SCIENTIFIC WORKSHOP 2016

INFLAMMATION &

IMMUNITY IN HEALTH AND DISEASE

CeMM, Vienna, May 12th - 13th 2016

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EU-LIFE scientific workshop

Inflammation & Immunity in Health and Disease

CeMM, Vienna, 12-13th May 2015

INTRODUCTION Goal This third EU-LIFE scientific workshop aims to bring together basic scientists and clinicians/translational researchers from EU-LIFE institutes (and collaborators) to explore and discuss inflammation and immunity in health and disease. We aim to foster a discussion on how to strengthen the interaction of basic and translational scientists with clinicians to translate research findings to diagnostic and therapeutic applications. The workshop will also be an opportunity to forge new scientific collaborations within EU-LIFE and found the basis for future joint activities and projects. Format Each EU-LIFE institute has invited a “tandem of speakers”, often one being a basic researcher and the second a clinical/translational researcher. The constellation of these “tandems” may vary: a) one or both speakers may work at the EU-LIFE center or partnering institute/university/hospital/company, and ideally they are engaged in a cooperative project; b) the basic researcher and the clinical/translational scientist do not have an ongoing cooperation but their work is relevant to each other. Each tandem of speakers will have 30-minutes to present their work jointly and highlight opportunities and challenges of their collaboration. The presentation will be followed by a 10-minute discussion. The meeting will also include the opportunity to present their work for junior researchers. Each EU-LIFE institute invited up to four PhDs students or postdocs to submit an abstract. To ensure maximum visibility of these presentations, we selected 12 of these abstracts for short presentations at the meeting (4 min speed talk + 1 min discussion). All other abstracts will be presented as posters at the conference dinner in Vienna’s Museum of Natural History. Organizers and hosts: Andreas Bergthaler and Stefan Kubicek with the EU-LIFE Translational Working group.

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GENERAL INFORMATION GETTING TO CeMM CeMM is located on the campus on the campus of the general hospital of Vienna (AKH)

Address: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences Lazarettgasse 14, AKH BT 25.3 1090 Vienna, Austria Transfer Vienna Airport (VIE) to City: CAT - City Airport Train http://www.cityairporttrain.com/, one way ticket: € 11 Taxi - Airportdriver http://www.airportdriver.at/en/airport-transfer, online one way pre-booking: € 33; It takes about 35-40 min. to get from the Vienna Airport to CeMM. For public transportation in Vienna, information on journeys and prices can be found on the following website: http://www.wienerlinien.at/eportal3/; costs for a single use ticket: € 2,20 and for a 24-hours ticket: € 7,60 More information about Vienna, including sightseeing recommendations: https://www.wien.info/en MEETING LOCATION The meeting will be held in CeMM’s 8th floor lecture hall. Presentations of this scientific meeting will be made available as live-stream to all EU LIFE institutes.

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HOTELS We have reserved several rooms including breakfast at the following hotels in Vienna. By mentioning “EU-LIFE”, the meeting participants will get a better price. Booking deadline is March 15, 2016 for all three hotels (to guarantee the special deals).

Mercure Hotel Josefshof http://josefshof.com/en/hotel Josefsgasse 4-6, 1080 Vienna, T: +43-1-40419, reservierung@josefshof.com Single Room: € 102,10 / Double Room (can also be booked for single use): € 122,20 4 star hotel near Vienna City Hall/Rathaus, walking distance to CeMM: 1,5 km Check-in after 2.00 pm, Check-out until 12.00 25Hours Hotel http://www.25hours-hotels.com/en/museumsquartier/home/home.html Lerchenfelder Strasse 1-3, 1070 Vienna, T: +43-1-52151-820, Events.Wien@25hours-hotels.com Single Room: € 184,00 / Double Room (can also be booked for single use): € 199,00 4 star hotel near Museumsquartier, walking distance to CeMM: 1,8 km Check-in after 3.00 pm, Check-out until 12.00 Hotel Ambassador http://www.ambassador.at/default-en.html Kärtner Strasse 22, 1010 Wien, T: +43-1-96161-0, sales@ambassador.at Double Room Vienna Classic (also for single use): € 230,00 5 star hotel near St. Stephen’s Cathedral/Stephansdom, walking distance to CeMM: 2,5 km Check-in after 2.00 pm, Check-out until 12.00

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AGENDA

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Thursday, May 12th 2016 13.00 Registration 13.30 Welcome 14.00 Session 1: Autoimmunity

14.00-14.40

Janna Saarela (FIMM) Filtering through population bottlenecks: variants causing primary immunodeficiency and autoinflammation in a population isolate Mikko Seppänen (Helsinki University Hospital) Clinical exome sequencing challenges and strengths

14.40-14.45 Dimitrios Tsiantoulas (CeMM) Deficiency of secreted IgM antibodies causes increased plasma IgE levels that promote atherosclerosis

14.45-14.50 Lars Vereecke (VIB) A20/TNFAIP3: Master regulator of intestinal immune homeostasis

14.50-15.30 Kim Bak Jensen (BRIC) Ole Haagen Nielsen (Herlev Hospital) Inflammatory bowel disease - from clinical manifestations to molecular mechanisms of tissue repair

15.30-15.35 Patrick Ejlerskov (BRIC) Involvement of IFN-beta signaling pathway in Parkinson's disease

15.35-15:40 Alba Manresa (BRIC) Role of RhoA in T-cell adhesion and migration in an experimental model of multiple sclerosis

15.40-16.00 Coffee Break 16.00 Session 2: Cancer immunology I

16.00-16.40

Olivier Lantz (Institut Curie) Characterization of the immune response towards uveal melanoma in different clinical setting Emanuela Romano (Institut Curie) Fc-mediated depletion of Tregs via ipilimumab-dependent ADCC in advanced melanoma patients

16.40-16.45 Christine Sedlik (Institut Curie) Spatial T cell dysfunction between tumor and draining lymph nodes

16.45-16.50 Ruby Alonso Ramirez (Institut Curie) “Infectious" tolerance transforms tumor antigen specific naive CD4 T cells into induced Tregs in a spontaneous lung tumor model

16.50-17.30 Thomas Blankenstein (MDC) Il-Kang Na (Experimental and Clinical Research Center of MDC and Charité) Adoptive T cell therapy of cancer

17.30-18.10 Pia Kvistborg (NKI) What T cells see on human cancer Seth Coffelt (NKI) Immune cell dominoes: falling in line to promote breast cancer metastasis

18.10-18.15 Mario Bunse (MDC) Improved T cell engineering using viral and transposon-based vectors encoding optimized TCR genes and redirected miRNAs

18.15-18.55

Jo Van Ginderachter (VIB) Functionally distinct macrophages and dendritic cell populations in the tumor microenvironment Max Mazonne (VIB) Hypoxic TAMs in cancer progression

19:30 Conference dinner including poster session Vienna Museum of Natural History Burgring 7, 1010 Wien

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Friday, May 13th 2016 9.00 Session 3: Cancer immunology II

9.00-9.40 Rahul Roychoudhuri (Babraham Institute) Making all the wrong decisions: Transcription factors in tumour immunosuppression Marc Veldhoen (Babraham Institute) Environmental influences on T cell immunity

9.40-10.20

Bill Keyes (CRG) The senescence associated secretory phenotype instructs stem cell function and regulation Clemens Schmitt (MDC/Charité) The dangerous liaison of therapy-induced senescence, senescence-associated cancer stemness and treatment failure

10:20-10:25 Ella Karjalainen (FIMM) Identifying Novel Treatment Strategies for AML through Stratified Biologically Relevant Drug Responses

10:25-10:30 Muntasir Mamun Majumder (FIMM) Precision medicine strategies for high-risk multiple myeloma to address variability in drug response and predicting treatment outcome

10.30-11.00 Coffee Break

11.00-11.40

Luca Mazzarella (IEO) Combined metabolic and epigenetic modulation in the treatment of Acute Myeloid Leukemia Anna Giulia Sanarico (IEO) Metabolic alterations in FLT3-ITD acute myeloid Leukemia

11.40 Session 4: Infection

11.40-12.20 Michael Trauner (Medical University of Vienna, Austria) Bile acids and inflammation Andreas Bergthaler (CeMM) Metabolic-Inflammatory Crosstalk in Viral Infection

12.30-13.35 Simona Saluzzo (CeMM) Homeostatically driven type-2 II pathways shape the lung pulmonary immune environment

12.35-13.40 Chloé Abels (VIB) Hepatic embryo-derived Kupffer cells regulate the onset of immune response against Listeria monocytogenes infection and co-exist with two other monocyte-derived macrophage populations

12.40-12.45 Alexandre Bignon (Babraham Institute) Mucosal administration of the B subunit of Escherichia coli heat-labile enterotoxin promotes tolerance by enhancing regulatory T cells and tolerogenic dendritic cells

12.45-13.30 Lunch

13.30-14.10 Miguel Soares (Instituto Gulbenkian de Ciência) Tissue damage control in immune mediated inflammatory diseases Luis Ferreira Moita (Instituto Gulbenkian de Ciência) Sepsis: the need for tolerance not complacency

14.10-14.50 Pavel Plevka (CEITEC) Structure and genome release of Myoviridae phage with double layered baseplate Mary O'Connell (CEITEC) ADAR1 is crucial for discriminating between ‘self’ and ‘non-self’ cellular RNAs

14:50 Wrap-up 15:00 End of meeting

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ABSTRACTS TANDEM TALKS

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SESSION 1: AUTOIMMUNITY Filtering through population bottlenecks: variants causing primary immunodeficiency and autoinflammation in a population isolate Janna Saarela Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland The population history of Finland, characterized by a restricted number of founders, isolation and several population bottlenecks, has caused enrichment of certain rare disease causing variants and losses of others, as part of a phenomenon called the Finnish Disease Heritage. Accordingly, rare founder mutations cause the majority of observed Finnish cases in these mostly autosomal recessive disorders that consequently are more frequent in Finland than elsewhere. Here I describe two examples of gene defects causing PID in Finland. First, exome sequencing of a multi-case Finnish family with a Hyper IgM type 2 phenotype identified a rare, homozygous, variant (c.416T>C, p.Met139Thr) in the AICDA gene, found to be significantly enriched in the Finnish population compared with other populations of European origin (38.56-fold, p <0.001). Further screening of all currently known Finnish patients with HIGM2 showed them to be homozygous for the same variant. Furthermore, all carriers of the p.Met139Thr in Finnish population cohorts originated from the eastern and northeastern parts of Finland, shared more of their genome identity by descent (IBD) than Finns in general (p<0.001), and carried a 207.5kb ancestral haplotype containing the variant. Based on these finding we suggest HIGM2 as a novel Finnish Disease Heritance disorder. The second example describes identification of a novel PID gene by NGS screening of three unrelated Finnish kindreds with immunodeficiency and autoinflammation. In all 13 affected individuals, we identified heterozygous variants in NFKB1, encoding for the NF-κB family members p50/p105. Patients harboring a p.I553M variant presented with antibody deficiency, infection susceptibility, and multi-organ autoimmunity. In addition to hypogammaglobulinemia, patients with a p.H67R change suffered from autoinflammatory episodes including aphthae, gut disease, febrile attacks and small vessel vasculitis characteristic of Behcet's disease. The main symptom in patients with a p.R157X stop-gain variant was a severe hyperinflammatory response to routine surgery. Functional analyses indicated that both missense variants led to a reduction in p50/p105 function, whereas the p.R157X variant likely leads to rapid elimination of the truncated transcript. The p.H67R variant reduced nuclear entry of p50, which in luciferase-reporter assays led to a decrease in transcriptional activity. The p.I553M mutation, in turn, reduced phosphorylation of the NF-κB inhibitor p105 decreasing its stability during TNF induction. Both missense mutations also led to altered protein-protein interactions in affinity purification mass spectrometry. These findings suggest that a subset of autoinflammatory diseases may be caused by rare, monogenic variants and highlight the importance of careful homeostasis between components of the NF-κB pathway.

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Clinical exome sequencing challenges and strengths Mikko Seppänen Rare Disease Center, Helsinki University Hospital (HUH), Helsinki, Finland From the known protein-protein interactions it has been predicted that, instead of the known approximately 350 primary immunodeficiency (PID) and bone marrow failure (BMF) genes, there are over 3100 potential genes that could cause PID. For many of these, both gain and loss of function seem plausible, and the data on protein-protein interactions is still far from comprehensive. When diagnosing PIDs in isolated populations like the genetic Finns, these predicted numbers seems fully likely. Finns have relatively little “background noise” when filtering for potential novel PID candidate genes. Both the clinical phenotypes and the known or suspected gene candidates after whole exome and/or genome sequencing differ strongly from those found from better studied populations. This has led to a situation where a substantial number of patients harbor likely novel diseases or phenotypes caused by known or novel candidate genes. The rate limiting steps are of course effective and reliable variant filtering as well as gathering enough functional data to prove that the candidate gene in fact causes the disease in patients or even large kindreds. Similarly as in other genetic isolates, such as the populations of Middle Eastern ancestry, studying PIDs in the Finns has already led to identification of novel PID causing genes. However, creation of a comprehensive map of genes involved in rare disorders, such as PIDs and bone marrow failure, will require European wide and even a global collaboration.

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Inflammatory bowel disease - from clinical manifestations to molecular mechanisms of tissue repair Ole Haagen Nielsen1 and Kim B. Jensen2 1. Department of Gastroenterology, Medical Section, Herlev Hospital, University of Copenhagen, DK-2730 Herlev, Denmark 2. BRIC - Biotech Research and Innovation Centre, University of Copenhagen, Ole Maaloes Vej 5, DK-2200 Copenhagen N, Denmark Inflammatory bowel disease is an umbrella term of a number of distinct disorders characterised by uncontrolled inflammation in the gastrointestinal tract of which ulcerative colitis and Crohn’s disease are the most prevalent entities. Ulcerative colitis is confined to the colon, whereas Crohn’s disease may affect any part of the GI tract, and the prevalence of both diseases is increasing world-wide possibly due to yet unknown factors in the environment. A compromised barrier function of the epithelium in the GI tract causes additional problems due to the direct exposure to the commensal microbiota. Currently, patients are treated with glucocorticoids, immunomodulators (i.e. thiopurines and methotrexate) as well as biologics (i.e. TNF-α inhibitors and α4β7 anti-integrins) and oral administered small molecules and antisense therapeutics are expected in the near future to control the intestinal inflammation and allow the GI tract to restore a normal barrier function. We will discuss how understanding clinical manifestations of inflammatory bowel disease as well as the process of normal epithelial tissue repair will provide new insights into disease development and help us to identify novel therapeutic strategies for a more tailored approach to target the disease in the future.

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SESSION 2: T-CELLS IN CANCER IMMUNOLOGY Characterization of the immune response towards uveal melanoma in different clinical setting Olivier Lantz on behalf of the Institut Curie Uveal melanoma translational research group Clinical Immunology laboratory, Institut Curie, Paris, France. Once metastatic the prognosis of uveal melanoma (UM) is very poor. In the absence of active conventional or targeted therapy, immunotherapy would be very attractive. However, only anecdotic responses to anti-check point blockers have been reported. UMs do not harbor somatic mutations that would be the source of neoantigens. Yet, immune infiltrates are found in both primary tumors and metastatic lesions. In addition, increased proportion of oligoclonal effector CD4 and CD8 T cells are found in the blood of metastatic patients suggesting the existence of an anti-tumor response (1). Herein, characterized the immune response observed in the blood of patients either at the time of primary treatment or when operated for liver metastasis. We observed the presence of effector CD4 T cells at the time of diagnosis in a fraction (≈20%) of the 85 patients we studied. The correlation with genomic anomalies is ongoing. At the time of liver metastasectomy, effector CD4 T cells were observed in the blood of about half of the metastatic patients. We also characterized the immune infiltrated in the liver lesions of 15 patients using both immunochemistry and cytometry. In comparison with adjacent "healthy" liver, we observed an increased proportion of Tregs and CD8 T cells with a concomitant decrease in MAIT and NKT cell numbers. By immunochemistry, T cells were found in both the periphery and inside the tumors. Altogether, these results suggest that contrary to what is usually assumed uveal melanoma tumor cells are seen by the immune system. The antigen specificity of the effector T cells observed in UM patients will be characterized in further work and may allow the development of therapeutic vaccine strategy. The probable presence of tumor reactive T cells in UM patients also justifies the use of therapeutic schemes including anti-checkpoint blockers. Peguillet et al. Cancer. Res. 2014. 74(8):2204-16

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Fc-mediated depletion of Tregs via ipilimumab-dependent ADCC in advanced melanoma patients Emanuela Romano Institut Curie, Paris, France Enhancing immune responses with immune-modulatory monoclonal antibodies (mAbs) directed to inhibitory immune-receptors is a promising modality in cancer therapy. Clinical efficacy has been demonstrated with antibodies blocking inhibitory immune checkpoints such as CTLA-4 or PD- 1/PD-L1. Treatment with ipilimumab, a fully human CTLA-4 specific mAb, showed durable clinical efficacy and improved overall survival in metastatic melanoma; its mechanism/s of action, however, are only partially understood. Recent studies in melanoma mouse models revealed that the anti-tumour activity of CTLA-4 blockade is mediated by FcgRIV-expressing macrophages in the tumour microenvironment (TME) via in-trans depletion of tumour-infiltrating Tregs. We speculated that a similar mechanism might operate in melanoma patients responding to ipilimumab. To investigate this hypothesis, we interrogated peripheral blood mononuclear cells (PBMCs) and matched melanoma metastases from 15 patients responding (R) and 14 non-responding (NR) to ipilimumab. Our findings show, for the first time, that ipilimumab leads to the depletion in vitro of regulatory T cells (Tregs) via an antibody-dependent-cellular-cytotoxicity (ADCC) mechanism, selectively mediated by FcgRIIIA (CD16)-expressing, non-classical monocytes. In contrast, classical monocytes, lacking the FcgRIIIA expression, are unable to deplete Tregs in an ADCC assay. Interestingly, patients responding to ipilimumab displayed significantly higher baseline peripheral frequencies of non-classical monocytes than non-responder patients. Evaluation of matched melanoma metastases from pre- and post-ipilimumab time-points by IHC revealed that, in the TME, responders had the highest CD68+/CD163+ macrophage ratios at baseline, and showed decreased infiltration of Tregs after treatment. Notably, baseline Treg infiltration was comparable between the two groups. Our results provide novel mechanistic insight into the clinical activity of ipilimumab, highlighting the contribution of the tumour stroma into the final outcome of antibody-based immunomodulatory therapies and suggest non-classical monocytes as a potential biomarker of response.

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Adoptive T cell therapy Thomas Blankenstein Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13092 Berlin, Germany Charité Centrum für Tumormedizin, Institut für Immunologie, Campus Berlin-Buch, Lindenberger Weg 80, 13125 Berlin Adoptively transferred T cells have been shown to reject large established tumors. In such models, T cells recognize the tumor antigen as foreign. The task is to generate human T cell receptors (TCR) that recognize human tumor-associated (self) antigens as foreign and use these TCRs for gene therapy. We use mice with a humanized TCR repertoire to isolate therapeutic TCRs. Adoptive T cell therapy of cancer Il-Kang Na Department of Hematology, Oncology and Tumor Immunology, Charité University Medicine CVK, Berlin, Germany Experimental and Clinical Research Center (ECRC), Berlin, Germany Adoptively transferred T cells require successful infiltration into the tumor tissue and activation on site for efficient tumor eradication. Using a dual-luciferase transgenic mouse, which enables us to concurrently and longitudinally visualize migration and activation of T cells in vivo, we compare activation and effector function of T cell subpopulations when targeting a tumor-specific or a tumor-associated antigen and test combinatorial drug and adoptive T cell therapies to prevent tumor relapse.

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What T cells see on human cancer Pia Kvistborg The Netherlands Cancer Institute Human tumors contain large numbers of mutations, of which many hundreds can be present within expressed genes. As the resulting altered protein sequences are foreign to the immune system, immune recognition of such 'neo-antigens' is likely to be of significant importance to the activity of clinically used immunotherapeutics such as anti-CTLA-4 and anti-PD-1 in melanoma. However, the vast majority of the mutations in human cancers are unique to individual patients and, because of this, broadly applicable approaches to link the consequences of DNA damage in human cancer to tumor-specific T cell activity have long been lacking. Using in-house developed technologies for monitoring of T cell activity, we have recently demonstrated the feasibility of cancer exome-driven analysis of both tumor-specific CD8+ T cell reactivity and CD4+ T cell reactivity in human melanoma. The data obtained demonstrate that T cell recognition of the consequences of DNA damage is a common feature in human melanoma. Furthermore, based on the distribution of mutation loads in other major human cancer types, we propose that also in many other human tumors, the repertoire of mutant antigens provided by DNA damage (the ‘neo-antigen space’) will suffice to allow T cell recognition. Collectively, these data indicate that mutational load may form a biomarker in cancer immunotherapy, and that the development of ‘personalized immunotherapies’ that exploit cancer genome information to target patient-specific mutant antigens should be explored.

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Immune cell dominoes: falling in line to promote breast cancer metastasis Seth Coffelt (NKI) & Karin E. de Visser The Netherlands Cancer Institute Metastasis remains the primary cause of death for breast cancer patients. This is due in large part to the lack of knowledge about the mechanisms underlying cancer spread. Metastatic disease is still largely unexplored, poorly understood and incurable. Emerging evidence indicates that immune cells are critical regulators of the different steps of the metastatic cascade. Recently, we showed that mammary tumors in K14-cre;Cdh1F/F;Trp53F/F mice elicit a systemic inflammatory cascade to dampen anti-tumor T cells and maximize metastasis formation. This cascade is initiated by tumor microenvironment-derived IL1β that activates IL17-producing γδ T cells, leading to G-CSF-dependent neutrophil expansion and repolarization. In turn, these neutrophils suppress CD8+ T cells, allowing disseminated cancer cells to go unnoticed. Current efforts are underway to determine how genetic makeup of different mammary tumors regulates the γδ T cell – IL17 – neutrophil axis. Together, these data provide further insight into how immune cells participate in metastasis and uncover several putative targets for metastatic breast cancer.

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Making all the wrong decisions: Transcription factors in tumour immunosuppression Rahul Roychoudhuri Lymphocyte Signalling and Development Laboratory, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT, UK. Through functional diversification, cells of the innate and adaptive immune system either drive or constrain immune reactions within tumours. Thus, while the immune system has a powerful ability to recognize and kill cancer cells, this function is often suppressed, preventing clearance of disease. The transcription factor (TF) BACH2 regulates the differentiation and function of multiple innate and adaptive immune lineages, but its role in controlling tumor immunity is not known. We have found that BACH2 is required to establish immunosuppression within tumors. Growth of tumours was markedly impaired in Bach2-deficient mice and coincided with intratumoural activation of both innate and adaptive immunity. However, augmented tumor clearance was dependent upon adaptive immunity. Analysis of tumor-infiltrating lymphocytes in Bach2-deficient mice revealed high frequencies of CD4+ and CD8+ effector cells expressing the inflammatory cytokine interferon (IFN)-γ. T cell activation correlatedwith reduction in the frequency of intratumoral CD4+ Foxp3+regulatory T (Treg) cells. Mechanistically, BACH2 promoted tumor immunosuppression through Treg-dependent inhibition of intratumoural CD8+ T cells. These findings demonstrate that BACH2 is a key component of the molecular program of tumor immunosuppression and identify a new target for development of therapies aimed at reversing immunosuppression in cancer.

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Environmental influences on T cell immunity Marc Veldhoen Lymphocyte Signalling and Development Laboratory, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT, UK. Epithelial barrier sites, such as the gastrointestinal tract and skin, show a remarkable compartmentalisation with respect to T lymphocytes and their function. The top most layer, the intraepithelial compartment and epidermis contain a mix of innate-like T cells capable of making interferon gamma (IFNγ) whilst the deeper tissue layers, the lamina propria and dermis, are enriched for interleukin (IL)-17 producing T cells. We have previously shown that particular environmental factors, such as those derived from the diet, are able to directly affect Th17 as well as intraepithelial lymphocytes (IELs) via the arylhydrocarbon receptor. For some time we have been probing the requirements for Th17 cell differentiation. Although IL-6 and IL6R/Stat3 signalling is a requirement for Th17 cell polarisation and although TGFβ and TGFβR are reportedly required, no known downstream TGFβR signalling pathway, such as the SMADs, are essential of Th17 development. We hypothesised if alternative pathways are required for Th17 cell development. We will present data on an alternative pathway with unexpected results. Recent observations showed a strong correlation between tumours and Th17 cell numbers. This observation in combination with the sites where Th17 cells are enriched made us hypothesise if alterations in metabolic availability in the microenvironment could influence Th17 cell differentiation. We will present new data on how metabolic disturbances specifically influence Th17 cell differentiation, with implications for chronic inflammatory disorders and cancer.

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SESSION 3: INNATE PATHWAYS IN CANCER IMMUNOLOGY The senescence associated secretory phenotype instructs stem cell function and regeneration Bill Keyes Centre for Genomic Regulation (CRG), Barcelona Senescence is a form of cell cycle arrest that prevents the proliferation of damaged cells through the activation of tumor suppressor networks. As a result, senescence is described as a tumor suppressive mechanism. However, even while arrested, senescent cells can interact extensively with their microenvironment through the secretion of a variety of proteins termed the senescence-associated secretory phenotype (SASP). This secretome is largely composed of growth factors, cytokines/chemokines and extracellular-matrix remodeling proteins secreted by the arrested cells, and can function to reinforce the arrest, or recruit immune cells to clear the senescent population. Conversely however, in certain conditions, the SASP can favor tissue growth and repair, such as during wound healing and as we recently demonstrated, during embryonic development. In some cases, the SASP can even promote tumor growth. These functions suggest a broader more complex physiological role for senescence and the SASP than currently understood. Here we describe that the induction of oncogene-induced senescence (OIS) in primary mouse keratinocytes leads to a dramatic increase in stem cell gene expression. Interestingly, this signature can also be induced in normal cells upon exposure to the SASP, suggesting that secreted factors by the senescent cells induce stem cell gene expression. In order to examine stem cell functionality in these cells, we performed full-thickness skin grafting with senescent or SASP-treated cells. Interestingly, cells transiently exposed to the SASP acquired full-stem cell capacity, and could regenerate skin and hair follicles in the recipient host. However, transplantation of fully senescent cells induced the formation of papilloma, a pre-malignant skin lesion that is maintained by aberrantly proliferating stem cells. Together, our data suggests that the SASP is in fact a regenerative mechanism that instructs stemness, but that if left uncurtailed, leads to tumor formation through aberrant regenerative mechanisms.

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The dangerous liaison of therapy-induced senescence, senescence-associated cancer stemness and treatment failure Clemens A. Schmitt and colleagues Charité – University Medical Center, Medical Department of Hematology, Oncology and Tumor Immunology, and Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany Cellular senescence is a stress-responsive cell-cycle arrest program that terminates further expansion of (pre-)malignant cells. Interestingly, many key signaling components of the senescence machinery also operate as critical regulators of stem cell functions (collectively termed ‘stemness’), among them the p53 axis and the transcriptionally repressive lysine 9-trimethylated histone H3 (H3K9me3) mark. We report here our experimental approach to address two pivotal questions: (I) May chemotherapy-induced senescence evoke stem cell-related functionalities in malignant cells of murine and human origin? (II) Is cellular senescence a potentially reversible program, thereby allowing cells to execute their stem-like capacity in a division-coupled manner? Gene expression and functional analyses comparing chemotherapy-induced senescent vs. non-senescent Bcl2-overexpessing (and, thus, apoptosis-blocked) Eµ-myc transgenic B-cell lymphomas unveiled massive upregulation of an adult tissue stem cell signature, activated Wnt signaling, and de novo expression of distinct stem cell markers in senescence. We previously demonstrated that the H3K9-targeting histone methyltransferase Suv39h1, like p53, operates in a senescence-essential fashion in these murine B-cell lymphomas. Utilizing conditional Suv39h1- and p53-based, genetically switchable ‘matched pair’ models of senescence to mimic spontaneous escape (‘previously senescent’, PS), we found PS cells to re-enter the cell-cycle with strongly enhanced and Wnt-dependent clonogenic growth when compared to their equally chemotherapy-exposed but never senescent (NS) counterparts. In vivo, these PS lymphoma cells presented with a much higher tumor initiation potential, which was neutralized upon pharmacological or genetic Wnt inhibition. Strikingly, temporary enforcement of senescence in a p53-regulatable leukemia model reprogrammed non-stem bulk PS leukemia cells into self-renewing leukemia-initiating stem cells, while equally chemotherapy-exposed NS bulk cells did not acquire stemness potential. Our data, further supported by consistent findings in various human cancer cell lines and primary tumor samples, uncover senescence-associated stemness as an unexpected, cell-autonomous feature that exerts its detrimental potential upon escape from the cell-cycle block. These findings indicate a hitherto unappreciated role of senescence in stress-evoked tissue damage but also raise concerns about the long-term benefit of senescence-inducing cancer therapies, and provide new mechanistic insights into the plasticity of the “cancer stem cell” condition. In turn, we present synthetic lethal targeting of senescence-associated stemness as a conceptually novel, outcome-improving anticancer treatment strategy.

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Functionally distinct macrophage and dendritic cell populations in the tumor microenvironment. Jo A. Van Ginderachter Myeloid Cell Immunology Lab, VIB; Cellular and Molecular Immunology Lab, Vrije Universiteit Brussel, Brussels, Belgium Tumor-associated macrophages (TAM) are exposed to multiple microenvironmental cues in tumors, which collaborate to endow these cells with protumoral activities. Given the inherent plasticity of macrophages, we hypothesized that different macrophage types might exist in distinct tumor regions. We showed that the myeloid infiltrate in tumors is complex and encompasses three ontogenically distinct tumor-associated dendritic cell (TADC) populations and at least two major TAM subsets, designated as MHC-IIlow MMRhi and MHC-IIhi MMRlow TAM, both of which were derived from tumor-infiltrating Ly6Chi monocytes. MHC-IIlow TAM express higher levels of prototypical M2 markers and reside in more hypoxic regions, as shown by in vivo molecular imaging using MMR-specific nanobodies. Employing different in vivo strategies, our results show that hypoxia is not a major driver of TAM subset differentiation and polarization per se, but rather specifically fine-tunes the phenotype of M2-like MHC-IIlow TAM. We now identified M-CSFR signaling as an important regulator of monocyte and macrophage differentiation in tumors, which seems especially important for regulating the MHC-IIlow TAM population. Conversely, GM-CSFR signaling rather fine-tunes the phenotype of MHC-IIhi TAM. Ongoing work in our lab is further exploring the distinct functions and effector molecules of these TAM subsets and strategies to target them in vivo. The identification of functionally distinct tumor-associated DC (TADC) subpopulations could prove essential for the understanding of basic TADC biology and for envisaging targeted immunotherapies. We demonstrated that multiple mouse tumors as well as human tumors harbor ontogenically discrete TADC subsets. Monocyte-derived TADC are prominent in tumor antigen processing, but lack strong T-cell stimulatory capacity due to NO-mediated immunosuppression. Pre-cDC-derived TADC have lymph node migratory potential, whereby cDC1 efficiently activate CD8+ T cells and cDC2 induce Th17 cells. Mice vaccinated with cDC2 displayed a reduced tumor growth accompanied by a reprogramming of pro-tumoral TAM and a reduction of MDSC, while cDC1 vaccination strongly induces anti-tumor CTL. Our data might prove important for therapeutic interventions targeted at specific TADC subsets or their precursors.

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Influence of Macrophage Metabolism on Vessel Shape: Implications for Cancer Max Mazzone VIB Cancer typically has an inflammatory nature. Of all immune cells invading the tumor, the role of macrophages remains controversial because of their functionally distinct differentiation states. Classically activated (M1-like) macrophages and alternatively activated (M2-like) macrophages represent the two extremities of a continuum, in which the former are proinflammatory and tumoricidal, whereas the latter display pro-angiogenic and protumoral activities. This heterogeneity reflects the plasticity and versatility of these cells in response to microenvironmental signals. The tumor environment is constituted of cancer cells and stromal cells that coexist under different oxygen tensions and the influence of soluble factors. A plethora of (hypoxia-induced) cytokines and chemokines can define the macrophage phenotype and their positioning within the tumor. We have elucidated an elegant mechanism by which macrophages are recruited and retained in avascular hypoxic niches of the tumor, where they exert their immunosuppressive and pro-angiogenic activity. Hypoxia by itself indeed strongly affects macrophage function, as the cells are forced to adapt and alter their metabolism, which is accompanied by a drastic change in their transcriptome and proteome. We have found that the resultant metabolic changes drive macrophages towards a pro-angiogenic phenotype in an attempt to restore oxygenation. Soluble factors derived from the tumor exacerbate this macrophage shift by further enforcing this metabolic adaptation. As a consequence, tumor blood vessels are abnormal and dysfunctional, thus perpetuating hypoxia, which in turn fosters metastasis. By using genetic tools in mice, we show that inhibiting macrophage recruitment in hypoxic niches, or altering their metabolic and energetic state can break this feed-forward loop, resulting in normalized blood vessels and thus, strongly reducing metastatic dissemination.

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Combined metabolic and epigenetic modulation in the treatment of Acute Myeloid Leukemia Luca Mazzarella1, Tiphanie Durfort1, Rani Pallavi1, Elena Mylona1, Paolo Falvo1, AnnaGiulia Sanarico1, Massimo Stendardo1, Luciano Giaco’1, Anna Russo1, Lucilla Luzi1, Massimiliano Mazza1, Giulia de Conti1, Paola Vianello2, Pierluigi Rossi1, Elena Ceccacci1, Mohamed Elgendy1, Marco Giorgio1, Ciro Mercurio2,3, Saverio Minucci1,2 and Pier Giuseppe Pelicci1 1Dept of Experimental Oncology and 2Academic Drug Discovery Program and Drug Development Program, Dept of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139, Milan, Italy 3Genextra Group, DAC s.r.l. Via Adamello 16, 20139, Milano, Italy Introduction. There is increasing interest in the therapeutic modulation of metabolic pathways in cancer. Tumor cells preferentially use aerobic glycolysis to meet their energetic demands, but can switch to alternative substrate usage when specific nutrients are limiting. The molecular basis of metabolic adaptation is poorly understood. Recently, the histone demethylase LSD1 (Lysine-Specific Demethylase 1) has been implicated in the control of oxidative phosphorylation (OXPHOS) in adipocytes through its interaction with NRF1 (Nuclear Respiratory Factor 1), a master regulator of metabolic gene transcription (1). We hypothesized that LSD1 could regulate metabolic adaptability and be a therapeutic target upon metabolic modulation through Caloric Restriction (CR) in Acute Myeloid Leukaemia (AML) and specifically in APL (Acute Promyelocytic Leukaemia), which we showed to be sensitive to body fatness in the clinic (2). Methods. APLs were generated in mice expressing the PML-RARa fusion under the control of the Cathepsin G promoter (3). Primary leukemias were transplanted into recipients subjected to 30% CR or Standard Diet (SD). We scored the effect of CR alone or in combination with the LSD1 inhibitor IEO368 (4) on mouse survival, Leukemia Initiating Cell (LIC) frequency and epigenomic, transcriptomic and metabolic parameters. Results. Compared to SD controls, CR-fed recipients experienced an initial dramatic decrease in the total leukemic burden accompanied by cell cycle slowdown (“adaptation phase”); this was followed by a delayed disease progression that brought animals to death (“terminal phase”) (median survival 91 vs 51 days, p=0.038). Limiting-dilution transplantation of CR-conditioned leukemias revealed increased frequency of LICs (estimated frequency 1/3064 cells in SD vs 1/947 in CR, p=0.003) and increased aggressiveness (median survival reduced to 49 vs 70.5 days with 5000 cells injected, p<0.0001). Thus, CR limits the expansion of leukemic cells but enriches for cells with increased ability to regrow. RNAseq of leukemic cells purified during the terminal phase (but not earlier) showed that a dramatic transcriptional reprogramming in CR, characterized by upregulation of genes controlling OXPHOS, Krebs’ cycle and nucleotide and protein biosynthesis, and downregulation of insulin signaling and glucose transporters. In particular, superenhancer-associated genes were uniformly downregulated in CR, suggesting enhancer "decommissioning", a process dependent on LSD1 in embryonic stem cells. Strikingly, co-treatment of leukemic mice with CR and our LSD1 inhibitor IEO368 (4) resulted in macroscopic and microscopic eradication of disease. LSD1 inhibition alone was also effective but did not produce bona fide disease eradication. Importantly, some of the features of the CR-LSD1 interaction could be modeled by combining LSD1 and an IGF1/Insulin inhibitor. In vivo, this combination was synergistic and led to durable responses (median survival 121 vs 50 days in untreated controls, p=0.0143, vs 65.5 and 78.5 days with Insulin/IGF1 Inhibitor and IEO368 respectively). Conclusion: the combination of LSD1 inhibition and insulin/IGF1 signaling reduction by pharmacological or dietary intervention appears as an extremely effective therapeutic strategy and deserves further investigation. Preclinical studies are ongoing to verify its applicability to other models of cancer.

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Metabolic alterations in FLT3-ITD acute myeloid Leukemia Anna Giulia Sanarico, Luca Mazzarella, Paolo Falvo, Luciano Giaco', Tiphanie Durfort, PierGiuseppe Pelicci Dept of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139, Milan, Italy Mutations in the tyrosine kinase FLT3, usually by insertion of Internal Tandem Duplications (ITD) in the region encoding the autoinhibitory juxtamembrane domain, are associated with poor prognosis in Acute Myeloid Leukemia (AML). Attempts to target FLT3 by direct kinase domain inhibitors have provided small albeit significant survival advantages in clinical trials. However, mutations involving drug-binding sites rapidly appear and confer resistance. Therefore, a better understanding of FLT3 biology is needed to develop suitable treatments. We previously showed that in a subtype of AML, Acute Promyelocytic Leukemia (APL), increased body mass is associated with worse outcome. Intriguingly, we found that overweight or obese patients had a 4-fold higher incidence of FLT3 mutations. Promotion of FLT3ITD-associated leukemia by obesity was confirmed in transgenic FLT3ITD mice, in which long term high fat diet (HFD) resulted in significantly accelerated leukemic mortality. These findings suggested that the obese “milieu” might provide a selective advantage for FLT3-mutated clones, prompting us to investigate specific metabolic requirements induced by FLT3ITD. Analysis of syngeneic cell lines overexpressing WT or ITD FLT3 showed that FLT3ITD generates significant ER stress that cannot be alleviated through reduction in protein biosynthesis rate due to persistently elevated mTOR signaling. Multiple adaptive pathways including SREBP1/2-dependent lipid biosynthesis are modulated at the transcriptional level and required for FLT3ITD cell survival, providing a possible mechanistic link with obesity. The net result of this metabolic rewiring is an unbalanced redox microenvironment that consumes reduced glutathione; depletion of glutathione or ablation of glutathione-replenishing enzymes induced rapid cell death, revealing a potential new avenue for FLT3ITD targeting in leukemia.

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SESSION 4: INFECTION Bile acids and inflammation Michael Trauner, M.D., and Claudia Fuchs, PhD Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Austria; michael.trauner@meduniwien.ac.at Bile acids (BAs) have been traditionally viewed as pro-inflammatory molecules as a result of their detergent activities attacking cell membranes and capability to stimulate the secretion of cytokines and chemokines. Accumulation of BAs in various (particularly cholestatic) liver diseases is a major driver of hepatic inflammation, fibrogenesis and carcinogenesis. Moreover, systemic BA accumulation may also damage extrahepatic organs and tissues such as kidney (e.g. cholemic nephropathy). BA-induced tissue damage and inflammation has been closely linked to their hydrophobicity and thereby potential to damage cell membranes. Furthermore, BAs can act as signalling molecules with hormonal actions mediated through activation of dedicated BA receptors such as the nuclear BA receptor farnesoid X receptor (FXR: NR1H4) and the membrane-bound BA receptor TGR5 (also called GPBAR1 or M-BAR/BG37). Via these two receptors BAs exert anti-inflammatory effects. FXR has anti-inflammatory and immunomodulatory actions and controls intestinal permeability as well as gut microbiota. FXR also interacts with NF-κB signalling. Moreover, TGR5 activation by BAs inhibits pro-inflammatory cytokine release and reduces phagocytic activity in rabbit alveolar macrophages, human monocytic leukemia cells and isolated rat Kupffer cells.

Our laboratory focuses on the metabolic, anti-inflammatory and immunomodulatory actions of BA receptor (FXR, TGR5) ligands, as well as endogenous polyhydroxylated bile acids (as potential ligands of nuclear receptors such as RORγt) and anti-inflammatory effects of modulating key bile acid transporters within the enterohepatic circulation (ASBT, NTCP, and BSEP). As such mice lacking BSEP are metabolically preconditioned with a more hydrophilic bile pool that protects them from cholestatic liver and bile duct injury. Preliminary data suggest that this could be mechanistically linked to inhibition of of RORγt/Th17 cell signaling. Furthermore we investigate the function of the synthetic bile acid 24-norursodeoxycholic acid (norUDCA), (a novel side-chain shortened derivate of UDCA) which undergoes cholehepatic shunting (instead full enterohepatic circulation) resulting in 'ductular targeting' to inflammed bile ducts/ductules and hepatic enrichment. As such, norUDCA (but not “conventional” UDCA) reverses sclerosing cholangitis in the experimental Mdr2/Abcb4 knockout mouse cholangiopathy model while UDCA aggravates bile infarcts in cholestatic conditions with (complete or partial) biliary obstruction. Moreover, norUDCA (but not UDCA) inhibits granuloma formation and hepatic fibrosis in a mouse model of Schistosoma mansoni infection as world-leading cause of hepatic fibrosis and portal hypertension. The anti-inflammatory properties of norUDCA were directed to MHC class II protein expression on dendritic cells and macrophages and norUDCA reduced T-lymphocyte proliferation and serum levels of pro-fibrogenic Th2 cytokines IL-13 and IL-4. These properties may also contribute to anti-inflammatory and anti-fibrotic effects of norUDCA. Based on these encouraging experimental data in preclinical models, norUDCA is currently undergoing clinical development in humans for PSC and NASH.

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Metabolic-Inflammatory Crosstalk in Viral Infection Andreas Bergthaler CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria The molecular mechanisms of how viral infections cause tissue damage and clinical disease are poorly understood and cannot solely be attributed to classical immune effectors. Instead, the involved inflammatory processes are increasingly being appreciated as tightly integrated with metabolic pathways. By taking an integrated approach of virology, mouse immunology, pathology and systems biology, we unveiled a novel link between the antiviral cytokine interferon and redox metabolism. Virus-induced interferon was found to regulate the expression of key antioxidant genes, leading to the intracellular accumulation of reactive oxygen species, the death of hepatocytes and liver pathology. Ongoing research in our laboratory focuses on the molecular interface of metabolism and inflammation in chronic viral infection. We investigate the impact of the multi-cytokine milieu on systemic metabolism, tissue damage and infection-associated cachexia. This systemic perspective aims towards a better pathophysiological understanding of infectious and inflammatory diseases.

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Tissue damage control in immune mediated inflammatory diseases Miguel Soares Inflammation Laboratory, Instituto Gulbenkian de Ciência, Oeiras, Portugal

Damage control refers to actions made towards minimizing the extent of damage associated with a given emergency situation. Depending on context, damage control may refer to emergency procedures dealing with the sinking of a ship or to surgery procedures dealing with severe trauma or even to a company in Marvel Comics, which repairs damaged property arising from conflicts between super heroes and villains. By extension, “tissue” damage control refers to adaptive responses that minimize the extent of tissue damage and dysfunction associated with the pathogenesis of immune mediated inflammatory conditions, including in infectious diseases1,2. Presumably, tissue damage control is regulated by a number of evolutionarily conserved stress- and damage-responses associated with the induction of overlapping profiles of gene expression1. This argues for the existence of a core number of evolutionarily conserved genes regulating tissue damage control1. Moreover, this might explain why overlapping stress- and damage-responses confer protection against apparently unrelated forms of stress and damage, a phenomenon known as hormesis. A subgroup of these evolutionarily conserved genes regulates iron metabolism and control the participation of iron in the production of free radicals leading to oxidative stress and tissue dysfunction. In support of this notion, immune mediated inflammatory diseases are often associated with deregulated iron metabolism and oxidative stress3,4. Here I will discuss how the expression of stress responsive genes controlling iron metabolism exert anti-oxidant effects that confer tissue damage control in different infectious diseases5-7.

References 1 Soares, M.P. et al. (2014) Tissue damage control in disease tolerance. Trends Immunol

35 (10), 483-494 2 Medzhitov, R. et al. (2012) Disease tolerance as a defense strategy. Science 335

(6071), 936-941 3 Gozzelino, R. et al. (2010) Mechanisms of cell protection by heme oxygenase-1. Annu

Rev Pharmacol Toxicol 50, 323-354 4 Soares, M.P. and Weiss, G. (2015) The Iron age of host-microbe interactions. EMBO

Rep 16 (11), 1482-1500 5 Pamplona, A. et al. (2007) Heme oxygenase-1 and carbon monoxide suppress the

pathogenesis of experimental cerebral malaria. Nat Med 13 (6), 703-710 6 Larsen, R. et al. (2010) A central role for free heme in the pathogenesis of severe

sepsis. Sci Transl Med 2 (51), 51ra71 7 Ferreira, A. et al. (2011) Sickle Hemoglobin Confers Tolerance to Plasmodium Infection.

Cell 145 (April 29), 398–409

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Sepsis: the need for tolerance not complacency Luis Ferreira Moita Innate Immunity and Inflammation Laboratory, Instituto Gulbenkian de Ciência, Oeiras, Portugal Sepsis is a life-threatening condition that arises as a systemic inflammatory response syndrome to an infection. Its uncontrolled progression can in frequent cases lead to multiple organ failure, which is still associated with high mortality rates. Modern antibiotics made clear that the infection is only an initiating, and not always necessary, event of this syndrome as many patients with sepsis die despite effective eradication of the inciting pathogen. This observation critically contributed to a paradigm shift that focused the pathogenesis of sepsis on the host and not on the pathogen. However, therapeutic strategies based on the inhibition of proinflammatory critical mediators of sepsis or immunostimulation have so far failed to improve sepsis outcome and, therefore, this condition urgently needs transformative therapeutic ideas and strategies. We argue that the induction of disease tolerance, a defence strategy that minimises the impact of an infection on organ function without directly affecting the pathogen burden, is perhaps the missing but essential element to add to the current components of sepsis care and treatment.

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Structure and genome release of Myoviridae phage with double layered baseplate Pavel Plevka CEITEC Central European Institute of Technology, Brno, Czech Republic

Bacteriophages from the family Myoviridae use double-layered contractile tails to infect bacteria. Contraction of tail sheath enables tail tube to penetrate through bacterial cell wall and serve as channel for transport of phage genome into cytoplasm. However, the mechanisms controlling the tail contraction and genome release of phages with “double layered” baseplates were unknown. We used cryo-electron microscopy to show that binding of Twort-like phage phi812 to Staphylococcus aureus cell wall requires 210° rotation of receptor-binding complexes within its baseplate. The rotation disrupts interaction of the receptor-binding complexes with tail sheath and, hence, triggers its contraction. However, the tail sheath contraction of myoviridae phages is not sufficient to induce genome ejection. We show that end of phi812 dsDNA genome is bound to one protein subunit from a connector complex that also forms interface between phage head and tail. The tail sheath contraction induces conformational changes of tail neck and connector that result in disruption of the DNA binding. The genome penetrates into the neck, but is stopped at a bottleneck before the tail tube. Subsequent structural change of the tail tube induced by its interaction with S. aureus cell is required for the genome release.

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ADAR1 is crucial for discriminating between ‘self’ and ‘non-self’ cellular RNAs Mary O'Connell CEITEC Central European Institute of Technology, Brno, Czech Republic

How a cell can distinguish between self and non-self nucleic acids is critical as failure to discriminate correctly can cause autoimmune disorders. Recent technological developments have revealed that the abundance and diversity of endogenous RNAs in the cell is greater than previously anticipated. Therefore the cell must a rigorous system to prevent autoimmune reactions. Our group were the first to demonstrate that the deamination of adenosine to inosine in dsRNA marks endogenous RNA as ‘self’1. In humans only 0.4% of RNA editing events catalysed by ADARs result in recoding while the majority, which is over 100 million editing events occur within transcripts encoding Alus or SINEs in other organisms. Mutations in ADAR1 have been shown to cause the autoimmune disorder Aicardi Goutiѐres syndrome (AGS) 2. Patients with AGS display heightened levels of type-I interferon (IFN) and IFN stimulated genes (ISGs). We have characterised the editing activity of these ADAR1 mutants identified in the AGS patients and find that they have reduced editing activity that is consistent with clinical observations of the disease. Mice lacking Adar1 also have an immune phenotype with heighten levels of IFN and ISGs1. We have rescued the embryonic lethality of Adar1-/- mice to birth by generating a double homozygous mutant between Adar1 and Mavs (Mitochondrial antiviral-signaling protein); an important protein in the innate immune RLR (RIG-I like receptors) pathway. We propose that ADAR1 plays a major role in the modification and hence the discrimination of endogenous cellular dsRNAs and in the absence of ADAR1, cellular RNAs aberrantly stimulate an innate immune response which leads to autoimmune disease phenotypes. This immune function of ADARs is evolutionary conserved and occurs in Drosophila which has a very different immune system to vertebrates, lacking both IFN and the dsRNA sensor proteins. In addition Drosophila encode the orthologue of ADAR2 not ADAR1 as it has been lost in Diptera. Thus this evolutionary conservation of ADARs role in innate immunity is highly significant. 1. Mannion N. et al. The RNA editing enzyme ADAR1 is a key component of innate immune responses to RNA. Cell Reports 9, 1482-9 (2014). 2. Rice, G.I. et al. Mutations in ADAR1 cause Aicardi-Goutieres syndrome associated with a type I interferon signature. Nat Genet 44, 1243-8 (2012).

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ABSTRACTS SHORT TALKS

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Deficiency of secreted IgM antibodies causes increased plasma IgE levels that promote atherosclerosis Dimitrios Tsiantoulas1,2, Ilze Bot3, Mate Kiss1,2, Barbara Bartolini-Gritti1,2, Laura Göderle1,2, Maria Ozsvar-Kozma1,2, Thomas Perkmann2, Karsten Hartvigsen1,2, Andreas Bergthaler1, Daniel H. Conrad4, Johan Kuiper3, Ziad Mallat5, Hassan Jumaa6, Christoph J. Binder1,2 1CeMM, Research Center for Molecular Medicine, Vienna, 1090, Austria 2Dept. of Laboratory Medicine, Medical University of Vienna, Vienna, 1090, Austria 3Division of Biopharmaceutics, LACDR Leiden University, Leiden, 9502, The Netherlands 4Dept. of Microbiology and Immunology, Virginia Commonwealth University, Virginia, 23284 USA 5Division of Cardiovascular Medicine, University of Cambridge, Cambridge, CB2 OSZ, UK 6Institute of Immunology, University Hospital Ulm, Ulm, 89081, Germany Atherosclerotic cardiovascular disease is the major cause of death worldwide. Deficiency of secreted IgM antibodies (sIgM-/-) in atherosclerosis-prone low density lipoprotein receptor deficient (Ldlr-/-) mice results in accelerated atherosclerosis. However, the underlying mechanisms have not been elucidated. We demonstrate that secreted IgM antibodies dampen antigen induced B cell receptor (BCR) signalling in vitro, which is the major driver of B cell development. In line with this, B cells in sIgM-/- mice display elevated BCR signaling as judged by increased levels of phosphorylated Btk and Syk kinases. Furthermore, sIgM-/- mice exhibit disturbed B cell development, which is characterized by robustly decreased CD23+ B cells. Because CD23, which is the low affinity receptor of IgE, has been shown to facilitate the clearance of IgE immunoglobulins, we quantified the plasma IgE levels in Ldlr-/-sIgM-/- mice. IgE levels were >4 fold increased in the plasma of Ldlr-/-sIgM-/- compared to Ldlr-/- mice fed an atherogenic diet for 16 weeks (744±158 vs 166±38 ng/ml; P<0.0001). To investigate whether the accelerated atherosclerosis is due to increased IgE levels, Ldlr-/-sIgM-/- and Ldlr-/- mice were fed an atherogenic diet for 6 weeks, during which they were treated weekly with a neutralizing anti-IgE Ab that targets free IgE or a control Ab, respectively. While Ldlr-/-sIgM-/- mice treated with the control Ab displayed increased atherosclerosis compared to Ldlr-/- mice, this enhanced lesion formation was fully reversed by anti-IgE treatment (10.6±1.3 vs 7.7±0.7 vs 6.5±0.6 104µm2; P<0.05). Furthermore, Ldlr-/-sIgM-/- mice displayed increased numbers of perivascular neutrophils and activated mast cells, which were also reversed in mice treated with anti-IgE Ab. Our data suggest a previously unsuspected therapeutic option against the proinflammatory effect of secreted IgM deficiency in atherosclerosis, which is mediated due to increased IgE levels. In conclusion, we identified a novel property of secreted IgM that is characterized by their capacity to regulate B cell development and humoral immunity homeostasis through decreasing BCR signaling.

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A20/TNFAIP3: Master regulator of intestinal immune homeostasis Lars Vereecke VIB The transcription factor NF-κB is indispensable for intestinal immune homeostasis, but contributes to chronic inflammation and inflammatory bowel disease (IBD). A20, an inhibitor of both NF-κB and apoptotic signalling, was identified as a susceptibility gene for multiple inflammatory diseases, including IBD. Using tissue specific A20 knockout mice, we dissected the contribution of A20 to maintaining intestinal immune homeostasis. Despite absence of spontaneous intestinal inflammation in intestinal epithelial cell (IEC) specific A20 knockout mice, we found additional myeloid-specific A20 deletion to synergistically drive intestinal pathology through cell-specific mechanisms. A20 ensures intestinal barrier stability by preventing cytokine-induced IEC apoptosis, while A20 prevents excessive cytokine production in myeloid cells. Combining IEC and myeloid A20 deletion induces ileitis and severe colitis, characterized by IEC apoptosis, Paneth and goblet cell loss, epithelial hyperproliferation and intestinal microbiota dysbiosis. Continuous epithelial cell death and regeneration in an inflammatory environment sensitizes cells for neoplastic transformation and the development of colorectal tumours in aged mice. However, myeloid specific A20 knockout mice do not spontaneously develop intestinal pathology and are strongly protected in experimental colitis models. The pro-inflammatory environment in myeloid specific A20 knockout mice induces strong expansion of ‘myeloid derived suppressor cells’ (MDSCs) which suppress both innate and adaptive immunity in a tissue dependent manner. Although A20 defects are associated with multiple human inflammatory and auto-immune pathologies, myeloid-specific A20 deficiency results in protection from colitis. These studies underscore the delicate role of A20 in balancing intestinal immunity.

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Involvement of IFN-beta signaling pathway in Parkinson's disease Patrick Ejlerskov BRIC Only around 5% of all Parkinson’s disease (PD) patients are known to have familial mutations associated with the disease whereas the etiology of the vast majority of patients, termed sporadic (s)PD, is unknown. We have shown that mice lacking the immunoregulatory cytokine interferon-β (IFN-β) or IFN-α/β-receptor (IFNAR) spontaneous development α-synuclein-positive Lewy bodies in the brain, reduction in dopaminergic neurons, motoric disturbances and progressive dementia, all hallmarks of Parkinson’s disease with dementia (PDD). However, within the IFN-β/IFNAR signaling pathway no gene variants are strongly associated with PD, thus we hypothesized that weakly associated mutations in various genes within the same signaling pathway would cause the same pathogenesis. Gene set enrichment analysis (GSEA) of already published microarray data from patients with sPD and sPDD revealed that the Cytokine-cytokine receptor and JAK-STAT signaling pathways, both pathways directly regulated by IFN-β/IFNAR signaling, were among the top 3 pathways associated with sPDD. Target gene meta-analysis of GWAS dataset from sPD indicated that the heterogeneity of sPD was associated with a wide range of sequence variants in several IFN-β/IFNAR associated genes. Differential gene regulation of IFN-β/IFNAR associated genes were confirmed in an independent patient cohort with healthy controls, sPD and sPDD, where a pronounced increase in protein inhibitor of STAT2 (PIAS2) gene was observed in sPDD, suggesting its involvement in disease progression to dementia. In support, blocking IFN-β mediated signaling in SHSY5Y cells affected neuronal development and electrophysiological properties negatively. Collectively, our data show an association between defective IFN-β/IFNAR signaling and dementia in sPD, thus targeting this pathway could hold a therapeutic potential.

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Role of RhoA in T-cell adhesion and migration in an experimental model of multiple sclerosis Alba Manresa BRIC Aim: Using the mouse model Experimental Autoimmune Encephalomyelitis (EAE), we investigate whether specifically knocking out the small GTPase RhoA in T-cells has an impact on CNS inflammation and the subsequent degeneration and disease severity. Our primary objective is to clarify the role of RhoA in the inflammatory reactions in peripheral and central tissues in EAE, the mouse model for multiple sclerosis (MS). / Methods: EAE is induced by subcutaneous injection of an emulsion of MOG35-55 or vehicle in Complete Freud’s Adjuvant. The study is conducted on female mice lacking RhoA expression in T-cells. Mice are evaluated daily using a score ranging from 0 to 5 where 0 corresponds to symptom-free and 5 to moribund or dead. Mice are terminated when they reach clinical score below or equal to 4, and blood and tissue samples are taken for flow cytometry and histological analysis. / Results: Our results show that mice knock-out for RhoA show no disease symptoms or a milder form of the disease with a much later onset compared to their wild-type littermates. Mice heterozygous for the deletion of RhoA in T-cells present a disease pattern similar to their wild-type littermates, but with decreased disease severity. / Conclusions: Our preliminary findings suggest that the expression of RhoA plays an important role in the onset and progression of EAE, which might be due to its role in the leukocyte adhesion and migration into the CNS, one of the key steps in the development of EAE and MS.

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Spatial T cell dysfunction between tumor and draining lymph nodes Núñez NG1, de la Rochere P1, Nadan AT2, Viel S1, Niborski L1, Loirat D1, Milder M1, Meseure D2, Amzallag N1, Sastre-Garau X2, Sedlik C1, Amigorena S1, Piaggio E1*. 1INSERM U932; 2:Département de Biologie des Tumeurs. Institut Curie, Paris, France In human breast cancer, invasion by tumor cells of the draining lymph nodes (LNs) contributes to disease progression and has predictive value. We compared the immune profile of Non-invaded (NI), Invaded (I) LNs, and Tumor (T) samples from untreated luminal breast cancer. Flow cytometry analysis indicated that compared to NI LNs, in T and I LNs there were significant higher frequencies of CD8+ T cells and regulatory T (Treg) cells with conserved in vitro suppressive function. Also, in T and I LNs the frequency of naïve CD8+, CD4+ Tconv and Treg cells were significantly lower, compensated by an increase in the effector memory compartment. In the T and I LNs there was a significant increase of all CD4+ T cell subpopulations expressing ICOS, GITR, OX-40 and PD-1. Of note, CD8+ T cells in T expressed higher frequencies of the inhibitory checkpoints CTLA-4, KLRG-1 and 2B4, and in I LNs there was a significantly increased frequency of PD1+ CD8+ T cells compared to NI LNs. To understand whether T and I TDLNs represent sites of immune activation or tolerization, we ex-vivo stimulated T cells using total NI, I LNs and T. CD4+ and CD8+ T cells from T showed the lowest proliferation index. Also, higher levels of IFN-γ, IL-6 and IL-17F were detected in I compared to NI LNs and T; and no differences were observed for IL-17A, IL-13 and IL-10. Interestingly, T supernatant showed the highest concentration of TGF-b. Overall, these data suggest that although T cells in the T seem dysfunctional, LN invasion by tumor cells seems to locally prime T cells, inducing an accumulation of more cytotoxic CD8+ T cells and an increased production of pro-inflammatory CKs, all critical parameters characteristic of an effective anti-tumor immune response.

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"Infectious" tolerance transforms tumor antigen specific naive CD4 T cells into induced Tregs in a spontaneous lung tumor model Ruby Alonso Ramirez Institut Curie During tumor development, the immune system is facing with persistent exposure to tumor-associated antigens, frequently in a non-inflammatory context, favoring the establishment of tolerance. Passive (ignorance, anergy or deletion of tumor specific T cells) or active mechanism mediated by regulatory T cells (Tregs) may be involved in tolerance. CD4 T cells are the main source of Tregs but also display indirect or direct anti-tumor activity. Because of the long life span of immune-dominant MHC-II/peptide complexes, transplantable tumor systems are not suitable to study the relationship between CD4 T cells and tumors. / In this work, we studied a genetically induced ("spontaneous") tumor model of lung adenocarcinoma expressing a MHC-II restricted cytoplasmic antigen, luciferase fused to HY:DBY. We showed that the tumor antigen reaches the lymph node and activates naive CD4 T cells (from Marilyn TCR transgenic RAG KO mice, HY:DBY specific T cells) to proliferate and recirculate. Transcriptome analysis of tumor specific CD4 T cells showed an enrichment of Treg specific genes starting at the early stages of tumor development and lasting afterward. We formally demonstrated de novo induction of tumor antigen specific Treg from uncommitted naïve CD4 T cells. Finally, depletion of the host Treg compartment before the transfer of tumor specific naive CD4 T cells impaired the conversion of the latters into iTreg and favored their activation into full-blown effector cells able to migrate to the tumor sites. Thus, host Tregs confer infectious tolerance to naive tumor specific CD4 T cells arriving into the tumor draining lymph node reinforcing tumor immune suppression.

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Improved T cell engineering using viral and transposon-based vectors encoding optimized TCR genes and redirected miRNAs Mario Bunse MDC Engineered T cells for adoptive T cell therapy (ATT) are generated by transgenic expression of a TCR or chimeric antigen receptor. However, expression of inhibitory receptors or endogenous TCR chains can compromise the functionality and specificity of the therapeutic T cells. To further complement genetic engineering of T cells, we developed viral and transposon-based vectors expressing redirected miRNAs in addition to the antigen receptor. Employing miRNAs targeting endogenous TCRs improved surface expression of transgenic TCRs and reduced potentially self-reactive mixed TCR dimers composed of endogenous and transgenic TCR chains. Importantly, applying a viral RNAi-TCR replacement vector in ATT prevented lethal mixed TCR dimer-dependent autoimmunity in mice. To generate human engineered T cells, we developed a Sleeping Beauty (SB) transposon vector encoding a codon-optimized MAGE-A1-specific TCR that was isolated from a transgenic mouse model. Using the hyperactive SB100X transposase, we achieved stable TCR expression in 45-50% of the T cells without selection. Nonetheless, almost all cells still expressed endogenous TCRs on their surface. Both TCR silencing by miRNAs and usage of TCR constant regions harboring mouse-derived sequences and two additional cysteines reduced endogenous TCR expression. Best results were achieved by combining these strategies. As further miRNA targets, we chose inhibitory receptors characteristic for exhausted T cells that may limit the functionality of tumor-infiltrating lymphocytes (TILs). In a mouse model, in which TILs isolated after cancer relapse expressed PD-1, LAG-3 and TIM-3, we currently analyze ATT using TCR-engineered T cells expressing miRNAs targeting inhibitory receptors to prevent outgrowth of therapy-induced tumor variants.

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Identifying Novel Treatment Strategies for AML through Stratified Biologically Relevant Drug Responses Ella Karjalainen FiMM Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by / impaired differentiation and uncontrollable accumulation of immature myeloid / progenitor cells in the bone marrow. Due to the heterogeneous nature of AML, relapse / rates are high and overall survival poor. Our approach is to stratify AML cases based / on biologically and therapeutically relevant drug responses: cell viability, cell / differentiation and immune signaling. The stratified drug responses will be integrated / with the genetic and transcriptomic background of the AML patients in the hopes that / our findings could be translated into clinically relevant biomarkers and personalized / treatment options for AML patients.

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Precision medicine strategies for high-risk multiple myeloma to address variability in drug response and predicting treatment outcome Muntasir Mamun Majumder1, Raija Silvennoinen2,3, Pekka Anttila3, David Tamborero4, Samuli Eldfors1, Bhagwan Yadav1, Riikka Karjalainen1, Heikki Kuusanmäki1, Juha Lievonen3, Alun Parsons1, Minna Suvela1, Esa Jantunen2, Kimmo Porkka3,5, Caroline A. Heckman1 1Institute for Molecular Medicine Finland FIMM, University of Helsinki, Helsinki Finland 2Department of Medicine, Kuopio University Hospital, Kuopio, Finland 3Department of Hematology, Helsinki University Central Hospital Cancer Center, Helsinki, Finland 4Research Unit on Biomedical Informatics, Dept. of Experimental and Health Sciences, University Pompeu Fabra, Barcelona, Spain; 4Department of Hematology, Helsinki University Central Hospital Cancer Center, Helsinki, Finland; 5Hematology Research Unit, University of Helsinki, Helsinki, Finland. Disease and patient heterogeneity presents a major challenge for predicting treatment response and outcome for patient with multiple myeloma (MM). Despite new drugs and increased survival of many patients, high-risk multiple myeloma (HRMM) patients are challenging to treat and their outcome remains poor. To address variability in response and design informed treatment selection for HRMM patients we assessed ex vivo sensitivity of 50 MM patient samples to 308 approved and investigational drugs. With the results we i) classified patients based on their ex vivo drug response profile; ii) identified and matched potential drug candidates to specific cytogenetic markers or disease state; iii) determined if ex vivo drug sensitivity stratification can predict patient outcome, and iv) assessed the predictive value of drug sensitivity testing to guide treatment decision for RRMM patients. Based on their drug sensitivity profiles, MM patients can be stratified into four distinct subgroups with varied outcomes. Although cells from del(17p) patients exhibited resistant profiles, HDAC and BCL2 inhibitors were effective for this HR group. Cells from t(4;14) patients, however, were exceptionally sensitive to immunomodulatory drugs, proteasome inhibitors and several other targeted drugs. Three patients treated based on their drug profile showed good response to the selected treatments. Our results demonstrate that a precision medicine approach by ex vivo drug testing may be effectively applied to potentially achieve therapeutic benefit for MM in a clinical setting.

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Homeostatically driven type-2 II pathways shape the lung pulmonary immune environment. Simona Saluzzo CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria The lungs provide oxygen for the entire body from birth throughout life and accommodate a complex immunological milieu. Here, we investigated the homeostatic role of IL-33 in newborns and discovered the immediate upregulation of IL-33 on the first day of life which was followed by a wave of IL-13 producing type-2 innate lymphoid cells (ILC2s). This expansion of activated ILC2s coincided with the appearance of alveolar macrophages and their early polarization to an IL-13-dependent M2 phenotype. ILC2s continued to maintain alveolar macrophages in an M2 phenotype during adult life at the cost of an enhanced susceptibility to Streptococcus pneumoniae infection. ILC2s specifically contributed to lung quiescence in homeostasis by polarizing both tissue resident alveolar macrophages and transplanted macrophage progenitors towards an M2 phenotype. These data enhance our understanding of the neonatal, post-natal and adult lung immune environment, highlight the homeostatic role of ILC2s in setting the activation threshold in the lung, and underline their implications in anti-bacterial defenses.

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Hepatic embryo-derived Kupffer cells regulate the onset of immune response against Listeria monocytogenes infection and co-exist with two other monocyte-derived macrophage populations Chloé Abels1,2, David Torres3, Arnaud Kohler3, Charlotte Scott4, Martin Guilliams4, Jo A. Van Ginderachter1,2, Patrick De Baetselier1,2, Veronique Flamand3, Alain Beschin1,2 1. Cellular and Molecular Immunology Unit, Vrije Universiteit Brussel (VUB), B-1050 Brussels, Belgium 2. Myeloid Cell Immunology Laboratory, VIB Inflammation Research Center, 9052 Ghent, Belgium 3. Institute of medical immunology, Université libre de Bruxelles (ULB), rue des Professeurs Jeener et Brachet 12, 6041 Gosselies 4. VIB Inflammation Research Center, UGent, 9052 Ghent Listeriosis is a foodborne disease caused by the ingestion of the intracellular bacterium Listeria monocytogenes affecting mainly the liver and the brain in immunocompromised persons. Severe disease also occurs in pregnant women, resulting in abortion. In experimental listeriosis, contradictory results were reported on the role of liver resident MF (Kupffer Cells, KC) due to the inability to correctly distinguish resident embryonic KC from infiltrating monocyte-derived macrophages. In this context, we have identified a Clec4f marker that is specifically expressed in KC and used this marker to generate a mouse model of selective diphtheria toxin-mediated depletion of tissue-resident KC. Using these mice, we found a protective role of KC during L. monocytogenes infection. KC-depleted mice became highly susceptible to infection, with significantly reduced survival rates and increased bacterial loads in the liver and spleen. The inability of KC-depleted mice to control the infection was associated with an altered innate myeloid cell response and inadequate adaptive type 1 immune response in the liver and the spleen. In the absence of KC, T and B cells numbers were reduced, and antigen-specific CD8 T cells were impaired in their IFN-g synthesis capacity, coinciding with a drastic reduction in DCs, in part attributed to a blockage in DC precursor. These results indicate an important interplay between liver-resident KC and other non-parenchymal hepatic antigen-presenting cells. By addressing the fate of hepatic MFs in infected liver-shielded CD45.1-->CD45.2 chimera, we evidence the existence of 3 MF populations, bone marrow derived CD45.1+ Clec4f- Tim4- moMFs and Clec4f+ Tim4- moKCs, and CD54.2+ Clec4f+ Tim4+ embryonic KCs. Whether these populations fulfil different functions is under investigation.

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Mucosal administration of the B subunit of Escherichia coli heat-labile enterotoxin promotes tolerance by enhancing regulatory T cells and tolerogenic dendritic cells Alexandre Bignon Babraham The mucosal immune system is a complicated and tightly regulated system of suppression and controlled inflammation. Although the exact mechanisms governing this regulation remain to be elucidated, evidence exists for the involvement of different cell types, including CD4+ regulatory T cells (Treg) and tolerogenic dendritic cells (DCs). The Escherichia coli heat-labile enterotoxin is a hetero-oligomeric AB5 toxin composed of a toxic enzymatic A subunit and five identical non-toxic B subunits (EtxB). Mucosal administration of EtxB to mice, protects type 1 diabetes or collagen-induced arthritis. Amelioration of these autoimmune phenotypes by EtxB could be recapitulated by the transfer of splenocytes from EtxB treated animals, interestingly when these splenocytes were devoid of CD4+ T cells they could not mediate tolerance, suggesting a role for EtxB modulating suppressive Treg cells. In addition, one study has shown that in vitro EtxB treatment increases viability in DCs and gives them a lower activation status based on the reduced expression of MHC-II, CD80 and CD86 suggesting a immature phenotype. These studies suggest that EtxB modulates tolerance although the mechanisms of mucosal tolerance induction by EtxB remain to be established. In order to determine the mechanism by which EtxB induces tolerogenic environment at mucosal sites, the effects of EtxB treatment on both cellular actors important in the establishment of mucosal tolerance, Treg cells and DCs, have been investigated in vivo and in vitro. Our results show that intra-nasal administration of EtxB increased the proportion of activated and suppressive Treg cells and tolerogenic DCs in the lung. Interestingly, in vitro experiments show that EtxB promotes functional tolerogenic bone marrow derived DCs that are unable to activate naïve CD4+ T cells.

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ABSTRACTS POSTERS

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A cellular screen identifies two FDA-approved drugs, ponatinib and pazopanib, as inhibitors of necroptosis Astrid Fauster CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria Necroptosis is a pro-inflammatory form of programmed necrotic cell death mediated by receptor interacting serine/threonine kinase (RIPK)3 and its substrate mixed-lineage kinase domain-like protein (MLKL). Necroptotic cell death has been shown to contribute to the pathophysiology of several disorders, including myocardial infarction, stroke and atherosclerosis. However, no inhibitors of necroptosis are currently available for clinical use. / Here, we performed a phenotypic screen for inhibitors of TNF-induced necroptosis in FADD-deficient Jurkat cells using a representative panel of FDA-approved drugs. We identified the two anti-cancer agents ponatinib and pazopanib as potent and specific inhibitors of necroptosis. To unravel the precise molecular mode of action of the identified drugs, we applied both an unbiased chemical proteomics approach, as well as a series of more targeted, biochemical assays for target deconvolution. We thus validated ponatinib as the first necroptosis inhibitor targeting both RIPK1 and RIPK3, whereas our data point to RIPK1 as the main functional target mediating the protective effect of pazopanib. In order to obtain a global picture of the protein network underlying necroptotic cell death, we furthermore employed forward genetic screening in a haploid human cell line and mass spectrometry-based pathway mapping. Detailed understanding of the necroptotic signaling network is expected to open further avenues for targeted modulation and therapeutic intervention in clinical settings of pathological necroptosis. In this regard, the identification of necroptosis inhibitors among already approved drugs, combined with the elucidation of their precise molecular mode of action, provides a valuable chemical basis for the development of potent anti-necroptosis agents.

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Neuronal IFN-beta–induced PI3K/Akt-FoxA1 signaling is essential for generation of FoxA1+Treg cells Yawei Liu1, Patrick Ejlerskov1, Louise Munk Rasmussen1 & Shohreh Issazadeh-Navikas1,*

1Neuroinflammation Unit, Biotech Research & Innovation Centre (BRIC), Health Science Faculty, University of Copenhagen, Copenhagen Biocentre, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark *Corresponding author: Shohreh Issazadeh-Navikas, Professor, Neuroinflammation Unit, Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen Biocentre, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark. / E-mail: shohreh.issazadeh@bric.ku.dk Tel +45-353 25649 Neurons reprogram encephalitogenic T(enc) cells to become regulatory T(reg) cells either FoxP3+Tregs or FoxA1+Tregs. We reported previously that neuronal ability to generate FoxA1+Tregs was central to preventing neuroinflammation in experimental autoimmune encephalomyelitis. Mice lacking the cytokine interferon (IFN)β were defective in generating FoxA1+Tregs and hence developed chronic neuroinflammation. Neuron-induced FoxA1+Tregs were capable of preventing chronic and demyelinating EAE in mice lacking IFNβ. Here we show that lack of neuronal-IFNβ prevented their ability to reprogram Tenc cells to FoxA1+Tregs. We identified that IFNβ activated neuronal PI3K/Akt signaling. Phosphorylated Akt consequently bound to transcription factor FoxA1, which then translocated to the nucleus and induced neuronal program death-ligand1 (PDL1) expression. Conversely, inhibition of PI3K/Akt, or FoxA1 and PDL1 knock-down blocked neuronal ability to generate FoxA1+Tregs. Our study identified crucial molecular players central for neuronal ability to reprogram pathogenic T-cells and to generate FoxA1+Tregs, which could be a therapeutic target to prevent neuroinflammation.

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Dissecting the molecular and cellular mechanisms responsible for tissue repair in inflammatory bowel disease Shiro Yui BRIC Ulcerative colitis is a life-long inflammatory disease in the large bowel, where affected individuals suffer from reduced quality of life associated with a significant socio-economical burden. Patients are broadly treated by palliative immune-suppressive therapy. In order to establish curative therapies, we need to understand the pathophysiology of the disease. First and foremost, we need to obtain insight into the normal wound healing process, which is poorly understood. The hippo pathway via YAP/TAZ has been implicated in the process of tissue repair although mechanistic insight into the pathway remains unexplored. By adapting DSS injury model, we have developed methods to isolate the regenerating intestinal epithelium during early stages of wound repair. Transcriptional profiling of the repairing epithelium reveals a strong inflammatory profile, but also a significant enrichment of genes normally associated with active YAP/TAZ signaling. Interestingly, the repairing epithelium adopts a fetal-like state during this regeneration process associated with the expression of specialized matrix molecules. In vitro these matrix molecules are capable of inducing similar transitions in droplet culture systems demonstrating that the matrix composition during wound repair is a driver in cellular reprogramming. Importantly, these changes are reversible both in vitro and in vivo but only during a limited time window, as the change in cellular identity is associated with compromised stem cell self-renewal. / These results provide new insight into the process of intestinal wound repair and how compromised stem cell self-renewal in the repairing epithelium constitutes a major problem as epithelial cells over time lose their ability to revert into the adult homeostatic state.

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The pitfalls of dissecting host genetic versus environmental impacts on gut microbiota composition and colitis susceptibility Michail Mamantopoulos1,2, Filip Van Hauwermeiren1,2, Liesbet Martens1,2, Yvan Saeys1,2, Mohamed Lamkanfi1,2 and Andy Wullaert1,2 1Inflammation Research Center, VIB, Ghent, Belgium 2Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium Inflammatory Bowel Diseases (IBD) are chronic intestinal disorders thought to arise at least partially from exacerbated immune responses to the gut luminal microbiota. Genome-wide association studies have identified a plethora of host genetic susceptibility loci for the development of IBD. On the other hand, IBD patients often display an altered phylogenetic composition of the gut microbiota, a condition termed dysbiosis. These observations suggested that host genetic impacts on the gut microbiota composition may contribute to IBD susceptibility. Several genetic mouse models support this hypothesis, as they show that particular host genetic defects provoke dysbiosis and as such elicit more inflammation in colitis models. For instance, mice lacking the pattern recognition receptor Nlrp6 were shown to be more sensitive to Dextran Sodium Sulphate (DSS)-induced colitis owing to a disturbance of the gut microbiota composition. However, interpretation of such intestinal host-microbiota interactions is complicated by the fact that besides host genetics also housing conditions and maternal inheritance of microbiota impacts on DSS colitis susceptibility. We therefore aimed to thoroughly dissect genetic and environmental contributions to colitis susceptibility in Nlrp6-/- mice. For this purpose, we designed an experimental setup subjecting Nlrp6-/- and WT mice from distinct breeding schemes and housing conditions to gut microbiome sequencing and DSS-induced colitis in order to delineate the contributions of host genetics, housing conditions, maternal inheritance or gut microbiota composition in DSS-susceptibility of Nlrp6-/- mice. The results of these experiments evaluating Nlrp6 effects on the gut microbiota will be presented on the meeting.

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The transcription factor BACH2 promotes tumor immunosuppression. Francis Grant Babraham The immune system has a powerful ability to recognize and kill cancer cells, but its function is often suppressed within tumors, preventing clearance of disease. The transcription factor (TF) BACH2 regulates the differentiation of multiple innate and adaptive cellular lineages, but its role in controlling tumor immunity has not been elucidated. We demonstrate that BACH2 is required to establish immunosuppression within tumors. Tumor growth was markedly impaired in Bach2-deficient mice and coincided with intratumoral activation of both innate and adaptive immunity. However, augmented tumor clearance in the absence of Bach2 was dependent upon the adaptive immune system. Analysis of tumor-infiltrating lymphocytes from Bach2-deficient mice revealed high frequencies of rapidly proliferating effector CD4+ and CD8+ T cells that expressed the inflammatory cytokine IFN-γ. Effector T cell activation coincided with a reduction in the frequency of intratumoral Foxp3+ Tregs. Mechanistically, BACH2 promoted tumor immunosuppression through Treg-mediated inhibition of intratumoral CD8+ T cells and IFN-γ. These findings demonstrate that BACH2 is a key component of the molecular program of tumor immunosuppression and identify therapeutic targets for the reversal of immunosuppression in cancer.

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Characterization of H‐Y specific T cells infiltrating into tumor versus non‐tumor tissue expressing both the H‐Y antigen. Martin Szyska1, Stefanie Herda1, Stefanie Althoff1, Andreas Heimann1, Daniele D ́Abundo1, Dang, Tra My1, Josefine Russ1, Thomas Blankenstein2, Il‐Kang Na1,3

1Experimental and Clinical Research Center (ECRC), Berlin, Germany 2Max Delbrück Center for Molecular Medicine (MDC), Berlin, Germany 3Department of Hematology, Oncology and Tumorimmunology, Charité, Berlin, Germany Cancer immunotherapy is an emerging field that has shown promising results in recent clinical trials. In adoptive T cell transfer (ATT), infused T cells target tumor-associated antigens resulting in a graft versus tumor (GvT) immune response. However, the lack of known tumor-specific antigens links GvT effects to harmful graft versus host disease (GvHD) and limits the applicability and efficacy of ATT. We have generated a transgenic mouse bearing a dual‐luciferase reporter enabling us to visualize migration (via constitutive Renilla‐luciferase) and activation (via NFAT‐driven Click Beetle-luciferase) of adoptively transferred T cells via in vivo bioluminescence imaging (BLI). We have crossed these mice with TCR transgenic Marilyn (CD4) and MataHari (CD8) mice, whose T cells exclusively recognize the male histocompatibility antigen H-Y. In order to characterize disparate on-target T cell responses against tumor (GvT) versus off‐tumor tissues (GvHD), we analyzed T cell migration and activation in H‐Y+ tumor bearing female versus male mice via bioluminescent imaging. Our BLI demonstrated a rapid T cell migration into H‐Y+ tumors followed by a transient T cell activation (1-2 days) resulting in a tumor size reduction in both sexes. In male recipients, infiltration of T cells into the skin and a longer‐lasting activation could be observed. Ex vivo analyses revealed that tumor-infiltrating T cells expressed low levels of the T cell activation markers CD25 and CD69, but high levels of the T cell inhibitory receptors PD‐1, TIM‐3 and LAG-3. Interestingly, these exhaustion markers could not be observed in those T cells infiltrating the skin. We hypothesize that the tumor microenvironment uniquely influences infiltrating antigen-specific T cells. Further analyses are planned to unravel differences in phenotype and signaling pathways of antigen‐specific T cells in tumor versus non-tumor tissue in order to develop new strategies enabling the dissection of GvT from GvHD.

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Macrophage metabolism controls tumor blood vessel morphogenesis and metastasis Mathias Wenes VIB Hypoxic tumour-associated macrophages (TAMs) acquire angiogenic and immunosuppressive properties and display a specific metabolic fingerprint. Yet, if and how metabolism influences these functions remain unknown. Here, we found that REDD1, a negative regulator of the energy/nutrient sensor mTOR, is synergically upregulated by hypoxia and tumour-derived stimuli. mTOR inhibition by REDD1 sustains the angiogenic capacity of TAMs, partly contributing to aberrant blood vessel formation. Genetic deletion of REDD1 rewires the metabolism of hypoxic macrophages towards enhanced glycolysis leading to NADH build-up and, hence, increased mitochondrial ROS production. ROS signalling in REDD1 knockout macrophages stabilizes NF-κB, promoting an angiostatic phenotype. Accordingly, REDD1-deficiency in TAMs leads to the formation of smoothly aligned, pericyte-covered blood vessels, which prevents hypoxia and metastasis in several tumours. Tuning-down glycolysis in REDD1 knockout TAMs by targeting the glycolytic activator PFKFB3 re-establishes abnormal angiogenesis and metastases. Our data provide a mechanism whereby metabolic switches in hypoxic macrophages restore tissue oxygenation through vascular remodelling.

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BACH2 regulates CD8 + T cell differentiation by controlling access of AP-1 factors to enhancers Mohammed Firas Sadiyah Babraham T cell antigen receptor (TCR) signaling drives distinct responses depending upon the differentiation state and context of CD8 + T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity following viral infection. BACH2 was recruited to enhancers where it limited expression of TCR-driven genes by attenuating the availability of activator protein 1 (AP-1) sites to Jun family signal-dependent TFs. In naïve cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs.

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Autoinducer-2 influences the streptomycin-treated gut microbiota Jessica Thompson1, Ana Rita Oliveira1, Carles Ubeda2 and Karina Xavier1 1Instituto Gulbenkian de Ciência, Oeiras, Portugal; 2Departamento de Genómica y Salud, Centro Superior de Investigación en Salud Pública, Valencia, Spain jthompson@igc.gulbenkian.pt The mammalian gut is home to a complex, densely populated bacterial community, which is intimately linked to the balance between host health and disease. Whilst the microbiota provides immense benefits for the host: influencing digestion, immune development and resistance to pathogens; community imbalances (dysbiosis) are linked with increased susceptibility to many infectious and non-infectious diseases. Despite the huge potential to be gained from understanding the mechanisms driving maintenance, disruption and restoration of this community, they remain largely uncharacterised. Bacteria are known to communicate and regulate group behaviours within and between species through small signalling molecules in a density-dependent manner. One molecule, Autoinducer-2 (AI-2), is produced by many different species including some which inhabit the gut, and has been shown to mediate interspecies communication in vitro. We investigated whether AI-2 plays a role in shaping the gut microbiota by constructing mutant strains affected in the synthesis, uptake or degradation of AI-2 in the gut commensal, Escherichia coli. Using these strains, AI-2 could be accumulated in or depleted from the gut of gnotobiotic mice. These mutants were then used to manipulate AI-2 levels in the gut of streptomycin-treated mice and the effects of both antibiotic treatment and signal manipulation upon microbiota composition assessed. Streptomycin induced a dysbiosis characterised by changes in bacterial abundance, decreased diversity and a shift from roughly equal proportions of bacteria from the Bacteroidetes and Firmicutes phyla to a dominance of the community by the Bacteroidetes (>90%). Increased AI-2 availability changed the overall structure of this antibiotic-treated community, leading to a small but significant increase in the ratio of Firmicutes to Bacteroidetes. These data show that AI-2 signalling influences the composition of the gut microbiota; we are now investigating whether these changes can modulate the associated metabolome and community recovery antibiotic insult or protection from infection.

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Adaptive immunity increases the pace and predictability of evolutionary change in commensal gut bacteria João Barroso-Batista, Jocelyne Demengeot, Isabel Gordo. Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal João Batista <jbatista@igc.gulbenkian.pt> Co-evolution between the mammalian immune system and the gut microbiota is believed to have shaped the microbiota?s astonishing diversity. Here we test the corollary hypothesis that the adaptive immune system, directly or indirectly, influences the evolution of commensal species. We compare the evolution of Escherichia coli upon colonization of the gut of wild-type and Rag2-/- mice, which lack lymphocytes. We show that bacterial adaptation is slower in immune-compromised animals, a phenomenon explained by differences in the action of natural selection within each host. Emerging mutations exhibit strong beneficial effects in healthy hosts but substantial antagonistic pleiotropy in immune-deficient mice. This feature is due to changes in the composition of the gut microbiota, which differs according to the immune status of the host. Our results indicate that the adaptive immune system influences the tempo and predictability of E. coli adaptation to the mouse gut.