Invited Speaker Abstracts BSI 2014 Opening Ceremony & Keynote Address 127 Inflammasomes in health and disease R. A. Flavell Immunobiology, HHMI, Yale University School of Medicine, New Haven, CT, USA Dysregulation of the immune system and host-microbiota interaction has been associated with the development of a variety of inflammatory as well as metabolic diseases such as obesity and diabetes. Recent studies in our lab have elucidated the important function of inflammasomes as steady-state sensors and regulators of the gut microbiota. Mice with a disrupted inflammasome pathway have been shown to develop a colitogenic microbial community, which resulted

in exacerbation of chemical-induced colitis and diet-induced steatohepatitis, obesity and type 2 diabetes. These disease phenotypes have been associated with dysbiosis resulting from the expansion of “pathobionts” which are believed to be causally driving pathogenesis. A key issue is to identify and isolate such problematic organisms. I will discuss a new way we have developed to identify and isolate such organisms from mice and humans. Using this approach, we have shown that predicted “pathobionts” from human patients with inflammatory bowel diseases (IBD) that we isolated by this approach can drive susceptibility to severe IBD in germ free mice, whereas predicted harmless microbes from the same patients do not. These data suggest a significant involvement of such microbes in human disease. These approaches should be useful to the study of human microbiota driven diseases.

This section includes all abstracts received by our publication cutoff date.

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2 Abstracts

Generation, Properties & Clinical Relevance of Antibodies in Autoimmune Disease and Allo-Transplantation 54 The clinical relevance of alloantibodies in transplantation D. Roelen Department of Immunohematology and Blood Transfusion, LUMC, Leiden, The Netherlands The introduction of new sensitive assays for the detection of HLA antibodies on the basis of their binding to isolated HLA molecules has an enormous impact on the decision making process with respect to donor selection for (sensitised) patients. In the past, when only complement-dependent cytotoxicity was used as a tool to define HLA alloantibodies, the presence of donor-specific antibodies (DSA) before transplantation was considered a contra-indication for (renal) transplantation with that donor. The interpretation of the current DSA results is far more difficult and leads to a lot of discussions and controversy. The problems associated with the use of solid phase assays for clinical decision making and possible solutions are discussed.

113 Alloantibodies in renal transplantation – therapeutic strategies M. R. Clatworthy Department of Medicine, University of Cambridge, Cambridge, UK There is a growing awareness in solid organ transplantation of the potential deleterious effects of antibody on allografts. In addition to the challenges to short- and long-term graft survival encountered when transplanting individuals with pre-formed ABO or human leucocyte antigen (HLA) antibodies, it is now well-established that the appearance of de novo donor-specific antibodies is associated with acute antibody-mediated rejection (ABMR). Such antibodies, and indeed some non-HLA antibodies, may also contribute to long-term allograft attrition, in the guise of chronic ABMR. This presentation will consider potential therapeutic strategies to tackle the problem of alloantibodies in transplantation. This includes the use of immunomodulators that might reduce the occurrence of de novo donor-specific antibodies, those that might limit established antibody production by targeting plasma cells, and therapies that block the effector functions of antibody.

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Abstracts 3

Infection & Allergy: Modulation & Mechanisms 88 Non-redundant roles for basophils in allergy and protective immunity H. Karasuyama Immune Regulation, Tokyo Medical and Dental University, Tokyo, Japan Basophils are the least common granulocytes, representing 90%), will be used at a near intensive care unit or patient testing site and will be operated by relatively untrained staff. The system will include panels of antibodies that will denote either the absence or presence of bacterial infections through the upregulation of distinct molecules on the surface of blood neutrophils. The novelty of the test is that it does not require samples from suspected sites of infection and that it is using the main biological response to bacterial infections as a diagnostic target. Rapidly identifying bacterial

172 Sooner is better: identifying early host responses to enterohemorrhagic E. coli and Shiga toxins D. Stearns-Kurosawa Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, MA, USA How many patients come to the emergency room with a moderate fever, high blood leukocyte count, rapid heart rate and malaise? Short of obvious trauma, many patients present with these non-specific symptoms and the diagnostic and prognostic puzzles are often complicated by non-specific tests. New biomarkers like certain damage associated molecular patterns (DAMPs) that report cell injury may distinguish sterile from infectious sepsis aetiology with levels corresponding to severity. In our studies of toxigenic enterohemorrhagic E. coli infection, early biomarkers of cell injury, inflammation and coagulopathy reflect Shiga toxin-induced cell injury during the time frame when clinical presentation is relatively non-specific. Molecular biomarkers have long been sought as a panacea for when clinical testing falls short, but their true value likely lies less for diagnosis and more as a partner for monitoring disease severity and response to therapy.

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8 Abstracts

Plenary 1: Memories of Time and Place: Immune Cells on the Move and in Tissues 107 In vitro and in vivo analysis of immunological synapses M. L. Dustin NDORMS, Kennedy Institute of Rheumatology, Headington, UK CD8 T cells undergo different modes of interaction at different stages of differentiation. In this talk I will explore interactions of CD8 T cells during early phases of priming, in premitotic effector phases and in tissues with fully differentiated CTL in the context of viral, bacterial and parasitic infection. Notable features include specialized cytotoxic function in premitotic CD8 T cells during infection and the distinct behaviour of perivascular CD8 T cells in the meninges during viral and parasitic infections.

Tissue Resident Memory CD8+ T cells: quantity, function, and protection D. Masopust University of Minnesota Medical School, USA The quantity, migration patterns, and function of memory CD8 T cells relate to their protective efficacy upon anamnestic viral infection. This presentation will focus on a recently defined lineage of non-recirculating memory T cells within the mucosa, resident memory CD8 T cells (TRM), and related technical and conceptual issues. Recent data will be presented indicating that (i) isolation methods fail to extract most CD8+ TRM from mucosal tissues and biases

against certain subsets, (ii) memory subset homing to inflammation does not conform to previously hypothesized migration patterns, and (iii) TRM greatly outnumber recirculating cells within mucosal tissues. Functions for mucosal TRM will be described; as local sensors of previously encountered antigens that broadcast innate-like alarm signals that activate local humoral, cell-mediated, and innate immunity and confer an antiviral state that can confer near sterilizing immunity against a viral challenge within the mouse female reproductive tract. Applications for vaccines will be discussed. 110 Trained immunity: a memory for the innate immune responses M. Netea Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands The inability of innate immunity to build an immunological memory, considered one of the main characteristics differentiating it from adaptive immunity, has been recently challenged by studies in plants, invertebrates and mammals. The increasing evidence for immunological memory within innate immunity is the focus of intense investigation. Long-term reprogramming of innate immunity, which induces adaptive traits and has been termed “trained immunity”, characterises prototypical innate immune cells such as natural killer cells and monocytes, and provides protection against reinfection in a T/B-cell-independent manner. Both specific signalling mechanisms and non-specific epigenetic reprogramming have been implicated in mediating these effects. This concept represents a paradigm change in immunity and its putative role in resistance to reinfection may represent the next step in the design of future vaccines.

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Abstracts 9

Immunometabolism Control in Chronic Inflammatory Diseases 38 The impact of carbohydrate metabolism on immunology A. Haschemi Medical University of Vienna, Wien, Austria A phenotypic shift in cells is often associated with reprograming of cellular bio-energetics and is likely to account for the specific demand of precursor molecules, available bio-energy and appropriate redox-balance to adopt their function. Investigating the metabolism of immune cells, especially of macrophages, has a very long tradition. However, recent publications indicate that there are several functional interfaces of cellular metabolism, protein signalling and macrophage function. In this presentation I will mainly focus on the adaptation of primary carbohydrate metabolism, including glycolysis and the pentose phosphate pathway, and how these may create a bio-energetic environment to properly sustain inflammation. Specifically, I will present data on the recently characterised sedoheptulose kinase (CARKL) and how the endogenous regulation of this kinase participates in immune cell activation by regulating both the cellular carbon-flux and the redox-state. Understanding the functional interactions between metabolism and protein signalling will provide crucial insights into the phenotypes of macrophages and their activation process. Currently the main challenge is to link metabolic processes with protein-signalling events, gene-expression and their functional consequences for e.g. macrophages. In the long term, we need to decipher the causalities of these cellular processes to generate even more comprehensive models of immunology in health and disease.

145 Metabolic control of regulatory T-cell function V. De Rosa Consiglio Nazionale delle Ricerche (IEOS-CNR), Napoli, Italy Regulatory T lymphocytes (Treg) are essential for maintaining immune tolerance and homeostasis. Although different mechanisms contribute to Treg cell activity in various biological settings, the expression of the forkhead-box-P3 (FoxP3) transcription factor is necessary for Treg cell function. The array of Treg cell types that control immune response is still very debated and there is considerable interest to identify the molecular pathways underlying generation and stability of Treg cells. Extensive experimental evidence has suggested a correlation between the occurrence of autoimmune disorders and the functional impairment in T cell subsets involved in the control of peripheral immune tolerance such as Treg cells. In multiple sclerosis (MS), an alteration of suppressive function and/or a numerical deficit in Treg cell compartment has been described. The transcription factor FoxP3 has a key role in the development and proper function of Tregs. Recent studies have shown the link between metabolic programmes and lymphocyte activation. To this aim, we evaluated FoxP3 expression in activation-induced regulatory T cells (a-iTregs) generated in vitro from CD4+CD25- in MS patients and healthy subjects, respectively. With this approach, we evaluated whether metabolic changes may influence the FoxP3 expression and the suppressive activity of regulatory T cells. All these data should help the comprehension of the mechanisms that control the immune system function and its alteration during autoimmune diseases.

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10 Abstracts

Peripheral Tissues in the Control of Adaptive Immune Responses 31 Epigenetic control of the decidual immune response A. Erlebacher Department of Pathology, NYU School of Medicine, New York, NY, USA How the fetus and placenta avoid rejection by the maternal immune system during pregnancy is a question that has fascinated both reproductive biologists and immunologists alike since the time it was first articulated as a paradox of transplantation immunology nearly 60 years ago. These mechanisms also have major implications for complications of human health, as their disruption has been anticipated to underlie various complications of human pregnancy. Using the mouse as a model organism, our laboratory has identified several key mechanisms that explain the unique immunological status of the fetus and placenta. These include the entrapment of dendritic cells within the decidua, which is the specialised stromal tissue that surrounds the conceptus, and the minimisation of decidual macrophage and dendritic cell tissue densities. More recent work has focused on the intrinsic inflammatory characteristics of decidual stromal cells. We have found that these cells engage a developmental programme that epigenetically silences the expression of key T cell-attracting chemokines. As a result, activated T cells are not able to accumulate at the maternal-fetal interface to pose a threat to fetal survival. Epigenetic mechanisms also appear to limit the general ability of decidual stromal cells to mount an inflammatory response. These results reveal a novel feature of the maternal-fetal interface within broad implications for the immunology of pregnancy. Supported by grants from the NIH, the March of Dimes and the American Cancer Society.

39 Alveolar macrophages determine outcome in MRSA pneumonia

These findings suggest that airway macrophages provide a critical anti-inflammatory role in the setting of MRSA pneumonia and that strategies to prevent their depletion through necroptosis would help to prevent inflammatory airway damage.

40 Mechanisms of epidermal and intradermal routes of immunisation B. Combadie`re Immunity and Infection, INSERM U1135, Cimi Paris, Paris, France Vaccination is one of the most effective and affordable ways to save 2–3 millions lives each year. However, critical issues on effectiveness and acceptability for vaccines are still discussed. Innovative vaccines are explored to conquering infectious diseases (Hepatitis, Dengue, HIV, Malaria and TB). Therefore, two aspects have been added, which are the prediction of intensity of immune responses through systems biology approaches and the development of alternative routes of immunisation for increased efficacy. Most of the current vaccines are administered by intramuscular (IM) or subcutaneous (SC) routes, However, alternative routes are regaining popularity. Several factors strongly justify the use of these methods. The rationalisation of targeting of the different cutaneous layers, i.e. the epidermis, dermis or hypodermis as well as our advances in knowledge about the plasticity of antigen-presenting cells (LC, DC) allow us today to propose rational immunisation procedures. We have previously demonstrated that vaccination by this route activates CD8 T cells in a human clinical Phase I study using conventional flu vaccine. One can thus question the impact of vaccine compounds and specific skin APC targeting, on the intensity, the nature and the quality of immune responses and consequently on the control of infectious diseases. We have studied the mechanism of immunisation using a particle-based vaccine including the role of LC and dermalDC as well as the recruitment of inflammatory cells and their impact on different arms of immunity. The vaccination field is at a turning point with ideas, however, increasingly relies on basic research in understanding and translating human immunity.

A. Prince Pediatrics, Columbia University, New York, NY, USA Staphylococcus aureus evokes an intense proinflammatory response in the lung associated with substantial morbidity and mortality. The recruitment of neutrophils is a major characteristic of staphylococcal infection, but models of MRSA pneumonia fail to demonstrate a correlation between numbers of neutrophils and outcome. USA300 MRSA express multiple toxins that target leukocytes. As the population of neutrophils in the setting of acute pneumonia is maintained from bone marrow derived stores, we postulated that macrophages might be targeted by MRSA toxins and not readily replenished. By performing clodronate depletion experiments, we found that airway macrophages are critical in the initial stages of acute MRSA pneumonia in a murine model of infection. A collection of USA300 mutants lacking specific leukotoxins were compared to the WT strain demonstrating that human macrophages as well as THP-1 cells undergo necroptosis in response to agr-dependent staphylococcal toxins. In the murine model, the airway macrophage population was acutely depleted by toxin-induced RIP1/RIP3-mediated necroptosis, a highly inflammatory mechanism of death. In contrast to the neutrophil population, lost macrophages were not immediately replenished. Rip3-/- mice had significantly improved MRSA clearance and preserved lung architecture. The macrophage population in the Rip3-/- mice had increased expression of CD200R and CD206 markers, characteristic of an immunomodulatory phenotype. The Rip3-/mice also had decreased production of IL-6, TNF, KC and IL-1b.

98 There and back again: modelling the regulation of immune responses by cutaneous DC M. E. Polak*,† *Innate Immunity Group, University of Southampton, Southampton, UK, †Institute for Life Sciences, University of Southampton, Southampton, UK As an immuno-competent site, the skin harbours a dense network of dendritic cells (DCs), including epidermal DCs, and Langerhans’ cells (LCs), able to induce either immune activation or tolerance. LC function can be modified by cytokine signals from keratinocytes, such as tumour necrosis factor alpha (TNF-a) in psoriasis and thymic stromal lymphopoietin (TSLP) in AD, resulting in alteration of the type of adaptive immune responses induced. Detailed understanding of human LCs biology is lacking. We have recently documented the potency of TNF-a to promote LC immunogenic function, including induction of co-stimulatory molecules and enhancement of cross-priming efficiency. Utilising an ex vivo skin challenge model we have shown that while both TNFa and TSLP matured LCs were able to activate T lymphocyte proliferation and IFNc release, TSLP significantly augmented LC potential to prime Th2 responses (n = 3, p < 0.01).

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Abstracts 11 Utilising genome-wide comparison of the transcriptional profiles of skin LCs and dermal DCs exposed to epidermal cytokines, we have delineated a “gene signature” of human LCs, coding for antigen capture, intracellular trafficking and formation of immunoproteasome. Functional interference of caveolin abrogated LCs superior ability to cross-present antigens to CD8+ T lymphocytes, highlighting the importance of identified networks to biological function. In contrast to TNF-a, TSLP maintained the transcriptome of LCs in situ, lacking the antigen processing associated signature. Consistently, the ability of TSLP-matured LCs to stimulate antigen-specific CD8 T cells was diminished. Our work demonstrates the use of a systems immunology approach for identification of candidate molecular targets for the treatment of cutaneous inflammation.

112 Tissue-specific migration receptor controlling cutaneous memory T cells B. Moser Cardiff University, School of Medicine, Cardiff, UK Localisation of memory T-cells to human skin is essential for longterm immune surveillance and the maintenance of barrier integrity. Defects in the immune surveillance system as found in immunosup-

pressed transplant patients and elderly people, is frequently associated with uncontrolled skin infections and cancer. We have reported that the chemokine receptor CCR8 is highly expressed by skin memory T-cells, including the recently described resident memory T-cell (TRM) population, but is largely absent on peripheral blood mononuclear cells. In healthy skin, CCR8 is found on lymphocytes (ab Tcells, gd T-cells, NK cells) but not macrophages and DC. Of note, its expression during na€ıve T-cell activation is fully dependent on “skin factors” produced during culture of intact epidermal tissue or skinderived keratinocytes but not dermal fibroblasts or skin-unrelated epithelial cells. We propose a model whereby the tissue environment plays a fundamental role in generating long-lived memory T-cells with tissue-selective homing properties. During memory T-cell generation signals 1 (TCR triggering) and 2 (co-stimulation) contribute to T-cell expansion and differentiation whereas signal 3 (tissue factors) instills tissue-homing preferences. This model applies to immune surveillance (memory) T-cells but not short-lived effector T-cells that respond to inflammatory migration cues produced at sites of inflammation and disease. We discuss our recent investigations into the molecular mechanisms underlying CCR8 expression in ab T-cells from both humans and mice. We believe these studies address a shortfall in vaccination research, namely the molecular pathway(s) directing the generation of tissue-specific memory Tcells.

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12 Abstracts

Signalling & Transcriptional Regulation in Innate Immunity

139 Holistic analysis of transcriptional cascades induced by inflammatory stimuli

847 Control of inflammatory TLR signaling in macrophages: a tale of two TRAF members

S. T. Smale Microbiology, Immunology, and Molecular Genetics, UCLA, Los Angeles, CA, USA

S.-C. Sun Department of Immunology, University of Texas MD Anderson Cancer Center, Houston, TX, USA

Macrophages and many other cell types respond to diverse stimuli by activating the transcription of hundreds of genes in a defined temporal, stimulus-specific and cell type-specific manner. The transcriptional cascade is typically orchestrated by several signal transduction pathways, which target transcriptional regulators and coregulators; these factors target genes that are assembled into diverse chromatin states, with the chromatin state of a gene potentially conferring susceptibility or resistance to activation. A major goal of our research is to understand how signalling pathways, transcriptional regulators, and chromatin structure act in concert to direct a stimulus- and cell type-specific transcriptional cascade. To achieve this goal, a better understanding of the molecular logic through which a transcriptional cascade is elicited in a single cell type by a single stimulus is needed. We therefore are focusing much of our effort on understanding the initial response of mouse bone marrow-derived macrophages to lipid A stimulation. These studies derive great benefit from our ability to accurately and quantitatively monitor transcriptional kinetics of each lipid A-induced gene using RNA-seq analysis of nascent, chromatin-associated transcripts. By incorporating ChIP-seq data sets and binding site motif analyses, and focusing initially on careful scrutiny of genes that are induced most potently, insights have been obtained that are being used to inform studies of genes induced with smaller magnitudes. The insights provided by these studies are allowing us to better understand how different stimuli generate distinct responses in specific physiological settings and disease states.

Macrophages are an integral part of the innate immune system and have a crucial role in mediating host defense against infections as well as inflammatory responses. The activation of macrophages relies on pattern-recognition receptors (PRRs) that detect various molecular patterns associated with the invading pathogens. One major family of PRRs is the toll-like receptors (TLRs), which stimulate cascades of signaling events that lead the induction of proinflammatory cytokines and antimicrobial proteins. The TLR-stimulated proinflammatory cytokine induction involves activation of several transcription factors, including NF-jB, AP1, and IRF5; however, how the proinflammatory TLR signaling is negatively regulated is still poorly understood. Using animal models, we found that both TRAF2 and TRAF3 serve as crucial negative regulators of the proinflammatory TLR signaling. Ablation of either TRAF2 or TRAF3 promotes TLR-stimulated proinflammatory cytokine expression in macrophages and exacerbates colitis in an animal model of inflammatory bowel disease. I will discuss how these TRAF members exert this novel function in the control of TLR signaling.

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Abstracts 13

The Microbiota and the Immune System 93 Cancer as a disease of the metaorganism G. Trinchieri Cancer and Inflammation Program, National Cancer Institute, Frederick, MD, USA Commensal microorganisms colonise barrier surfaces of all multicellular organisms, including those of humans. For more than 500 million years commensal microorganisms and their hosts have coevolved and adapted to each other. As a result, the commensal microbiota affects many immune and non-immune functions of their hosts, and de facto the two together comprise one metaorganism. The commensal microbiota communicates with the host via biologically active molecules. Recently, it has been reported that microbial imbalance may play a critical role in the development of multiple diseases, such as cancer, autoimmune conditions and increased susceptibility to infection. The commensal microbiota not only may affect the development, progression and immune evasion of cancer but it has also important effects on the response to cancer immune- and chemo-therapy. Myeloid cells are a major component of the tumour microenvironment where they play a dual role inducing anti-tumour immune responses but mostly promoting immune evasion, tumour progression and metastases formation. Myeloid cells respond to environmental factors including signals derived from commensal microbes that modulate their function and reactivity thus impacting the response to cancer therapy.

106 Microbial colonisation early in life impacts innate and adaptive immune regulation K. McCoy Department of Clinical Research, University of Bern, Bern, Switzerland Microbial colonisation of mucosal surfaces is initiated during birth as soon as the newborn leaves the sterile uterine environment. Establishment of stable bacterial consortia then occurs over time through a dynamic process that is heavily shaped by exposure to environmental bacteria and external factors. Composition of the commensal microbiota early in life likely plays an important role in development of the immune system and influences susceptibility to a variety of immune-mediated diseases. Epidemiological studies suggest that alterations within the intestinal microbiota are associated with increased incidence of allergic and autoimmune disorders, including food allergy and type 1 diabetes. Studies using germ-free and gnotobiotic animal models start to provide insight into the mechanisms involved in the protective capacity of the commensal microbiota. We found that germ-free mice and mice with a low diversity microbiota developed elevated serum IgE levels. More complex microbiotas could prevent IgE induction but only if a critical level of diversity was reached early in life. Immune dysregulation in germ-free and low complexity microbiota colonised mice resulted in exaggerated oral-induced systemic anaphylaxis. We are continuing to dissect out the immune regulatory effects of microbial composition and diversity early in life and aim to identify the key innate sensors of microbial-derived signals.

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14 Abstracts

Type 17/23 Responses in Human Inflammatory Diseases, New Understanding and New Treatments 92 The IL-17 and CD161 family of players: their role in childhood arthritis L. R. Wedderburn Rheumatology, UCL Institute of Child Heath, London, UK Th17 cells are known to be involved in the pathogenesis of several autoimmune conditions including psoriasis and some forms of human arthritis, and blockade of IL-17 and IL-23 is increasingly considered as a therapeutic option in these conditions. In childhood arthritis (juvenile idiopathic arthritis, JIA) we have shown that the frequency of Th17 cells is correlated with severity of arthritis, and reciprocally related to the frequency of regulatory T cells (Treg) at the disease site. We have also shown that CD161 is expressed both on Th17 cells, but also on a subset of Th1 cells, and a proportion of Treg cells, in the joint. When synovial Th17 effector cells undergo plasticity in the joint to become poly-functional cytokine producers, expression of CD161 is maintained, and these ‘ex- Th170 cells then produce GMCSF in addition to the typical Th1 cytokines. In this talk we will consider the lineage, transcriptional and functional relationships between Treg, Th17 and Th1 cells in JIA and the translational implications of these findings.

ª 2014 The Authors. © 2014 John Wiley & Sons Ltd, Immunology, 143 (Suppl. 2), 1–40

Abstracts 15

Unconventional T Cells Subsets: Activation & Function 33 Role of CD1 and MR1-restricted T-cells in diabetes A. Lehuen*,† *Institut Cochin, Paris, France, †Paris Descartes University, Paris, France Innate-like T cells, such as NKT and Mucosal Associated Invariant (MAI)T cells, act as a first line of T cell responders controlling the outcome of immune responses. Our studies these past years have revealed the critical role of iNKT cells in the prevention of type 1 diabetes. This autoimmune disease is caused by the destruction of pancreatic beta cells by effector autoreactive T cells. In the context of LCMV infection and/or after specific activation by alpha-GalCer, iNKT cells promote the tolerogenic function of plasmacytoid dendritic cells and the induction of Foxp3 regulatory T cells. Upon infection by Coxsackie virus, which has been involved in the aetiology of diabetes, iNKT cells induce a shift of pro-inflammatory macrophages into regulatory macrophages producing suppressive enzyme. Recent advances in understanding the aetiology of obesity and type 2 diabetes (T2D) have established the involvement of the immune system. Similarly to iNKT cells, MAIT cells express an invariant TCRalpha chain, Valpha7.2 in humans and Valpha19 in mice associated with a limited number of beta chains. These innatelike T cells recognise bacterial ligands presented by the non-polymorphic MR1 molecule. Since obesity and T2D are associated with low-grade inflammation, activation of immune cells and alteration in gut microbiota, we have undertaken the analysis of MAIT cells in obese and T2D patients. Our data reveal major alterations of MAIT cells in the blood of obese and T2D patients as compared to healthy lean control individuals, suggesting their potential role in these pathologies.

44 Unconventional mechanisms of effector gamma-delta T cell differentiation B. Silva-Santos Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal The accidental discovery of gamma-delta T cells thirty years ago paved the way to the characterisation of unconventional T cell populations that add significantly to the biology of their conventional counterparts. The production of large amounts of pro-inflammatory cytokines like IL-17 and IFN-gamma contribute greatly to the nonredundant roles of gamma-delta T cells in infection, cancer and autoimmunity. We and others have demonstrated that gd T cells differentiate in the murine thymus into IL-17 versus IFN-gamma producing subsets that can be segregated on the basis of CD27 expression. We have been unravelling the molecular cues that determine commitment to each functional gamma-delta T cell subset. Our results show differential involvement of TCR/CD3 and CD27 signals in the development of IL-17 versus IFN-g producers. Downstream of such receptors, we have dissected the transcriptional and epigenetic networks that regulate effector gamma-delta T cell fate. This revealed significant differences with the molecular programmes (chromatin modifications, transcription factors, microRNAs) involved in conventional ab T cell differentiation. I will also discuss the physiological implications of effector gamma-delta T cell subsets in tumour development and in malaria pathogenesis, where IL-17 and IFN-gamma can play striking protective or pathogenic roles. I

will highlight the lineage-specific mechanisms and biological contributions of these unconventional lymphocytes that have been conserved alongside ab T cells and B cells for over 450 million years of evolution.

102 New insights into cell stress sensing by human gamma/delta T cells M. Bonneville Cancer Research Center Nantes Angers, INSERM, U892, Nantes Cedex, France Vc9Vd2 T lymphocytes are a major cd T cell subset in adults that makes up 0.5 to 5% of the peripheral lymphoid pool. Vc9Vd2 T cells recognise a broad set of activated, transformed or infected cells in vitro. Accordingly they are expanded in vivo in a variety of infectious or tumour contexts. This broad reactivity pattern is primarily accounted for by recognition of small non-peptidic phosphorylated compounds, referred to as “phosphoantigens” (P-Ag), which correspond to metabolites of the mevalonate pathway used by mammalian cells and the DOXP pathway used by micro-organisms. How exactly P-Ag are sensed by Vc9Vd2 T cells remains unknown. Recent studies indicate that BTN3A1, a member of the B7 receptor superfamily related to butyrophilins, plays a key role in TCR-mediated activation of Vc9Vd2 T cells by P-Ag in both tumour and infectious contexts. The possible mechanisms underlying BTN3A1-dependent sensing of cell dysregulation by Vc9Vd2 T cells and their relationship to cell stress sensing strategies developed by other human gd T cell subsets wiill be presented and discussed.

103 Understanding and exploiting the NKT:CD1d system in disease M. Exley*,† *Manchester Collaborative Centre for Inflammation Research (MCCIR), University of Manchester, Manchester, UK, †Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA CD1d-restricted ‘Natural Killer T’ cell (NKT) populations enhance anti-pathogen and anti-tumour immunity. NKT cells are required for optimal resistance to certain infections and presence of Th1biased NKT cells is specifically associated with cancer patient survival. Patient NKT defects are reversible in vitro, so we are investigating their correction in models and the clinic, based on our NKT cell mAb. In the Tramp prostate cancer model, a tumour cell-based vaccine augmented by NKT stimulation was therapeutically active in advanced disease. NKT cell restoration in stage IV melanoma patients was well-tolerated with signs of immune activation. Progression-free survival of the majority of these highly-selected patients was over a year. There are further distinct NKT populations in human as well as murine bone marrow and adipose, where they can suppress inflammation (e.g. Graft-versus-Host Disease and Type 2 Diabetes). We have found that NKT cells are highly enriched in adipose, but depleted in obesity. However, NKT can still protect against metabolic effects of obesity. Finally, where NKT cells are found to be refractory, CD1d mAbs, as well as blocking NKT:CD1d interaction, potently stimulate CD1d+ antigen-presenting cell maturation. We are working towards control of NKT and other regulatory lymphocytes in a range of diseases and vaccines.

ª 2014 The Authors. © 2014 John Wiley & Sons Ltd, Immunology, 143 (Suppl. 2), 1–40

16 Abstracts 146 Harnessing invariant NKT-cells to enhance antigen specific immune responses V. Cerundolo MRC Human Immunology Unit, University of Oxford, Oxford, UK We aim to gain a better understanding of the mechanisms that control the cell-cell interplay required for optimal activation of tumourspecific immune responses and to apply this knowledge to the development of better treatment of cancer patients. Our current studies are focused on two major themes (i) Kinetic and functional analyses of the activation of invariant NKT (iNKT) cells to harness their ability to enhance antigen-specific immune responses; and (ii) Analysis of inflammatory responses upon inflammasome and TLR signalling events. The unique position of iNKT cells at the interface of the innate and adaptive immune responses has led to their intensive investigation as a means of augmenting the immune response in vaccination strategies both against infections and cancer.

ª 2014 The Authors. © 2014 John Wiley & Sons Ltd, Immunology, 143 (Suppl. 2), 1–40

Abstracts 17

Plenary 2: Beyond “-omics”: Translating Large Datasets into Molecular Mechanisms of Disease 50 T cell metabolism and differentiation D. Cantrell College of Life Sciences, University of Dundee, Dundee, UK mTORC1 (mammalian target of rapamycin complex 1) is a major integrator of nutrient, antigen receptor and cytokine signalling in CD8+ cytolytic T cells (CTL) and controls T cell differentiation. Despite its importance in T cell biology, the full impact of mTORC1 inhibition on CTL protein expression is not fully understood. Using transcriptional profiling and an unbiased quantitative mass spectrometry approach, we report for the first time the consequences of mTORC1 inhibition on the proteome of primary CTL. In these experiments, the high coverage of the murine CTL proteome supplied us with invaluable insight into the abundancy and isoform expression of more than 6700 proteins in CTL. The data also reveal how mTORc1 programmes the transcriptional and proteome landscape of T cells and in particular controls the expression of key metabolic regulators, adapter proteins and transcription factors. These experiments have highlighted the power of high resolution analysis of proteomes to uncover critical signalling checkpoints that control T cell differentiation.

immune cells, their response to challenge, and their interactions with tissues. Therefore, we are conducting a large-scale, high throughput screening of approximately 800 knockout mouse lines generated by the Wellcome Trust Sanger Institute (WTSI) to assess the impact of genes on many facets of immunobiology. Funded by the Wellcome Trust and led by our team at King0 s College London, the consortium includes partners from WTSI, Imperial College, Albert Einstein College of Medicine and the Universities of Oxford, Cambridge, and Manchester. We analysed the immune cell compartments of spleen, mesenteric lymph nodes, bone marrow, blood and serum in order to identify genes regulating the immune system at steady state. Furthermore, we investigated responses to chemical stress (DSS colitis) and to viral, bacterial and nematode infections (Salmonella, influenza and Trichuris muris) that collectively mimic major aspects of human exposure to the environment. The study is an open-source programme with data deposited into the public domain for follow-up by those bestequipped to do so. We shall consider issues relating to the implementation, interpretation, and data-dissemination in relation to an open-source high throughput screen; the insights into immunobiology already emerging after 1 year of operations; and how the community can realise the opportunity to benefit from these insights.

132 Understanding cellular heterogeneity 95 The many routes to immune-malfunction – multiparameter phenotyping of a knockout mouse library L. Abeler-Do¨rner King’s College London, London, UK The immune system is by now irrefutably implicated in processes beyond anti-microbial defense, including immuno-protective efficacy against cancers and roles in thermo-adaptation and adipose tissue regulation. And yet, we lack a full understanding of the molecules and pathways underpinning the development and regulation of

S. A. Teichmann EMBL-EBI, Cambridge, UK From techniques such as microscopy and FACS analysis, we know that many cell populations harbour heterogeneity in morphology and protein expression. With the advent of high throughput single cell RNA-sequencing, we can now quantify transcriptomic cell-to-cell variation. I will discuss technical advances and biological insights into understanding cellular heterogeneity in T cells and ES cells using single cell RNA-sequencing.

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18 Abstracts

Plenary 3: Autoinflammation: Function & Dysfunction of the Inflammatory Cascade 115 Gene hunting in autoinflammation of the young: not just a research tool P. Brogan UCL Institute of Child Health, London, UK Monogenic inflammatory diseases are an expanding group of hereditary diseases. Many monogenic inflammatory symptoms have a significant impact on quality of life including disability and potentially life-threatening complications. Most children will experience recurrent attacks of fever with associated symptoms of painful inflammation often of joints, eyes and skin. Over many years chronic inflammation impairs growth, adversely affects education, can be associated with hearing and visual impairment, and in the long-term can lead to kidney failure and death due to a complication called reactive amyloidosis, where excessive inflammatory proteins accumulate in organs causing them to fail. Current genetic testing in routine practice is time-consuming, labour intensive, costly and causes significant diagnostic delay. Furthermore, most genes known to be associated with monogenic inflammatory diseases are not currently available for routine testing in the NHS. Next generation genetic sequencing technologies, currently only available in research settings, are now increasingly being used in routine clinical practice to improve diagnosis and inform treatment decisions. When all conventional treatments have been tried and failed, haematopoietic stem cell transplantation might be an option, but invariably requires some understanding of the molecular basis for the disease to be justified. This presentation highlights clinical cases where a gene hunting strategy involving next generation sequencing was used to rapidly secure a molecular diagnosis leading to targeted therapy and improved outcome. The case is thus made that gene hunting in this way is no longer just a research tool, but is essential for the clinical care of these patients.

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Abstracts 19

Can Immunological Advances Enhance Vaccine Design?

and in response to infection. In this presentation, I will discuss the potential contribution of NK cells to vaccine-induced immunity, and how this is affected by host genotype, age and infection status.

61 NK cells as potential effectors of vaccine-induced immunity E. Riley London School of Hygiene & Tropical Medicine, London, UK A better understanding of the essential, protective mechanisms induced by vaccination would allow the development of more effective vaccines and the identification of correlates of immunity for rapid assessment of vaccine efficacy. Vaccine developers have traditionally made use of only a rather limited set of immunological parameters, focusing almost exclusively on T cell and antibody responses, very few of which have been validated as reliable correlates of immunity. More recently, however, transcriptomic approaches have reminded us of the importance of innate immune responses in initiation of long-term protective immunity, and of the interplay between innate and adaptive responses. Natural killer (NK) cells are traditionally regarded as innate immune cells but their activation by antigen-specific CD4+ T cell-derived IL-2 and by crosslinking of the low affinity IgG receptor, CD16, by antigen-antibody immune complexes, also places them at the heart of the adaptive response. The heightened responsiveness of NK cells during postvaccination recall responses suggests that NK cells may play a role in protection from vaccine preventable diseases, particularly as NK cells respond more quickly than T cells upon re-exposure to vaccine antigens. However, it is also becoming clear that NK cells are extremely heterogeneous, both phenotypically and functionally; that this is, in part, genetically determined; and that NK cells differentiate with age

79 Systems vaccinology: its promise and challenge for vaccine design H. I. Nakaya*,† *Clinical and Toxicological Analyses, University of S~ao Paulo, S~ao Paulo, Brazil, †Pathology, Emory University, Atlanta, GA, USA Systems vaccinology has recently emerged as an interdisciplinary field that combines systems-wide measurements, networks and predictive modelling in the context of vaccinology. For centuries, vaccines were developed empirically and with very little understanding of the mechanisms by which they mediate protective immunity. With the technological revolutions that occurred in the past decades, we are now able to access and integrate information about all the components within a biological system (e.g., genes, proteins, cells) and use it to compute and predict that system’s behaviour. When applied to vaccinology, systems biology approaches can help us to understand the mechanisms by which vaccines stimulate protective immunity, and also to identify predictors of vaccine-induced immunity. This seminar highlights how systems vaccinology has led to the discovery of certain signatures that can predict the immunogenicity of influenza and yellow fever vaccines, and how its application towards basic immunology research may provide novel mechanistic insights about immune regulation.

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20 Abstracts

Immune Frailty & Defense Against Infections 72 The impact of CMV infection on immunity and survival in older humans G. Pawelec University of T€ ubingen, T€ ubingen, Germany Immunosenescence, defined as the age-associated decline of the immune system, is characterised by impaired protective immunity resulting in increased susceptibility of the elderly to infectious diseases and their decreased responsiveness to vaccination. Recent studies suggest that a latent infection with human Cytomegalovirus, a ubiquitous beta herpes virus is associated with accelerated ageing of the immune system, especially in the adaptive arm. Whether infection with CMV and the imprinting of the adaptive immune system by this virus have any clinical implications is still controversial. Our data, to be presented in this talk, demonstrate a negative impact of a latent infection with CMV on Influenza vaccination outcome as well as memory T-cell responses to Influenza in the elderly. We also have evidence that the immunosenescent phenotype associated with CMV and the type of immune surveillance against this virus can have implications on healthy ageing and mortality in the very elderly and potentially deleterious sequelae in the younger elderly.

143 Fitness and survival consequences of immune variation in a wild mammal population T. N. McNeilly Disease Control, Moredun Research Institute, Penicuik, UK Unlike laboratory animals which exist in highly controlled environments, wild populations are exposed to a wide range of different types of pathogen to which they have to generate and regulate appropriate immune responses. Furthermore, natural populations exhibit considerable genetic and environmental diversity which will contribute to variation in immune responsiveness. To understand the consequences of this immune variation, it is necessary to study populations with detailed demographic and parasitological data, and to be able to accurately quantify immune parameters within individuals. Recently, we have employed a number of immunological tools developed by the veterinary immunology community to study a population of free living Soay sheep on the St Kilda archipelago, which has been subjected to individual based monitoring for almost three decades. This has allowed us to explore the fitness and survival

consequences of variation in both humoral and cellular immunity in significant detail. This presentation will outline some findings of this work, including a strong positive association between anti-nematode immunoglobulin G antibodies in summer and subsequent over-winter survival, providing a rare example of the fitness benefits of antihelminth immunity under natural conditions, and a positive association between levels of naive CD8+ T cells and survival in adult sheep. Such studies provide an insight into both the selective forces maintaining immunological variation and the consequences of that variation for host population dynamics.

173 NK cells in ageing. A role for CMV in age-associated changes in NK cells? R. Solana Immunology, IMIBIC – Hospital Universitario Reina Sofia – Universidad de Cordoba, Cordoba, Spain Natural Killer (NK) cells are innate lymphoid cells that play an important role in the response against virus infection and tumours. Age-associated changes in NK cells have been reported in human and mice, including alterations in maturation, NK cell peripheral distribution and function. We have studied NK cell frequency and NK cell subsets in healthy young and old individuals by using CD56 and CD16 markers and the expression of NK cell differentiation markers. Considering the relevance of CMV on T cell immunosenescence and its impact on NK cell function, young donors were stratified according to CMV serostatus. All elderly donors were CMV seropositive. NK cell frequency and NK cell subsets were not significantly affected by CMV serostatus. The percentage of NK cells observed in elderly subjects, who were all CMV seropositive, was significantly increased when compared with young donors. The percentage of immature CD56bright NK cells was significantly decreased in old individuals. The percentage of mature CD56dimCD16+ NK cells was not significantly altered by ageing or CMV seropositivity, whereas an expansion of CD56CD16+ dysfunctional NK cells was found in the elderly. CMV seropositivity and ageing were associated with the increased expression of CD57 on all NK cells except on CD56bright cells. The expression of CD94/NKG2C dimers on mature CD56dimCD16+ NK cell subset and on the dysfunctional CD56CD16+ NK cells was mainly observed in the CMV seropositive donors. These results support that NK cell changes observed in the elderly are related not only with age but also with exposure to CMV.

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Immune Responses at Mucosal Surfaces 45 Regulation of virally induced lung inflammation by type I interferons C. Johansson NHLI/Respiratory Infections, Imperial College London, London, UK Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infections in infants, as well as in the elderly and immunocompromised individuals. Type I IFNs (IFN-a/b) are produced very early upon direct recognition of the virus and then signal through the IFN-a/b receptor to induce expression of more than 300 genes. These encode proteins important for limiting viral replication, for amplifying production of type I IFNs and for directing adaptive immune responses. The cellular source of type I IFNs and how these cytokines regulate the balance between host resistance and immunopathology during RSV infection is poorly understood. Although all cell types can make type I IFNs, we have found a discrete population of lung cells to be the main cellular source following RSV infection of mice. We show that its production of type I IFNs is strictly dependent on cytosolic receptors of the RIG-I family. Furthermore, we uncover a profound role for type I IFN production by this population in the initiation of the entire inflammatory response to RSV infection. Our findings shed light on the mechanisms underlying RSV-induced inflammation and how antiviral immune responses are initiated during lung infections.

125 Dendritic cells in decision making between tolerance and inflammation in the respiratory mucosa A. Ray University of Pittsburgh, Pittsburgh, PA, USA Loss of immune tolerance to antigens induces inflammatory diseases like asthma and autoimmune diseases. Dendritic cells (DCs) play a central role in regulating immune tolerance and activation but the molecular axis within DCs that dictates immune outcome is unknown. We have recently shown the importance of lung CD103+ DCs in airway tolerance to low dose inhaled antigen, which mimics ambient antigen inhalation. We also showed that CD103+ DCs from tolerised mice express high levels of aldh1a2, the key enzyme for retinoic acid production, which promotes Foxp3 expression in CD4+

T cells in the context of TGF-b. We will discuss our new insight into a mechanism that regulates aldh1a2 expression selectively in CD103+ DCs and also simultaneously prevents expression of pro-inflammatory cytokine genes in all lung DCs and macrophages to minimise both destabilisation of Tregs and induction of unwarranted effector T cell responses. We believe this mechanism plays a central role in immune homeostasis that prevents induction of allergic responses and potentially many autoimmune diseases.

148 Dendritic cell subsets in mucosal immune responses W. Agace*,† *Immunology Section, Lund University, Lund, Sweden, †Section of Immunology and Vaccinology, Danish Technical University, Copenhagen, Denmark Dendritic cells (DCs) are distributed throughout the intestinal mucosa, either diffusely within the intestinal lamina propria (LP), or localised within gut associated lymphoid tissues, including Peyer’s patches and solitary isolated lymphoid tissues. Together these cells are believed to play an essential role in promoting tolerance to self and innocuous luminal antigen in the steady state as well as protective immunity to mucosal pathogens. We have previously described a subset of intestinal DCs in the intestinal mucosa of mice and humans that express the integrin alphaE(CD103)beta7. In the mouse these CD103+ DCs represent the major migratory DC populations in the intestinal mucosa and are thought to play an important role in initiating T cell responses to luminal antigen in the draining mesenteric lymph nodes. Intestinal CD103+ DCs can be divided into two major subsets; CD103+CD11b- and CD103+CD11b+ DCs, both of which appear to derive from pre-DCs. However, the transcription factors (TFs) required for the generation and maintenance of CD103+CD11b- DCs differs from that of CD103+CD11b+ DCs. Thus intestinal CD103+CD11b- DCs are developmentally dependent on the transcription factors IRF8, Id2 and Batf3, and as such appear closely related to splenic and lymph node resident CD8+ DCs. In contrast CD103+CD11b+ DCs, which represent the dominant migratory DC subset in the small intestinal mucosa, develop independently of these transcription factors and are dependent on the TF IRF4 for their survival. Here I will discuss our recent findings regarding the role of IRF8 dependent intestinal DCs in mucosal immune homeostasis.

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22 Abstracts

Recent Developments in Anti-Fungal Immunity

131 NK cell-mediated protection from Candida albicans

71 C-type lectins in anti-fungal immunity

S. LeibundGut-Landmann Institute of Microbiology, ETH Z€ urich, Z€ urich, Switzerland

G. Brown University of Aberdeen, Aberdeen, UK

Candida albicans is a clinically relevant fungal pathogen that causes many deaths per year due to nosocomial infections in immunocompromised individuals. Protection from invasive candidiasis is controlled by innate immune mechanisms. Using a mouse model of systemic candidiasis we have recently revealed a novel role for NK cells in this process. We showed that NK cells are recruited to the infected kidney and promote the candidacidal activity of neutrophils by secreting GM-CSF. This activity is under the control of IL-23p19, which is induced in response to the fungus by CD11c+ mononuclear phagocytes in a Syk-dependent manner. Furthermore, we identified an unexpected mode of regulation of NK cells by IL-17, which acts indirectly during the development of these cells in steady state to promote the functional competence of NK cells. Together, these data reveal a novel mechanism of IL-17- and NK cell-dependent defense against C. albicans.

The last few decades has seen a tremendous increase in our understanding of the mechanisms underlying the development of protective anti-fungal immunity. Key among these discoveries is the identification of pattern recognition receptors (or PRRs) expressed by immune cells which recognise conserved fungal components, such as beta-glucans and mannans. Recognition of these structures by PRRs, particularly by members of the C-type lectin receptor (CLR) family, triggers intracellular signalling cascades that initiate a variety of cellular and inflammatory responses, and induce the development of pathogen-specific adaptive immunity. We now understand that innate recognition by CLRs is essential for the development of protective immunity to these pathogens. In this presentation, I will cover the key developments in our understanding of the function and roles of these receptors, highlighting recent achievements.

137 Two faces of IL-17 signalling: anti-fungal host defense versus autoimmunity S. L. Gaffen Division of Rheumatology & Clinical Immunology, University of Pittsburgh, Pittsburgh, PA, USA IL-17 is a pro-inflammatory cytokine that mediates immunity to fungi (e.g., Candida albicans) but also promotes immunopathology in autoimmune disease. This talk will focus on the source(s) of IL-17 during oral candidiasis, and the downstream mechanisms of IL-17-mediated anti-fungal immune responses.

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Translating Immune Mechanisms into Clinical Opportunities 29 Preventing infectious disease pandemics with T cells A. Lalvani Imperial College London, London, UK Tuberculosis and influenza are major respiratory infections at pandemic scale. Both are caused by intracellular pathogens that induce strong cellular immune responses. TB is characterised by a long and variable incubation period, known as latent TB infection, which progresses to active, infectious TB in a proportion of people, particularly those with suppressed cellular immunity. Early treatment of latent infection is key as it prevents progression to disease and onward transmission of infection. The challenge lies in detecting latent infection and pinpointing those at highest risk of progression. Interrogation of the multiple T cell responses evoked by latent infection can yield immune biomarkers that could transform diagnosis, screening and pre-emptive treatment of latent TB. Whilst cognate antibody responses to influenza virus surface proteins provide long-lasting (homotypic) protective immunity to the homologous strain, new reassortant viruses with antigenically shifted surface proteins circumvent this. This leads to global spread of novel reassortant virus through antibody-na€ıve populations, resulting in pandemics, as in 2009 with pH1N1. However, the core proteins of influenza virus, which are recognised by CD8 T cells, remain relatively constant among influenza viruses and over time. Whilst crossreactive CD8 T cells induced by previous seasonal influenza are unable to prevent initial infection per se, they might limit influenza symptoms and viral shedding. Testing this hypothesis could yield a correlate of protective immunity against influenza disease but is only possible during a pandemic. The identification and implications of such a correlate for developing universal influenza vaccines to mitigate future pandemics will be discussed.

63 The immunobiology of mesenchymal stromal cells F. Dazzi King’s College London Faculty of Life Sciences and Medicine, London, UK Mesenchymal stromal cells (MSC) have been shown to exert a potent anti-proliferative effect which results in the inhibition of immune responses. Such an effect is not cognate-dependent because it can still be observed using MSC from third-party donors fully mismatched for the MHC haplotype of the responder T cells. MSCinduced unresponsiveness also lacks of any selectivity, because MSC are equally effective at inhibiting proliferation of memory and na€ıve T cells, do not preferentially affect CD4+ or CD8+ subsets and have similar effects on natural killer cells. MSC immunosuppressive activity is licensed by a set of inflammatory molecules which promote the secretion of enzymes consuming essential aminoacids. MSC activity is not confined by a direct action on effector cells but also involve the recruitment of a number of regulatory networks. Whilst MSC expand and activate regulatory T cells, they also have the capacity to interfere with the maturation and function of antigen-presenting cells. These properties have been extensively tested in animal models and in the clinical setting. It appears that the infusion of MSC have dramatic effects on a variety of auto- and allo-immune diseases. In the clinical setting MSC have been shown to improve conditions like autoimmune diseases, graft rejection and severe graft-ver-

sus-host disease. The mechanisms of their therapeutic effects remain to be fully elucidated but they involve a number of synergistic molecules that are triggered only under conditions of acute inflammation. This notion impacts on patient selection and will be extensively discussed.

68 The long and winding road towards clinical transplantation tolerance G. Lombardi King’s College London, London, UK The only effective treatment for end-stage organ failure is the use of organ transplants. However, success is limited due to the vigorous immune responses triggered by donor antigens and to the morbidity and mortality associated with the use of immunosuppressive drugs. One way forward is to develop strategies to induce tolerance. Data from experimental rodent models and from monitoring human transplant patients suggest that tolerance induction is associated with the presence of regulatory T cells (Tregs). Adoptive transfer of Tregs in animal models has suggested that this strategy can promote tolerance. The results of few clinical applications of polyclonal Tregs in the treatment of diabetes and to prevent GvHD have already demonstrated the safety of this strategy. In the last few years we have established in the laboratory a clinical protocol for the expansion of human polyclonal Tregs. This protocol has recently moved to the new GMP facility at Guy’s Hospital and two clinical trials have just started for the treatment of patients receiving kidney (The One Study) and liver (ThRIL) transplants. However, we have demonstrated in different animal models that mouse and human Tregs specific for the graft are superior compared to polyclonal Tregs in protecting from graft rejections. A new GMP facility with a cell sorter to enrich for antigen-specific Tregs is under development. Altogether the results obtained from our studies have important implications for Treg therapy in preventing transplant rejection and autoimmune disease.

84 Autologous tolerogenic dendritic cells for rheumatoid and inflammatory arthritis (AuToDeCRA) J. Isaacs Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK Rheumatoid arthritis (RA) is a chronic autoimmune disease which results from a breakdown of immune tolerance. Despite their efficacy, current RA therapeutics, including biologic and non-biologic DMARDs, generally require chronic administration with the associated risk of adverse effects. The concept of therapeutic tolerance states that it should be possible to reprogramme unwanted immune responses, including autoimmunity, and reset the immune system to self-tolerance. Although the concept has been achieved in animal models of autoimmunity and transplantation it has not yet been convincingly demonstrated in humans. We have developed a potentially tolerogenic therapy, autologous tolerogenic dendritic cells (tolDC), and have completed an ascending dose, randomised, controlled, un-blinded phase I study in patients with inflammatory arthritis. This presentation will briefly describe the background to the study, and the route to regulatory approval for a clinical trial of a cellular therapy. The results of the clinical trial will be presented.

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24 Abstracts

Tumour Microenvironment 85 Tumour-associated stroma: a mechanism of immune evasion? J. Shields MRC Cancer Unit, University of Cambridge, Cambridge, UK Non-cancerous cells within tumours form a supportive network commonly known as the tumour stroma. Lymphatic vessels found within stromal zones link a tumour to downstream lymph nodes and the immune system, yet in many cases the anti-tumour immune response is ineffective or dysfunctional. The mechanisms by which a tumour evades destruction whilst being directly connected to the immune system still remains unclear, although it is becoming apparent that stromal components have the potential to directly contribute. A subset of cancer-associated fibroblasts (CAF) share characteristics with lymph node stroma: a site of immune decision making within lymph nodes. One of these shared characteristics is the expression of podoplanin. Clinical studies have correlated its presence with poor prognosis, and in mouse models podoplanin-expressing stroma was also associated with enhanced tumour progression and altered immune populations. Using fibroblasts derived from normal and tumour tissues modified to express varying podoplanin levels, we are working towards identifying the mechanisms by which supporting cells within a tumour operate in favour of a developing tumour. Combining live confocal microscopy, tumour models and co-culture systems, we show that CAFs are able to engulf and process tumour debris and soluble antigen, reminiscent of the lymph node stroma, and modulate T cell viability and function in an antigen-specific manner. In summary, CAFs have the potential to protect a developing tumour via direct interaction and adaptation of the anti-tumour immune response in situ.

87 In vivo discovery of targets for cancer immunotherapy K. W. Wucherpfennig Department of Cancer Immunology & AIDS, Harvard Medical School, Boston, MA, USA

ling T cell function in immunosuppressive tumors are not well understood. We show that such inhibitory mechanisms can be systematically discovered in the tumour microenvironment. We devised an in vivo pooled shRNA screen in which shRNAs targeting negative regulators became highly enriched in tumours by releasing a block on T cell proliferation upon tumour antigen recognition. Such shRNAs were identified by deep sequencing of the shRNA cassette from T cells infiltrating tumour or control tissues. One of the target genes was Ppp2r2d, a regulatory subunit of the PP2A phosphatase family: In tumours, Ppp2r2d knockdown inhibited T cell apoptosis and enhanced T cell proliferation as well as cytokine production. Key regulators of immune function can thus be discovered in relevant tissue microenvironments.

130 Manipulating the tumour microenvironment to enhance antibody therapeutics S. A. Beers Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton Faculty of Medicine, Southampton, UK Monoclonal antibodies (mAb) as drugs have become established in the treatment of a variety of cancers – transforming the outcome of patients. Despite their success in the clinic, responses can vary markedly and indeed a number of cancers have proven resistant to mAb therapy demonstrating a real need for new and improved treatment strategies. Successful antibody therapy appears to rely predominantly on Fcc receptor expressing effector cells such as macrophages. Tumour associated macrophages are frequently present in high numbers in tumours and in close proximity to malignant target cells thereby offering great potential as a mAb effector cell population. Understanding the key mechanisms of resistance to antibody drugs will be instrumental to opening up these immunotherapeutic strategies to more patients. One such potential mechanism of resistance is the adverse immunosuppressive effects of the tumour microenvironment on effector cell activity. Here, the potential issues of harnessing tumour associated macrophages as antibody effectors will be discussed as well as means presented by which they may be manipulated to enhance their effector capacity.

Recent clinical trials showed that targeting of inhibitory receptors on T cells induces durable responses in a subset of cancer patients, despite advanced disease. However, the regulatory switches control-

ª 2014 The Authors. © 2014 John Wiley & Sons Ltd, Immunology, 143 (Suppl. 2), 1–40

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Generation, Properties & Clinical Relevance of Antibodies in Autoimmune Disease and AlloTransplantation 42 Goodpasture Autoantigen: structure, assembly and clues about etiology and pathogenesis B. Hudson Medicine, Vanderbilt University Medical Center, Nashville, TN, USA Goodpasture’s autoimmje disease is distinguished by pathogenic auto-antibodies that target the a345 collagen IV scaffold of basement membranes of renal glomeruli and pulmonary alveoli. Under normal conditions, this scaffold provides critical extracellular support for tissues, while the binding of auto-antibodies induces an acute inflammatory response leading to organ failure and death. The underlying disease mechanisms have been steadily deciphered over the past 30 years, with seminal advancements being the determination that auto-antibodies drive the observed pathology and the discovery of the target autoantigen, the a3 chain of collagen IV scaffolds. The alpha-3 chain assembles together with the alpha-4 chains and alpha5 chain into a complex a345 network that functions as a scaffold by providing structural integrity to tissues and tethering numerous molecules that influence the behavior of epithelial cells. The autoantigen is fastened together by a newly described sulfur-nitrogen chemical bond (=S=N-), sulifilimine bond, uncovering potential clues to the etiology and pathogenesis of this autoimmune disease, Science (2009). The enzyme that forms the bond was recently identified, Nature Chemical Biology (2012), and the evolutionary origin of the sulfilimine bond was determined, Proceedings of the National Academy of Sciences (2014). A recent discovery revealed that the trace element bromine is required for bond formation and that bromine

is an essential trace element for tissue genesis throughout the animal kingdom, Cell (2014). The presentation will highlight advancements in the structure and assembly mechanism and potential clues about etiology and pathogenesis of Goodpasture’s autoimmune disease.

133 The pathogenesis of immune thrombocytopenia (ITP) J. W. Semple St. Michael’s Hospital, Toronto, ON, Canada Immune thrombocytopenia (ITP) is a bleeding disorder in which autoantibodies and/or cytotoxic T cells are directed against an individual’s own platelets and/or megakaryocytes which leads to enhanced peripheral destruction and/or reduced bone marrow production, respectively. The cause of this disease is unknown and most research in ITP has concentrated on the characterization of the anti-platelet autoantibodies. In the last 25 years, however, a relatively explosive body of research literature has arisen to shed new light on the complex immunopathogenesis of ITP. It is associated with three interrelated areas of autoimmunity pertaining to environmental e.g. infectious influences, antigen presenting cell (APC) function and T cell abnormalities. It appears that the processes of apoptosis and deficiencies of CD4+ T regulatory cells are central events that lead to tolerance breakdown and induction of platelet autoimmunity. This lecture will update our understanding of the humoral and cell-mediated immunology of ITP and will highlight the recent literature which suggests that inflammatory sequences lead to an environment that significantly dysregulates autoreactive T cells leading to the production of anti-platelet autoimmunity.

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26 Abstracts

Macrophages: Heterogeneity of Origin and Function 62 Tissue-resident macrophages originate from yolk sac-derived erythro-myeloid progenitors E. Gomez Perdiguero King’s College London, London, UK Most hematopoietic cells in adults renew from adult hematopoietic stem cells (HSC). In contrast, tissue ‘resident’ macrophages develop in the absence of the transcription factor Myb and can self-maintain in adult tissues independently of HSC. However, the developmental origin of the tissue-resident macrophage progenitors and the qualitative and quantitative contribution of HSC and putative non-HSC progenitors to macrophages are still unclear. Our recent work demonstrates that the vast majority of adult tissue-resident macrophages in liver (Kupffer cells), brain (microglia), epidermis (Langerhans

cells), and lung (alveolar macrophages) originate from a Tie2+ cellular pathway generating Csf1r+ erythro-myeloid progenitors (EMP) distinct from HSC. These progenitors are generated in the extraembryonic yolk sac (YS) from embryonic day (E) 8.5 to E10.5 but are not restricted to the YS compartment, since they colonise the nascent fetal liver before E10.5. They give rise to fetal erythrocytes, macrophages, granulocytes and monocytes until at least E16.5. Subsequently, HSC-derived cells replace erythrocytes, granulocytes and monocytes, whereas Kupffer cells, microglia, and Langerhans cells are not replaced in 1-year old animals. Other tissue-resident macrophages, such as alveolar macrophages, may be progressively replaced in aging mice while YS-derived macrophages in the intestine do not persist in adulthood (Bain et al., 2014). The combination of different fate mapping experiments identifies a sequence of yolk sac EMPderived, followed by HSC-derived hematopoiesis in the fetal liver, and identify yolk sac EMP as a common origin for tissue macrophages.

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Abstracts 27

Plenary 1: Memories of Time and Place: Immune Cells on the Move and in Tissues 58 Development and maintenance of human tissue resident memory T cells D. L. Farber Columbia Center for Translational Immunology, Columbia University, New York, NY, USA Immune responses occur in diverse anatomical sites, including protective responses to pathogens and dysregulated immune responses in autoimmune diseases. In humans, our understanding of T cell activation and differentiation into effector and memory T cells derives largely from studies of peripheral blood, though the majority of T cells are found in lymphoid and mucosal sites. We have established a novel tissue resource to obtain multiple tissues from individual organ donors in collaboration with the New York Organ Donor Network (NYODN). We present here a multidimensional, quantitative analysis of human T cell compartmentalization and maintenance over six decades of life in blood, lymphoid and mucosal tissues from 56 individual organ donors. Our results reveal that the distribution and tissue residence of na€ıve, central and effector memory, and terminal effector subsets is contingent on both differentiation state and tissue localization. Moreover, T cell homeostasis driven by cytokine or TCR-mediated signals is dependent on CD4+ or CD8+ T cell lineage, subset differentiation and tissue localization, and cannot be inferred from blood. T cell clonal analysis by T cell receptor sequencing reveals that memory CD4+ T cells are resting and com-

partmentalized in tissues, while memory CD8+ T cells are more clonally expanded and distributed in multiple sites. Our data provide an unprecedented spatial and temporal map of human T cell compartmentalization and maintenance, supporting distinct pathways for human T cell fate determination and homeostasis.

120 Never confuse movement with action: the role of CD8+ T cells in leishmaniasis P. Kaye University of York, York, UK The leishmaniases represent a spectrum of diseases caused by diverse parasite populations, and cell mediated immunity represents both the basis of host protection and also drives the strikingly diverse immunopathologies associated with these diseases. CD8+ T cells are well known to play a role in disease outcome and in some cases in pathology, and have become potential targets for therapeutic intervention, through reversal of the exhausted phenotype that accompanies some forms of chronic leishmaniasis, or through CD8-targeted therapeutic vaccination. However, the evidence for the importance of CD8+ T cells has often been correlative and drawn from static data that identifies these cells within the lesion site. Here, we will discuss data obtained from dynamic intra-vital imaging of CD8+ T cell responses during experimental visceral leishmaniasis and how we have adopted in silico modelling approaches to predict and further elucidate the function of CD8+ T cells at inflammatory sites.

ª 2014 The Authors. © 2014 John Wiley & Sons Ltd, Immunology, 143 (Suppl. 2), 1–40

28 Abstracts

Regulating Immunity: Tricks and Treats of the Immune System

147 Transcriptional network controlling development of Tregs and Th17 cells

67 Signalling and secretion at the immunological synpase

V. Kuchroo Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

G. Griffiths University of Cambridge, Cambridge Institute for Medical Research, Cambridge, UK Cells of the immune system need to communicate with each other in order to generate an effective immune response. One way in which they do so is by forming an “immunological synapse” a term first used to describe the directed secretion of cytokines “into a small space between two cells” by Seder and Paul in 1994. The immunological synapse provides a remarkable example of cell polarity, with membrane receptors, cell cytoskeleton and secretory organelles focused towards the point of interaction. But what are the events that lead to secretion at the immunological synapse? Using high resolution multi-colour imaging in 4D we have now studied these events and the movies capturing CTL killing their targets in 3D provide a new temporally resolved model providing new insights into the mechanisms underlying secretion at the immunological synapse.

Interleukin (IL)-17-producing T cells (TH17) are a distinct subset of T cells that play a crucial role in the induction of autoimmune tissue injury. In contrast to the effector T cells, CD4+CD25+, Fox-P3+ regulatory T cells (T-regs) inhibit autoimmunity and protect against tissue inflammation. TGF-b1 is a critical differentiation factor for the generation of T-regs and using Foxp3-GFP “knock-in” mice we have shown that IL-6, an acute phase protein induced during infection, inflammation and injury inhibits the generation of Foxp3+ T-reg cells and induces proinflammatory Th17 cells. Accumulating data suggests that there are three distinct steps in Th17 differentiation: Induction, Amplification and Stabilization mediated by distinct cytokines and loss of any of the cytokines (TGF-b, IL-6, IL-21 or IL-23) in the pathway results in a defect in generation of Th17. Using expression profiling at very high temporal resolution, novel computational algorithms and innovative nano-wire based “knock-down” approaches, we have developed a regulatory network that governs the development of Th17 cells. In addition to high density temporal microarray analysis, we have now performed single-cell RNA-seq of Th17 cells in order to characterize cellular heterogeneity, identify subpopulations, functional states and learn how gene expression variation affects Th17 effector functions. By utilizing novel computational tools for processing and analyzing the resulting dataset (~730 Th17 cells), we have identified novel regulators of Th17 cells that do not affect Th17 differentiation but affect pathogenic vs. non-pathogenic functional states of Th17 cells.

ª 2014 The Authors. © 2014 John Wiley & Sons Ltd, Immunology, 143 (Suppl. 2), 1–40

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Unlocking the Host Response: The Key to Early Diagnosis of Infection? 144 Identifying the molecular mechanisms behind the host immune response to bacterial LPS M. Triantafilou Institute of Infection and Immunity, Cardiff University, School of Medicine, Cardiff, UK The innate immune system utilises a trimolecular complex of receptors, comprising of TLR4, CD14 and MD2, in order to sense bacterial products. Even though the components of the mammalian LPS “sensing machinery” have been identified, our understanding of the

initial events that take place as LPS engages its receptor(s) still unclear. An important question that still remains is what is the stochiometry of TLR4 in the “LPS-sensing apparatus”. In the current study, we set out to investigate the specific oligomeric state of TLR4 on living cells before and after stimulation by bacterial LPS using Single particle fluorescent imaging (SPFI), which is a non-invasive biophysical method. Our data demonstrates that TLR4 exists as monomers or dimmers on unstimulated cells, and upon ligation by LPS, it is induced to form stable homodimers and tetramers that are crucial for cell activation. This study demonstrates for the first time, the stochiometry of TLR4 within the “LPS-sensing apparatus” and shows that ligand binding seems to induce oligomer formation enabling stable ectodomain receptor-receptor interactions therefore allowing downstream signalling to occur.

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30 Abstracts

Immunometabolism Control in Chronic Inflammatory Diseases 73 Vascular and adipose tissue interface in Crohn’s disease G. Randolph Washington University at St. Louis, St. Louis, MO, USA Cardinal characteristics of Crohn0 s disease include transmural inflammation, skip lesions characterized by the separation of inflamed and diseased intestinal segments with normal regions of intestine, and so-called creeping fat, in which a correlation with the intensity of the disease is the appearance of fat that no longer stops at its usual boundary along the anti-mesenteric border but continues

over the intestinal wall. Though the mesentery is central to blood and lymph circulation in the intestine, little attention has been paid to the mesentery in Crohn0 s disease. I will present data to reveal striking alterations in the mesenteric venous vasculature of CD specimens, including smaller veins, notable masses of venous smooth muscle observed in vein lumens, with highly disorganized muscle cells in the vein wall and as single cells and small clusters in the mesenteric adipose tissue. Such vein walls possess altered mRNA transcripts governing intercellular adhesion, muscle proliferation, innervation, infiltration of natural killer (NK) cells, and expression of transcripts associated with NK-mediated vascular remodeling. The data suggest that venous vasculopathy is central to Crohn0 s disease and I will discuss how this vasculopathy might explain the cardinal features of the disease and comprise new therapeutic targets.

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Can Immunological Advances Enhance Vaccine Design?

118 Rational and practical hurdles in vaccine development for respiratory disease

77 How to improve the efficacy of TB vaccines: lessons from the MVA85A phase IIb efficacy trial

P. J. Openshaw Respiratory Medicine, Imperial College London, London, UK

H. Fletcher London School of Hygiene & Tropical Medicine, London, UK Despite the global use of the bacillus Calmette-Guerin (BCG) vaccine there are 9 million new cases of tuberculosis (TB) each year and an urgent need for improved vaccines. Better understanding of the vaccine induced immune response is important for the design of improved vaccines. Samples collected by the Jenner Institute, University of Oxford, during 10 years of clinical testing of the viral vector vaccine MVA85A (a candidate TB vaccine) have been used for the characterisation of the antigen specific immune responses following vaccination. The immune response to MVA85A is predominately mediated by a CD4+ antigen specific T cell response that is highly polyfunctional. Latent TB infection (LTBI) does not impact on the magnitude or the quality of the immune response to MVA85A, although the magnitude of the response is lower in infants and children compared to adults. Using stored PBMC from South African infants enrolled into a Phase IIb efficacy trial of MVA85A (a candidate TB vaccine) we have further explored immune responses in BCG and MVA85A vaccinated infants. There was no significant efficacy with MVA85A in this clinical trial but we have been able to use samples from case and control infants to further explore immune correlates of TB disease risk. Understanding why TB vaccination fails to protect in endemic populations will help guide the development of more effective vaccination strategies in the future.

Respiratory viral diseases are amongst the commonest cause of morbidity and mortality in children, in those with existing lung diseases (such as asthma and chronic bronchitis) and in elderly and debilitated adults. The common cold agents remain a largely unmet global challenge; there are many vaccines available for influenza, but these need improvement in durability and breadth. RSV is a major cause of hospitalisation in infancy and winter deaths in the elderly, but there is no vaccine available. Several immunological issues have delayed the development of vaccines, especially the poorly protective response to mucosal infections and the enhancement of disease by some vaccines. Advances in knowledge of the immune system, of the virus and its antigenic properties combined with new vaccine technologies are now injecting new hope into the field and have given rise to many promising vaccine approaches. Some of these may be optimal for use in children, while others may be more appropriate for pregnant women or vulnerable older adults. With a multi-pronged approach to prevention, it may be possible to destabilise community circulation of some respiratory viruses and thus to significantly lessen their impact.

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32 Abstracts

Immune Frailty & Defense Against Infections 48 Long-term impact of CMV infection: insights from the Belfrail study C. Mathei Public Health & Primary Care, KU Leuven, Leuven, Belgium It is well established that CMV infection impacts markedly on the immune system. Less is known about the lifetime effects of CMV on immune functioning and health. We investigated the relationship between CMV infection, T-cell phenotyping, humoral response to CMV, functional performance and mortality among the very elderly. Contrary to the findings of studies in younger populations, CMV infection was not associated with impaired functional performance in the Belfrail cohort. Among CMV-infected individuals, however, a na€ıve-dominated CD4:CD8 ratio phenotype, defined as having a ratio >5 was associated with impaired physical functioning, whereas individuals with a ratio 5. The very elderly seem to represent a distinct phenotype with respect to their susceptibility to the adverse effects of CMV. This may be the result of a survival effect. Even among these survivors, a great deal of heterogeneity is still observed, both in terms of the immune response to CMV and the impact of CMV on health. Our results suggest that the long-term impact of CMV follows from the complex interaction between the virus and the nature of the host’s immune response, which seems to differ depending on age and gender and mostly likely genetic makeup.

832 Targets for improving vaccine-mediated protection against influenza in older adults J. E. McElhaney Advanced Medical Research Institute of Canada, Sudbury, ON, Canada Age-dependent changes in the cellular immune response have been mainly described in CD8+ T cells, with relative sparing of CD4+ T cells. We have shown that following influenza vaccination, late effector memory CD8+ T-cell subsets in older vs. young adults develop limited cytolytic activity upon challenge with influenza virus. In contrast, CD4+ T-cells in older adults demonstrate cytolytic activity in response to influenza challenge and share similar phenotypic and functional characteristics with virus-specific T cell subsets from young adults. Thus, CD4+ T cells may provide a compensatory response to influenza infection when CD8+ T cells become compromised during the aging process. Our recent work has focused on strategies to improve the T cell response to influenza in the development of new influenza vaccines. To target both innate and adaptive immune mechanisms, we have combined TLR ligands as adjuvants with seasonal influenza vaccine in older adult PBMC to enhance the response to live influenza virus challenge. Specifically, TLR4 ligand was shown to stimulate the production of pro-inflammatory cytokines by myeloid dendritic cells. PBMC stimulated with vaccine/ TLR4 ligand showed a significant enhancement of the cell-mediated immune response. Key cytokines produced in response to influenza vaccine (IL-2) and TLR4 ligand (IL-6) appear to be important in restoring the cytolytic CD8 T cell response by improving the proliferation and preventing apoptosis of the critical mediators of killing of virus-infected cells. These results offer insights into how the cellmediated immune response to split-virus influenza vaccines could be designed for improved protection in older adults.

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Immune Responses at Mucosal Surfaces 78 Plasticity and function of human ILC subsets H. Spits Cell Biology & Histology, Academic Medical Center, Amsterdam, The Netherlands Group 1 ILC comprise a heterogeneous group of subsets that share the capacity to secrete large amounts of IFNc. Conventional NK cells were initially classified in group 1 ILC. Recently however, the concept is emerging that ILC should be divided in cytotoxic ILC (NK cells) and non-cytotoxic ILC which can be considered as the innate equivalents of CD8+ and CD4+ T cells. In my presentation I will give an overview of human ILC and in particular of non-cytotoxic ILC1. These cells express CD127 and Tbet and lack cNK markers as CD94, perforin and Eomes. These cells were compared with cNK cells and the recently identified intra epithelial ILC1 subset. I will furthermore show data on the plasticity of CD127+ ILC1 and discuss the possible consequences of this plasticity for innate immunity and tissue homeostasis.

89 Inflammasome activation in the intestinal epithelium regulates protection and pathology in the gut K. J. Maloy Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Inflammasomes are multi-molecular platforms triggered following sensing of infection or danger by NOD-like receptors (NLR). In myeloid cells, inflammasomes drive caspase-1 activation, which leads to the processing and secretion of the inflammatory cytokines IL-1b and IL-18, as well as cell death (pyroptosis). Although implicated in host innate defence, genetic polymorphisms in NLR or associated cytokine pathways have been linked to chronic inflammatory disorders, including inflammatory bowel disease (IBD). In addition, the expression and functions of NLR and inflammasomes in non-haematopoeitic cells have not been extensively characterized. We report that activation of both NLRP3 and NLRC4 inflammasomes in intestinal epithelial cells (IEC) is required for protection from the intestinal pathogen Citrobacter rodentium. Inflammasome activation in IEC acted early during infection to limit bacterial colonization of the epithelium and limited subsequent pathology. We further show that, during steady state, IEC constitutively produce IL-18 that acts directly on IL-18R1-expressing CD4+ T cells to limit colonic Th17 cell differentiation. In addition, we found that IL-18R1 signaling was critical for Foxp3+ Treg cell mediated control of intestinal inflammation, by promoting expression of key Treg effector molecules. Thus, inflammasome activation in IEC has key roles in intrinsic protection from pathogens and in regulation of adaptive T cell responses in the gut.

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Plenary 3: Autoinflammation: Function & Dysfunction of the Inflammatory Cascade 75 Autoinflammatory diseases and the inflammasome: lessons from patients and mice H. Hoffman Pediatrics, UC San Diego, La Jolla, CA, USA The autoinflammatory diseases are a relatively new immune disease classification characterized by systemic and tissue specific inflammation due to innate immune dysregulation. The identification of genes responsible for the hereditary fever disorders has resulted in better understanding of the pathophysiologic mechanisms of these diseases. Many of these disorders have been linked to inflammasome function and interleukin-1B activity including the cryopyrin associated periodic syndromes. Translational studies utilizing in vitro and in vivo studies in humans and mice have uncovered novel pathways and led to improved therapy.

109 Genetically-complex autoinflammatory diseases: Still0 s disease and Behcßet0 s disease

Mendelian inheritance, genetically-complex diseases are those that arise from a combination of multiple genetic and environmental factors. The concept of autoinflammation originally emerged from the identification of monogenic, activating lesions of the innate immune system as the molecular basis of the hereditary periodic fever syndromes. In addition to these rare, monogenic forms of autoinflammation, genetically-complex inflammatory diseases, like Still’s disease and Behcßet’s disease, also fulfill the literal definition of autoinflammatory diseases - namely the development of apparently unprovoked episodes of inflammation without identifiable exogenous triggers and in the absence of autoimmunity. Interestingly, genetic studies of both of these diseases have found the MHC complex to be a strong disease susceptibility locus. In Still’s disease, variants of HLA-DR and HLA-DQ in the class II locus influence disease risk, while variants of HLA-B and HLA-A in the class I locus are the predominant risk factors for Behcßet’s disease. In both cases, these associations seem to implicate T lymphocytes and thereby adaptive immunity in disease pathogenesis. Although the clinical presentations of Still’s disease and Behcßet’s disease are both indicative of autoinflammatory disease, emerging evidence has implicated both innate and adaptive immune mechanisms in these diseases, blurring the line between autoinflammation and autoimmunity. These diseases act to establish a paradigm for concerted innate and adaptive immune dysfunction among the group of genetically-complex autoinflammatory diseases.

M. J. Ombrello Translational Genetics and Genomics Unit, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD, USA In contrast to monogenic diseases, which are caused by highly penetrant genetic variants of a single gene and which usually demonstrate

ª 2014 The Authors. © 2014 John Wiley & Sons Ltd, Immunology, 143 (Suppl. 2), 1–40

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Recent Developments in Anti-fungal Immunity 51 Immunity to pulmonary aspergillosis in solid organ transplantation D. Armstrong-James Respiratory Sciences, Imperial College London / NHLI, London, UK Solid organ transplant recipients are at high risk of opportunistic fungal infections. However, in contrast to stem cell transplant recipient, neutropaenia is not a key driver for fungal infection in organ transplantation. Our goal is to define the mechanistic basis for susceptibility to pulmonary aspergillosis in organ transplant. The main

drug used as transplant immunosuppression is tacrolimus-FK506, a calcineurin inhibitor that blocks T cell mediated calcineurin-NFAT responses. However our studies indicate that tacrolimus also has major effects on macrophages in the lung. Furthermore, phagocytosis of Aspergillus fumigatus by human alveolar macrophages leads to activation of calcineurin-NFAT signalling, which is required to coordinate appropriate immune responses in the airway. Thus, calcineurin inhibitors used to block T cell mediated responses in transplantation also have major effects on innate fungal immunity. Our studies reveal a new role for the calcineurin pathway in endosomal signalling that is likely to be relevant to a range of opportunist pathogens.

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Translating Immune Mechanisms into Clinical Opportunities 74 T cell engineering for immunotherapy of cancer H. Stauss UCL/Institute of Immunity and Transplanation, London, UK Adoptive T cell transfer has been used to control cancer growth and prevent disease from viral reactivation in immunosuppressed patients. The selection of antigen-specific T cell populations with appropriate effector function remains a major bottleneck for T cell therapy. The transfer of genes encoding T cell receptor (TCR) chains and regulatory molecules provides an opportunity to reliably produce therapeutic T cells of desired specificity and function.

(TCR) gene therapy does not require the patients’ own immune system to mount immune responses against cancer antigens, but can instead be used to genetically enhance cancer immunity in patients who are unable to control cancer. Furthermore, this type of biological therapy with genetically enhanced immune cells is expected to have lasting benefits as the therapeutic immune cells can expand and persist in patients for several years, thus providing lasting protection. We have developed therapeutic TCR specific for the Wilms Tumour Antigen 1 (WT1) and the LMP2 antigen of Epstein-Barr Virus (EBV). WT1 is an embryonic transcription factor that is not expressed in most adult tissues, but switched on in most types of leukaemia and many solid cancers. The LMP2 antigen is expressed in EBV-associated nasopharyngeal carcinoma and EBV-lymphomas. The preclinical validation of these TCRs will be presented, as well as strategies to further enhance the anti-cancer activity of TCR gene engineered T cells.

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Plenary 2: Beyond “-omics”: Translating Large Datasets into Molecular Mechanisms of Disease 90 Datasets, pathways and the biology of outcome in immunemediated disease K. Smith University of Cambridge, Cambridge, UK Western medicine has developed by classifying disease into defined diagnostic categories, and modern genetics and genomics has largely been occupied trying to uncover the genetic variants that drive their development. Even within a specific diagnostic category, however, the clinical course a disease takes can vary greatly between individuals. Thus to a patient with immune-mediated disease, for example, long-term outcome can be far more important than the specific diagnosis they are given. To investigate what controlled long term patient outcome we recruited patients with ANCA-associated vasculitis, SLE, Crohn’s

disease and ulcerative colitis at diagnosis. We then performed a comprehensive RNA expression analysis of separated leucocyte subsets and correlated this with prospective clinical follow-up data over a median of 6 years. We found a CD8 T cell transcription signature that predicts outcome, but is not associated with diagnosis, in these important immune-mediated diseases. A candidate gene study based on pathways identified by this signature in Crohn’s disease revealed a novel pathway driven by FOXO3 that regulates inflammation and is associated with long-term outcome, but not diagnosis, in a number of conditions. This presentation will explore new data extending this work that addresses the specific immunological mechanisms driving long-term outcome in immune-mediated disease, and the genetics that underpins this. Evidence will be presented suggesting that the biology underlying long-term disease outcome, or prognosis, is distinct from that driving specific diagnosis, and represent an under-investigated but clinically relevant aspect of disease pathogenesis.

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38 Abstracts

Signalling & Transcriptional Regulation in Innate Immunity

autophosphorylating Ser181. We have identified some new substrates for IKKb.

81 IRF5 controls macrophage-neutrophil axis in inflammation

References:: Emmerich et al (2013). Proc. Natl. Acad. Sci. USA 110, 15247– 15252. Zhang et al (2014). Biochem. J. 461, 531–537.

I. Udalova Kennedy Institute, University of Oxford, Oxford, UK Macrophages are heterogeneous immune cells that have diverse origins, regulatory complexity and function and are essential for the initiation and resolution of pathogen- or tissue damage-induced inflammation. The current challenges in the field are to untangle control mechanisms governing these functions. Previously we have shown that the transcription factor IRF5 is involved in establishing inflammatory macrophage phenotype. We have recently characterised the genome-wide sites of IRF5 recruitment in these macrophages and discovered the global rules of IRF5 involvement in regulation of inflammatory genes. These studies also highlighted the role of IRF5 in orchestrating the expression of the chemokine family members. The functional role of the IRF5-imposed control on neutrophil recruitment to the sites of inflammation will be discussed.

116 A new twist to the activation of NFjB P. Cohen MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Lys63-linked and Met1-linked (or linear) ubiquitin have been implicated in the activation of the canonical IKK complex by inflammatory stimuli. Lys63-linked ubiquitin binds to the TAB2/3 components of the TAK1 complex, inducing kinase activation, while Met1-linked ubiquitin binds to the NEMO component of the IKK complex. We will demonstrate that the Met1-linked ubiquitin formed upon activation of MyD88 signaling network are attached covalently to Lys63-linked ubiquitin, that the IL-1-stimulated formation of Met1-linked ubiquitin is dependent on the prior formation of Lys63-linked ubiquitin, and that HOIP, the catalytic component of LUBAC (the linear ubiquitin assembly complex), interacts with Lys63-linked ubiquitin specifically. These observations may explain, in part, why Lys63-linked ubiquitin is the preferred substrate for LUBAC in the MyD88 signaling network. The formation of Lys63 /Met1-linked hybrid ubiquitin chains co-localizes the TAK1 and IKK complexes to the same ubiquitin chains, facilitating the TAK1-catalysed activation of the IKKs. We demonstrate that Met1-linked ubiquitin is required for IKK activation using MEFs from knock-in mice in which wild type HOIP is replaced by an inactive mutant and in which NEMO is replaced by a polyubiquitin-binding defective mutant. We further demonstrate that the IL-1-stimulated activation of IKKb occurs via a three-step process. The binding of Met1-linked ubiquitin to NEMO permitting TAK1 to phosphorylate IKKb at Ser177, which primes IKKb to complete the activation process by

128 Regulation of inflammatory signaling by A20 R. Beyaert Inflammation Research Center, Ghent University - VIB, Gent, Belgium The deubiquitinase A20 (also known as TNFAIP3) is a potent antiinflammatory protein that is known to interfere with ubiquitindependent signaling. Mutations in A20 or the A20 locus are associated with chronic inflammation, autoimmunity and B cell lymphoma in humans. A20 negatively regulates NF-kB and cell death signaling in response to multiple cytokine and pattern recognition receptors in different cell types. However, the potential effect of A20 on other signaling pathways as well as their relative contribution in A20’s anti-inflammatory function is largely unknown. Recent findings showing the regulation of multiple signaling pathways by A20 as well as their cell-specific role in the anti-inflammatory function of A20 will be presented.

847 Control of inflammatory TLR signaling in macrophages: a tale of two TRAF members S.-C. Sun Department of Immunology, University of Texas MD Anderson Cancer Center, Houston, TX, USA Macrophages are an integral part of the innate immune system and have a crucial role in mediating host defense against infections as well as inflammatory responses. The activation of macrophages relies on pattern-recognition receptors (PRRs) that detect various molecular patterns associated with the invading pathogens. One major family of PRRs is the toll-like receptors (TLRs), which stimulate cascades of signaling events that lead the induction of proinflammatory cytokines and antimicrobial proteins. The TLR-stimulated proinflammatory cytokine induction involves activation of several transcription factors, including NF-kB, AP1, and IRF5; however, how the proinflammatory TLR signaling is negatively regulated is still poorly understood. Using animal models, we found that both TRAF2 and TRAF3 serve as crucial negative regulators of the proinflammatory TLR signaling. Ablation of either TRAF2 or TRAF3 promotes TLR-stimulated proinflammatory cytokine expression in macrophages and exacerbates colitis in an animal model of inflammatory bowel disease. I will discuss how these TRAF members exert this novel function in the control of TLR signaling.

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The Microbiota and the Immune System 69 T-bet as a key regulator of mucosal immunity G. Lord Experimental Immunobiology, King’s College London, London, UK The nature of the interface of the immune system with the host microbiome is one of the most fundamental scientific questions currently being addressed. The predominant site of this interaction occurs at mucosal surfaces in the bowel, although other sites such as the lung are also important. Dysregulation of this symbiotic relationship causes pathology, most notably inflammatory bowel disease (IBD) and inflammation-associated colonic cancer. Other diseases such as diabetes, obesity and arthritis have also been linked to an aberrant host-commensal relationship, highlighting the critical importance of understanding this complex interaction. We have previously shown that the transcription factor (TF) Tbet (T-box expressed in T cells, Tbx21) is a central mediator of this host-commensal interaction via expression in colonic mucosal dendritic cells. The molecular understanding of this model has been underpinned by the mapping of transcription factor binding sites using genome wide location analysis, identifying transcriptional de-repression of TNF-a as a key initiating event. We recently identified that the effector functions of a newly discovered population of innate lymphoid cells (ILCs) are controlled by T-bet and have resolved the microbial drivers of mucosal inflammation at a specific bacteriological level. ILCs are thought to be one of

the most important mediators of mucosal homeostasis in both health and disease and T-bet is a critical molecular link between ILCs and dendritic cells (DCs) in the causation of IBD. The recent discovery that the genetic architecture predisposing to IBD in humans maps to the innate immune system emphasizes the disease relevance of these data.

99 The lung microbiome in health and disease M. F. Moffatt Imperial College London, National Heart and Lung Institute, London, UK Molecular techniques have revolutionized the practice of standard microbiology. Initiatives such as the Human Microbiome Project are providing vital insights in to the role that microbiota have to play in health and disease. Previously considered sterile, the healthy lung is now recognised to harbour a community of bacteria and fungi. The composition of this microbiome can be dramatically affected by disease and therapies. An overview of the emergence of knowledge about the lung microbiome will be given with examples from a number of important lung diseases. Consideration of the challenges in conducting lung microbiome studies will be highlighted as will the lines of investigation possible when dealing with samples with rich metadata.

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Type 17/23 responses in Human Inflammatory Diseases, New Understanding and New Treatments 94 Induction and regulation of IL-17+ T cells in human chronic inflammatory disease L. Taams King’s College London, London, UK IL-17A (henceforth called IL-17) is a pro-inflammatory and osteoclastogenic cytokine, which can be produced by various cell types, including T cells. Results from in vitro studies, experimental models and clinical trials indicate that IL-17 plays a role in the immunopathology of several inflammatory diseases, including psoriasis, psoriatic arthritis (PsA) and rheumatoid arthritis (RA). We and others have shown that IL-17 producing CD4+ T cells (Th17 cells) are present at increased levels in the blood, synovial tis-

sue and synovial fluid of patients with RA, and that the presence of these cells in the joint correlates with clinical parameters of active disease. In addition, we found that both in vitro activated (LPS-stimulated) and in vivo activated CD14+ cells from the inflamed RA joint are potent drivers of IL-17 production in CD4+ T cells. PsA is an inflammatory arthritis that is genetically, radiologically, serologically and clinically distinct from RA. Our recent work shows that the inflamed joints of patients with PsA are enriched for IL-17 producing CD8+ T cells (Tc17) and that these cells correlate with clinical parameters of disease. These data suggest that Tc17 cells may constitute a hitherto unrecognized pathogenic immune cell population in PsA. Our current work is aimed at investigating what drives and regulates IL-17 producing T cells in human inflammatroy disease. Recent data will be discussed at the meeting.

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Abstracts of the British Society for Immunology Annual Congress, 1-4 December 2014, Brighton, UK.

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