi cells are not required for inflammation-induced formation of lymphoid tissues. Surprisingly, we also demonstrated that proinflammatory signals rescue the development of most lymph nodes in the absence of these cells. Moreover, we demonstrate that certain lymphoid tissues such as fat associated lymphoid clusters develop independently of the presence of both LTi cells and LTab signaling.

755 Type-2 innate lymphoid cells (ILC2) in immunity, allergy and asthma A. N. J. McKenzie MRC Laboratory of Molecular Biology, Cambridge, UK Innate lymphoid cells (ILC) are recently identified members of the lymphoid lineage with emerging roles in mediating immune responses, and regulating tissue homeostasis and inflammation. We have demonstrated that ILC2, in addition to the classical Th2 cells, produce the type-2 cytokines IL-4, IL-5, IL-9, and IL-13, and play critical roles in the initiation of the type-2 response. Notably, ILC subsets appear to be particularly prevalent at mucosal surfaces, which are constantly exposed to infectious agents in the external environment. The fact that ILC2 rapidly secrete high levels of IL-5 and IL13, resulting in eosinophilia and mucus hypersecretion, suggests that these cells have evolved to provide a potent innate effector response to combat intestinal parasitic helminth infections. ILC2 require the transcription factors GATA3 and RORa for their development from lymphoid precursors. Ongoing studies using new reagents, including ILC2-deficient mice, are aimed at dissecting the roles of ILC2 in infection, asthma and allergy, and their interactions with innate and adaptive immune responses. Supported by grants from MRC, Wellcome, the American Asthma Foundation and Janssen.

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Parallel Session: Modulation of Host Response by Infection 736 Analysis of the role of the immune response in human cancers mediated by small DNA tumour viruses G. E. Blair University of Leeds, Leeds, UK Cell immortalisation and transformation is a consequence to the host cell of infection by certain viruses. These events are mediated by virus-encoded proteins and can lead to the development of cancers in animals and humans. Viruses that contain small DNA genomes are associated with a number of human cancers, such as cervical and head and neck cancers (mediated by human papillomaviruses, HPV) and certain skin cancers such as Merkel cell carcinoma (mediated by a recently-discovered human polyomavirus termed Merkel cell polyomavirus). In addition, other small human DNA viruses, such as the adenoviruses, immortalise and transform human cells although they are not currently associated with any identified human cancer. As well as subverting the cell cycle and suppressing apoptosis, these viruses modulate pathways in the cell that are involved in antigen presentation and immune cell recognition of the virus-transformed cell. These viruses and immune-associated pathways will be reviewed and recent research using whole-cell transcriptome (RNA-Seq) analysis will be described that aims to reveal further components involved in immune evasion by virustransformed cells.

763 T cell suppression by measles virus: modulating the thresholds? S. Schneider-Schaulies Univeristat Wurzburg, Wurzburg, Germany Abrogation of T cell expansion is a hallmark of measles virus (MV) induced immunosuppression, and this can be effectively induced on exposure of human or murine T cells to the MV glycoprotein complex independently of infection. Activation of the phosphatidylinositol-3-/Akt kinases and their downstream effectors were identified as prime targets of MV T cell inhibition, which was characterized by the failure to progress beyond the G1 phase, formation of unstable conjugates with DCs and physical paralysis as reflected by the loss of spreading functions and collapse of microvillar protrusions. The receptor structure used by MV in this setting is unknown, but appears to activate neutral and acid sphingomyelinases (NSMase and ASMase) to cause ceramide release. While ablation of ASMase efficiently rescued MV-induced physical paralysis, that of NSMase partially corrected for the loss of proliferative responses indicating that SMases target different steps in T cell activation. Most interestingly, genetic ablation of NSMase per se enhanced T cell spreading and early expansion. These findings indicate that at least NSMase acts to raise the threshold of early T cell activation which may, when activated in a timely of spatially inappropriate manner by MV, contributes to T cell inhibition.

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12 Abstracts

Plenary Session 2: Diseases from the Developing World: Closer to Home than You Think 762 The influence of vector transmission and neutrophils on the immune response to Leishmania major D. L. Sacks*, F. L. Ribeiro-Gomes† & N. C. Peters* *National Institute of Health, Bethesda, MD, USA, †NIAID, National Institute of Health, Bethesda, MD, USA Following inoculation of Leishmania major by sand fly bite or by needle, neutrophils are the predominant recruited and infected cells in the early stages of infection in the skin, and depletion of neutrophils hastens the development of Th1 mediated immunity. We assessed the interaction of neutrophils and DCs in vivo and in vitro and the consequences of this interaction for the anti-Leishmania T cell response. Following injection of RFP-L. major into the ear dermis of C57B/6 mice, the RFP+DCs recovered from the skin at early time points were positive for neutrophil markers, suggesting that the DCs became infected via uptake of infected neutrophils. Following neutrophil depletion, the RFP+DCs remained negative for these markers, and early OT-II CD4+ T cell priming in response to infection with OVA-L. major was markedly enhanced. In vitro, the uptake of dermal-derived infected neutrophils by bone marrow DCs completely inhibited their ability to cross present OVA to OT-I CD8+ T cells. A role for the receptor tyrosine kinase MerTK in mediating the apoptotic cell-induced inhibition of DC function was shown. These data suggest that L. major exploits the massive neutrophilic response to sand fly bite and the apoptotic cell clearance function of DCs to inhibit early T cell priming in order to establish infection and promote disease.

764 Protective and pathogenic immunity in dengue virus infection G. Screaton Imperial College, London, UK Dengue is a mosquito borne virus infection occurring in tropical and subtropical countries. There are estimated to be around 400 million infections annually of which approximately one quarter are clinically apparent. The majority of these result in a self-limited, but non the less unpleasant febrile illness, dengue fever. One to five percent of infections lead to a more severe disease, dengue haemorrhagic fever, which is characterized by a severe vascular leak, hypovolaemia and in extreme cases shock and haemorrhage. Dengue exists as four highly divergent serotypes differing in sequence by some 30–35%; infection with one serotype does not provide protection against the other three. In endemic areas serotypes frequently co-circulate and repeat infections are common. Interestingly, severe disease is much more common in secondary as opposed to primary infections, implying a role of the acquired immune system in disease pathogenesis. Understanding this immune enhancement of disease is crucial for the design of safe and effective vaccines. Through clinical collaborations in Thailand and Vietnam we have been studying the immune response to dengue in cohorts of infected children and will describe components of T cell and antibody mediated immunity and their potential to enhance or protect from dengue infection.

ª 2013 The Author(s). © 2013 Blackwell Publishing Ltd, Immunology, 140 (Suppl. 1), 1–18

Abstracts 13

Parallel Session: Autoimmunity 737 TNF receptor family members in the control of the T cell response J. Borst, Y. Xiao, N. Babala, T. Ahrends, V. Peperzak, E. Veraar & H.van Eenennaam The Netherlands Cancer Institute, Amsterdam, The Netherlands In this lecture, the molecular basis of T cell costimulation will be reviewed. Costimulatory TNF receptor family members contribute to T cell priming, effector function, memory formation and memory quality. They deliver certain specific survival signals to T cells during priming in lymphoid tissue and during the effector phase in non-

lymphoid tissue. Overall, there is a delicate division of labor between cytokines and costimulatory molecules to ensure T cell survival throughout the consecutive phases of the T cell response. Costimulatory molecules are essential to counteract apoptotic cell death in the contraction phase and elegantly work together to form and maintain the memory T cell pool. Whereas the CD28 costimulatory receptor appears to act as a signal amplifier for the TCR, TNF receptor family members not only contribute quantitatively, but also provide unique qualitative input into T cells and affect CD4 and CD8 T cells in different ways. One important aspect is memory T cell programming, a phenomenon that may take place during priming, in a beautifully orchestrated contact between the dendritic cell, the CD4 and the CD8 T cell. We will outline how agonism for TNF receptor family members can be employed to optimize anti-tumor immunity.

ª 2013 The Author(s). © 2013 Blackwell Publishing Ltd, Immunology, 140 (Suppl. 1), 1–18

14 Abstracts

Parallel Session: Innate Immune Defence Mechanisms in the Lung

734 Dendritic cells, innate lymphocytes and defence of mucosal surfaces in infection

767 Pathogenic potential of type I IFN in influenza infection

G. Belz Walter + Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia

A. Wack*, S. Davidson† & S. Crotta† *NIMR, London, UK, †MRC National Institute for Medical Research, London, UK Influenza symptoms vary from mild to severe disease or even death. Type I interferon (IFNab) is recognized to have antiviral function in vitro, but its role in restricting influenza infection in vivo is less clear. We show here that mouse strains highly susceptible to influenza virus showed significantly higher levels of IFNab, compared to resistant mouse strains. In the resistant mouse genetic background, IFNab receptor (IFNabR) deficiency slightly increased morbidity, mortality and lung damage. In contrast, IFNabR deficiency in highly susceptible strains leads to markedly reduced mortality, lung damage and inflammation. Plasmacytoid dendritic cells (pDCs), the main source of systemic IFNab, are more enriched in infected lungs of susceptible compared to resistant strains. pDC depletion from susceptible mice lowered influenza-induced IFNab levels, ameliorated disease and reduced inflammation, suggesting that excessive pDCderived IFNab can exert pro-inflammatory, pathogenic effects. The upstream causes and downstream mechanisms of excessive IFN responses to influenza virus infection will be discussed. Our findings have important implications for prediction and treatment of severe influenza. Funding by the Medical Research Council UK is gratefully acknowledged.

Respiratory and intestinal infections remain a significant global public health challenge. Resolution of infection requires rapid detection of infection and depends on initiation of an immune cascade in which virus-specific B and T cells are amplified to control and clear virus together with mechanisms to activate repair of damaged mucosal epithelium. Populations of innate lymphocytes form a critical interface between the multitude of microorganisms and antigens encountered by the body on a daily basis, and dendritic cells that are key initiators of adaptive immune responses. We have endeavoured to delineate the molecular regulation of these cell types during their development and in response to infection. This has revealed key roles for specific transcription factors in individual cell types necessary for mucosal protection.

ª 2013 The Author(s). © 2013 Blackwell Publishing Ltd, Immunology, 140 (Suppl. 1), 1–18

Abstracts 15

Parallel Session: Metabolic Control of Immunity in Health and Disease 760 Glucose uptake and metabolic programming of T cell subsets J. C. Rathmell, V. A. Gerriets, A. N. Macintyre & A. G. Nichols Duke University, Durham, NC, USA Lymphocyte activation leads to a rapid transition from a quiescent state to rapid proliferation and differentiation. We have examined this metabolic reprogramming and found that CD4 T cell subsets are metabolically distinct. The effector T cell fates (Th1, Th2, Th17) activate a highly glycolytic program. Regulatory T cells (Treg), in contrast, utilize a more oxidative metabolism and utilize lipids as a

major fuel. These metabolic distinctions may allow new understanding and approaches to manipulate immunity. To directly target T cell metabolic pathways we have examined Glut1 regulation and Glut1 conditional knockout animals. Glut1 is a member of the glucose transporter family, of which T cells express several members. Interestingly, normal resting peripheral T cells do not rely on Glut1. Upon activation, however, Glut1-deficient T cells fail to induce glucose uptake and metabolism and do not grow and proliferate. Glut1-deficient Treg, in contrast, appear normal in vivo. These data show that activated T cells specifically rely on Glut1 to generate effectors while Treg are capable of utilizing other metabolic pathways. Understanding mechanisms that regulate T cell Glut1 and glucose metabolism, therefore, may provide new tools to modulate immunity the balance of T cell effector and regulatory populations.

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16 Abstracts

Parallel Session: Neuroimmunology – Immune/Nerve Cell Interactions 733 Mast cells, glia and neuroinflammation: partners in crime? S. D. Skaper*, L. Facci† & P. Giusti† *University of Padua, Padua, Italy, †Universita degli Studi di Padova, Padova, Italy Recognition that an extensive communication exists between the immune system and the central nervous system (CNS) is one of the more fundamental recent advances in neuroscience. Glia, and microglia in particular, elaborate pro-inflammatory mediators to participate in this communication network and play key roles in CNS disorders from neuropathic pain and epilepsy to neurodegenerative diseases. Microglia respond also to pro-inflammatory signals released from other non-neuronal cells, mainly those of immune origin, like mast cells. The latter are found in most tissues, are resident in the CNS, and traverse the blood-spinal cord and blood-brain barriers where barrier compromise results from CNS pathology. Recognition of mast cell – glia communication opens new perspectives for development of therapies to target neuroinflammation by differentially modulating activation of non-neuronal cells normally controlling neuronal sensitization – both peripherally and centrally. Mast cells and glia possess endogenous homeostatic mechanisms/molecules which are up-regulated following tissue damage or stimulation of inflammatory responses. Such molecules include N-palmitoylethanolamine (PEA), which may have a key role in maintaining cellular homeostasis in the face of external stressors provoking, for example, inflammation. PEA is efficacious in mast-cell mediated experimental models of acute and neurogenic inflammation. Here we provide an overview of recent progress relating to the pathobiology of neuroinflammation, the role of microglia, neuro-immune interactions involving mast cells, and the possibility that mast cell-microglia cross talk may contribute to the exacerbation of acute symptoms of chronic neurodegenerative disease and accelerate disease progression, as well as promote pain transmission pathways and thereby provide a target for therapeutic intervention.

745 Can we switch microglia phenotype to foster neuroprotection? D. Giunti University of Genoa, Genoa, Italy Microglia, the resident innate immune cells in the brain, are highly active and continuously survey their microenvironment for ‘danger signals’ interacting dynamically with surrounding cells. Upon CNS injury, microglia become activated, undergoing morphological and

functional changes from so-called ‘classically activated’, with a highly pro-inflammatory, neurotoxic profile, to ‘alternatively activated’ associated with a beneficial, less inflammatory, neuroprotective profile. Microglia activation has been demonstrated in most neurological diseases of diverse etiology and has been implicated as a contributor to neurodegeneration. The possibility to promote their neuroprotective phenotype has therefore become a therapeutic goal. This presentation will focus on the role of microglia in multiple sclerosis, a prototype of inflammatory neurodegenerative disease, and the effect of currently approved or on-trial anti-inflammatory therapeutic strategies that might mediate neuroprotection at least in part through fostering a switch in microglia’s functional phenotype from a detrimental to a protective one. In particular, the effect of mesenchymal stem cells and their potential role in neuroprotection through their ‘calming’ effect on microglia via release of CX3CL1 will be discussed, and recent data on the signaling pathway through which the newly approved dimethyl fumarate modulates microglial activation will be presented.

754 Immune intervention in neurodegenerative diseases M. A. Lynch Trinity College Institute of Neuroscience, Dublin, Ireland Most neurodegenerative diseases, and animal models of disease, share a number of characteristics, among which are inflammatory and oxidative changes. A key element in driving these changes is glial activation, particularly activation of microglia. However, like macrophages, microglia adopt different activation states, at least in vitro; IFNg induces increases in expression of TNFa and iNOS which are markers of classical activation, whereas IL-4 upregulates mannose receptor and arginase 1 which identify the alternative activation state. We have compared age-related changes in these markers in the hippocampus of wild type mice and mice which overexpress amyloid precursor protein and presenilin 1 (APP/PS1 mice) and have shown that development of cognitive dysfunction is accompanied by a shift in the balance between markers of classical activation and alternative activation and with increased accumulation of amyloid-b. The evidence suggests that increased blood brain barrier permeability in aged and APP/PS1 mice permits the infiltration of IFNg-producing Th1 cells providing the trigger for classical activation. Consistently, adoptive transfer of Th1 cells induces microglial activation in the hippocampus of APP/PS1 mice and this is attenuated by treatment of mice with anti-IFNg antibody. Data will be presented which further support the pivotal role of infiltrating cells in driving the neuroinflammation that develops with age in APP/PS1 mice, and which suggest that strategies directed at preventing infiltration may be useful in reducing amyloid pathology.

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Abstracts 17

Parallel Session: The Ageing Immune System: Consequences and Interventions 732 Thymus generation and regeneration G. Anderson University of Birmingham, Birmingham, UK The thymus provides a specialised microenvironment that supports the generation of self-tolerant MHC restricted CD4+ and CD8+ Tcells from blood-borne progenitors. However, T-cell production in the thymus is not constant. The progressive loss of thymus function as a result of age-related thymus atrophy results in a decline in newly produced T-cells, resulting in an increasingly oligoclonal T-cell repertoire and increased susceptibility to infectious disease. Moreover, thymus atrophy limits the efficiency of T-cell reconstitution following bone marrow transplantation for the treatment of cancers. Importantly, thymic atrophy is linked to a decline in cortical and medullary thymic epithelial cell (TEC) compartments that drive intrathymic T-cell development and selection. Strategies aimed at regenerating, or replacing, TEC populations are likely useful in enhancing T-cell mediated immunity, both in the elderly and following ablative therapies. To provide a starting point in harnessing the potential of TEC progenitors in this setting, we have defined a TEC progenitor subset that can generate both cortical and medullary TEC populations in vivo. Additional in vivo studies show that induced Pleuripotent Stem (iPS) cells can correct the thymus deficiency of athymic nude mice, providing a manipulable source of cells which with to study TEC generation under defined experimental conditions.

741 B cells and humoral immunity in older adults D. Dunn-Walters Kings College London, London, UK Diversity of repertoire is a necessity for effective function of the immune system. We have previously shown that repertoire diversity can be compromised in old age and that this correlates with poor health. We have also investigated the repertoire changes during vaccination in older people and have seen that age-related perturbations of the repertoire can be seen at different stages of the response. Closer analysis, using high throughput sequencing, shows that the IgA response in particular seems to be much delayed, to the extent that there is not much expansion of IgA cells at day 7 after vaccination in old compared to young, and where the young group had resolved the IgA expansions by day 28, the old group still had significant Ig gene expansion. Cell sorting to investigate age-related changes in different B cell subsets (by CD27 and IgD phenotype) has indicated that the repertoire of IgG memory B cells also changes with age, such that it acquires characteristics of IGHV use that we previously reported were distinguishing features of so-called IgM memory cells. These differences indicate an age-related change in the dynamics of the response and in the selection pressures on the B cell repertoire.

ª 2013 The Author(s). © 2013 Blackwell Publishing Ltd, Immunology, 140 (Suppl. 1), 1–18

18 Abstracts

Parallel Session: The Dynamics of Immune Cell Recognition and Signalling

768 Spatiotemporal organisation of the T cell signalling system as a regulator of function

740 Using super-resolution microscopy to watch immune cells kill

C. Wuelfing Bristol University, Bristol, UK

D. M. Davis University of Manchester, Manchester, UK

The spatiotemporal organisation of a signalling system determines how signalling information is processed inside live cells by controlling the efficiency of signalling interactions. In the activation of primary T cells by antigen presenting cells (APC) we have imaged >60 elements of T cell activation and found them to be dynamically distributed throughout the activating T cell in a highly diverse fashion. Identifying groups of related signalling intermediates, signal initiation and core signalling preferred the centre of the T cell:APC interface, while signal amplification was localized in a newly discovered transient lamellum. Adaptor proteins provided bi-directional links between these two dominant signalling areas. The observed signalling distributions were driven by cell biologically distinct structures, a large protein assembly at the interface centre, a large invagination, the actin-supported interface periphery as extended by smaller individual lamella, and the whole-interface actin-driven lamellum, as characterised here. Actin dynamics with their control by more than ten imaged core regulators drove signalling distributions through regulation of the underlying structures. A highly undulating T cell/ APC interface, as characterised by electron microscopy, imposed substantial constraints on T cell organization. By understanding how signalling processes fit into this spatiotemporal context, their function can be efficiently elucidated.

Cell-contact dependent regulation of immune cell responses plays a vital role in balancing the need for rapid and efficient responses to a wide variety of pathological challenges, while at the same time maintaining self-tolerance. Over the last decade, much research has studied how immune cell interactions are often accompanied by the segregation of proteins into micrometer- and submicrometer-scale domains at an immune synapse. The emerging new paradigm is that interactions between immune cell receptors, kinases and adaptors are at least in part controlled by transient interactions between supramolecular assemblies. This is a significantly different concept from a linear cascade of individual protein-protein interactions depicted in textbook diagrams of immune receptor signaling pathways. Here, I will present new data using high- and super-resolution imaging techniques that reveal novel insights into molecular recognition by human Natural Killer cells and how specific effector functions are realized.

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Abstracts of the Annual Congress of the British Society for Immunology. December 2-5, 2013. Liverpool, United Kingdom.

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