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the 30th Annual Conference of the 21-23 September 2016 Amsterdam, The Netherlands www.emds2016-amsterdam.nl Dendritic cells and macrophages: Distant relatives or just close neighbours? Photo Source: Sriram Subramaniam National Cancer Institute (NCI). Welcome in Amsterdam “To be or not to be a macrophage or a dendritic cell…is that the question?” We wish everybody fruitful discussions! 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 2 Contents Welcoming address 4 Local Organizing Scientific Committee and Conference Agency 5 The European Macrophage and Dendritic Cell Society (EMDS) 6 Sponsors 7 General Information 8 Conference Venue 9 Party information 10 Invited Speakers 12 Scientific Programme 13 Abstracts of oral presentations 17 Abstracts of poster presentations 33 Participants Contact List 82 Schedule of Poster Walks 87 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 3 A word of welcome from the conference chairs Dear colleagues and friends, On behalf of the Organizing and Scientific Committee it is our great pleasure to welcome you to the 30 th Annual Meeting of the European Macrophage and Dendritic Cell Society (EMDS). We are delighted that you decided to join us in Amsterdam. Amsterdam is a great small metropole. The city has a rich historic culture, many museums, wide gastronomic diversity, good transportation and the nightlife of a big city. It is however small enough to see most things by foot, or by bike, which is the most popular form of transportation in the Netherlands. Going around by boat is a great way to see the city from the perspective of its extensive canals. Do not miss any of Amsterdam’s famous museums, such as the Rijksmuseum, - the home of the Nightwatch by Rembrandt -, the Van Gogh Museum or the Anne Frank House, which is located close to the Rode Hoed, where the EMDS is organized. Additionally, Amsterdam is a hotspot of science in the Netherlands. Three top institutes – the VU University Medical Center/Cancer Center Amsterdam, Academic Medical Center, University of Amsterdam and Sanquin Research – have joined forces to organise this exciting 30th edition of the annual EMDS meeting. Basic aspects, such as control of APC function, metabolism, signalling, heterogeneity, antigen processing & presentation, and microbe-host crosstalk will be discussed. Additionally, many clinical aspects of APC dysfunction in chronic inflammatory disorders and cancer will be addressed as well as the possibility to target dendritic cells and/or macrophages in therapeutic strategies. We are honoured by the 13 eminent scientists who have accepted the invitation to present their latest scientific insights during this meeting, and feel privileged that 115 abstracts have been submitted. Over 200 participants from 13 countries, including Australia and China, will participate in the meeting, many of which will share their data in either an oral presentation or during the poster walks. There is ample time to interact and exchange views during the coffee breaks, lunches and the congress party. After all, ‘To be a macrophage or a dendritic cell…’ Does it really matter? We wish you an enjoyable, satisfying, interesting scientific and socially interactive meeting. On behalf of the local Organizing and Scientific Committee, Marjolein van Egmond Yvett e van Kooyk 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 4 Local Organizing and Scientific Committee Anja ten Brinke Sanquin Marjolein van Egmond VUmc-CCA Tanja de Gruijl VUmc-CCA Joke den Haan VUmc-CCA Marieke van Ham UvA, Sanquin Esther de Jong AMC, UvA Yvette van Kooyk VUmc-CCA Arjan van de Loosdrecht VUmc-CCA Sandra van Vliet VUmc-CCA Conference Agency Congres Office De Vlinder, Nieuw Bergen, The Netherlands [email protected] 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 5 The European Macrophage and Dendritic Cell Society (EMDS) The European Macrophage and Dendritic Cell Society (EMDS) has emerged from the activities of the former European Macrophage Study Group (EMSG), a loose association of scientists interested in basic and clinical aspects of monocytes, macrophages, dendritic cells and other myleoid cells in man and experimental animal models. The group was constituted in 1992 as a result of a successful series of annual conferences called THE MACROPHAGE. The annual macrophage conference originated from meetings in the Upper Rhine area organized by scientists from the universities and research centers of Strasbourg, Freiburg and Basel, but rapidly grew to a European format. On April 28, 1999, the European Macrophage Society (EMS) was founded in Regensburg. At the end of year 2000, the members of the EMS decided to rename the society as European Macrophage and Dendritic Cell Society (EMDS) in order to better emphasize the two main streams of research within the Society. The EMDS currently has 485 members from 34 countries. For more information and registration as a member, please see our website: www.macrophage.de Council of the EMDS Advisory Board of the EMDS President Guenter Weiss, Innsbruck, Austria Reinhard Andreesen, Regensburg, Germany Robert HJ Beelen, Amsterdam, The Netherlands Christian Bogdan, Erlangen, Germany Patrick De Baetselier, Brussels, Belgium Antonio Celada, Barcelona, Spain Thomas Decker, Vienna, Austria Jean-Claude Drapier, Gif-sur-Yvette, France Hans D. Flad, Freiburg, Germany Regine Landmann, Basel, Switzerland Michael Rehli, Regensburg, Germany Silvano Sozzani, Brescia, Italy Andreas Spittler, Wien, Austria Alexander Steinkasserer, Erlangen, Germany Jo Van Ginderachter, Brussels, Belgium Loems Ziegler-Heitbrock, Gauting, Germany Officers Amaya Puig Kröger, Madrid, Geert Raes, Brussels, Belgium Maciek Siedlar, Krakow, Poland Treasurer Ulrike Schleicher, Erlangen, Germany Secretary Luigi Varesio, Genova, Italy 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 6 Thanks to our sponsors Platinum Gold Bronze Sponsors 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 7 General Information Registration desk The registration desk is located on the ground level of the ‘Rode Hoed’. Opening hours: Wednesday, September 21st: 08:00 am-17:30 pm Thursday, September 22nd: 8:00 am- 17:30 pm Friday, September 23rd: 8:00 am- 13:30 pm Oral presentations Exactly 15 minutes, including discussion, are allocated for each oral presentation. Please stay within the allocation time. Oral presenters are requested to upload their presentation 20 minutes before the start of their session on the computer of the Rode Hoed, which is present on the first balcony. There is no possibility of using your own computer during the presentation. Poster presentations The posters session will take place at the 2 balconies of the Rode Hoed. Please set up your poster before the start of the first session of the day of your poster presentation, and remove the poster at the end of the day. Poster boards are 2 meters high and 1 meter wide, and will be numbered. Material to attach the poster to the poster boards will be available. Posters can be viewed during the coffee breaks and lunch. Additionally, please be on time for the scheduled poster walk in which you have the opportunity to present your poster in 3 minutes, after which it can be discussed. The poster walk are scheduled: Wednesday, September 21st: 11:30-12.30 pm Thursday, September 22nd: 11:00-12:00 am Coffee breaks and lunch Coffee/ tea and lunch will be served on the ground floor and the first balcony. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 8 Conference Venue De Rode Hoed Keizersgracht 102, 1015 CV Amsterdam Central Station Congress venue Rode Hoed WIFI network: derodehoed Password: rodehoed1987 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 9 Party information Conference Party with EMDS and Society of Leukocyte Biology Travel Awards and Walking Dinner at the “IJ-kantine” As local organizing committee we have selected a program for you by which you can experience the different aspects of Amsterdam, from the historical canals to the trendy and industrial northern shore of the IJ. At 17.45 hrs. at the dock in front of the Rode Hoed (the conference venue) a canal boat will pick us up. After a cruise through the canals of Amsterdam, we will cross the IJ were we will depart the boat. We will arrive at the IJ-kantine around 1900. IJ-kantine is located at northern shore of the IJ. From the industrial and spacious place of the IJkantine you can enjoy the beautiful view looking out over the harbor of Amsterdam and the water of the IJ. You can feel yourself far away while being close to the city center. At the IJ-kantine a walking dinner will be served, giving you plenty of time for socializing and dancing with the other conference participants. Dinner and the first 3 drinks are included. Together with DJ John Valk we will try to make it a conference party you will not easily forget. The party ends around 2400. Return to the city center of Amsterdam is your own responsibility and will not be offered by the conference organization. From the NDSM dock, situated immediately next to the IJ-kantine, a free ferry departs to the Center of Amsterdam and will take you in about 10 minutes to the dock at the back of the Central station. The ferry is leaving 2 times an hour at the half and whole hour, with the last one leaving exactly at 2400. Address: Restaurant-Café IJ-kantine MT. Ondinaweg 15-17 1033 RE Amsterdam (NDSM-WERF) 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 10 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 11 Invited speakers Joachim Schultze, Limes Institute, University of Bonn, Bonn, Germany Menno de Winther, Amsterdam Medical Center, University of Amsterdam, Netherlands Marc Raaijmakers, Erasmus Medical Center, Rotterdam, Netherlands Edward Pearce Max Planck Institute of Immunobiology and Epgenetics, Freiburg, Germany Mihai Netea, Radboud University Medical Center, Nijmegen, Netherlands Paul Kubes, Snyder Institute for Chronic Diseases, University of Calgary, Canada Marjolein van Egmond, VU University Medical Center, Amsterdam, Netherlands Peter Cresswell, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, USA Esther de Jong, Amsterdam Medical Center, University of Amsterdam, Netherlands Muzlifah Haniffa, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK Sjoerd van der Burg, Leiden University Medical Center, Leiden, Netherlands Kris Thielemans, Free University, Brussels, Belgium Benjamin Marsland, University Hospital of Lausanne, Lausanne, France 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 12 EMDS 2016 Program Wednesday 21 September 8.30 Registration 9.15 Opening by Marjolein van Egmond 9.30- 11.00 Session I: Control of APC function (chairs: Joachim Schultze and Joke den Haan) 9.30- 10.00 Joachim Schultze next? The multi-dimensional model of macrophage activation: What’s 10.00-10.15 Carla Ribeiro Distinct signaling by C-type lectin receptors dictates HIV-1 restriction by E3 ligase TRIM5α in human dendritic cell subsets 10.15-10.30 Gera Goverse Interaction between enteric glia and myeloid cells as critical players in intestinal immune homeostasis 10.30-11.00 Menno de Winther 11.00-11.30 Coffee break and poster viewing 11.30- 12.30 Guided posterwalks 12.00-13.00 EMDS council meeting 12.30-13.30 Lunch and poster viewing 13.30-15.00 Session Epigenetic pathways regulating macrophage function II: Chronic inflammatory disorders (chairs: Marc Raaijmakers and Arjan van de Loosdrecht) 13.30-14.00 Marc Raaijmakers The inflammatory niche in myelodysplastic syndromes 14.00-14.15 Roham Parsa BAFF-secreting neutrophils drive plasma cell responses during emergency granulopoiesis 14.15-14.30 Arjan Boltjes CD141+ conventional dendritic cells (cDC) are enriched at the site of autoimmune inflammation and display a regulatory phenotype. 14.30-14.45 Monika Linke Active mTORC1 signaling induces macrophage granuloma formation and sarcoidosis progression 14.45-15.00 Manon E. Wildenberg Benzimidazole anti-helminthics promote anti-TNF induced regulatory macrophage formation and potentiate therapeutic effect in a mouse model of inflammatory bowel disease 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 13 15.00 Coffee break and poster viewing 15.30-17.15 Session III: Metabolism and Signaling (chairs: Edward Pearce and Sandra van Vliet) 15.30-16.00 Mihai Netea Immunometabolic circuits in innate immune responses 16.00-16.15 Leonard R Pelgrom by dendritic cells Distinct metabolic requirements for T helper 1 and 2 polarization 16.15-16.30 Hweixian Leong Penny Warburg metabolism in tumor-conditioned macrophages promotes metastasis in human pancreatic ductal adenocarcinoma 16.30-16.45 Veronika Grau Alpha-1 antitrypsin inhibits ATP-dependent release of monocytic IL-1 via CD36, phospholipase A2 and nicotinic acetylcholine receptors 16.45-17.15 Edward Pearce activation Metabolic reprogramming during plasmacytoid dendritic cell 17.15 End Thursday 22 September 9.00- 10.30 Session IV: Macrophage function in health and disease (chairs: Paul Kubes and Anja ten Brinke) 9.00- 9.30 Paul Kubes A novel macrophage recruitment paradigm 9.30-9.45 Geert Raes Imaging of myeloid cell behaviour 9.45-10.00 Aoife Kelly Human peripheral blood monocytes and intestinal macrophage populations activate TGF-β via expression of the integrin αvβ8 10.00-10.30 Marjolein van Egmond Enlisting myeloid cells in the fight against cancer 10.30-11.00 Coffee break and poster viewing 11.00- 12.00 Guided posterwalks 12.00-13.00 Lunch and poster viewing 13.00- 14.30 Session V: Antigen processing and presentation (chairs: Peter Cresswell and Marieke van Ham) 13.00- 13.30 Peter Cresswell Mechanistic elements of conventional MHC class I antigen processing and cross-presentation 13.30-13.45 Katrien van der Borght Tissue necrosis is a driver of organ directed autoimmunity through activation of dendritic cells 13.45-14.00 Andrew S MacDonald A central role for Type I IFN in Th2 induction by dendritic cells 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 14 14.00-14.15 Dieke van Dinther melanoma 14.15-14.30 Martijn Verdoes In vivo imaging of tumor-associated macrophages and subcellular compartment characterization in dendritic cells using novel quenched fluorescent cysteine cathepsin probes 14.30-15.00 Coffee break and poster viewing 15.00 – 16.45 Session VI: Development of a macrophage-based vaccination strategy for Functional heterogeneity of APCs (chairs: Muzlifah Haniffa and Yvette van Kooyk) 15.00 – 15.30 Esther de Jong development DCs, neutrophils and T cells: trinity for human Th17 cell 15.30-15.45 Rieneke van de Ven Correlating the ILT4/HLA-G inhibitory pathway to an immune suppressive microenvironment in sentinel lymph nodes draining head and neck squamous cell carcinoma 15.45-16.00 Anouk Zaal The anaphylatoxin C5a regulates the pro-inflammatory potential of human 6-sulfo LacNAc dendritic cells (slanDC) through crosstalk with TLR-induced ERK/p38/CREB signalling 16.00-16.15 Dorine Sichien IRF8 controls survival and function in terminally differentiated conventional and plasmacytoid dendritic cells respectively 16.15 – 16.45 Muzlifah Haniffa health and disease Functional heterogeneity of human mononuclear phagocytes in 16.45-17.45 General Assembly (EMDS members only) 17.45-19.00 Canal cruise 19.00-24.00 Conference Party with EMDS and Society of Leukocyte Biology Travel Awards and Walking Dinner at the “IJkantine” Friday 23 September 9.00- 10.45 Session VII: Clinical applications and tumor immunology (chairs: Kris Thielemans and Tanja de Gruijl) 9.00- 9.30 Sjoerd van der Burg Chemotherapy driven modulation of intratumoral myeloid cells to improve immunotherapy 9.30-9.45 Mario Kuttke Myeloid PTEN deficiency impairs tumor immune surveillance via immune checkpoint inhibition 9.45-10.00 Camilla Salvagno Therapeutic synergy between macrophage blockade and chemotherapy in breast cancer elicits neutrophil-dependent therapy resistance 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 15 10.00-10.15 Rebekka Wehner Accumulation of tolerogenic human 6-sulfo LacNAc+ dendritic cells in renal cell carcinoma is associated with poor prognosis 10.15- 10.45 Kris Thielemans 10.45-11.15 Coffee break 11.15- 12.45 Session Dendritic cell and mRNA based immunotherapy VIII: Host-microbe crosstalk (chairs: Benjamin Marsland and Esther de Jong) 11.15-11.30 Ulrike Schleicher Suppression of arginase 1 by TNF facilitates the production of NO by type 2 NO synthase and is a novel mechanism by which TNF confers protection against Leishmania major parasites in vivo 11.30-11.45 Melissa Newling IgG opsonization of viruses functions as an endogenous suppressor of type I and III IFN-related anti-viral immunity by human myeloid cells through FcγRIIa 11.45-12.00 Joris K. Sprokholt Follicular T helper cell differentiation induced by Dengue virus through RIG-I like receptor and Type I Interferon receptor crosstalk 12.00-12.15 Late breaking abstract Diana Dudziak Human Lympoid tissue dendritic cells are less influenced by their microenvironment their counterparts in non-lymphoid tissues 12.15- 12.45 Benjamin Marsland 12.45 Closing remarks and awarding prizes by Marjolein van Egmond 13.00 End Implications of the microbiota in airway inflammatory responses 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 16 Abstracts Oral presentations Wednesday September 21st Poster number 2101 Carla Ribeiro Distinct signaling by C-type lectin receptors dictates HIV-1 restriction by E3 ligase TRIM5α in human dendritic cell subsets. 1 1 1 1 Carla M. S. Ribeiro , Ramin Sarrami-Forooshani , Laurentia C. Setiawan , Esther M. Zijlstra-Willems , John 1 2 2 1 1 L. van Hamme , Wikky Tigchelaar , Nicole van der Wel , Neeltje A. Kootstra , Sonja I. Gringhuis and 1 Teunis B. H. Geijtenbeek 1 Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, 2 Amsterdam, the Netherlands Department of Cell Biology & Histology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands Mucosal Langerhans cells (LCs) act as barriers against HIV-1 infection. HIV-1 capture by the C-type lectin receptor (CLR) langerin prevents infection of LCs. However, the underlying molecular mechanism of langerin-mediated HIV-1 restriction remains unknown. Here we show that E3 ligase TRIM5α potently + restricts HIV-1 infection of LCs but not of submucosal DC-SIGN dendritic cells (DCs). TRIM5α associates at steady-state with langerin via adaptor protein LSP-1 and mediates the recruitment of autophagic molecule Atg5 to TRIM5α-Atg16L1-HIV-1 capsid complexes upon HIV-1 exposure in LCs. The TRIM5αmediated autophagy-activating scaffolds targets langerin-HIV-1 capsid complexes into autophagic vesicles for degradation in LCs. Notably, HIV-1 infection of DCs leads to disassociation of TRIM5α from the DCSIGN signalosome and abrogates TRIM5α-mediated autophagic degradation. Remarkably, HIV-1 restriction by TRIM5α was thus far considered to be reserved for rhesus TRIM5α in monkeys but our data strongly indicate that human TRIM5α is a cell-specific restriction factor dependent on CLR signalling. Hence, our data strongly suggest that HIV-1 binding to either langerin or DC-SIGN dictates TRIM5α restriction or viral escape, respectively. Funding: Dutch Scientific Organization NWO (VENI 863.13.025) 2102 Gera Goverse Interaction between enteric glia and myeloid cells as critical players in intestinal immune homeostasis. G. Goverse, M. van Winge, M. Stakenborg, E. Labeeuw, M. Hao, P.J. Gomez-Pinilla, P. VandenBerghe, G.E. Boeckxstaens and G. Matteoli KU Leuven, Translational Research Center for Gastrointestinal Disorders (TARGID), Department of Clinical and eperimental medicine, Leuven, Belgium In the gastrointestinal tract a balance between immune activation and tolerance is essential to preserve intestinal homoeostasis. Recently we have demonstrated that stimulation of the enteric nervous system (ENS) has potent anti-inflammatory effects on macrophages (MFs). In the current study, we investigate if also enteric glial cells (EGCs), the major cellular constituent of the ENS, may have immunomodulatory properties. Immunohistochemical analysis revealed that EGCs are in close contact with MFs within all layers of the intestine. Moreover, we found that glial-secreted molecules were able to decrease expression of pro-inflammatory cytokines in MFs in response to LPS stimulation. In addition, typical antiinflammatory MF markers, such as IL-10, MRC-1 and MSR-1, were increased in MFs stimulated with EGC supernatant. Furthermore, analysis of the EGCs phenotype in vivo during inflammation revealed expression of several monocyte-chemoattractant proteins, as well as genes involved in the synthesis of 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 17 the tolerogenic molecule retinoic acid. In conclusion, we showed that EGCs exert immunomodulatory effects on MFs, suggesting that these interactions might be crucial to maintain intestinal immune homeostasis and prevent immune-mediated diseases such as inflammatory bowel disease. Funding: FWO postdoctoral fellow 2108 Roham Parsa BAFF-secreting neutrophils drive plasma cell responses during emergency granulopoiesis. Dr. Roham Parsa , Mr. Harald Lund , Dr. Anna-Maria Georgoudaki , Dr. Xing-mei Zhang, Dr. André Ortlieb Guerreiro-Cacais , Mr. David Grommisch , Mr. Andreas Warnecke , Dr. Andrew L Croxford , Dr. Maja Jagodic , Prof. Burkhard Becher , Dr. Mikael C. I. Karlsson, Prof. Robert A. Harris Prolonged infections or adjuvant usage can trigger emergency granulopoiesis, leading to dysregulation in neutrophil blood counts. However, the impact of emergency granulopoiesis on T and B cell function remains largely unknown. To address this question we used a mouse model of neutropenia and studied immune activation following adjuvant administration. The initial neutropenic state fostered an environment of increased DC activation and T cell-derived IL-17 production. Interestingly, neutropenic LysM-DTA mice exhibited striking emergency granulopoiesis and amplified neutrophil recruitment to the lymph nodes that was dependent on IL-17-induced prostaglandin activity. The recruited neutrophils secreted BAFF that highly accelerated plasma cell generation and antigen-specific antibody production. Reduction of neutrophil functions via G-CSF neutralization significantly diminished plasma cell formation, directly linking emergency granulopoiesis with the humoral immune response. We conclude that neutrophils are capable of directly regulating T cell-dependent B cell responses in the lymph nodes. 2109 Arjan Boltjes CD141+ conventional dendritic cells (cDC) are enriched at the site of autoimmune inflammation and display a regulatory phenotype. 1 1 2 2 1 Arjan Boltjes , Michal Mokry , Bas Castelijns , Menno Creyghton and Femke van Wijk y 1 Laboratory for Translational Immunology, Wilhelmina Children’s Hospital, University Medical Center 2 Utrecht (UMC Utrecht), Utrecht, The Netherlands Hubrecht Institute, Utrecht, The Netherlands Dendritic cells (DC) are crucial in initiating and shaping immune responses. However, so far, little is known about the heterogeneity and functional specialization of human DC subsets, especially in (local) inflammatory conditions. In synovial fluid (SF), derived from inflamed joints of juvenile idiopathic arthritis (JIA) patients, we found a remarkable enrichment of CD141+ cDC. We therefore compared SF CD1c+ cDC, CD141+ cDC, and inflammatory monocytes based on phenotype, transcriptome, epigenome (enhancer activity determined with H3K27ac Chip-sequencing), and function. Phenotypically, SF CD1c+ cDC resembled monocytes, based on monocyte-associated markers, while CD141+ cDC selectively expressed CLEC9A and CD26. RNA-seq confirmed related transcriptional profiles between SF CD1c+ DC and SF monocytes. CD1c+ cDC were clearly superior in inducing T-cell proliferation, while all APC subsets were similarly capable of cross-presentation. Monocytes constitutively produced pro-inflammatory cytokines like IL-6 and TNF, but upon TLR induction also CD1c+ cDC showed a pro-inflammatory profile, which was confirmed by transcriptome and epigenome data. In contrast, CD141+ cDC revealed a more quiescent signature, and functional data with DC-subset co-cultures suggest a regulatory function. In conclusion, while CD1c+ cDC and monocytes at the site of inflammation show a related proinflammatory profile, enriched CD141+ cDC possess regulatory features, which emphasizes the differential programming of these subsets, especially in inflammation. Funding: VIDI grant form the Netherlands Organization for Scientific Research (NWO, ZonMw) 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 18 2110 Monika Linke A Active mTORC1 signaling induces macrophage granuloma formation and sarcoidosis progression. 1 1 1 1 2 1 Monika Linke , Ha Thi Thanh Pham , Karl Katholnig , Thomas Schnöller , Anne Miller , Florian Demel , 1 1 3 3 4 5 Andrea Preitschopf , Boris Kovacic , Birgit Niederreiter , Stephan Blüml , Peter Kuess , Veronika Sexl , 6 1 7 2 8 Mathias Müller , Mario Mikula , Wolfram Weckwerth , Arvand Haschemi , Martin Susani , Markus 1 9 1 Hengstschläger , Michael J Gambello , Thomas Weichhart 1I 2 nstitute of Medical Genetics, Medical University of Vienna, 1090 Vienna, Austria Department of 3 Laboratory Medicine, Medical University of Vienna, 1090 Vienna, Austria Division of Rheumatology, 4 Internal Medicine III, Medical University of Vienna, 1090 Vienna, Austria Department of Radiation 5 Oncology, Medical University of Vienna, 1090 Vienna, Austria Institute of Pharmacology and Toxicology, 6 University of Veterinary Medicine, 1210 Vienna, Austria Biomodels Austria and Institute of Animal 7 Breeding and Genetics, University of Veterinary Medicine, 1210 Vienna, Austria Department of Ecogenomics and Systems Biology, Vienna Metabolomics Center (VIME), University of Vienna, 1090 8 Vienna, Austria Clinical Institute of Pathology, Medical University of Vienna, 1090 Vienna, Austria 9 Department of Human Genetics, Emory University, Atlanta, Georgia 30322, United States Aggregation of hypertrophic macrophages constitutes the basis of all granulomatous diseases such as tuberculosis or sarcoidosis and is decisive for disease pathogenesis. However, intrinsic pathways driving granuloma initiation and maintenance still remain elusive. Here we show that activation of mTORC1 in macrophages by deletion of Tsc2 was sufficient to induce hypertrophy and proliferation resulting in excessive granuloma formation in vivo. Remarkably, Tsc2-deficient macrophages formed mTORC1dependent hypertrophic granulomatous structures in vitro and showed constitutive proliferation mediated by the neo-expression of cyclin-dependent kinase 4 (CDK4). Moreover, mTORC1 promoted metabolic reprogramming via CDK4 towards increased glycolysis, while simultaneously inhibiting NF-κB signaling and apoptosis. Inhibition of mTORC1 rapidly induced apoptosis and completely resolved granulomas in myeloid Tsc2-deficient mice. Notably, in human sarcoidosis, mTORC1 activation, macrophage proliferation, and glycolysis were identified as hallmarks that correlated with clinical disease progression. Collectively, TSC2 maintains macrophage quiescence and prevents mTORC1-dependent granulomatous disease with clinical implications for sarcoidosis. Funding: T.W. is supported by grants from the Austrian Science Fund (FWF) grant FWF-P27701-B20, the Else-Kröner-Fresenius-Stiftung (P2013_A149), and the Herzfelder’sche Familienstiftung. M.L. is supported by the [DOC] Doctoral Fellowship Programme of the Austrian Academy of Sciences. V.S. is funded by FWF SFB F28 and SBF F47; M.Mü. is funded by FWF SFB F28. M.Mi. is supported by the FWF grant P25336-B13. 2111 Manon E. Wildenberg Benzimidazole anti-helminthics promote anti-TNF induced regulatory macrophage formation and potentiate therapeutic effect in a mouse model of inflammatory bowel disease. 1,2 2 3 2 2 Manon E. Wildenberg , Alon D. Levin , Alessandro Ceroni , Zhen Huo , Pim J. Koelink , Theodorus B.M. 2 2 1 3 1 Hakvoort , Felicia M. Bloemendaal , Johannan F. Brandse , Alison Simmons , Geert R.A.M. D’Haens , 3 1,2 Daniel Ebner and Gijs R. van den Brink 1 Tytgat Institute for Intestinal and Liver Research, Academic Medical Center, Amsterdam, The Netherlands Department of Gastroenterology and Hepatology, Academic Medical Center, Amsterdam, The Netherlands 3 Nuffield Department of Medicine, University of Oxford, Oxford, UK Introduction 2 The clinical benefit of anti-TNF in IBD correlates with their capacity to induce immunosuppressive macrophages in vitro and in vivo. The aim of this study was to screen a library of 1600 existing drugs for their capacity to potentiate this induction and thus enhance clinical efficacy of anti-TNF. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 19 Methods: Induction of CD14+CD206+ macrophages was analyzed by flow cytometry. Positive hits were validated in vitro and in the T cell transfer model of colitis. Results: Ninety-eight compounds potentiated anti-TNF mediated macrophage induction, including the commonly used anti-helminthic drug albendazole. Anti-TNF + albendazole combination treated macrophages were phenotypically comparable to macrophages induced by anti-TNF alone (CD14+, CD206+, CD163+), but immunosuppressive capacity was enhanced. In vivo, albendazole + anti-TNF combination therapy increased the presence of regulatory macrophages in the colon. Consequently, combination therapy was clinically superior in the treatment of experimental colitis compared to either compound alone. Conclusions: The anti-helminthic drug albendazole enhances the induction of regulatory macrophages by anti-TNF In vitro and the clinical efficacy in a colitis model. Given the good safety profile of this drug, antiTNF plus albendazole combination therapy may enhance clinical response in IBD. Funding: European Crohn’s and Colitis Organization, Dutch Society for Gastoenterology (NVGE) 2128 Leonard R Pelgrom Distinct metabolic requirements for T helper 1 and 2 polarization by dendritic cells. Leonard R. Pelgrom, Alwin J. van der Ham, Leonie Hussaarts, Bruno Guigas, Maria Yazdanbakhsh & Bart 1 Everts * *Corresponding author 1 Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands Dendritic cells (DCs) play central role in the activation and polarization of T cell responses. We recently found that toll-like receptor signalling promotes a shift to glycolysis to support the anabolic demands of murine DC activation and effective priming of T cell responses. However, the metabolic requirements for polarization of distinct T helper cell (Th) responses by DCs remain poorly defined. Through genome wide expression analysis of human monocyte-derived DCs stimulated with helminth-derived antigens (Schistosoma mansoni derived egg antigen [SEA] and omega-1) or pharmacological compounds (rapamycin) that are known to prime Th2 responses, we identify suppression of genes involved in glycolysis as a key characteristic of Th2-polarizing DCs. Conversely, stimuli that promote Th1 responses enhance glycolysis in DCs. Furthermore, consistent with these observations, when glycolysis is blocked, DCs fail to induce Th1 responses and instead favour Th2 polarization. On the other hand, DCs that have been conditioned with SEA rely on mitochondrial fatty acid oxidation to promote effective differentiation of Th2 cells. These findings suggest that DC-driven polarization of different T cell responses is dependent on the activation of distinct metabolic programs in DCs and highlights that metabolic manipulation of DCs could hold promise as a novel therapeutic approach to control immune-polarization in disease settings. 2129 Hweixian Leong Penny Warburg metabolism in tumor-conditioned macrophages promotes metastasis in human pancreatic ductal adenocarcinoma. 1 1 2 1 1 1 Hweixian Leong Penny , Je Lin Sieow , Giulia Adriani , Wei Hseun Yeap , Peter See Chi Ee , Boris San Luis , 1 3 4 4 4 5 Bernett Lee , Terence Lee , Shi Ya Mak , Ying Swan Ho , Kong Peng Lam , Choon Kiat Ong , Ruby YJ 6 1 1 2,7 1 Huang , Florent Ginhoux , Olaf Rotzschke , Roger D. Kamm , Siew Cheng Wong 1 Singapore Immunology Network (SIgN), Biomedical Sciences Institute, A*STAR, 8A Biomedical Grove, 2 Immunos, Singapore 138648 BioSystems and Micromechanics IRG, Singapore-MIT Alliance for Research 3 and Technology (SMART), 1 CREATE Way, Singapore 138602 Raffles Institution, 1 Raffles Institution Lane, 4 Singapore 575954 Bioprocessing Technology Institute, A*STAR, 20 Biopolis Way, Centros, Singapore 5 138668 NCCS-VARI Translational Research Laboratory, National Cancer Centre, 11 Hospital Drive, 6 Singapore 169610 Centre for Translational Medicine NUS Yong Loo Lin School of Medicine, CSI Singapore, 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 20 7 14 Medical Drive, Level 11, MD6, Singapore 117599 Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA Patients with pancreatic ductal adenocarcinoma (PDAC) face a clinically intractable disease with exceptionally high rates of metastasis. Although Epithelial-to-mesenchymal transition (EMT) is pronounced at inflammatory foci within the tumor, the immunological mechanisms promoting tumor dissemination remain unclear. It is well established that tumors exhibit the Warburg effect, a preferential use of glycolysis for energy production to support rapid growth. We hypothesized that the metabolic pathways utilized by tumor-infiltrating macrophages are altered in PDAC, conferring a pro-metastatic phenotype. Human peripheral blood monocytes were cultured with conditioned media generated from normal pancreatic or PDAC cell lines to obtain steady-state and tumor-associated macrophages (TAMs), respectively. Compared with steady-state macrophages, TAMs augmented vascular network formation, promoted extravasation of tumor cells out of blood vessels, and induced higher levels of EMT. This prometastatic phenotype correlated with a pronounced glycolytic signature. Inhibiting glycolysis in TAMs with a single competitive inhibitor to Hexokinase II (HK2), 2-deoxyglucose (2DG), was sufficient to disrupt this pro-metastatic phenotype, reversing the observed increases in TAM-supported angiogenesis, extravasation, and EMT. Our results indicate a key role for metabolic reprogramming of tumor-infiltrating macrophages in PDAC metastasis, and highlight the therapeutic potential of using pharmacologics to modulate these metabolic pathways. 2130 Veronika Grau Alpha-1 antitrypsin inhibits ATP-dependent release of monocytic IL-1 via CD36, phospholipase A2 and nicotinic acetylcholine receptors. 1 1 1 1 1 1 Robin Siebers , Katrin Richter , Anna Zakrzewicz , Bijan Fink , Sigrid Wilker , Mira Küllmar , Andreas 1 2 1 3 4 Hecker , Nupur Aggarwal , Winfried Padberg , J. Michael McIntosh , Sentot Santoso , Sabina 2,5 1,5 Janciauskiene , Veronika Grau 1 Laboratory of Experimental Surgery, Department of General and Thoracic Surgery, Justus-Liebig2 University, Giessen, Germany Department of Pulmonology, Hannover Medical School, Hannover, 3 4 Germany Department of Biology, University of Utah, Salt Lake City, Utah, USA Institute for Clinical 5 Immunology and Transfusion Medicine , Justus-Liebig-University, Giessen , Germany. Member of the German Centre for Lung Research High systemic levels of IL-1beta, a potent cytokine involved in host defense, cause life-threatening inflammatory diseases. Monocytes/macrophages synthesize a cytoplasmic pro-form of IL-1beta in response to triggers such as lipopolysaccharide. Extracellular ATP originating from the cytoplasm of damaged cells is a typical signal inducing inflammasome-dependent maturation and release of IL-1beta. Ample evidence suggests that the anti-protease alpha-1 antitrypsin (AAT) and IL-1beta regulate each other via largely unknown mechanisms. Here we demonstrate that AAT dose-dependently inhibits ATPinduced IL-1beta release by monocytic U937 cells and primary human monocytes, whereas ATPindependent release is unimpaired. Evidence is provided that AAT signals via long-chain fatty acid receptor CD36 that activates calcium-independent phospholipase A2 and leads to the secretion of a small soluble factor X. Factor X acts as an agonist of nicotinic acetylcholine receptors containing subunit alpha9 that, in turn, efficiently inhibit ion channel functions at ATP receptor P2X7. In conclusion, we suggest a novel triple-membrane-passing signaling pathway, by which AAT controls ATP-induced IL-1beta release from human monocytes. AAT might be a potent medicament for the prevention of trauma-associated systemic inflammatory response syndrome. Funding: LOEWE Program of the State of Hesse (Non-neuronal cholinergic systems), by the German Centre for Lung Research, by the Excellence Cluster Cardio-Pulmonary Systems, and by the German Research Foundation to V.G. (GR 1094/7-1). 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 21 Oral presentations September Thursday 22nd 2144 Geert Raes Imaging of myeloid cell behaviour. 1,2,3 4 5 6,7 Fang Zheng , PhD, Harris Perlman , PhD, Patrick Matthys , PhD, Tony Lahoutte , MD/PhD, Serge 2 3 2,3 2,3 Muyldermans , PhD, Shemin Lu , PhD, Patrick De Baetselier , PhD, Steve Schoonooghe *, PhD, Nick 2,6 2,3 Devoogdt * PhD and Geert Raes *, PhD Department of Biochemistry and Molecular Biology, Health Science Center, Xi'an Jiaotong University, 2 Xi’an, China; Research group of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, 3 Belgium; Laboratory of Myeloid Cell Immunology, VIB Inflammation Research Center, Ghent, Belgium; 4 Division of Rheumatology, Feinberg School of Medicine, Northwestern University, Chicago, IL60611,USA; 5 6 Laboratory of Immunobiology, Rega Institute, Katholieke Universiteit Leuven, Leuven, Belgium; In Vivo 7 Cellular and Molecular Imaging Center, Vrije Universiteit Brussel, Brussels, Belgium; Department of Nuclear Medicine, UZ Brussel, Vrije Universtiteit Brussel, Brussels, Belgium. * S.S, N.D. and G.R. share senior authorship Single-photon emission computed tomography combined with micro-CT (SPECT/µCT) imaging using Nanobodies against complement receptor of the Ig superfamily (CRIg), found on tissue macrophages such as synovial macrophages, has promising potential to visualize joint inflammation in experimental arthritis. Here, we further addressed the specificity and assessed the potential for arthritis monitoring. Methods: 99m Signals obtained with Tc-labelled NbV4m119 Nanobody were compared in joints of wild type (WT) -/versus CRIg mice with collagen-induced arthritis (CIA) or K/BxN serum transfer-induced arthritis (STIA). In addition, SPECT/µCT imaging was used to investigate arthritis development in STIA and in CIA under 99m dexamethasone treatment. Results: Tc-NbV4m119 accumulated in inflamed joints of WT, but not CRIg /mice with CIA and STIA. Development and spontaneous recovery of symptoms in STIA was reflected in 99m initially increased and subsequently reduced joint accumulation of Tc-NbV4m119. Dexamethasone 99m treatment of CIA mice reduced Tc-NbV4m119 accumulation as compared to saline control in most 99m joints except knees. Conclusions: SPECT/µCT imaging with Tc-NbV4m119 allows specific assessment of inflammation in different arthritis models and provides complementary information to clinical scoring for quantitatively and non-invasively monitoring the pathological process and the efficacy of arthritis treatment. 2145 Aoife Kelly Human peripheral blood monocytes and intestinal macrophage populations activate TGF-β via expression of the integrin αvβ8. 1,2,3 1,2,3 1,2,3 Aoife Kelly *, Elinor Shuttleworth *, Tom Fenton , Catherine Smedley 1,2,3 Travis * Both authors contributed equally to this work 1 1,2,3 , Scott 4 Levison , Mark 2 Manchester Collaborative Centre for Inflammation Research, Wellcome Trust Centre for Cell-Matrix 3 4 Research, Manchester Immunology Group, University of Manchester, UK. Central Manchester University Hospital NHS Foundation Trust, Manchester, UK. Transforming growth factor beta (TGF-β) plays a key role in maintaining intestinal homeostasis, acting to control inflammation and promote tissue repair. The cytokine is secreted as a latent complex, requiring activation to be functional. Although recent work has identified activators of TGF-β in mice, how the cytokine is activated to regulate human immune responses is poorly understood. Here, we show that human CD14+ classical monocytes activate high levels of TGF-β, which is not apparent in murine monocytes. Mechanistically, we show that activation of TGF-β by human monocytes requires expression of the integrin, αvβ8. When monocytes are differentiated to macrophages, integrin αvβ8 expression levels and TGF-β-activating function is elevated on tolerogenic versus inflammatory macrophages. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 22 Interestingly, we find that integrin αvβ8 is expressed by a proposed anti-inflammatory human intestinal macrophage subset expressing CD163 and CD206, with this macrophage population decreased in patients with active Crohn’s disease. Thus, our data suggest that expression of integrin αvβ8 by human monocytes may play an important role in the induction and function of anti-inflammatory macrophages in the intestine via TGF-β activation. 2201 Katrien van der Borght Tissue necrosis is a driver of organ directed autoimmunity through activation of dendritic cells. Katrien A Van der Borght(1,2), Charlotte L Scott(1,4), Veronica Nindl(5), Ann Bouché(3), Liesbet Martens(1,2,4), Dorine Sichien(1,4), Justine Van Moorleghem(1,2), Manon Vanheerswynghels(1,2), Sofie De Prijck(1,4), Gert Van Isterdael(1,2), Burkhard Ludewig(5), Thierry Gillebert(2), Martin Guilliams(1,4), Peter Carmeliet(3,7) and Bart N Lambrecht (1,2,6,7_ Katrien A Van der Borght(1,2), Charlotte L Scott(1,4), Veronica Nindl(5), Ann Bouché(3), Liesbet Martens(1,2,4), Dorine Sichien(1,4), Justine Van Moorleghem(1,2), Manon Vanheerswynghels(1,2), Sofie De Prijck(1,4), Gert Van Isterdael(1,2), Burkhard Ludewig(5), Thierry Gillebert(2), Martin Guilliams(1,4), Peter Carmeliet(3,7) and Bart N Lambrecht (1,2,6,7) (1)VIB Inflammation Research Center, VIB and Ghent University, Ghent, Belgium(2)Department of Internal Medicine, Ghent University, Ghent, Belgium(3)Vesalius Research Center, VIB and KULeuven, Leuven, Belgium(4)Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium(5)Institute of Immunobiology, Kantonal hospital St. Gallen, St. Gallen, Switzerland(6)Department of Pulmonary Medicine, ErasmusMC, Rotterdam, The Netherlands Peripheral tolerance is crucial for avoiding activation of self-reactive T cells to tissue restricted antigens. Autoimmunity can be triggered by sterile tissue injury, but it is unclear how immune cells get activated to respond to self-antigen. A good example of sterile injury is acute myocardial infarction (MI). We hypothesized that tissue necrosis is an activator of dendritic cells (DC) that control tolerance to self+ antigens. DC subsets of the murine healthy heart consisted of IRF8-dependent XCR-1 conventional + + (c)DC1, IRF4-dependent CD172α cDC2 and CD64 monocyte-derived DCs, that shared a common gene signature with DC subsets elsewhere in the body. In steady state, cardiac self-antigen α-myosin was presented in the heart-draining mediastinal lymph node (mLN) by cDC1s, driving the proliferation of + antigen-specific CD4 TCR-M T cells, and expansion of Treg cells. Following MI, all DC subsets infiltrated the infarcted heart, while only cDCs migrated to the mLN. Here, cDC2s induced TCR-M proliferation and differentiation into IL-17/IFNγ producing effector cells. RNA sequence analysis revealed that MI changed the heart DC gene expression profile, inducing genes involved in migration and T cell activating capacity. Thus, tissue necrosis is a major driver of autoimmunity to tissue restricted self-antigen via activation of DCs. 2202 Andrew S MacDonald A central role for Type I IFN in Th2 induction by dendritic cells. 1,7 2,8 2 1 3 Lauren M. Webb , Rachel J. Lundie , Jessica G. Borger , Sheila L. Brown , Lisa M. Connor , Adam N. R. 1 4 5 1 2 Cartwright , Annette M. Dougall , Ruud H. P. Wilbers , Peter C. Cook , Lucy H. Jones , Alexander T. 1 6 1 4 3 Phythian-Adams , Cecilia Johansson , Daniel M. Davis , Benjamin G. Dewals , Franca Ronchese and 1 Andrew S. MacDonald . 1 Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester, U.K. 3 University of Edinburgh, Edinburgh, U.K. Malaghan Institute of Medical Research, Wellington, New 4 5 Zealand. University of Liege, Belgium. Wageningen University and Research Centre, Wageningen, The 6 7 Netherlands. Imperial College London, London, U.K. Current address: Cornell University, Ithaca, U.S.A. 8 Current address: Monash University, Victoria, Australia. 2 Although dendritic cells (DCs) are vital for Th2 induction against helminths or allergens, relatively little is known about how they are activated and function in response to Th2-polarizing antigens. We have 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 23 discovered a previously unappreciated role for Type I IFN (IFN-I) in the activation and function of DCs following exposure to strongly Th2-polarizing antigens. So far, IFN-I has chiefly been associated with antiviral immunity, while its role in Th2 settings is much less clear. DCs cultured with total egg antigens from the parasitic helminth Schistosoma mansoni, or the dominant immunostimulatory component of S. mansoni eggs omega-1, produced IFN-I. IFN-I was also detected in response to the common allergen house dust mite (HDM). DCs lacking the IFN-I receptor displayed dramatically impaired Th2 induction in vivo, but unimpaired ability to support CD4+ T cell polarization in vitro. Further, Th2-promoting DCs depended on IFN-I signaling for optimal activation, efficient migration to the draining LN, and effective localization within the T cell zone. Additionally, challenge of mice with S. mansoni eggs or HDM in the absence of the IFN-I receptor resulted in significantly reduced Th2 cytokine induction in vivo. Together, our data suggest a key and unexpected role for IFN-I responsiveness in enabling Th2 induction by DCs in vivo. Funding: Medical Research Council UK 2203 Dieke van Dinther Development of a macrophage-based vaccination strategy for melanoma. Dieke van Dinther (1), Henrike Veninga (1), Leoni Hoogterp (1), Mirjam Revet (1), Ellen G.F. Borg (1), Hakan Kalay (1), Jan Wouter Drijfhout (2), Yvette van Kooyk (1), Joke M.M. den Haan (1) (1) Department of Melcular Cell Biology and Immunology, Vumc, Amsterdam, The Netherlands (2) Departement of Immunohematology and Blood Transfusion, Leiden University Medical Centre Leiden, the Netherlands. Melanoma causes the majority of skin cancer related deaths, but because it is highly immunogenic, treatment with immunotherapies such as immune blockade inhibitors might work synergistically with vaccination strategies. As an in vivo vaccination strategy we propose to target melanoma antigens (Ag) to a specific subset of Ag-capturing macrophages in the spleen that are characterized by the expression of CD169. Previously, we have shown that these CD169+ macrophages transfer Ag to B cells and DCs for the induction of strong humoral responses and T cell responses, respectively. In the current study we compare the cytotoxic T cell response after targeting Ag to CD169 in the presence or absence of CD4 T cell help and a humoral response. We conjugated protein or peptide to anti-CD169 antibodies and then monitored (i) the primary, (ii) the recall and (iii) the anti-tumor immune responses after immunization with these conjugates. Both protein and peptide targeting to CD169 resulted in strong primary and recall immune responses and preliminary data on the protective immunity against melanoma show promising results. In conclusion, this macrophage-based vaccination strategy stimulates strong immune responses and can be applied to raise protective immunity against melanoma. Funding: KWF 2204 Martijn Verdoes In vivo imaging of tumor-associated macrophages and subcellular compartment characterization in dendritic cells using novel quenched fluorescent cysteine cathepsin probes 1,2 3 1 Martijn Verdoes, * Kristina Oresic Bender, Carl Figdor, Matthew Bogyo. 3 1 Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboudumc and 3 Radboud University, Institute for Molecules and Materials, Nijmegen, The Netherlands. Departments of Pathology, Stanford School of Medicine, United States. *Correspondence: [email protected] 2 Cysteine cathepsins (CCTS) are highly expressed by myeloid immune cells. They are involved in antigen presentation, migration as well as tumor progression. Several studies suggest that CCTS regulate the activity of their family members and have seemingly redundant as well as very specific functions. This suggests a high degree of control of localization of their individual proteolytic activities. However, no tools 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 24 existed for live cell activity imaging of specific CCTS with respect to their family members. We developed a quenched activity-based probe (qABP) specific for cathepsin S. In combination with a complementary panreactive qABP we generated a set of tools which enable us to perform dual color live cell activity localization studies. Using this set of qABPs we identified sub-cellular compartments containing exclusively cathepsin S activity in live dendritic cells. Furthermore, we use these qABPs to perform noninvasive and multiphoton intravital imaging of mouse models of cancer and we found that within the tumor microenvironment a subpopulation of macrophages with a pro-tumor, M2 type phenotype are the major source of CCTS activity. We are currently investigating the functional characterization of this subpopulation of tumor resident myeloid cells, as well as designing molecules to manipulate these cells in vivo. Funding: Institute for Chemical Immunology, ERC Starting Grant 2210 Rieneke van de Ven Correlating the ILT4/HLA-G inhibitory pathway to an immune suppressive microenvironment in sentinel lymph nodes draining head and neck squamous cell carcinoma. 1 2 1 3 4 2 A.M Heeren , A.G.M. Stam , E.S. Jordanova , S. van Weert , K.H. Karagozoglu , T.D. de Gruijl , C.R. 3 5 2 Leemans , E. Bloemena and R. van de Ven * 1 2 3 4 Departments of Gynecology, Medical Oncology, Otolaryngology-Head and Neck surgery, Oral and 5 Maxillofacial Surgery and Oral Pathology, Pathology, VU University medical center – Cancer Center Amsterdam, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. The sentinel lymph node (SLN) is the first tumor-draining LN and consequently the first site after the primary tumor where tumor-mediated immune suppression can hamper the development of an efficient anti-tumor immune response. Presence of the inhibitory molecule immunoglobulin-like transcript 4 (ILT4) on tolerogenic dendritic cells (DC) has been described to result in induction of regulatory T cells (Treg). In SLN (n=12) draining head and neck squamous cell carcinomas (HCSCC) we studied by flow cytometry whether the presence of ILT4 and its ligand the non-classical MHC class I molecule HLA-G, correlated with immune suppression. Migratory DC, lymph node resident conventional DC (LNR-cDCs) and CD14+ macrophages (MFs) in the SLN all expressed HLA-G. ILT4 expression was highest on CD14+ MFs. However, increased expression of ILT4 (mean fluorescence index) on LNR-cDC was found to correlate with increased frequencies of activated Treg (aTreg) (Pearson r= 0.84, p<0.001). The same was true for HLA-G expression levels on this subset (Pearson r=0.79, p<0.01). Our data suggest that in HNSCC, the ILT4/HLA-G pathway may contribute to an immune suppressive environment within the SLN, with increased aTreg frequencies. Interference with this pathway might result in reduction of immune suppression and improved anti-tumor reactivity. 2211 Anouk Zaal The anaphylatoxin C5a regulates the pro-inflammatory potential of human 6-sulfo LacNAc dendritic cells (slanDC) through crosstalk with TLR-induced ERK/p38/CREB signalling. Anouk Zaal, Miranda Dieker, Manon Oudenampsen, Annelies W. Turksma, Suzanne N. LissenbergThunnissen, Diana Wouters, S. Marieke van Ham, Anja ten Brinke Sanquin Research, Dept Immunopathology, Amsterdam, The Netherlands, and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands Activation of antigen presenting dendritic cells (DC) and the complement system are essential events in the immune defence against pathogens and occur rapidly upon pathogen invasion. Recently, we and others demonstrated crosstalk between complement and DC, as the complement activation product C5a inhibited induction of pro-inflammatory cytokines during human DC maturation by TLRs. Here, we demonstrate that this crosstalk is important in 6-sulfo LacNAc dendritic cells (slanDC), comprising the most pro-inflammatory DC subset found in human. Investigation of the mechanism through which C5a 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 25 regulates pro-inflammatory cytokine production by DC elucidated that C5aR and TLR exhibit extensive crosstalk in the ERK/p38/CREB signalling pathways. Acceleration of TLR-induced CREB phosphorylation by C5a was key in this regulatory effect of C5a on TLR-mediated DC maturation. Enhanced CREB phosphorylation induced production of IL10, which inhibited pro-inflammatory cytokine production via negative feedback signalling. Importantly, the regulatory effect of C5a on pro-inflammatory DC activation affected T cell immunity by leading to decreased Th1 and cytotoxic T cell responses. The finding that the pro-inflammatory effector function of slanDC can be down modulated by activation products of the complement system highlights the existence of intricate regulatory feedback interactions between various arms of the immune system. It also emphasizes the need for extensive immune monitoring upon application of complement modulating compounds in chronic immune-mediated diseases. 2212 Dorine Sichien IRF8 controls survival and function in terminally differentiated conventional and plasmacytoid dendritic cells respectively. 1,2 1,2 3,4 1,3 1,4,5 Dorine Sichien , Charlotte L. Scott , Liesbet Martens , Matthias Vanderkerken , Sofie Van Gassen , 1,3 6 1,2 1,7 1,3 Maud Plantinga , Thorsten Joeris , Sofie De Prijck , Leen Vanhoutte , Manon Vanheerswynghels , 1,3 1,3 1,3 1,3 6,8 Gert Van Isterdael , Wendy Toussaint , Filipe Branco Madeira , Karl Vergote , William W. Agace , 9 1,3 10-12 1,3,4 1,3,13 Björn E. Clausen , Hamida Hammad , Marc Dalod , Yvan Saeys , Bart N. Lambrecht ,* and Martin 1,2 Guilliams ,* 1 VIB Inflammation Research Center, Lab of Immunoregulation, Ghent University, Ghent, Belgium. 3 Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium. Department of 4 Respiratory Medicine, University Hospital Ghent, Ghent, Belgium. VIB Inflammation Research Center, 5 Data mining and Modelling for Biomedicine Group, Ghent University, Ghent, Belgium. Department of Information Technology, iMinds, Ghent University, Ghent, Belgium.6Section of Immunology and 7 Vaccinology, Danish Technical University Veterinary Institute, Copenhagen, Denmark. Department of 8 Medical Genetics, University Hospital Ghent, Ghent, Belgium. Department of Experimental Medical 9 Science, Immunology Section, Lund University Institute for Molecular Medicine, University Medical 10 Center of the Johannes Gutenberg-University Mainz, 55131 Mainz, Germany. Centre d’Immunologie de Marseille-Luminy, UNIV UM2, Aix-Marseille Université, Parc Scientifique et Technologique de Luminy, 11 12 Marseille, France. INSERM, U1104, Marseille, France. CNRS, UMR7280, Marseille, France. 13 Department of Pulmonary Medicine, Erasmus MC, University Medical Center, Rotterdam, The Netherlands. *These authors shared supervision over this work. Correspondence should be addressed to B.N.L. ([email protected]) or M.G ([email protected]). 2 IRF8 has been proposed to be essential for early development of monocytes, plasmacytoid dendritic cells (pDCs) and type 1 conventional dendritic cells (cDC1s) but remains highly expressed in differentiated DCs. Transcription factors that are required to maintain the identity of terminally differentiated cells were fl/fl designated “terminal selectors”. Using BM chimeras, conditional Irf8 mice and various promotors to target Cre recombinase to different stages of monocyte and DC development, we have identified IRF8 as a terminal selector of the cDC1 lineage controlling survival. In monocytes, IRF8 was necessary during early but not late development. Unexpectedly, complete or late deletion of IRF8 had no effect on pDC development or survival but altered their phenotype and gene expression profile leading to increased T cell stimulatory function but decreased type 1 I nterferon production. Thus, IRF8 differentially controls the survival and function of terminally differentiated monocytes, cDC1s and pDCs. Funding: D.S. is supported by a fellowship grant of FWO. B.N.L is supported by the University of Gent MRP program “Group-ID”, by a European Research Council consolidator grant, by an Interuniversity Attraction Pole grant, and by several FWO grants. CLS was supported by a Marie Curie Intra-European Fellowship (IEF) as part of Horizon 2020. M.G. is supported by a Marie Curie Reintegration grant, an Odysseus grant, and several FWO grants of the Flanders Government. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 26 Oral presentations September Friday 23rd 2219 Mario Kuttke Myeloid PTEN deficiency impairs tumor immune surveillance via immune checkpoint inhibition 1 1 1 1 1 1 1 1 1 Kuttke M. , Sahin E. , Pisoni J. , Percig S. , Vogel A. , Kraemmer D. , Hanzl L. , Brunner J. S. , Paar H. , 2 2 2 3 3 4 4 Soukup K. , Halfmann A. , Dohnal A. M . , Steiner C. W. , Blüml S. , Basilio J. , Hochreiter B. , Salzmann 4 4 5 6 4 1 M. , Hoesel B. , Lametschwandtner G. , Eferl R. , Schmid J. A. and Schabbauer G. 1 Institute for Physiology, Centre for Physiology and Pharmacology, Medical University of Vienna, A-1090 2 3 Vienna, Austria; St. Anna Children’s Cancer Research Institute, A-1090 Vienna, Austria; Department of 4 Rheumatology, Internal Medicine III, Medical University of Vienna, A-1090 Vienna, Austria; Institute for Vascular Biology and Thrombosis Research, Centre for Physiology and Pharmacology, Medical University 5 6 of Vienna, A-1090 Vienna, Austria; Apeiron Biologics AG, A-1030 Vienna, Austria; Institute of Cancer Research, Internal Medicine I, Medical University of Vienna, A-1090 Vienna, Austria. In our study we are investigating the effects of the myeloid PI3K/PTEN pathway on tumor immune surveillance. resulted in protection of myeloid conditional knock-out mice (myPTEN-/-) in models of acute infection and inflammation. A reduction in pro-inflammatory responses could, however, cause an increase in tumor burden. Addressing this question, we induced colitis associated colon cancer in myPTEN-/- mice and found enhanced tumor burden and a reduction in survival. This was accompanied by increased numbers of splenic cross-presenting dendritic cells expressing immune checkpoint regulators. This resulted in a reduced proliferation of KO-mice derived T-cells. Our data were further substantiated by findings using the B16 melanoma model. In this model myPTEN-/mice showed a decreased T-cell activation and a reduced melanoma cell killing capacity. Taken together, our findings show that genetic deletion of PTEN in cells of myeloid origin results in an increase in splenic APCs expressing immune checkpoint regulators causing a decrease in tumor immune surveillance. Therefore, our study suggests that PI3K-inhibitors, which are currently tested as anti-cancer drugs, might have additional beneficial effects on immune cells. Funding: Austrian Science Fund projects P24802 (to GS) and SFB-F54 (to GS ans JS). 2220 Camilla Salvagno Therapeutic synergy between macrophage blockade and chemotherapy in breast cancer elicits neutrophil-dependent therapy resistance. 1 1 1 1 1 2 Camilla Salvagno , Metamia Ciampricotti , Seth B. Coffelt , Kelly Kersten , Cheei-Sing Hau , Sander Tuit , 3 4 1 5 2 6 Katharina Wartha , Ji-Ying Song , Kim Vrijland , Jos Jonkers , Joachim L. Schultze , Carola Ries and Karin E. 1 de Visser 1 2 Division of Immunology, Netherlands Cancer Institute, The Netherlands, LIMES-Institute, Germany, 4 5 Actelion Pharmaceuticals, Switzerland, Department of Experimental Animal Pathology, Division of 6 Molecular Pathology, Netherlands Cancer Institute, The Netherlands, Roche Innovation Centre Penzberg, Germany 3 Tumor-associated macrophages (TAMs) limit the anti-cancer efficacy of chemotherapy in many pre-clinical models. Clinical trials are evaluating novel drugs that target TAMs in cancer patients. To maximize the success of these compounds, it is critical to understand which drug combinations provide the best anticancer response, and to gain insights into the resistance mechanisms. Using the conditional 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 27 F/F F/F K14cre;Cdh1 ;Trp53 mouse model for de novo mammary tumorigenesis, we show that targeting TAMs by CSF-1 receptor (CSF-1R) blockade improves the anti-cancer efficacy of cisplatin, but not of docetaxel. CSF-1R blockade induced a strong type I interferon response which was necessary for the therapeutic synergy between cisplatin and CSF-1R blockade. Moreover, this treatment evoked a compensatory neutrophil response limiting the synergistic anti-cancer effect of this combination. Combined depletion of macrophages and neutrophils further improved the anti-cancer efficacy of cisplatin, which was partially + dependent on CD8 T cells. In conclusion, these data highlight the importance for optimally matching chemotherapeutics with immunomodulatory drugs. In addition, we show that CSF-1R blockade combined with cisplatin elicits a rewiring of the inflammatory tumor microenvironment resulting in neutrophildependent therapy resistance. Funding: EU project: FP7 MCA-ITN 317246 2221 Rebekka Wehner Accumulation of tolerogenic human 6-sulfo LacNAc+ dendritic cells in renal cell carcinoma is associated with poor prognosis. 1 2 3 3 4 Rebekka Wehner , Marieta Toma , Anja Kloß1, Kati Erdmann , Susanne Füssel , Barbara Seliger , Dorothee 5 5 6 3,7 2,7 1,7 Brech , Elfriede Noessner , Knut Schäkel , Manfred Wirth , Gustavo Baretton & Marc Schmitz 1 2 Institute of Immunology, Medical Faculty, Dresden University of Technology, Dresden, Germany Institute 3 of Pathology, University Hospital of Dresden, Dresden, Germany Department of Urology, University 4 Hospital of Dresden, Dresden, Germany Institute for Medical Immunology, Martin Luther University 5 Halle-Wittenberg, Halle (Saale), Germany Institute of Molecular Immunology, Helmholtz Center Munich, 6 German Research Center for Environmental Health, Munich, Germany Department of Dermatology, 7 University Hospital, Heidelberg, Germany National Center for Tumor Diseases, University Hospital of Dresden, Dresden, Germany + 6-sulfo LacNAc dendritic cells (slanDCs), representing a myeloid human blood DC subset, produce proinflammatory cytokines, display cytotoxic activity and efficiently stimulate NK cells and T cells. Recently, it has been shown that slanDCs accumulate in metastatic lymph nodes from cancer patients. Therefore, slanDCs may contribute to antitumor immunity. Here, we analyzed slanDCs in 263 clear cell renal cell carcinoma (ccRCC) and 227 tumor-free tissues and found increased frequencies of slanDCs in the tumor. slanDCs were also detectable in metastatic lymph nodes and distant metastases. Remarkably, a higher density of slanDCs in ccRCC was correlated with a reduced survival of tumor patients. Freshly prepared ccRCC-infiltrating slanDCs were immature, do not express pro-inflammatory cytokines but the anti-inflammatory cytokine IL-10. Further studies revealed that primary ccRCC cells efficiently impair slanDC-induced T-cell and NK-cell activation. In conclusion, higher slanDC numbers in ccRCC tissues are associated with poor prognosis. The tolerogenic phenotype of slanDCs leading to an insufficient activation of antitumor immunity may represent a novel immune escape mechanism of ccRCC. These observations may have implications for the design of therapeutic strategies that harness tumor-directed functional properties of DCs. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 28 2240 Ulrike Schleicher Suppression of arginase 1 by TNF facilitates the production of NO by type 2 NO synthase and is a novel mechanism by which TNF confers protection against Leishmania major parasites in vivo. 1 1 1 2 3 Ulrike Schleicher , Katrin Paduch , Andrea Debus , Peter J. Murray , Renato Ostuni , and Christian Bogdan 1 1 Mikrobiologisches Institut - Klinische Mikrobiologie, Immunologie und Hygiene, Friedrich-AlexanderUniversität (FAU) Erlangen-Nürnberg and Universitätsklinikum Erlangen, 91054 Erlangen, Germany 2 Departments of Infectious Diseases and Immunology, St. Jude Children's Research Hospital, Memphis, TN 3 38105, USA. [email protected] Department of Experimental Oncology, European Institute of Oncology (IEO), 20139 Milan, Italy Mice deficient for tumor necrosis factor (TNF) are highly susceptible to infections with the intracellular pathogen Leishmania major despite an intact Th1 immune response and a sustained expression of type 2 NO synthase (NOS2) in the infected tissues. Here, we demonstrate that in macrophages and dendritic cells TNF is a potent negative regulator of IL-4-induced arginase (Arg) 1, which competes with NOS2 for the common substrate L-arginine. TNF did not suppress the phosphorylation or nuclear translocation of STAT6, the key transcription factor for the induction of Arg1 by IL-4, but reduced the acetylation of histone residues critical for the opening of the promoter and regulatory region of the Arg1 gene locus. In vivo, TNF-deficiency caused a hyperexpression of Arg1 leading to an increased frequency of Arg1/NOS2 double-positive cells and an impaired release of NO at the site of infection. A similar phenotype was seen in non-healing L. major-infected BALB/c mice. Deletion of Arg1 in hematopoietic and endothelial cells unleashed the production of NO in these mice and protected them from an otherwise lethal course of infection, although their Th2 response was maintained. These data identify a novel mechanism by which TNF confers protection against intracellular pathogens. Funding: German Research Foundation (RTG1660, CRC1181) 2241 Melissa Newling IgG opsonization of viruses functions as an endogenous suppressor of type I and III IFN-related anti-viral immunity by human myeloid cells through FcγRIIa. 1,2* 3* 1,2 3 4 Melissa Newling , Lisa T.C. Vogelpoel , Willianne Hoepel , Esther W.M. Taanman-Kueter , Dirk Eggink , 3 1 3# 1,2# Martien L. Kapsenberg , Dominique L.P Baeten , Esther C. de Jong , Jeroen den Dunnen 1 Amsterdam Rheumatology and Immunology Center, Department of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands 2 Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, 3 Amsterdam, The Netherlands Department of Cell Biology and Histology, Academic Medical Center, 4 University of Amsterdam, Amsterdam, The Netherlands Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands *M.N. and L.T.C.V. contributed equally to this study, sharing first authorship #E.C.dJ. and J.dD. contributed equally to this study, sharing last authorship While type I and type III interferons (IFNs) are fundamental for antiviral immunity, prolonged expression of these IFNs is also detrimental to the host. Therefore, type I and III IFN expression is tightly controlled upon viral infection, with high levels in the first few days followed by a strong and rapid decline. However, the endogenous mechanisms responsible for this suppression are still largely unknown. Here, we hypothesized that virus-specific IgG antibodies, which only emerge during late-phase of infection, function as an environmental cue for suppression of type I/III IFN responses. Strikingly, we indeed observed that IgG opsonization of model viruses influenza and RSV strongly suppressed type I/III IFN production by various human myeloid cells, including DCs, macrophages, and LCs. Consequently, IgG opsonization also suppressed expression of numerous IFN-stimulated genes (ISGs), while other anti-viral genes such as IL12, IL-27 and CD70 were unaffected. We identified Fc gamma receptor IIa (FcγRIIa) as the main receptor 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 29 responsible, which strongly and selectively suppressed type I/III IFN gene transcription via cross-talk with TLR3/8 and RIG-I/MDA5. Taken together, we have identified IgG opsonization of viruses as a novel endogenous suppression mechanism of type I and III IFN responses by the myeloid cell compartment. 2242 Joris K. Sprokholt Follicular T helper cell differentiation induced by Dengue virus through RIG-I like receptor and Type I Interferon receptor crosstalk. Joris K. Sprokholt, Tanja M. Kaptein, John L. van Hamme, Ronald J. Overmars, Sonja I. Gringhuis, Teunis B.H. Geijtenbeek Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Follicular T helper cells (TFH) are fundamental in orchestrating effective antibody-mediated responses that are critical for immunity against viral infections and effective vaccines. However, it is unclear how virus infection leads to TFH induction. Here, we show that dengue virus infection of human dendritic cells (DCs) drives TFH formation via crosstalk between RIG-I like receptors (RLRs) and type I interferon (IFN) receptor (IFNAR). DENV replication triggers activation of the RLRs RIG-I and MDA5 leading to IFN-β production and IFNAR signaling. Notably, our data show that IKK-epsilon activation by RLRs crosstalks with IFNAR signaling to specifically activate transcription factor ISGF3 (interferon-stimulated gene factor 3). ISGF3 drives the + expression of IL-27p28 and subsequent IL-27 secretion by DCs is crucial for the induction of CXCR5 PD+ + 1 Bcl-6 TFH cells. Blocking IFNAR signaling or silencing RLRs using RNA interference abrogated IL-27 secretion by DENV-infected DCs and blocked TFH formation. Furthermore, RLR activation in DCs by synthetic ligands also induced IL-27 secretion and TFH polarization. Hence, we have identified a pivotal role for RLRs in antibody-mediated responses by inducing TFH formation and we have identified an innate mechanism by which DENV-specific antibodies can develop during disease or vaccination. Our results have the potential to improve vaccine development. 2243 Diana Dudziak Late breaking abstract: Human Lympoid tissue dendritic cells are less influenced by their microenvironment their counterparts in non-lymphoid tissues. 1, ‡ 2, ‡ 1 1 1 Gordon F. Heidkamp , Jil Sander , Christian H. K. Lehmann , Lukas Heger , Nathalie Eissing , Anna 1, † 1 1 3 4 4 Baranska , Jennifer J. Lühr , Alana Hoffmann , Anja Lux , Stephan Söder , Arndt Hartmann , Johannes 5 2 6 7 8 8 Zenk , Thomas Ulas , Naomi McGovern , Christoph Alexiou , Bernd Spriewald , Andreas Mackensen , 9 10 10 11 12 12 Gerold Schuler , Burkhard Schauf , Anja Forster , Roland Repp , Ariawan Purbojo , Robert Cesnjevar , 13, 14 6 2,6,15 3 2,15, ‡,* Evelyn Ullrich , Florent Ginhoux , Andreas Schlitzer , Falk Nimmerjahn , Joachim L. Schultze , 1, ‡,* Diana Dudziak 1 Friedrich-Alexander-University Erlangen-Nürnberg (FAU), University Hospital Erlangen, Dept. of 2 Dermatology, Laboratory of Dendritic Cell Biology, Erlangen, Germany LIMES – Life and Medical Sciences 3 Center, Genomics and Immunoregulation, University of Bonn, Bonn, Germany FAU, Chair of Genetics, 4 Dept. of Biology, Erlangen, Germany FAU, University Hospital Erlangen, Dept. of Pathology, Erlangen, 5 6 Germany Klinikum Augsburg Süd, Dept. of Otorhinolaryngology, Augsburg, Germany Singapore 7 Immunology Network (SIgN), A*STAR, Singapore, Singapore. FAU, University Hospital Erlangen, Dept. of Otorhinolaryngology, Head and Neck Surgery, Section for Experimental Oncology and Nanomedicine 8 (SEON), Else Kröner-Fresenius-Stiftung-Professorship, Erlangen, Germany FAU, University Hospital 9 Erlangen, Dept. of Hematooncology, Erlangen, Germany FAU, University Hospital Erlangen, Dept. of 10 Dermatology, Erlangen, Germany Sozialstiftung Bamberg, Dept. of Obstetrics and Gynecology, Bamberg, 11 12 Germany Städtisches Krankenhaus Kiel, Medical Dept. 2, Kiel, Germany FAU, University Hospital 13 Erlangen, Dept. of Pediatric Heart Surgery, Erlangen, Germany LOEWE Center for Cell and Gene Therapy, 14 Goethe University, Frankfurt, Germany Division for Stem Cell Transplantation and Immunology, Dept. for 15 Children and Adolescents Medicine, Hospital of the Goethe University, Frankfurt, Germany Single Cell Genomics and Epigenomics Unit at the University of Bonn and the German Center for Neurodegenerative 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 30 Diseases, Bonn, Germany.*Corresponding authors. [email protected]‡ these authors contributed equally. E-mail: [email protected]; Introduction: Dendritic Cells (DCs) are important regulators of immune responses. In our previous studies we showed differential antigen-presentation capacities of murine DC-subpopulations in an in vivo antigen targeting system. In contrast, the distinct functional roles of human tissue DC subpopulations remain largely unknown. We are focussing on a standardized, comparative characterization of DC subpopulations directly isolated from various lymphoid and non-lymphoid tissues in order to provide a deeper insight into human DC subset phenotypes. Moreover, we aim to clarify their different functional roles in the initiation of human immune responses. Methods: Human tissues (thymus, spleen, bone-marrow, tonsils, cord-blood, and peripheral blood) were received from otherwise healthy individuals. We performed 6-color confocal immunofluorescence analyses and up to 18-color FACS and cell-sort analyses for the study of 230 cell surface molecules. We further investigated the DCs’ antigen uptake properties and analyzed the RNA expression profile by microarray. Results: The comprehensive analysis of human DCs revealed both a subset- and tissue-specific expression profile of human DC subpopulations. The percentages of the three main DC subpopulations of CD1c+ DCs, CD141+ DCs, and pDCs were varying depending on the analyzed tissue, proposing different functional roles. The analysis of 230 cell surface molecules revealed a heterogeneous expression profile among the different human DC-subpopulations. Data extensions to non-lymphoid tissue DCs from skin and lungs revealed that DCs from those tissues are stronger influenced by their microenvironment as their counterparts in lymphoid tissues. Conclusion: With cutting-edge technologies we have characterized directly isolated human blood and tissue DC subpopulations and revealed tissue-specific phenotypical and functional differences. The here presented data will help to elucidate the implication of human DC subpopulations in the initiation of immune responses. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 31 Poster Sessions Session I Control of APC function Session II Chronic inflammatory disorders Session III Metabolism and signaling Session IV Macrophage function in health and disease Session V Antigen processing and presentation Session VI Functionals heterogeneity of APC Session VII Clinical applications and tumor immunology Session VIII Host- microbe crosstalk 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 32 Session I Control of APC function 2103 Anno Saris IMMUNE MODULATORY EFFECT OF PLATELETS ON DENDRITIC CELLS A. Saris1,2, P.F. van der Meer2, S.M. van Ham1, J.J. Zwaginga3, A. ten Brinke1, 1 Sanquin Research, Immunopathology, Amsterdam, the Netherlands, 2 Sanquin Blood Bank, Product and Process Development, Amsterdam, the Netherlands, 3 Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands Platelets are typically known for their role in primary hemostasis but are increasingly acknowledged for their role in modulating immune responses. Their effect on DC functionality are unknown. We therefore studied the effect of platelets on monocyte-derived dendritic cell (DC) maturation, the phagocytic capacity of the latter and most important their ability to stimulate antigen-specific T cells. Immature DCs cultured in the presence of platelets showed a major reduction in endocytosis (OVA and transferrin) and phagocytosis (zymosan and salmonella), as compared to DCs cultured without platelets. Additionally DCs were matured in absence or presence of platelets. DCs matured in the presence of platelets expressed less CD83 but more CD86, produced significantly less TNFα, IL6 and IL12p40 and were less capable to induce tetanus toxoid specific autologous T cell proliferation. Thus, platelets inhibit the capacity of immature DCs to phagocytose and platelets affect the maturation of DCs by altering expression of co-stimulation markers, inhibiting pro-inflammatory cytokine production and reducing DCs capacity to stimulate antigen specific autologous T cell proliferation. Hereby we demonstrate that platelets can indeed modulate the immune response by affecting DC function. 2104 Estafania Herdoiza Padilla miRNA-dependent Regulation of Phagocytosis in Human Macrophages Estefania Herdoiza Padilla, Christian U. Riedel Institute of Microbiology and Biotechnology, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, ermany One of the characteristic features of macrophages is phagocytosis, which is is a central and complex element of host defence by which infected or dead cells, extracellular pathogens as well as foreign particles are ingested and removed from the circulation. Depending on the cytokine microenvironment, macrophages can polarize into two main functional phenotypes: pro-inflammatory (also termed M1) macrophages and anti-inflammatory (also termed M2) macrophages. As reported previously be our own as well as other groups, M-CSF differentiated, M2-like human macrophages show higher phagocytosis of pathogenic and commensal bacteria and opsonized latex beads than M1-like human macrophages generated in the presence of GM-CSF. The phagocytic activity of macrophages must be tight regulated, however the genetic mechanisms underlying this difference in phagocytosis remain poorly understood. Over the last years, micro-RNAs (miRNA) have emerged as potent regulators of biological pathways by inducing gene silencing. The aim of our study is the identification of human miRNAs involved in the regulation of phagocytosis by a functional genome-wide high-content miRNA screen. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 33 2105 Alsya J Affandi miR-618 modulates the number and activation status of plasmacytoid dendritic cells in systemic sclerosis , 1,2 1,2 3 Marzia Rossato, PhD ; Alsya J. Affandi, MSc, ; Soley Thordardottir, MSc, ; Catharina G. K. Wichers, 1,2 1,2 1,2 1,2 BASc, ; Marta Cossu, MD, ; Jasper C. A. Broen, MD, PhD, ; Frederique M. Moret, PhD, ; Lara 4,5 1,2 1,2 1,2 Bossini-Castillo, PhD, ; Eleni Chouri, MSc, ; Lenny van Bon, MD, PhD, ; Femke Wolters, MD, ; 1,2 1,2 1,2 Wioleta Marut, PhD, ; Maarten van der Kroef, MSc, ; Sandra Silva-Cardoso, MSc, ; Harry Dolstra, 3 2 4 1,2 PhD, ; Jacob M. van Laar, MD, PhD, ; Javier Martin MD, PhD, ; Joel A. G. van Roon, PhD, ; Kris A. 1,2 6 1,2 Reedquist, PhD, ; Lorenzo Beretta, MD, PhD, ; Timothy R. D. J. Radstake, MD, PhD 1 Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands; 2 Department of Rheumatology and Clinical Immunology, University Medical Center Utrecht, Utrecht, The Netherlands; 3 Department of Medicine, Laboratory of Hematology, Radboud University Medical Center, Nijmegen, The Netherlands;4 Consejo Superior de Investigaciones Científicas (IPBLN-CSIC), Instituto de Parasitología y Biomedicina López-Neyra, PTS Granada, Granada, Spain; 5 Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK; 6 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico di Milano, Milan, Italy. Plasmacytoid dendritic cells (pDCs) constitute a critical source of type I interferons (IFNs) that can contribute to pathogenesis of autoimmune diseases. The molecular mechanisms leading to pDC dysregulation and a persistent type I IFN signature are largely unexplored, especially in systemic sclerosis (SSc). Objective: To investigate the epigenetic mechanisms mediated by microRNAs, potentially underlying pDC dysregulation and IFN-response in SSc. Methods: microRNA expression profiling and validation in multiple cohorts were performed in pDC isolated from healthy controls and SSc patients. In vitro experiments were used to identify the function of miR-618 and its impact on pDC biology. Results: miR-618 was upregulated in pDCs of SSc patients, including those with early onset. IRF8, a crucial transcription factor for pDC development and activation, was identified as a target of miR-618. miR-618 overexpression reduced the development of pDCs from CD34+ cells in vitro and enhanced their IFNα secretion, thus reflecting the pDC phenotype observed in SSc patients. Conclusion: miR-618 upregulation suppresses pDC development and increases their IFNα production, as seen in SSc patients. Thus, miR-618 potentially constitutes an important epigenetic target to regulate immune system homeostasis in SSc and other type I IFN diseases. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 34 2106 Anouk Zaal RNA-seq analysis reveals that C5a and LPS synergize at the transcriptional level in human dendritic cells. Anouk Zaal; Miranda Dieker; Kat Moore; Benjamin Nota; S. Marieke van Ham; Anja ten Brinke Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, University of Amsterdam, Academic Medical Center, Amsterdam, The Netherlands The complement component 5a (C5a) is a well-known chemo-attractant affecting the innate immune system. In several autoimmune diseases elevated C5a levels have been found. Clinical trials are performed using complement modulatory compounds to treat auto-immune diseases; however treatment is focused mainly on innate immunity, while adaptive immunity plays a very important role in the progression of these diseases. C5a indeed has been implicated in regulation of adaptive immune responses through modulation of antigen presenting cell (APC) function, however, studies in human APC have been incomplete. Therefore, the effect of C5a on the activation of human APC has to be clarified. We performed unbiased RNA-seq analysis to elucidate which APC functions are affected by C5a. We analyzed mRNA expression in human monocyte-derived dendritic cells (moDC) stimulated with C5a both in the absence and presence of LPS. GO term enrichment analysis revealed that C5a affects processes like Fc-receptor mediated signaling, phagocytosis and T cell immune responses. The latter may be established via the cytokine IL12, as the most strongly down regulated gene by C5a in LPS-stimulated moDC is the p40 subunit of this Th1 skewing cytokine. In addition, we found that there was only minimal overlap between genes affected by C5a in the absence or presence of LPS, indicating that C5a affects different processes depending on the crosstalk with TLR signaling. Analysis of gene expression profiles revealed that C5a and LPS synergized at the transcriptional level, leading to unique differential expression of 59 genes. We are currently further investigating these synergistically affected genes and various processes affected by C5a according to GO term enrichment analysis. Using this unbiased approach, we will further elucidate the modulating capacity of the innate complement system on DC-mediated adaptive immune responses. 2107 Jelena Popović Control of Dendritic Cell Differentiation by SWAP-70 and DEF6 1 1 Jelena Popović , Carlos M. Ocaña-Morgner , Rolf Jessberger 1 Institute of Physiological Chemistry, Medical School, MTZ, Dresden University of Technologye-mail: [email protected] We have recently showed that absence of protein SWAP-70 increases the expression of maturation markers of bone marrow-derived GM-CSF-stimulated dendritic cells (BMDC). Preliminary data shows that besides SWAP-70, the only other closely related protein DEF6 also controls the expression of maturation -/-/markers in BMDC. We have found that both single knockout (SKO) (Swap70 , Def6 ), and double -/-/knockout (DKO) (Swap70 Def6 ) show up-regulation of DC signature genes and also have a higher DC/Macrophages populations ratio compared to WT, which suggest a relationship between these proteins and differentiation under GM-CSF. Studies using Flt3-L cultures, however, show a reduction in the frequency of common dendritic cells (cDC) generated from bone marrow cells of SKO animals. Progenitor cell analysis of bone marrow of SKO and DKO animals showed no differences in frequency and total number of macrophage-DC progenitor (MDP) and common DC progenitor (CDP) cells when compared to WT. This study highlights the importance of SWAP-70 and DEF6 in the two in vitro differentiation models commonly used to generate DC. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 35 Session II Chronic inflammatory disorders 2112 Elisabeth Zinser Grb2 is important for T-cell development, T-helper cell differentiation and induction of EAE Daniel Radtke, Sonja M. Lacher, Nadine Szumilas, Lena Sandrock , Jochen Ackermann, Lars Nitschke and Elisabeth Zinser The small adaptor protein Growth factor receptor-bound protein 2 (Grb2) modulates and integrates signals from receptors on cellular surfaces in inner signalling pathways. In murine T cells, Grb2 is crucial fl/fl tg for amplification of TCR signalling. T cell specific Grb2 Lckcre Grb2 deficient mice show reduced T cell numbers due to impaired negative and positive selection. In this study, we found that T cell numbers in fl/fl tg Grb2 CD4cre mice were normal in the thymus and only slightly affected in the periphery. Ex vivo analysis of CD4+ T Helper cell (TH) populations revealed an increased amount of TH1 cells within the CD4+ fl/fl tg population of Grb2 CD4cre mice. Additionally, Grb2-deficient T cells showed a higher potential to differentiate into TH17 cells in vitro. To test if these changes in TH differentiation potential rendered fl/fl tg Grb2 CD4cre mice more prone to inflammatory diseases, we chose the murine T H1- and TH17-driven fl/fl experimental autoimmune encephalomyelitis (EAE) model. In contrast to our expectations Grb2 tg CD4cre mice developed a milder form of EAE. The impaired EAE disease can be explained by a reduced proliferation rate of Grb2-deficient CD4+ T cells upon stimulation with IL-2 or upon activation by allogeneic dendritic cells, as the activation of T cells by dendritic cells and following T cell proliferation are known to be crucial factors to induce EAE. In summary Grb2 deficient T cells show defects in T cell development, elevated TH1 and TH17 differentiation capacities and impaired proliferation after activation by dendritic cells which likely reduces clinical symptoms of EAE. 2113 Anoushka K. Molhoek How does ACPA affect Rheumatoid Arthritis on a molecular level? Anoushka K. Molhoek, Sandra J. van Vliet, Juan J. Garcia-Vellejo, René E.M. Toes and Yvette van Kooyk. Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands.Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands. Anti-Citrullinated Protein Antibodies (ACPA) are highly specific for Rheumatoid Arthritis (RA) and have been implicated in disease pathogenesis. ACPA are directed against citrullinated self-peptides and proteins, and when present in RA patients the predicted clinical outcome is worse. To date we have limited understanding on the mode of action of these APCA. The fragment antigen-binding (Fab) domain of ACPA was recently shown to be extensively glycosylated. Surprisingly, it contains an unique glycan structure which is structurally different from ‘conventional’ IgG. We hypothesize that the altered glycosylation of ACPA may affect their biological function resulting in a more immunogenic outcome through the interaction with glycan binding receptors on immune cells. As the unusual APCA glycans are monosialyted and disialylated, they may interact with sialic acid-binding Ig-type lectins at the cell surface, thereby exerting immunomodulatory effects. Therefore, our aim is to understand the role of the ACPA glycosylation by identifying the binding receptors and the cells that respond to ACPA in vitro and ex vivo using healthy donors and RA patient material. Funding: ZonMw 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 36 2114 Ivo S Hansen FcαRI Co-stimulation Promotes Intestinal Inflammation by Human CD103+ Mucosal Dendritic Cells Through Amplification of Cytokine Gene Translation 1,2 3 4 3 5 Ivo S. Hansen , Jochem H. Bernink , Fabricio Loayza Puch , Hergen Spits , Sebastiaan A.J. Zaat , Reuven 4 3 1 1,2 Agami , Esther C. de Jong , Dominique L.P. Baeten and Jeroen den Dunnen 1 Amsterdam Rheumatology and Immunology Center, Department of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands 2 Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, 3 Amsterdam, The Netherlands Department of Cell Biology and Histology, Academic Medical Center, 4 University of Amsterdam, Amsterdam, The Netherlands Division of Biological Stress Response, The 5 Netherlands Cancer Institute, Amsterdam, The Netherlands Department of Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands + Crucial for regulation of intestinal tolerance are CD103 DCs. However, upon infection of the lamina propria of the gut the local tolerogenic response should be converted into a pro-inflammatory response. Yet, the factors that control this inflammatory switch are still largely unknown. Here, we have identified formation of IgA immune complexes, as occurs when bacteria enter the lamina propria, as a critical factor + for induction of inflammation by human CD103 DCs. We demonstrate that IgA immune complexes, through FcαRI, selectively amplified the production of pro-inflammatory cytokines TNFα, IL-1β, and IL-23, which was mediated by synergy of FcαRI with various families of pathogen-sensing receptors. This synergistic upregulation of cytokines promoted inflammation through both Th17 induction and by activating human intestinal ILC3 cells. Furthermore, we identified that FcαRI-induced inflammation is orchestrated via inflammasome activation and amplification of cytokine gene translation. Taken together, we have identified a new role for IgA and its receptor FcαRI in promoting intestinal inflammation by + CD103 DCs. The identification of the molecular mechanism behind this inflammatory process could be important in the pathogenesis of inflammatory bowel disease (IBD) and may provide new targets for treatment of this disease. 2115 Victoria Saferding MicroRNA-146a an important regulator of local bone destruction in inflammatory arthritis 1 1 1 1 2 Victoria Saferding , Antonia Puchner , Eliana Goncalves-Alves , Melanie Hofmann , Emine Sahin , Silvia 1 3 4 2 1 1 Hayer , Phillipe Georgel , Marije M. Koenders , Gernot Schabbauer , Josef S. Smolen , Günter Steiner , 1 1 Kurt Redlich and Stephan Blüml 1 2 Division of Rheumatology, Internal Medicine III, Medical University of Vienna, Austria. Institute for 3 Physiology, Center for Physiology and Pharmacology, Medical University Vienna, A-1090 Vienna, Austria. INSERM UMR_S 1109, Fédération de Médecine Translationnelle (FMTS), Université de Strasbourg, Centre de Recherche en Immunologie et Hématologie, 1, Place de l’Hôpital 67085 Strasbourg Cedex France 4 Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands MicroRNA (MiR-) 146a is a key regulator of the innate immune response. Elevated expression of miR-146a has been detected in synovial tissue of rheumatoid arthritis patients, but its role in inflammatory arthritis is still elusive. To analyse the role of miR-146a in arthritis we used two different disease models hTNFtg and K/BxN serum transfer arthritis, disease severity was assessed clinically and histologically. The regulatory function of this miRNA in fibroblasts and immune cells was evaluated. Loss of miR-146a leads to increased clinical signs of serum transfer arthritis and higher serum levels of the –/– cytokines IL12 and IL6 in miR146a deficient mice. When we crossed miR-146a mice into hTNFtg mice, + + –/– we found a significant increase in CD11b and CD11c cells in the blood of miR-146a /hTNFtg mice. Histological examination revealed an increase in synovial inflammation and local bone destruction in the –/– tarsal joints of miR-146a /hTNFtg mice. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 37 -/- MiR-146a /hTNFtg mice showed elevated levels of IL-1β, TRAF6 and RANKL and decreased expression of OPG locally in the paws. Bone marrow transplants revealed a pivotal role for miR-146a in mesenchymal cells in c ontrolling local osteoclast generation and bone destruction. These data clearly demonstrate a negative regulatory role of the miR-146a in inflammatory arthritis. 2116 Eliane S Piket Establishing a role for miR-21 in macrophages in experimental autoimmune encephalomyelitis 1 1 Eliane Piket , Sabrina Ruhrmann , Maja Jagodic (PhD) 1 1 Department of Clinical Neuroscience, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden Multiple Sclerosis (MS) is a chronic inflammatory disease characterized by autoimmune destruction of myelin and neurons in the CNS. T cells have been ascribed the instructing role in MS. They are primed in the periphery by antigen presenting cells (APCs) such as macrophages and dendritic cells (DCs). Macrophages are the primary effectors since they contribute to MS lesion formation and axonal damage by secreting proinflammatory mediators and through phagocytosis. In the CNS microglia can exert similar functions as macrophages. Understanding basic mechanisms of MS can have important implications for other diseases that comprise components of neuroinflammation and neurodegeneration. MicroRNAs (miRNAs) are evolutionary conserved small non-coding RNAs that fine-tune protein levels of target genes. They are important in the regulation of most biological functions and commonly dysregulated in disease. Several lines of evidence, including our published and preliminary findings, strongly implicate miR-21 in the pathogenesis of MS and experimental autoimmune encephalomyelitis (EAE), an animal model for MS. MiR-21 is encoded in a risk locus for MS, it has been shown to be upregulated in MS as well as EAE, and targeting miR-21 in mice affects EAE development. -/- We studied the involvement of miR-21 in miR-21 and wild type mice and demonstrated, in line with the -/previous report, that there was an amelioration of disease in miR-21 mice compared to littermate controls. We further aimed to investigate the impact of miR-21 on differentiation and function of bone marrow derived macrophages (BMMs) as well as to establish relevant target genes to understand how mechanisms involving miR-21 are impacted. 2117 Katharine M Irvine CRIG identifies a novel population of highly phagocytic peritoneal macrophages in ascites fluid associated with disease severity in patients with cirrhosis and ascites. 1,2 1 2 2 3,4 2 Katharine M Irvine *, Xuan Banh , Victoria L Gadd , Kyle K Wojcik , Juliana K Ariffin , Sara Jose , Samuel 3 3 3,4 1,2 Lukowski , Gregory J Baillie , Matthew J Sweet , Elizabeth E Powell 1 Department of Gastroenterology and Hepatology, Princess Alexandra Hospital, Brisbane, Queensland, 2 Australia Centre for Liver Disease Research, School of Medicine, The University of Queensland, Brisbane, 3 Queensland, Australia Institute for Molecular Bioscience (IMB), The University of Queensland, Brisbane, Queensland, Australia 4IMB Centre for Inflammation and Disease Research, The University of Queensland, Brisbane, Queensland, Australia Infections are an important cause of morbidity and mortality in patients with decompensated cirrhosis and ascites. Hypothesising innate immune dysfunction contributes to susceptibility to infection, we investigated ascitic fluid macrophages. The expression of complement receptor of the immunoglobulin superfamily (CRIg) defined two phenotypically and functionally distinct peritoneal macrophage subpopulations. Macrophage sub-population distribution differed between patients, and over time, and an expansion of the highly phagocytic and microbicidal CRIg+ macrophages was associated with reduced disease severity. Transcriptional profiling by RNASeq revealed significant similarities between CRIg+ cells, human 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 38 Hi macrophages and mouse F4/80 resident peritoneal macrophages, and between CRIg- macrophages, Low human monocytes and mouse F4/80 monocyte-derived peritoneal macrophages. Regulating the numbers and/or functions of these macrophage populations could provide therapeutic opportunities to prevent infections in cirrhotic patients. In light of the recent discovery that peritoneal macrophages can be directly recruited to the injured liver in mice, peritoneal macrophage distribution and function may also have important implications for liver status in patients with chronic liver disease. 2118 Marjan A. Versnel Hyperreactivity of TLR7 in a subset of the patients with Sjögren’s syndrome 1 1 2 1,2 Iris L.A. Bodewes , Cornelia G. van Helden-Meeuwsen , Paul L. van Daele1, , P. Martin van Hagen , 1 Marjan A. Versnel 1 Department of Immunology, Netherlands 2 Department of Internal Medicine, Erasmus MC, Rotterdam, The Primary Sjögren’s Syndrome (pSS) is a systemic autoimmune disease characterized by dryness of mouth and eyes and the frequent presence of multiple extraglandular manifestations. We have shown that systemic IFN type I activation is related to disease activity and is present in 57% of the patients. Plasmacytoid dendritic cells are potent IFN type I producing cells upon stimulation of toll-like receptor (TLR)7 and TLR9. TLR7 is recognizing RNA and TLR9 DNA. Previously we found evidence for an upregulation of RNA-sensing receptors in pDCs and monocytes of IFN+pSS patients. We hypothesized that increased systemic IFN type I expression in pSS is due to a hyperreactivity of TLR7. Peripheral blood cells of 12 pSS patients were stimulated in vitro for 5 hours with a TLR7 agonist followed by qPCR analysis of MxA, an IFN type I induced gene. Hyperresponsiveness was defined as the MxA expression was higher than twice the mean of the controls +2SD. Results: Of the 8 IFN+pSS patients 3 exhibited a hyperreactive response, 4 were responding similar as controls and one IFN+pSS did not respond at all. The 4 IFN-pSS patients also were nonresponding. In conclusion, these data indicate a variable response to TLR7 stimulation in pSS and that a the sustained IFN type I activation in a subset of the pSS patients might be due to hyperreactivity of TLR7. 2119 Annelot C Breedveld Cross-talk of dendritic cells with immunoglobulin A activated neutrophils 1 1 A.C. Breedveld , R. Mebius & M. van Egmond 1 1 Department of Molecular Cell Biology and Immunology, VU medical center, Amsterdam, The Netherlands. Immunoglobulin A (IgA) is the most prevalent antibody at mucosal sites and a potent stimulus of neutrophils (PMN) via the IgA Fc receptor (FcαRI). In patients with ulcerative colitis mucosal infiltration of PMNs as well as interaction with DCs is seen. We hypothesize that mucosal immune responses are influenced by FcαRI induced PMN activation. The aim here is to investigate crosstalk of IgA activated PMN with DCs. RA-DCs and MoDCs were derived from monocytes after being cultured for 6 days either with or without retinoic acid (RA). After PMNs phagocytosed IgA coated beads (IgA PMN) they were co-cultured with DCs, after which maturation marker expression was assessed. Cell-cell interactions were analyzed with live cell microscopy and cytokine production in supernatants of co-cultures was measured. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 39 RA-DCs expressed more CD103 compared to MoDCs. After co-culture with IgA PMN, RA-DCs expressed less CD80, CD83 and HLA-DR compared to MoDCs. Cell interactions and bead transfer between IgA PMN and DCs were seen in live cell microscopy. IL-12 was produced by MoDCs after co-culture with IgA PMN, but not by RA-DCs. To conclude, IgA PMN interacted with, and transferred beads to DCs. However, IL-12 was not produced by RA-DCs, suggesting induction of immune tolerance. 2120 Peter L Delputte Development and characterization of species cross-reactive anti-sialoadhesin antibodies for specific macrophage targeting in inflammatory disorders 1 2 1 1 1 2 Marjorie De Schryver , Hanne Van Gorp , Louis Maes , Guy Caljon , Paul Cos , Hans Nauwynck , Peter 1 Delputte 1 Laboratory for Microbiology, Parasitology and Hygiene (LMPH)Department of Biomedical Sciences, 2 University of Antwerp Laboratory for Virology, Ghent University Sialoadhesin (Sn; Siglec-1; CD169) is a sialic acid binding receptor expressed on a subset of resident tissue macrophages, but also on inflammatory monocytes and macrophages. Interaction of Sn with sialic acid containing pathogens, like HIV-1 and Haemophilus influenza, was also observed. Due to the restricted expression pattern and the upregulation on macrophages during inflammatory disorders, Sn might be an interesting target for therapeutic approaches. Recently, we developed new anti-human Sn (hSn) and anti-mouse Sn (mSn) monoclonal antibodies (mAbs). The mAbs were shown, by immunofluorescence and Western blotting, to recognize both Sn expressed on the cell surface. Cross-reactivity was only seen between hSn and porcine Sn (pSn), but not with mSn. Using competitive binding assays, the mAbs could be clustered in different groups. All the new mAbs blocked sialic acid-dependent interactions with Sn, as shown by a red blood cell binding assay. Finally, all newly developed mAbs could clearly induce Sn internalization, a feature that can be used to deliver phenotype-modulation or cytotoxic drugs to macrophages. In conclusion, new cross-reactive mAbs, with characteristics that are interesting to target Sn and Snexpressing macrophage were developed, both for hSn and mSn. 2121 Felicia Bloemendaal The therapeutic efficacy of anti-TNF in inflammatory bowel disease is mediated by activating Fc-gamma receptors and can be improved by defucosylating the Fc region. 1 1 1 1 3 Felicia M. Bloemendaal , Alon D. Levin , 2, Manon E. Wildenberg , Pim J. Koelink , J. Sjef Verbeek , Jill 3 2 4 5 1,2 W.C. Claassens , Geert R.A.M. D’Haens , Brad L. McRae , Gestur Vidarsson , Gijs R. van den Brink 1 Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, the Netherlands Department of Gastroenterology and Hepatology, Academic Medical Center, The Netherlands 3 4 Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands Abbvie 5 Bioresearch Center, Worcester, MA, USA. Department of Experimental Immunohaematology, Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands. 2 INTRODUCTION Anti-TNF antibodies which are clinically effective in inflammatory bowel diseases induce macrophages with a regulatory phenotype which suppress immune activation and contribute to tissue healing. Here we examine the role of Fc signaling in anti-TNF mediated macrophage induction and the therapeutic effect of anti-TNF in intestinal inflammation. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 40 METHODS Anti-TNF efficacy was tested in a mixed lymphocyte reaction and the T cell transfer model of colitis. RESULTS In vitro, adalimumab induced CD206+ macrophages that mediated inhibition of T-cell proliferation. The induction was prevented by blocking FcγRIII. Increasing the binding affinity for FcγRIII by Fc defucosylation increased both induction of CD206+ macrophage and their immunosuppressive properties. In vivo, antiTNF therapy greatly improved colitis, but this effect was completely abrogated in mice lacking all activating Fc-gamma receptors. In line with the in vitro data, increasing Fc binding affinity of anti-TNF by defucosylation potentiated the therapeutic effect in vivo and increased the generation of intestinal CD206+ macrophages. CONCLUSION Activating Fc-gamma receptors are indispensable for the therapeutic effect of anti-TNF in IBD. Enhancing Fc binding affinity of anti-TNF by defucosylation, improves effectiveness in vivo and in vitro. Funding: Abbvie 2122 Felicia Bloemendaal high Anti-TNF skews M1 macrophages towards an IL-10 manner 1 1 IL-12/IL-23 1 low phenotype in an Fc dependent 2 Felicia M. Bloemendaal , Manon E. Wildenberg , Pim J. Koelink , Geert R.A.M. D'Haens and Gijs R. van 1, 2 den Brink 1 Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, the Netherlands Department of Gastroenterology and Hepatology, Academic Medical Center, The Netherlands 2 INTRODUCTION IgG1 anti-TNFs like infliximab but not the anti-TNF Fab’ fragment certolizumab can cause complete mucosal healing in IBD patients. Therefore, features other than TNF neutralisation might contribute to the effector mechanism. In the inflamed gut, macrophages gain an inflammatory phenotype producing little IL-10 and high levels of IL-12 and IL-23, three key cytokines in the pathology of IBD. We investigated the potency of anti-TNF dependent Fc signalling to reverse pro-inflammatory macrophage function. METHODS IFNγ induced M1 macrophages were generated and stimulated with certolizumab or infliximab (IFX) 10 ug/ml and LPS. Cytokines were measured by ELISA. RESULTS We found that anti-TNF reverses the pro-inflammatory cytokine profile of M1 macrophages in an Fc dependent manner by increasing IL-10 and repressing IL-12 and IL-23 production. This effect was blocked by inhibiting Syk, a signalling molecule downstream of activating Fcγ receptors. Moreover, the IFX induced phenotype could be mimicked by combining TNF neutralisation by certolizumab with non-specific complexed IgG1. Finally, in T-cell co-cultures, only IFX significantly suppressed Th1 and Th17 responses. CONCLUSION IgG1 anti-TNF antibodies are capable of converting pro-inflammatory M1 macrophages toward a less proinflammatory phenotype in an Fc dependent manner. 2123 Maria Faas The nuclear receptor Nr4a1 acts as microglia rheostat and serves as therapeutic target in autoimmunedriven CNS inflammation Tobias Rothe, Natacha Ipseiz, Maria Faas, Stefanie Lang, Francesc Perez-Branguli, Daniel Metzger, Hiroshi Ichinose, Beate Winner, Georg Schett, Gerhard Krönke UK Erlangen 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 41 In the central nervous system, microglia are essential for host defense and fulfill key homeostatic functions. It has been shown that different polarization states of microglia can contribute to and ameliorate neurodegenerative and neuro-inflammatory diseases. Our data identify Nr4a1, a nuclear receptor (NR), as key rheostat controlling the activation threshold and polarization of microglia. The expression of this NR in microglia was induced by ubiquitous neuronal-derived stress signals such as ATP. This contributed to the maintenance of a resting and non-inflammatory microglia phenotype. Global deletion as well as myeloid-specific deletion of Nr4a1 triggered the spontaneous and overwhelming activation of microglia. This activation resulted in increased nitric oxide and cytokine production and in an exacerbated and accelerated form of experimental autoimmune encephalomyelitis (EAE). Accordingly, the course of disease could be ameliorated by ligand-induced activation of Nr4a1. Our current data show that Nr4a1 regulates the activation of microglia and reveal Nr4a1 as a new possible target for the treatment of multiple sclerosis and other inflammatory CNS diseases 2124 Wioleta Marut Hypoxia induces CXCL4 secretion by plasmacytoid dendritic cells 1 1 1 1 1 Ottria, A. , Mocholi-Gimeno, E. , Affandi, A. , Tieland, R. , Radstake, T.R.D.J. and Marut, W. 1 1 University Medical Center Utrecht, Utrecht, The Netherlands Background Systemic Sclerosis (SSc) is fibrotic disorder characterized by deregulation of the immune system, Raynaud's phenomenon and vasculopathies that culminates in excessive fibrosis in multiple organs. In a previous study we identified a chemokine - CXCL4, produced by plasmacytoid dendritic cells (pDCs), as a # novel predictive biomarker of SSc . Here we aimed to find the molecular events leading up to CXCL4 secretion of pDCs. Methods pDCs were isolated from peripheral blood mononuclear cells of SSc patients and healthy controls (HCs). Mitochondrial reactive oxygen species (mtROS) generated by pDCs in vitro, both at the basal level and after being exposed to hypoxia and TLR9 ligand, was measured using MitoSOX Red reagent. CXCL4 concentration in the supernatant of pDCs’ was assessed using ELISA. The glycolytic metabolism was evaluated using the Seahorse method. Results 1 SSc pDCs have increased basal level of CXCL4 , mtROS and glycolysis when compared to HCs. pDCs kept under hypoxic condition recapitulated the SSc situation, secreting higher amounts of CXCL4, mtROS and increased glycolysis when compared to normoxic conditions or to stimulation with a wide plethora of other stimuli. These findings suggest that hypoxia might be the factor underpinning the pathogenic contribution of pDCs in SSc patients. Funding: NWO, Marie Curie 2125 Cristina Municio Rueda Methotrexate conditions macrophage functional polarization and negatively regulates TLR and TNF-αinduced inflammation Cristina Municio, Raquel García Campos and Amaya Puig-Kröger Rheumatoid arthritis (RA) is a chronic inflammatory disorder that primarily affects synovial joints. Macrophages critically contribute to inflammation and joint destruction in RA and display a transcriptomic and phenotypic pro-inflammatory polarization profile that resemble GM-CSF differentiated macrophages. We have already described that MTX, the anchor drug for RA treatment, selectively targets GM-CSFprimed macrophages (GM-MØ) through a thymidylate synthase (TS)/p53 axis. However, the exact mechanism underlying the anti-inflammatory actions of MTX remains inconclusive. We now show that 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 42 long-term low-dose MTX treatment reduces LPS, LTA, TNF-α and RA synovial fluid-induced IL-6 and other pro-inflammatory cytokines at the mRNA and protein level in GM-MØ. MTX treatment reduces LPSdependent IκBα degradation. Other mechanisms regulated by MTX that contribute to these effects will be discussed. These results suggest that low-dose MTX imposes a tolerant-like state in susceptible macrophages that renders them less responsive to a subsequent stimulation by pro-inflammatory stimuli like TNF-α or RA synovial fluid. 2126 Federica Raggi Exosomal-miRs in the synovial fluid to monitor arthritis progression 1 1 1 2 1 Federica Raggi ; Martina Morini ; Davide Cangelosi ; Federica Penco ; Alessandra Eva ; Maria Carla Bosco 1 and Luigi Varesio . 1 1 2 Laboratory of Molecular Biology, G.Gaslini Institute, Genova, Italy. Department of Pediatrics, University of Genova and Pediatria II, G Gaslini Institute, Genova, Italy. Juvenile Idiopathic Arthritis (JIA) is a chronic disease characterized by synovial inflammation. Exosomes in synovial fluid represent a mirror of pathogenesis progression. We studied the synovial fluid exosomalmiRs profile, relative to plasma from healthy donors. We found a large number of exosomes in synovial fluid compared to plasma samples. Fifteen exosomal-miRs, including miR-16 and -484, were commonly expressed in plasma and synovial fluid samples. Among miRs expressed in synovial fluid, we found miR210 known to be induced by low oxygen tension, in agreement with the hypoxic status of the synovial fluid microenvironment. Analyzing the synovial fluid cell composition, we demonstrated the presence of a high percentage of macrophages polarized towards a M1 (classically activated macrophages) phenotype (CD80+) and a low percentage of M2 (alternatively activated macrophages) polarized macrophages (CD206+). This macrophage profile correlated with high levels of proinflammatory miRs, such as miRs-155 and -125a-5p, and low levels of miRs associated with the M2 profile, including miRs-26a and -223. We demonstrated that exosomal-miRs reflect the hypoxic inflammatory status of synovial fluid. Synovial exosomal-miRs may represent new biomarker indicators of JIA progression. 2127 Federica Raggi Hypoxia-mediated proinflammatory reprogramming of human macrophages 1 1 2 2 3 Federica Raggi ; Simone Pelassa ; Daniele Pierobon ; Mirella Giovarelli ; Federica Penco Luigi Varesio 1 and Maria Carla Bosco 1 1 2 Laboratory of Molecular Biology, G.Gaslini Institute, Genova, Italy. Center for Experimental Research and 3 Medical Studies (CERMS), Torino, Italy Department of Pediatrics, University of Genova and Pediatria II, G Gaslini Institute, Genova, Italy Mononuclear phagocytes are recruited as primary monocytes (Mn) from the circulation to sites of infection, inflammation, and tumor growth, were they undergo terminal differentiation into macrophages (Mf). Mf can be polarized into M1 or M2 subsets, respectively expressing a proinflammatory or an antiinflammatory phenotype. A common feature of the pathologic environment is represented by hypoxia. The objective of this study was to assess the impact of hypoxia on M1/M2 polarization. M1 (CD80+) and M2 (CD206+) Mf were generated by culturing human Mn with M-CSF for 6 days and LPS or IL4 for the last 24h under normoxia (20%O2) or hypoxia (1%O2). We show that hypoxia amplifies M1 proinflammatory state and reprograms M2 towards a proinflammatory direction by triggering inflammatory and proangiogenic cytokine/chemokine production and upregulating the expression of TREM-1, an Ig-like receptor endowed with proinflammatory properties. TREM-1 activation further amplifies the proinflammatory responses of both Mf subsets. Mf cells are enriched in the hypoxic joints of children affected by Juvenile Idiopathic Artrithis (JIA). We demonstrate a high percentage of TREM-1+ M1 cells in JIA synovial fluid, confirming the role of the pathologic hypoxic environment in skewing Mf towards a M1like proinflammatory phenotype 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 43 Session III Metabolism and Signaling 2131 Maria Cristina Lebre Bioenergetics of human dendritic cell subsets: role of microenvironment 1 1 2 2 2 M. Cristina Lebre , Carla M. S. Ribeiro , Esther Taanman , Toni M. M. van Capel , Esther C. de Jong & 1 Teunis B. H. Geijtenbeek 1 Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, 2 Amsterdam, the Netherlands Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands Dendritic cells (DCs) can shape innate and adaptive immunity toward a variety of functions, which depends on functional plasticity, tissue location as well as DC subset. Importantly, the activation state of DCs correlates with specific types of cellular metabolism. However, it remains unclear whether at steadystate DC subsets from different locations display distinct bioenergetics, which might depend on their function. Here we show that monocyte-derived DC (moDCs), skin-derived immature and mature Langerhans cells (LCs) have increased mitochondrial oxygen consumption, basal respiration and maximal respiration + + compared to circulating blood CD1c and CD304 DCs. ATP production was higher in immature LCs and moDCs compared to other DC subsets while mature LCs have the lowest spare respiratory capacity. From all DC subsets studied, mature LCs display the highest glycolytic capacity. This finding is supported by the fact that in mature LCs glycolysis is predominant compared to oxidative phosphorylation. These data suggest that differences in metabolic programs of DC subsets might have implications in their specific function (e.g. increased glycolysis in moDCs results in enhanced cross-presentation). In addition, these data indicate that at steady-state the cellular microenvironment dictates the energy demands of a particular DC subset. 2132 Jonathan Jantsch Ferritin-mediated iron sequestration stabilizes hypoxia-inducible factor-1α upon LPS-activation in the presence of ample oxygen in dendritic cells 1 2 3 4 5 4 Isabel Siegert , Johannes Schödel , Manfred Nairz #, Valentin Schatz , Katja Dettmer , Christopher Dick , 2 6 4 2 7 Joanna Kalucka *, Kristin Franke , Martin Ehrenschwender , Gunnar Schley , Angelika Beneke , Jörg 8 8 9 6 7 1 Sutter , Matthias Moll , Claus Hellerbrand , Ben Wielockx , Dörthe M. Katschinski , Roland Lang , Bruno 10 11 12 5 1 3 Galy , Matthias W. Hentze , Peppi Koivunen , Peter J. Oefner , Christian Bogdan , Günter Weiss , 2 1,4 Carsten Willam , Jonathan Jantsch 1 Microbiologisches Institut – Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen and Friedrich-Alexander Universität (FAU) Erlangen-Nürnberg, 91054 Erlangen, Germany 2 Department of Nephrology and Hypertension, Universitätsklinikum Erlangen and Friedrich-Alexander 3 Universität (FAU), 91054 Erlangen, Germany Department of Internal Medicine VI, Infectious Diseases, Immunology, Rheumatology, Pneumology, Medical University of Innsbruck, 6020 Innsbruck, Austria 4 Institute of Clinical Microbiology and Hygiene, University Hospital of Regensburg and University of 5 Regensburg, 93053 Regensburg, Germany Institute of Functional Genomics, University of Regensburg, 6 93053 Regensburg, Germany Heisenberg research group, Department of Clinical Pathobiochemistry, Institute of Clinical Chemistry and Laboratory Medicine, University of Technology, 01307 Dresden, 7 Germany Institute of Cardiovascular Physiology, University Medical Center, Georg-August-University 8 Göttingen, 37073 Göttingen, Germany Department of Chemistry and Pharmacy, Inorganic Chemistry, 9 Friedrich-Alexander Universität (FAU) Erlangen-Nürnberg, 91058 Erlangen, Germany Department of 10 Internal Medicine I, University of Regensburg, 93053 Regensburg, Germany Division of Virus-associated 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 44 11 Carcinogenesis, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany European 12 Molecular Biology Laboratory, 69120 Heidelberg, Germany Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine, Oulu Center for Cell-Matrix Research, University of Oulu, 90014 Oulu, Finland Hypoxic and inflammatory conditions both activate transcription factors such as hypoxia-inducible factor (HIF)-1α and nuclear factor (NF)-κB, which play a crucial role in adaptive responses to these challenges. In dendritic cells (DC), lipopolysaccharide (LPS)-induced HIF1α accumulation requires NF-κB-signaling and promotes inflammatory DC function. The mechanisms that drive LPS-induced HIF1α accumulation under normoxia are unclear. Here we demonstrate that LPS inhibits prolyl hydroxylase domain enzyme (PHD) activity and thereby blocks HIF1α degradation. Of note, LPS-induced PHD inhibition was neither due to cosubstrate depletion (oxygen or α-ketoglutarate) nor due to increased levels of reactive oxygen species, fumarate, and succinate. Instead, LPS inhibited PHD activity through NF-κB-mediated induction of the iron storage protein ferritin and subsequent decrease of intracellular available iron, a critical cofactor of PHD. Thus, hypoxia and LPS both induce HIF1α accumulation via PHD inhibition but deploy distinct molecular mechanisms (lack of co-substrate oxygen versus deprivation of co-factor iron). Funding: JJ and ME were funded by the DFG (JA1993/1-1 and EH465/2-1). CB was supported by funds from the Emerging Field Initiative of the FAU Erlangen-Nürnberg (consortium "Metal Redox Inorganic Chemistry") and from the Interdisciplinary Center for Clinical Research (IZKF) of the Universitätsklinikum Erlangen (project A61). JJ, JS, GS and CW were supported by the Center for Kidney and Blood Pressure Research Regensburg-Erlangen-Nuremberg (REN). JS is a recipient of an Else Kröner-Fresenius Exzellenzstipendium (2014_EKES.11). AB is supported by the DFG funded IRTG1816. 2133 Mieke Gouwy CXCL4 and CXCL4L1 differentially affect monocyte survival and differentiation 1 1 1 1 1 2 2 M. Gouwy , P. Ruytinx , E. Radice , F. Claudi , K. Van Raemdonck , R. Bonecchi , M. Locati , J. Van Damme 1 and S. Struyf 1 Laboratory of Molecular Immunology, Rega Institute for Medical Research, Department of Microbiology 2 and Immunology, University of Leuven, Leuven, Belgium Humanitas Clinical and Research Center, Rozzano, Italy Under inflammatory conditions, macrophages can differentiate from peripheral blood monocytes under the influence of various growth factors, cytokines or infectious agents. Chemokines, in particular the platelet-derived CXCL4, are also involved in polarization and survival of monocytes. Indeed, CXCL4 induces the polarization into an M4 phenotype with a unique transcriptome. In this study we compared the effect + of CXCL4 and its variant CXCL4L1 on the differentiation of monocytes. CD14 monocytes were cultured in the presence of CXCL4, CXCL4L1 and M-CSF. The expression levels of several genes and surface receptors were analyzed and cytokines released were measured. In contrast to M-CSF and CXCL4, CXCL4L1 did not + induce monocyte survival. CXCL4L1-treated CD14 monocytes showed a different transcriptional profile compared to CXCL4-polarized macrophages. In contrast to CXCL4, CXCL4L1 (10 µg/ml) stimulated the release of CXCL8 and CCL2. On the other hand, CXCL4 enhanced production of CCL22 compared to + CXCL4L1- and M-CSF-stimulated CD14 monocytes. Furthermore, unlike CXCL4, CXCL4L1 increased expression of the inflammatory chemokine receptors CCR2, CCR5 and CXCR3. We can conclude that both CXCL4 and its variant CXCL4L1 exert a direct, but distinct effect on monocytes yielding differently polarized macrophage phenotypes. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 45 2134 Melanie Pieber AbstracTGFβ regulates persistent neuroinflammation by controlling Th1 polarization and ROS production via monocyte-derived dendritic cells Roham Parsa, Harald Lund, Melanie Pieber, Xing-Mei Zhang, et al., Tobias Suter and Robert A. Harris Intracerebral levels of Transforming Growth Factor beta rise rapidly at the onset of experimental autoimmune encephalomyelitis (EAE), a mouse model of Multiple Sclerosis (MS). We addressed the role of TGFβ responsiveness in EAE by targeting the TGFβ receptor in myeloid cells, determining that Tgfbr2 was specifically targeted in monocyte-derived dendritic cells (moDCs) but not in CNS resident microglia by using bone-marrow chimeric mice. TGFβ responsiveness in moDCs was needed for the remission phase Cre fl/fl since LysM Tgfbr2 mice developed a chronic form of EAE characterized by severe demyelination and infiltration of activated moDCs in the CNS. Tgfbr2 deficiency resulted in increased moDC IL-12 secretion that skewed T cells to produce IFN-γ which enhanced the production of moDC-derived ROS that promote oxidative damage and demyelination. We identified SNPs in the human NOX2 (CYBB) gene linked to the severity of MS, and significantly increased CYBB expression was recorded in PBMCs from both MS patients and MS severity risk allele rs72619425-A carrying individuals. We thus identify a novel myeloid cell-T cell activation loop in the CNS during chronic disease that could be therapeutically targeted. 2135 R.J. Eveline Li Exploration of crosstalk between CLRs and TLRs using Li Rui Jún Eveline, Kalay H., García-Vallejo J.J., Van Vliet S.J., Van Kooyk Y. Department of Molecular Cell Biology and Immunology, O|2 building, VU University Medical Centre, Amsterdam, The Netherlands Dendritic cells (DCs) possess a multitude of pattern recognition receptors (PPRs) to elicit tailor-made immune responses against invading pathogens. Last decennia, C-type Lectin Receptors (CLRs) gained much attention not only as endocytic PRRs, but also as immune modulators. Strikingly, several glycan ligands are shared among CLRs, yet each seems to propagate an unique signalling cascade. The glycans can impact on how DC polarize or suppress T cell responses. Glycans, such as High Mannose-, LewisX-, or LewisY-containing ligands displayed differential DC IL-10 and IL-12 expression profiles after concomitant LPS stimulation. On the contrary α2-3- or α2-6-sialic acid-containing ligands skew DCs towards TREG induction. These glycans target differential CLRs as DC-SIGN and Siglec, respectively. For more insight in immunogenic signalling of the DC-expressed CLR DC-SIGN, as well as the Siglec tolerogenic signalling, we coupled different glycans to a dendrimeric structure, offering multivalent ligand presentation. We used phosphoproteomics to investigate differences in DC protein phosphorylation upon specific glycan engagement, as well as next generation sequencing on a transcriptional level. Detailed analysis of these pathways will reveal how glycans contribute to an immunogenic or tolerogenic fingerprint by modifying DC through CLRs. 2136 Lea A Hampton-O’Neil Investigating the contribution of the Eph receptor family to macrophage island integrity Lea Hampton-O’Neil, Sonam Gurung and Ash Toye School of Biochemistry, Biomedical Sciences Building, University Walk, University of Bristol. UK. Erythropoiesis is one of the most efficient cellular processes in the human body producing approximately 2.5 million red blood cells every second. This process occurs in the bone marrow, whereby the differentiating erythroid cells (erythroblasts) bind a resident macrophage to form an erythroblastic island. However, it is unknown how the differentiating erythroblast is initially attracted to the macrophage to form an island, what then maintains the attraction between these cells and how the enucleated cell 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 46 (reticulocyte) disengages and then migrates to enter the circulation. Using isolated human bone marrow islands and reconstituted in vitro islands, we are investigating the contribution of the Eph receptor family, a tyrosine-kinase receptor family involved in contact inhibition of locomotion, to these processes. We have established which members of the family were present on each cell type throughout erythropoiesis and in macrophages, utilizing RT-PCR, cell surface proteomics, flow cytometry and western blotting. In the future, we hope to investigate the role of individual Eph receptor family interactions to island formation and integrity, using specific inhibitors and also by overexpression or knockdown of the different receptors. Funding: Welcome Trust, NHSBT 2137 Sandra C.S. Cardoso CXCL4 exposure triggers polarization of human DCs towards inflammatory cytokine production and increased antigen cross-presentation Sandra C. Silva Cardoso, Marta Cossu, Lotte Spel, Louis Boon, Marianne Boes*, Timothy R.D.J. Radstake* S.C.S. Cardoso, M. Cossu, T.R.D.J. Radstake: Department of Rheumatology & Clinical Immunology, University Medical Center Utrecht, The NetherlandsL. Spel, M. Boes: Department of Pediatrics, University Medical Center Utrecht, The NetherlandsS.C.S. Cardoso, M. Cossu, L. Spel, M. Boes, T.R.D.J. Radstake: Laboratory of Translational Immunology, University Medical Center Utrecht, The NetherlandsL. Boon: EPIRUS Biopharmaceuticals Netherlands BV, Utrecht, the Netherlands* equal contributions CXCL4 is a chemokine that is abundantly produced by activated platelets, as well as by a variety of immune cells. Beyond the crucial role on physiological processes, CXCL4 also contributes to pathological conditions including cancer, infection and inflammatory diseases such as Systemic Sclerosis (SSc). Chemokines are increasingly investigated for having additional immune modulatory functions beyond steering cell migration. Considering the central role of dendritic cells (DCs) in immune activation, we here studied the effect of CXCL4 on DC differentiation and function. To this end, we compared conventional monocyte-derived (mo)DCs (6-day culture with GM-CSF and IL-4) with those that had been additionally cultured in the continuous presence of CXCL4. We observed that in the steady state, CXCL4moDCs already express decreased levels of CD1a and CD1c but increased expression of CD141, CD83, CD86, and MHCI and II molecules compared to conventional moDCs. We found that CXCL4moDCs appear more sensitized for TLR-ligand triggering, as upon exposure to several TLR agonists they more vigorously upregulated CD86, CD83 and MHCI. Additionally, CXCL4moDCs secreted increased amounts of IL-12 and TNF in response to poly(I:C) and R848 (TLR3 and TLR7/8 ligands, respectively). Furthermore, while the internalization of BSA was comparable, CXCL4moDCs were less efficient at antigen processing. CXCL4 moDCs are however more potent at stimulating antigen-specific + CD8 T-cell responses, which we show using the CMV pp65 protein as model antigen and CMV-pp65+ specific CD8 T-cell clones. Our data supports that increased levels of CXCL4 found in the circulation and tissues of patients, may contribute to immune dysregulation through altering DC differentiation and function. Targeting CXCL4 using therapeutic intervention may benefit immune-mediated conditions modulated by CXCL4, including those patients suffering from SSc. Funding: S.C.S. Cardoso: PhD grant from the Portuguese Fundação para a Ciência e a Tecnologia (SFRH/BD/89643/2012) T. R. D. J. Radstake: ERC st arting grant & Dutch Arthritis Foundation 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 47 2138 Zrinka Oreskovic Effect of cytokine microenvironment on porcine dendritic cells activation in vitro 1, 2 1 1 Oreskovic Z. , Leva L. , Kudlackova H. , Faldyna M. 1 1 2 Veterinary Research Institute, Immunology Department, Brno, Czech Republic Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic Dendritic cells (DCs) represent a heterogeneous group of major antigen presenting cells playing a pivotal role in driving the activation and differentiation of naïve T lymphocytes into distinct cell types. Since the microenvironment in which DC are present is crucial for their activation, effect of interleukin-4 and interferon-gamma on porcine monocyte-derived DC (MDDC) was studied in vitro. MDDC generated from CD14+ peripheral blood monocytes were cultivated under influence of IL-4 or IFN-γ or left without an additional cytokine treatment. To induce the immune response, they were stimulated with protein KLH for 48h. Activation of dendritic cells was measured using quantitative RT-PCR, ELISA and western blot. This experiment clearly showed that both expression of certain cytokines (IL-1β, IL-10, IL-12, IL-23 and TGFβ) and therefore possible polarization of cell-mediated response were dependent on cytokine microenvironment DCs were previously exposed to. Also, it suggests that in vitro stimulation of DCs can be used as a model for further experiments in vaccine studies. Funding: Ministry of Agriculture of Czech Republic (RO0516,QJ1510218) and Ministry of Education, Youth and Sport of Czech Republic (LO1218). 2139 Anna Zakrzewicz Secretory leukocyte protease inhibitor (SLPI) suppresses ATP-mediated release of IL-1beta from human monocytes 1 1 2 3 1 Anna Zakrzewicz , Sigrid Wilker , Sabina Janciauskiene , Ritva Tikkanen , Winfried Padberg , Veronika 1,4 Grau 1 Laboratory of Experimental Surgery, Department of General and Thoracic Surgery, Justus-Liebig2 University Giessen, Giessen, Germany Department of Respiratory Medicine, Hannover Medical School, 3 Hannover, Germany Institute of Biochemistry, Medical Faculty, Justus-Liebig-University Giessen, Giessen, 4 Germany Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Giessen, Germany IL-1beta is a key pro-inflammatory cytokine produced by monocytes/macrophages that is associated with diverse inflammatory disorders. IL-1beta release typically requires two danger signals. LPS induces at first pro-IL-1beta synthesis, whereas extracellular ATP, a typical second signal triggers inflammasome activation, cleavage of pro-IL-1beta and release of mature IL-1beta. Mechanisms controlling IL-1beta maturation in the presence of both danger signals are of clinical interest but so far remained largely unexplored. We demonstrated that SLPI, a well-known anti-protease of the lung, dose-dependently and efficiently inhibits ATP-mediated release of IL-1beta from human monocytes. Using a panel of receptor-specific nicotinic antagonists we have provided evidence that nicotinic acetylcholine receptors containing subunits alpha7, 9 and 10 are involved in the SLPI-dependent inhibition of inflammasome activation. These results were further corroborated by siRNA silencing of these subunits. Moreover, we showed that this mechanism depends on Src kinase activity. Overexpression of dominant negative Src or application of PP2, a Src inhibitor, abolished the inhibitory effect of SLPI. The discovery of a novel anti-inflammatory mechanism mediated by SLPI may contribute to the development of therapies preventing inflammatory disorders. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 48 2140 Manon E. Wildenberg Autophagy regulates dendritic cell migration through Rac1 – implications for thiopurine therapy 1,2 1 1 2 1,2 Manon E. Wildenberg , Pim J. Koelink , Kay Diederen , Marileen M. Prins , Anje A. te Velde , Veerle J. 3 3 4 4 2 Nuij , Maikel P. Peppelenbosch , Max Nobis , Owen J. Sansom , C., Geert R. D’Haens and Gijs R. van den 1,2 Brink 1 Tytgat Institute for Intestinal and Liver Research, Academic Medical Center, Amsterdam, The Netherlands Department of Gastroenterology and Hepatology, Academic Medical Center, Amsterdam, The 3 Netherlands Department of Gastroenterology and Hepatology, Erasmus MC, Rotterdam, The Netherlands 4 Beatson Institute for Cancer Research, Bearsden, Glasgow, UK 2 A variant of ATG16L1 that reduces autophagy is associated with Crohn’s disease (CD), although the etiopathology remains unclear. We have shown a regulatory role for autophagy during DC - T cell interactions and as cytoskeletal regulation is a key element of this interaction, we here investigated the role of autophagy in DC cytoskeletal organization and function . low fl/fl Method: Autophagy DC were generated experimentally or cultured from CD11cCre-Atg5 mice or risk allele carriers. Correlation between ATG16L1 genotype and response to thiopurines was determined in two cohorts. low Results: Autophagy DC displayed loss of filopodia and increased membrane ruffling due to Rac1 hyperactivity. Consenquently, adhesion to various substrates was increased, while random as well as directional migration were strongly impaired in vitro and in vivo. Strikingly, migration could be restored by addition of the Rac1 inhibitor and common IBD drug 6-TG, suggesting this as an effector mechanism for this drug. Indeed, the ATG16L1 risk variant associated significantly with response to thiopurine treatment in patients with CD. Conclusions: Our results suggest that defective autophagy results in hyperactive Rac1 and impaired myeloid cell migration in CD patients and point to correction of migration defects as an effector mechanism for thiopurine treatment. 2141 Tiago F. Carvalheiro Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) modulates monocyte-derived dendritic cell differentiation. 1,2 1,2 2 1,2 Tiago Carvalheiro , Wioleta Marut , Linde Meyaard , Timothy R. D. J. Radstake . 1 Department of Rheumatology and Clinical Immunology, University Medical Center Utrecht, Utrecht, The 2 Netherlands. Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands. Background: LAIR-1 is an inhibitory receptor widely expressed with abundant availability of ligands. The LAIR-1 functions include the ability to regulate in vitro function of NK, T and B cells, but also cytokine mediated signals in dendritic cells (DCs) and monocytes. Aim: Assess the ability of LAIR-1 to modulate monocyte-derived dendritic cell differentiation and function. Materials and methods: Freshly isolated monocytes from healthy donors were differentiated in DCs with GM-CSF and IL-4, in presence of anti-LAIR1 agonist. At day 6 the phenotype was evaluated and IL-12, IL-10 and TNF-α gene expression was assessed after stimulation with TLR4 agonist and IFN-α. Results: LAIR-1 crosslinking during monocyte-DC differentiation results in morphologic changes but also in phenotypic modifications which includes downregulation of CD1a and CD1c and upregulation of CD141. The expression of co-stimulatory molecules like CD86 is enhanced as well as the MHC-I (HLA-ABC) expression. TLR4 stimulation of LAIR-1 differentiated DCs results in a reduced IL-12 and IL-10 gene expression while the presence of IFN-α leads to an increased expression of IL-12, IL-10 and TNF-α. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 49 Conclusion: LAIR-1 plays a role in DC differentiation resulting in changes in DC fate with possible implications in inflammation but also in tissue immune homeostasis. Funding: Tiago Carvalheiro is supported by a grant from the Portuguese national funding agency: Fundação para a Ciência e a Tecnologia (SFRH/BD/93526/2013). 2142 Zsófia Agod DC autophagy in response to LPS and IFN-γ is controlled by SLAMF5 via p38 and IRF8 Zsófia Agod; Árpád Lányi Department of Immunology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary SLAMF5 (Signaling lymphocytic activation molecule family 5; CD84) is a self-ligand transmembrane receptor expressed on haematopoietic cells. By ensuring prolonged T cell-B cell interaction SLAMF5 is required for the differentiation and optimal function of follicular T helper cells. However, its role in the regulation of dendritic cell (DC) functions is poorly understood. We found that when human monocytes were differentiated into monocyte-derived DCs (moDCs) in the absence of SLAMF5 their phenotype became reminiscent of DCs developing in the presence of autophagy inhibitors. As compared to controls, siRNA mediated knock down of SLAMF5 generated moDCs with diminished CD1a expression, which in response to LPS and IFN-γ activation produced significantly higher amounts of IL-1β and IL-23, while IL-12 production was decreased. Furthermore, LPS and IFN-γ-induced phosphorylation of p38 and the level of IRF8 were also diminished in SLAMF5-silenced moDCs compared to control cells. Likewise, conversion of LC3-I to LC3-II, an indicator for autophagy, was decreased as well. Our results suggest that SLAMF5 via enhancing p38 and IRF8 activity is a regulator of autophagy influencing the differentiation process and the responsiveness of DCs. Funding: PN-II-PT-PCCA-2013-4-1583; OTKA K 109444 2143 Mateus Tomczyk Cardiac macrophages in myocardial infarction – role of hemeoxygenase-1 M. Tomczyk, K. Szade, I. Kraszewska, K. Bukowska-Strakova, A. Jozkowicz, J. Dulak, A. Jazwa Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Acute myocardial infarction (MI) activates immune system. Well-timed resolution of inflammation is crucial for proper regeneration of wounded myocardium. Heme oxygenase-1 (HMOX1) is a potential therapeutic target in cardiovascular disease, due to its anti-oxidative and anti-inflammatory properties. The aim of this study was to investigate the role of HMOX1 in suppression of post-ischemic immune -/responses in a mouse model of MI. Hmox1 mice subjected to surgically induced MI had significantly +/+ lower ejection fraction than their wild-type Hmox1 littermates. Additionally, there were higher numbers ++ + of inflammatory (Ly6C CD43 ) monocytes in peripheral blood and increased expression of monocyte -/chemoattractant protein-1 (MCP-1) in the hearts of Hmox1 mice. Moreover, pro-inflammatory MHC+ + + ++ + + -/II Ly6C CD11c and MHC-II Ly6C CD11c cardiac macrophages were more abundant in post-MI Hmox1 +/+ -/+/+ than Hmox1 mice. Transplantation of Hmox1 bone marrow to Hmox1 recipients revealed that deprivation of HMOX1 in blood cells does not significantly aggravate recovery after MI. HMOX1 deficiency significantly impairs heart function following MI due to monocyte/macrophagemediated adverse cardiac remodeling and impaired repair potential. Funding: Supported by Sonata Bis grant no. 2014/14/E/NZ1/00139 and Preludium grant no. 1027.0641.1.2016 both from the Polish National Science Center. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 50 Session IV Macrophage function in health and disease 2146 Esther Heideveld Cultured human macrophages as a model for central macrophages in erythroblastic islands 1 2 2 1 Esther Heideveld , Maartje van den Biggelaar , Floris van Alphen , Marieke von Lindern , Emile van den 1 Akker 1 Sanquin Research, afdeling Hematopoiesis, Amsterdam, Nederland, en Landsteiner Laboratory, 2 Academisch Medisch Centrum, Universiteit van Amsterdam, Amsterdam, Nederland. Sanquin Research, Afdeling Plasma Proteins, Amsterdam, Nederland, en Landsteiner Laboratory, Academisch Medisch Centrum, Universiteit van Amsterdam, Amsterdam, Nederland. Bone marrow (BM) erythroblastic islands are ill-defined structures directing erythroid maturation and clearance of nuclei presumably via TAM-receptors on central macrophages. Research into the erythroid + island is hindered by the absence of an in vitro model. Here we report the differentiation of CD14 peripheral blood mononuclear cells into M2c-like macrophages that harbor characteristics of BM resident + macrophages. CD14 cells cultured in serum-free media supplemented with Stem cell factor, Erythropoietin and dexamethasone (Dex) differentiate into M2c-like macrophages expressing CD163, CD169, CD206, CXCR4 and HLA-DR, a process dependent on glucocorticoid receptor activation. Protein ontology after Mass spectrometry of Dex and non-Dex cultured monocytes showed Dex-mediated enrichment of lysosome, endocytosis and endothelial development (e.g. STAB1, IL13RA1, CD81, SLC1A3 and FKBP5). Increased TAM family members MerTK and AXL mRNA expression was also observed. Functionally, the M2c-like macrophages phagocytose zymosan and co-culturing erythroid cells and + + extruded nuclei with these macrophages lead to GPA (erythroid marker)/CD14 cell aggregates suggesting formation of erythroid islands. Interestingly, an M2C-like macrophage that expresses CD163, CD206 and CD169 was observed in human BM aspirates resembling Dex differentiated macrophages. We speculate that glucocorticoids induce a monocyte differentiation program to macrophages that may be used to model erythroblastic island-mediated erythropoiesis. Funding: Landdsteiner foundation for Blood Transfusion Research Landsteiner (LSBR: 1141) 2147 Stephen J Jenkins A mechanistic study of the role of gender in regulation of peritoneal macrophage homeostasis 1 1 1 Calum Bain , Doug Gibson , Philippa Saunders , Stephen J Jenkins 1 1 Centre for Inflammation Research, Queens Medical Research Institute, University of Edinburgh, Edinburgh, UK We recently demonstrated that murine embryonic peritoneal macrophages are replaced with age by + CCR2 bone marrow-derived cells in a process that is highly dependent on gender. Replacement of the embryonic cells is not due to their intrinsic exhaustion, suggesting that this population is actively displaced and outcompeted by recruited cells. Notably, monocytes recruited during homeostasis first + lo differentiate into MHCII F4/80 intermediate cells that exhibit a unique activation state before later hi + + acquiring an F4/80 MHCII Gata6 resident peritoneal macrophage phenotype. Here, we investigate the mechanism by which gender controls the half-life of resident peritoneal macrophages and explore the implications of this on their homeostatic functions in male and female mice. We also perform loss-of+ function experiments to identify factors that control survival and differentiation of the MHCII intermediate to understand how the longevity of this macrophage compartment impacts the balance between self-maintenance of resident cells and replacement from the bone marrow. Funding: MRC New Investigator Research Grant 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 51 2148 Pieter J.M. Leenen Aging and macrophage polarization: a model study 1,3 1 1 2 1 Adriaan van Beek , Joanne Hoogerland , Tim de Winter , Chiara Milanese , Adri van Oudenaren , Pier 2 3 1 Mastroberardino , Huub Savelkoul , Pieter Leenen * *corresponding author 1Dept. Immunology and 2Dept. Genetics, Erasmus MC, Rotterdam, 3Cell Biology and Immunology Group, Wageningen University, The Netherlands Aging is associated with systemic low-grade inflammation. Macrophages might play key roles in this process as they are immune sentinel cells and prime cytokine producers. We used a mouse model to study how aging affects generation of bone marrow-derived macrophages (BMDM) and their polarization into different phenotypes. BMDM were generated from young (6 wk), mature (4 mo) and old (≥18 mo) C57BL/6 mice. The yield of BMDM in 7d culture did not differ between age groups. Unpolarized BMDM from old mice expressed some markers, incl. F4/80, at slightly lower level; M1- and M2-associated markers were similarly regulated among groups. Also, similar activation-related profiles were observed for senescence marker p16/CDKN2a and autophagy marker p62/SQSTM1. In contrast, we found that LPS activation of BMDM from old mice led to a reduced increase in phosphorylated ribosomal S6, reflecting diminished mTOR pathway activation and ribosomal biogenesis. Energy metabolic profiles showed similarly high oxygen consumption rates (OCR) for M2-, and high extracellular acidification rates for M1-BMDM in all age groups. Unpolarized BMDM from mature and old mice demonstrated lower OCR compared to young BMDM. Together, these findings indicate relatively unaffected responsiveness to polarizing triggers of in vitro BM-derived macrophages from old mice. Funding: Top Institute Food and Nutrition (TIFN) 2149 David Haschka Different iron handling capabilities of human monocyte subsets 1 1 2 1 1 1 Haschka D *, Tymoszuk P *, Sopper S , Seifert M , Theurl I #, Weiss G # *,#These authors contributed equally to this work 1 Medical University of Innsbruck, Department of Internal Medicine VI, Infectious Diseases, Immunology, 2 Rheumatology, Pneumology, Innsbruck, Austria. Medical University of Innsbruck, Department of Internal Medicine V and Tyrolean Cancer Research Institute, Innsbruck, Austria. Because iron significantly effects pathogen growth and impacts on host responses we questioned whether the functional diversity of human monocyte subsets may be attributed to alterations of cellular iron metabolism. We re-analyzed existing microarray data of three published studies for differential expression of genes linked to iron metabolism. Expression of identified candidates was investigated on protein level by flow cytometry and on transcript level by fluorescence activated cell sorting and ensuing real-time PCR. Functional studies were done by adding substances altering iron metabolism and by infection assays. In the retrospective gene chip analysis we were able to identify a set of significantly regulated iron-related genes, including the sole known iron exporter ferroportin (FPN1). As confirmed by flow cytometry, FPN1 was up-regulated in classical monocytes, while transferrin receptor was mainly expressed on intermediate followed by classical monocytes. These results might be due to different erythrophagocytosis capabilities of the subsets and were reflected in varying bacterial loads upon infection with Salmonella. Our results strongly suggest different iron-handling phenotypes and thus varying intracellular iron availability in the three monocyte subsets, which likely impacts on the defense of intracellular pathogens. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 52 2150 Sudhanshu Bhushan Testicular interstitial fluid polarizes blood monocyte derived M1 macrophages towards the M2 phenotype Sudhanshu Bhushan, Ming Wang, Monika Fijak, Andreas Meinhardt Department of Anatomy and Cell Biology, Unit of Reproductive Biology, Justus-Liebig-University, Aulweg 123, 35385 Giessen, Germany Macrophages are important for the activation of innate immune responses and maintain organ homeostasis in a tissue specific manner. Testicular macrophages (TM) comprise the largest immune cell population in the testis and reside in the testicular interstitial space. TM are assumed to play an important role in maintaining testicular immune privilege. Numerous studies have indicated that the interstitial fluid (IF) surrounding TM has immunosuppressive properties, which may influence the phenotype of macrophages. However, the molecular mechanisms involved in this phenomenon are almost completely elusive. To investigate the influence of the testicular microenvironment on the establishment of the macrophage phenotype, we have treated blood monocyte with testicular interstitial fluid and characterized the macrophage phenotype by flow cytometry and immunofluorescence analysis. Our preliminary data reveal that IF induce the polarization of blood monocytes towards an M2 phenotype as evidenced by enhanced expression of the CD163 marker. In contrast, treatment with rat serum alone did not increase the expression level of CD163. Moreover, qRT-PCR data revealed that expression of M2 macrophage markers increased after IF treatment, whilst in contrast M1 macrophage markers decreased. Treatment of IF treated macrophages with LPS induced strong secretion of the anti-inflammatory cytokine IL-10, which lead to activation of the STAT3 signaling pathway in these cells. Of note, IF polarized macrophages attenuated LPS stimulated activation of the NF-κB signaling pathway by delaying degradation of IκBα. Taken together, these results suggest that the testicular microenvironment polarizes macrophages towards an immunosuppressive M2 phenotype, which may play an important role in the establishment and maintenance of the immune privilege of the testis. Funding: Deutsche Forschungsgemeinschaft project grant (AZ: BH 93/1-1) 2151 Mohammed Ghiboub SP140 is a Potential Mediator of Macrophage Activation in Inflammatory Bowel Disease Mohammed Ghiboub, Andrew Y.F. Li Yim, Jing Zhao, Nicky Harker, Anje A. te Velde, Peter Henneman, Rab Prinjha, David F. Tough, Wouter J. de Jonge. Academic Medical Center, Tytgat Institute for Liver and Intestinal Research, University of Amsterdam, 1105 BK Amsterdam, The Netherlands.Academic Medical Center, Department of Genome Diagnostics, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.Epinova DPU, Immuno-inflammation TA Unit, GSK Medicines Research Centre, Stevenage, Hertfordshire, SG1 2NY, UK Single nucleotide polymorphisms in the SP140 gene are significantly associated with Crohn’s disease (CD). In addition, in a recent 450K DNA-Methylation array screen, we identified two hypermethylated regions in SP140 in CD patients. Following on from these human genetic data, we are investigating potential functional links between SP140 and CD, focussing on its role in human macrophages. Non-polarised (M0) macrophages derived from primary human monocytes were polarized to inflammatory M1 or to regulatory M2 cells using IFNγ or IL-4 respectively. SP140 mRNA levels were measured before or after stimulation of these cells with LPS. At baseline, SP140 was expressed at significantly higher levels in M1 cells compared to M0 or M2 cells, while LPS induced an increase in SP140 mRNA in all macrophage subsets. Immunofluorescence staining of SP140 in tissue sections from healthy and inflamed colon (CD) showed an enhanced SP140 protein expression in inflamed tissue macrophages compared to macrophages in healthy tissue. Finally, SP140 knockdown in the human monocyte cell line 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 53 THP1 led to reduced CD14 mRNA expression and inhibited PMA-induced differentiation to M0 cells. Taken together, these data suggest a role for SP140 in monocyte-to-macrophage maturation and M1polarization, providing insight into the link between SP140 and IBD. Funding: EU 2152 Jules Petit Increased oxygen but not nitrogen radicals in trained carp macrophages Jules Petit, Annelieke Wentzel, Maria Forlenza, Geert F. Wiegertjes Department of Animal Sciences, Cell Biology and Immunology Group, Wageningen University, P.O. Box 338, 6700 AH, Wageningen, The Netherlands Little is known about the evolutionary conservation in fish of “trained immunity”, a form of immunity described in most detail in humans and mice. Given the evolutionary position of teleost fish as early vertebrates with a fully developed immune system, it is likely that innate immune cells of fish should possess the capacity for specific features of trained immunity, such as the ability to mount a heightened response to a secondary stimulus in a non-specific manner. We performed our studies on common carp, a very close relative of zebrafish which has become a popular organism for the study of vertebrate gene function. We modified our well-established in vitro model of head kidney-derived macrophages of carp, where the head kidney is the equivalent of bone marrow. In humans, a crucial receptor involved in trained immunity is NOD2. Using a NOD1/2 specific ligand (PGN from E. coli K12), we were able to train carp macrophages by stimulating for only 2 hours. After training, carp macrophages were rested for 6 days, subsequently we measured the response to B-glucans (zymosan). We observed a heightened response to a secondary stimulus in a non-specific manner, but not as increased nitrogen radical production but only as increase in oxidative burst. Gene expression of pro-inflammatory cytokines confirm the training of carp macrophages. Funding: NWO-FAPESP Programme Biobased Economy 2153 Annelieke S. Wentzel Conservation of macrophage polarization in fish Annelieke S. Wentzel, Jules Petit, Inge R. Fink, Maria Forlenza, Geert F. Wiegertjes Department of Animal Sciences, Cell Biology and Immunology Group, Wageningen University, P.O. Box 338, 6700 AH, Wageningen, The Netherlands Macrophages of higher vertebrates can express a range of activation states, with the extremes termed M1 and M2. Neither the evolutionary conservation of these activation states, nor the exact microbial and/or cytokine stimulants involved, have been examined in detail in lower vertebrates. We have shown that macrophages of teleost fish, including carp, have the ability to polarize into activation states typical of classical (M1) and alternative (M2) extremes upon stimulation with LPS, or exogenous cAMP, respectively. Upon this polarization, carp macrophages display functional profiles and several molecular markers that indicate the M1-M2 dichotomy could be an intrinsic property of macrophages which arose early in evolution. Owing to the more recent discoveries of IFN-y and lL-4/IL-13-like cytokines in teleost fish, we now can also study cytokine-dependent polarization of carp macrophages. Interferon-y amplifies LPSinduced polarisation into M1-like profiles of carp macrophages, including the induction of nitric oxide. Strikingly, IL-4/IL-13 appears to increase levels of arginase along with an activation profile that at least partly overlaps with that of cAMP-induced M2 macrophages. Thus, the chief macrophage M1 and M2 activation states appear to operate under the guidance of primordially conserved principles. Funding: Horizon 2020-funded EU project ParaFishControl and FP7-funded project NEMO 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 54 2154 Christine W. Bruggeman Human splenic red pulp macrophages have a distinctive FcγR expression pattern 1 1 2 1 Christine W. Bruggeman , S.Q. Nagelkerke , J. den Haan , R. van Bruggen1, T.K. van den Berg , T.W. 1,3 Kuijpers 1 Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Academic Medical 2 Center (AMC), University of Amsterdam, Amsterdam, The Netherlands Department of Molecular Cell 3 Biology and Immunology, Free University Medical Center (VUmc), Amsterdam, The Netherlands Emma Children's Hospital, Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands In immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA) respectively platelets and red blood cells are opsonized by autoantibodies and phagocytized by splenic red pulp macrophages, via interaction with receptors that recognize IgG (FcγRs). To better understand this process of phagocytosis, we developed a method to isolate macrophages from human spleen. We investigated the FcγR expression pattern of the red pulp macrophages and their contribution to the phagocytosis of opsonized cells in comparison to blood monocyte-derived macrophages. The FcγR expression of splenic macrophages was further confirmed by immunostaining of cryostat sections of human spleen tissue. We found that red pulp macrophages express FcγRIIa and FcγRIIIa and hardly if any FcγRI and FcγRIIb, in sharp contrast to monocyte-derived macrophages that mainly express FcγRI, FcγRIIa and FcγRIIb. To investigate the contribution of the different FcγRs in the phagocytosis of opsonized red blood cells, a phagocytosis assay was performed with and without blocking the different FcγRs, showing that FcγRI and FcγRIIa are the main contributors to phagocytosis of opsonized cells. Understanding the regulation of FcγR surface expression on spleen macrophages could be used to optimize the reatment of ITP and AIHA. 2155 Branislav, Krljanac Identification and selective gene targeting in M2 macrophages using a novel Rentla-Cre mouse model Branislav Krljanac*, Christoph Koch*, Ronald Naumann** & David Voehringer* *Department of Infection Biology, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU), 91054 Erlangen, Germany**Transgenic Core Facility, MPI of Molecular Cell Biology and Genetics, 01307 Dresden, Germany Alternatively activated (M2) macrophages (Mfs) contribute to protective immunity against helminths and tissue repair. Identification of M2 cells in tissues is difficult due to scarcity of specific mouse models. Rentla is strongly and specifically upregulated in murine M2 cells induced by IL-4/IL-13. To obtain a novel M2-reporter mouse model we generated Rentla-Cre BAC-transgenic mice. To visualize Rentla-expressing cells Retnla-Cre mice were crossed to the R26-tdtomato reporter strain. Retnla-Cre_R26-tdtomato mice exhibited Retnla_tomato protein expression in subsets of bone marrow-derived, peritoneal and small intestine Mfs, as well as in subsets of eosinophils and neutrophils and in type II pneumocytes. Sorting for Retnla_tomato+ cells revealed that a proportion of peritoneal Mfs and blood neutrophils actively produced Retnla mRNA. Lungs of mice infected with the helminth Nippostrongylus brasiliensis showed a specific accumulation of Rentla_tomato and PD-L2+ interstitial M2 Mfs, while alveolar Mfs remained largely Retnla_tomato- and PD-L2-negative. Thus, Retnla-Cre mice may serve as a valuable tool to visualize/isolate M2 cells allowing for a deeper understanding of their function under steady-state conditions, in defence against helminths and remodeling/wound healing responses. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 55 2156 Gertrud Vieten Autophagy as a characteristic feature of neonatal peritoneal macrophages 1 1 1 2 3 § § 1 G. Vieten , T. Winterberg , Y. Yu , S. Groos , J.K. Park , T. Ulas , J. Schultze , B. Ure , J.F. Kuebler 1 2 1 3 Department of Pediatric Surgery, Institute of Cell Biology in the Center of Anatomy, Department of § Nephrology, Hannover Medical School, Hannover, Germany, LIMES-Institute, University of Bonn, Bonn; Germany Purpose: A growing body of evidence indicates that autophagy is a key component in macrophage (MØ) development and polarization, thus affecting all areas of the immune response. Autophagy has also been shown to be activated in various tissues after birth. As neonatal MØs are distinct in phenotype and function compared to their adult counterparts, we hypothesized that autophagy could be a feature of naive neonatal MØs. Results: MØs were collected by peritoneal lavages from C57Bl/6 mice. Neonatal MØs displayed a high amount of cytoplasmic vesicular structures. Electron microscopy frequently showed autophagosomes, whereas this double membrane structure was largely absent in the adult counterparts. Besides this, transcriptome analysis revealed an increased homeostatic activity as well as a decrease in resolving phase genes. Conclusion: Besides the already described phenotypic features neonatal MØs appear to display an increased autophagic activity in combination with a tissue and developmental homeostatic “priming”. Although this seems to be well adapted to the major changes in the tissue microenvironment after birth, these changes could have effects on the immunological balance by down regulation of pro-resolving and anti-inflammatory reactivity. Funding: DFG KU 2759/1-1 2157 Selien Sanches Scriptaid and SBHA have no effect on functional recovery after spinal cord injury 1* 1* 1 1,2 1 Sanchez S. , Gou Fabregas M. , Lemmens S. , Dooley D. , Sommer D. , Hendrix S. 1 1 2 . Dept. of Morphology & Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium. . Laboratory of Experimental Hematology, Vaccine and Infectious Disease Institute (Vaxinfectio), University of Antwerp, Antwerp, Belgium After spinal cord injury (SCI), excessive inflammation and glial scar formation are important causes of secondary damage to the healthy tissue surrounding the lesion and thus exacerbate the underlying neurodegenerative events. During this inflammatory response monocyte-derived macrophages are major players in modulating functional recovery after SCI. Pro-inflammatory M1 macrophages dominate the spinal cord after injury and exert detrimental effects by secreting multiple pro-inflammatory and neurotoxic factors and by stimulating the formation of the inhibitory fibrotic scar. In contrast, antiinflammatory M2 macrophages may contribute to regeneration after SCI. In this context, epigenetic modulation of gene expression is emerging as a new possibility to overcome regenerative failure after SCI. We hypothesized that the histone deacetylase inhibitors (HDACi) induce a beneficial M2 immune switch, resulting in improved functional recovery after SCI. Our in vitro results show that the HDAC inhibitors scriptaid and SBHA are able to reduce IL-6 and NO production in bone marrow derived macrophages upon LPS treatment, which points towards an impaired M1 polarization. However, in vivo, scriptaid and SBHA showed no improved functional recovery after SCI. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 56 Session V Antigen processing and presentation Thursday September 22nd 2205 Marjorie De Schryver Characterization of monoclonal antibody-induced internalization of human and mice sialoadhesin on macrophages and the effect on phagocytosis M. De Schryver, A. Leemans, G. Caljon, D. Cappoen, L. Maes, P. Cos and P. Delputte Laboratory for Microbiology, Parasitology and Hygiene (LMPH)Department of Biomedical Sciences, University of Antwerp, Wilrijk Sialoadhesin (Siglec-1 or CD169; Sn) is a macrophage-specific receptor. During inflammation and certain diseases, an upregulation of Sn is observed on macrophages, but also on blood monocytes and dendritic cells. Due to the interesting expression pattern, Sn might be an interesting target for therapeutic approaches. It was shown that Sn is able to internalize after binding with specific mAb. Previous research was performed for porcine Sn. Since the cytoplasmic tail of Sn is not conserved between species and Sn does not contain any known signalization motif, mAb-induced internalization of human (hSn) and mice Sn (mSn) might be different. We studied mAb-induced internalization of hSn and mSn in primary cells and cell lines. Internalization of both hSn and mSn, in primary cells and cell lines, were time-dependent, dynamin-dependent and clathrinmediated. The internalized vesicles for primary cells were located mostly in the endosomes and barely in the lysosomes. In contrast internalized vesicles were also observed in lysosomes in cell lines. The effect of mAb-targeting was studied on the phagocytosis capacity of primary cells. The phagocytosis was significantly reduced using carboxylate-modified beads and this effect was observed for a long time after targeting. However, phagocytosis of heat inactivated pathogens was augmented. 2206 Cecilia I Forss Activation of murine and human dendritic cells by Aspergillus fumigatus 1 1 2 2 3 3 C. Forss , P. Cook , M. Bertuzzi , E. Bignell , M. Enerbäck , D. Cunoosamy , A. MacDonald 1 1 Manchester Collaborative Centre for Inflammation Research, University of Manchester, 46 Grafton Street, Manchester M13 9NT, United Kingdom 2 Manchester Fungal Infection Group, University of Manchester, 46 Grafton Stress, Manchester M13 9NT, United Kingdom 3 Respiratory, Inflammation and Autoimmunity iMed, AstraZeneca R&D, Pepparedsleden 1, Mölndal, Sweden Aspergillus fumigatus (Af) has the capacity to cause a range of responses, from low level inflammation in healthy people to life-threatening conditions in the immunocompromised. We have focused on understanding early events in the immune response towards Af, with an emphasis on dendritic cells (DCs). 4 h post exposure, ~50% of in vitro generated DCs were positive for Af spores, and they showed higher expression of CD40, CD80 and CD86 compared to cells that were spore negative. Interestingly, while Af induced intermediate up-regulation of DC activation markers, it induced only low levels of cytokine production. As the type of interaction between DCs and Af likely influences downstream activation and polarization of T cells, we are currently dissecting the impact of internalised vs. surface bound spores, compared to soluble fungal extracts, on activation of DCs in vitro. In parallel, we have used murine intranasal exposure to live Af to study Af uptake by, and activation of, pulmonary DC subsets in vivo. Data generated so far indicate a fungal life stage-dependent up-regulation of activation markers, accompanied 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 57 by induction of low-level secretion of inflammatory cytokines. Ongoing work is addressing the ability of Af activated DCs to influence T cell activation and polarization in vitro and in vivo 2207 Anno Saris The role of platelets in the induction of alloimmunization after platelet transfusion 1 2 3 3 1 3 A. Saris , P.F. van der Meer , A.B. Meijer , I. Peyron , S.M. van Ham , J.J. Zwaginga , A. ten Brinke 1 1 2 Sanquin Research, Immunopathology, Amsterdam, the Netherlands Sanquin Blood Bank, Product and 3 Process Development, Amsterdam, the Netherlands Sanquin Research, Immunopathology, Amsterdam, 4 the Netherlands Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands Platelet transfusion can elicit alloimmune responses leading to alloantibody formation. These alloantibodies can results in ineffective platelet transfusions due to rapid clearance of platelets opsonized with alloantibodies. Introducing universal leukoreduction of platelet concentrates, significantly reduced the frequency of alloimmunization, indicating the importance of WBCs in the induction of alloimmunization. However, a residual risk for alloimmunization remains and it is currently unknown if 6 this is due to the residual WBCs (<1*10 / transfusion) or if platelets can also induce alloimmunity. Therefore we investigated the involvement of platelets in the indirect pathway of alloimmunization. To do this, monocyte-derived dendritic cells (DCs) were incubated with allogeneic platelets. Confocal microscopy confirmed that platelets are internalized by DCs. Peptide elution of HLA-DR of these DCs demonstrated that platelet specific peptides were presented. Altogether indicating the involvement of platelets in the indirect pathway of alloimmunization after platelets transfusion. Currently we investigate the effect of storage and pathogen reduction techniques on phagocytosis and/or presentation of platelets by dendritic cells. 2208 Christoph Lippuner Are miRNAs involved in HLA-DR expression on the cell surface? -a miRNA screen Maja Houseman, Matthias Staiger, Zaira Leni, Frank Stüber, Christoph Lippuner Department of Anaesthesiology and Pain Therapy, University Hospital Bern, SwitzerlandandDepartment of Clinical Research, University Bern, Switzerland. Clinical studies prove that cell surface expression of HLA-DR is down-regulated in patients undergoing surgery. This is interpreted to indicate a suppression of the immune system. The amount of HLA-DR molecules available for antigen presentation is associated with morbidity in the perioperative setting, i.e. nosocomial infections. A better understanding of HLA-DR surface expression is of importance to possibly interfere with perioperative HLA-DR downregulation. The question, whether miRNAs are involved in CLIP-loaded and peptide-loaded MHC-II expression will be tested with a flow cytometry based high throughput screen (HTS) with 2048 miRNA mimics in MelJuSo cells. None of the screened miRNAs changed the amount of CLIP-loaded MHC-II molecules on the cell surface. In contrast, HTS identified 52 miRNAs that lead to an up- (up to 2.6 fold) or down-regulation (up to 5.0 fold) of HLA-DR surface expression. 16 of the miRNAs with the strongest impact on HLA-DR surface expression were selected and verified successfully. A lentiviral based system was used to express these miRNAs in monocyte derived dendritic cells. Analysis thereof will be presented. The results may lead to a better understanding of the mechanisms of antigen presentation. This might help to define new possible methods to support the immune system in critically ill patients in the future. Funding: Internal funding INSEL hospital 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 58 2209 Robert, Blomgran HIV interferes with Mycobacterium tuberculosis antigen presentation in human dendritic cells 1 1 2 3 Susmita K. Singh , Anna-Maria Andersson , Rada Ellegård , Cecilia S. Lindestam Arlehamn , Alessandro 3 2 1 1* Sette , arie Larsson , Olle Stendahl , and Robert Blomgran 1 2 Division of Medical Microbiology, and Division of Molecular Virology, Department of Clinical and 3 Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden. La Jolla Institute for Allergy and Immunology, Department of Vaccine Discovery, 9420 Athena Circle, La Jolla, CA, 92037, USA. HIV coinfection is the most prominent risk factor for progression of Mycobacterium tuberculosis (Mtb) infection into active tuberculosis disease (TB). The mechanisms behind the increased transition from latent to active TB in coinfected individuals have not been well elucidated at the cellular level. We hypothesized that HIV infection contributes to Mtb pathogenesis by interfering with the DC-mediated immune control. Human immature DCs coinfected with HIV/Mtb had decreased expression of HLA-DR and the costimulatory molecules CD40, CD80, and CD86. In addition, Mtb-infected DCs triggered a significant release of IL-6, IL-1β and TNFα, whereas coinfected DCs did not. DCs antigen presentation capacity was assessed by co-culturing DCs and autologous Mtb antigen-specific CD4+ T cells. IFN-γ release was significantly reduced when Mtb antigen-specific CD4+ T cells had been activated with coinfected DCs compared to Mtb-infected DCs, and this effect was attributed to Mtb antigen processing rather than peptide-MHC class II loading. Evaluating autophagy as a measure of vesicular processing/maturation further revealed that HIV efficiently blocks initiation of this pathway. Overall, our results demonstrate that HIV impairs Mtb antigen presentation in DCs, thereby suppressing an important cell linking innate and adaptive immune responses in TB. Funding: 521-2012-1807 and 348-2013-6588 (VR, The Swedish Research Council); 2014-0578 (HLF, The Swedish Heart-Lung Foundation) 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 59 Session VI Functional heterogeneity of APC’s 2213 Julia S Brunner Arginase I and Osteoclastogenesis 1 2 2 1 1 3 3 1 Brunner JS , Hofmann M , Saferding V , Vogel A , Paar H , Chen L , Cheng P , Schabbauer G , Blüml S 1 2 2 Medical University Vienna, Institute for Physiology, Vienna, Austria; Medical University Vienna, Dpt. of 3 Rheumatology, Vienna, Austria; Bio Cancer Treatment International Ltd., Hong Kong, China Osteoclasts (OCs) are giant, multi-nucleated cells that derive from the monocyte-macrophage lineage and are critically involved in bone turnover. They are further known as the main effector cells for development of age-related osteoporosis. While the role of Arginase I within certain myeloid lineages such as macrophages is well appreciated, its role within osteoclasts is relatively unknown. Our aim was therefore to investigate the importance of the enzyme in the context of osteoclastogenesis. We analyzed osteoclast formation in vitro in the presence and absence of recombinant Arginase I (recARG1) and its inhibitor norNOHA. In osteoclast differentiation assays, we show that Arginase I is strongly downregulated during osteoclastogenesis, suggesting involvement of this enzyme in OC differentiation. We observed that recArgI specifically inhibits RANKL-mediated terminal differentiation of OCs, but has no effect on MCSF dependent generation of OC precursors. In line, we could show that addition of recombinant Arginase I negatively regulated the expression of classic RANKL induced osteoclastic marker genes such as TRAP and Cathepsin K. We therefore propose that recARG1 might be a potent inhibitor of osteoclastogenesis and could prove itself to be useful for the treatment of osteoclast driven diseases, such as osteoporosis. 2214 Urszula Florczyk Heme oxygenase-1 impact on osteoclasts formation from macrophage precursors Florczyk U, Józefczuk E, Bukowska-Strakova K, Mendel M, Nowak W, Józkowicz A, Dulak J Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland Acting as antioxidant heme oxygenase-1 (HO-1) may be involved in osteoclastogenesis. We aimed to investigate the role of HO-1 in osteoclasts (OCL) differentiation from bone marrow (BM) cells and specifically from bone marrow macrophages (BMMs). BM cells were isolated from HO-1-deficient mice (HO-1-/-) and in sequence stimulated with M-CSF alone and M-CSF with RANKL to induce OCL formation. The lack of HO-1 diminished the expression of OCL markers such as NFATc-1, cathepsin K and calcitonin receptor as well as TRAP enzyme activity (vs. HO1+/+). Our preliminary data points at decreased macrophage/monocyte ratio in HO-1 deficient BM population treated with M-CSF (vs. HO-1+/+). It may suggest the influence of HO-1 on macrophage differentiation. On the other hand, in murine macrophage cell line, RAW264.7, induction of HO-1 by CoPPIX or hemin inhibited NFATc-1. Also, BMMs HO-1-/- harvested and plated in equal numbers with HO-1+/+ were more effective in formation of TRAP+ cells. It may suggest the influence of HO-1 on macrophage precursor abundance. However, no difference in overall survival of M-CSF-treated BM cells were detected pointing at the importance of other processes such as proliferation. In summary, HO-1 effect on osteoclastogenesis seems to be complex and presumably partially relies on the regulation of macrophage precursors. Funding: Supported by Iuventus Plus grant from the Ministry of Science and Higher Education (0244/IP1/2013/72). 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 60 2215 Nathalie van Leeuwen Transcriptional profiling reveals functional dichotomy between human slan-positive cells and conventional myeloid dendritic cells 1 2 1 3 Nathalie van Leeuwen-Kerkhoff , Kristina Lundberg , Theresia M. Westers , Shahram Kordasti , Tanja D. 4 2 1 de Gruijl , Malin Lindstedt and Arjan A. van de Loosdrecht 1 2 Department of hematology, VU university medical center, Amsterdam, The Netherlands; Department of 3 immunotechnology, Lund University, Lund, Sweden; Department of haematological medicine King's 4 College London and King's College Hospital, London, United Kingdom; and Department of medical oncology, VU university medical center, Amsterdam, The Netherlands Human 6-sulfo LacNac+ (slan) cells have been described both as part of the dendritic cell (DC) compartment as well as of the CD16+ monocytes. The aim of this study was to directly compare the DClike functionalities of slan+ cells with conventional DC subsets (cDC, CD1c+ and CD141+) in human peripheral blood (PB). Transcriptional profiling and subsequent functional assays were carried out on isolated cells. Their ability to up-regulate co-stimulatory molecules, secrete cytokines and induce T cell proliferation was assessed. Detailed genome-wide analysis showed clear transcriptional differences between cDC and slan+ cells in which cDC clustered together. Genes upregulated in slan+ cells were mainly enriched in complementrelated and immune response pathways. Functionally, slan+ cells showed impaired upregulation of costimulatory molecules. They mainly secreted IL-1β and IL-6, whereas cDC produced high levels of IL12p70. Lower percentages of T cell proliferation were found in co-cultures with slan+ cells compared to cultures with CD1c+ DC. In conclusion, our study confirms a distinct role for slan+ cells compared to cDC. While the cDC are able to induce and imprint functionality on primary T cell responses, slan+ cells harbor phenotypic and functional traits consistent with the amplification and support of inflammatory responses. 2216 Alsya J Affandi Plasmacytoid DCs altered biology in systemic sclerosis correlate with low expression of RUNX3 1,2,3 1,2,3 4,5 Alsya J. Affandi, MSc ; Jasper C. A. Broen, MD, PhD ; Lara Bossini-Castillo, PhD ; Wioleta Marut, 1,2 1,2,3 1,2 1,2 PhD ; Lenny van Bon, MD, PhD ; Marzia Rossato, PhD ; Renoud J. Marijnissen, PhD ; Soley 6 6 3 4 Thordardottir, MSc ; Harry Dolstra, PhD ; Maria Trojanowska, PhD ; Javier Martin MD, PhD ; Robert 3 1,2,3 Lafyatis, MD ; Timothy R. D. J. Radstake, MD, PhD 1 Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands; Department of Rheumatology and Clinical Immunology, University Medical Center Utrecht, Utrecht, The 3 Netherlands; Rheumatology Section, Department of Medicine, Boston University School of Medicine, 4 Boston, MA, USA; Consejo Superior de Investigaciones Científicas (IPBLN-CSIC), Instituto de Parasitología 5 y Biomedicina López-Neyra, PTS Granada, Granada, Spain; Wellcome Trust Sanger Institute, Hinxton, 6 Cambridge, CB10 1SA, UK; Department of Medicine, Laboratory of Hematology, Radboud University Medical Center, Nijmegen, The Netherlands. 2 Systemic sclerosis (SSc) is an autoimmune disease with unknown pathogenesis manifested by inflammation, vasculopathy and fibrosis in skin and internal organs. Type I IFN signature found in SSc propelled us to study plasmacytoid dendritic cells (pDCs) in this disease. Objective: To identify candidate pathway underlying pDC aberrancies in SSc and to validate its function on pDC biology. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 61 Methods: PCR-based transcription factor profiling and methylation status analyses, and SNP genotyping by sequencing were performed in pDCs from healthy or SSc patients. In vitro and in vivo experiments were used to study Runx3 function. Results: We found transcription factor RUNX3 to be downregulated in SSc pDCs. We identified a nonsynonymous SNP and observed a higher methylation status that correlated with RUNX3 level and disease susceptibility. In CD34-DC differentiation assay, RUNX3 knockdown lead to lower pDC number, as seen in SSc. Furthermore, (p)DCs from Runx3-/- mice exhibited enhanced cytokine expression upon TLR stimulation. Conclusion: The presence of an associated SNP and higher methylation status suggest two causal pathways underlying low RUNX3 expression in SSc pDC, that can contribute to enhanced cytokine production upon activation. This knowledge on altered pDC biology opens novel avenues for therapeutic intervention in SSc. 2217 Susanne van der Grein Vitamin D3-tolerized dendritic cells release extracellular vesicles that suppress proinflammatory cytokine production 1 1 1 1 1 Susanne van der Grein , Tom Driedonks , Tom Groot Kormelink , Henrike Jekel , Marca Wauben , Esther 1 Nolte-’t Hoen 1 Department of Biochemistry and Cell Biology; Faculty of Veterinary Medicine; Utrecht University; Utrecht, the Netherlands Immunogenic and tolerogenic DC transmit different signals to interacting T cells, thereby regulating the type and strength of T cell responses. Besides direct cell-cell contact and release of soluble mediators, the transfer of protein, lipid, and RNA-encoded messages via extracellular vesicles (EV) plays a role in this process. We investigated whether immunogenic and tolerogenic DC release EV that differ in molecular contents and how these EV affect T cell responses. Tolerogenic DC were generated by treating murine bone marrow-derived DC with 1α,25-dihydroxyvitamin D3 (VitD3). EV were quantified and characterized using high-resolution flow cytometry and added to in vitro DC-CD4+ T co-cultures to assess their effect on T cell activation and cytokine production. EV released by VitD3-DC display a disparate surface protein profile compared to EV from untreated DC, including a substantially reduced level of MHC class II. EV from VitD3-DC but not from untreated DC were able to inhibit production of the proinflammatory cytokine IL-17 from an IL-17-producing memory T cell population, but not from the naïve T cell pool during a Th17 induction regime. These data suggest that tolerogenic DC EV may aid in protecting peripheral tissues from potentially harmful inflammatory responses by raising the threshold for T cell reactivation. Funding: European Research Council 2218 Emiliano, Melandri A novel method for direct isolation of mouse cross-presenting dendritic cells with outstanding performance Frank Ralf Morrissey-Wettey, Emiliano Melandri, Andrzej Dzionek + Cross-presenting, CD8α conventional DCs (cDCs) play a pivotal role in the induction of protective CTLresponses that are vital for the eradication of cancers and viral infections. They have the unique ability to internalise dead cells for processing and cross-presentation on MHC class I molecules, which makes them + superior in the induction of cytotoxic T-cell responses. In the past, studies of CD8α cDCs have been hampered by their scarcity and the lack of specific cell surface markers. However, it was recently 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 62 demonstrated that cross-presenting cDCs in lymphoid and non-lymphoid tissues specifically express the two newly discovered receptors Clec9A and XCR1. The expression of the latter correlates with the cDCs’ cross-presentation potential (ref. 1). ® Combining the recombinant REAfinity™ anti-mouse XCR1 mAb with our MACS technology, we developed + a new single-step method for the fast and easy isolation of XCR1 cross-presenting cDCs with remarkable recovery and high purity, and without the need for laborious flow sorting. This new isolation method will facilitate further functional studies of the DC subset biology, which ultimately will help to develop new therapeutic strategies targeting DCs. No affiliation 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 63 Session VII Clinical applications and tumor immunology 2222 Niels Heemskerk Novel antibody therapy formats to treat colorectal liver metastases Niels Heemskerk, Marijn Bögels, Marjolein van Egmond VU University medical center, Department of Molecular Cell Biology and Immunology, OI2 Colorectal surgery is still the most successful treatment to extend cancer patient survival and quality of life. Nonetheless many patients develop liver metastasis, since surgery paradoxically increases the risk of liver metastasis development. Pre-operative monoclonal antibody (mAb) treatment prior to surgery may limit development of liver metastasis, since it was very effective in eliminating circulating tumor cells in experimental models. However, once liver metastases had been established, mAb therapy was ineffective, most likely due to an immunosuppressive environment that is found in many solid tumors. In this study, we aim to unravel how to overcome the immunosuppressive environment that hampers antibody treatment. Here we use the human FcRI transgenic C57BL/6 mice in combination with the fully syngeneic B16F10 mouse melanoma model to investigate the effects of IgA and IgG co-treatment on established liver metastases. We anticipate that IgA anti-tumor mAbs have the ability to trigger neutrophil mediated tumor cell killing in vivo, hereby releasing danger signals that potentially induce long-term adaptive immunity against colon metastasis. Additionally, the combination of IgA and IgG may enhance the killing of tumor cells by other immune cells through FCγ receptor signaling and release of pro inflammatory cytokines. Thus, enhancing the destructive capacity of innate and adaptive immune cells through induction of a pro-inflammatory tumor microenvironment may improve current therapeutic strategies for the treatment of solid cancers. Funding: WCR 2223 Carla M.A. van Alem Targeted delivery of liposomal prednisolone after renal ischemia reperfusion injury in the rat 1 1 2 1 1 3,4 1 C.M.A. van Alem , T. Bezhaeva , M. Boonstra , R.A. Lalai , A. Koudijs , J.M. Metselaar ,, M.E. Reinders , C. 1 1 van Kooten , J.I. Rotmans . 1 2 3 Nephrology, LUMC, The Netherlands. Surgery, LUMC, The Netherlands. Enceladus Pharmaceuticals, 4 Naarden, the Netherlands. Experimental Molecular Imaging, University Clinic, RWTH-Aachen University, Aachen, Germany. Background: Corticosteroids (CS) are often used to treat inflammatory kidney diseases but are associated with severe side effects. Targeted delivery of CS to phagocytic cells in the inflamed kidney might circumvent these. This study focuses on targeting liposomes to phagocytic cells in the kidney after ischemia reperfusion injury (IRI) in the rat. Methods: Rats were subjected to renal IRI and treated with a) fluorescent liposomes or b) 10 mg/kg liposomal prednisolone (LP), prednisolone (P), empty liposomes (EL), or saline (S). At 4 days, the uptake of liposomes was evaluated and the kidneys were analysed by histology and RT-PCR. Results: Fluorescent liposomes predominantly accumulated in the injured kidneys compared to the contralateral kidneys (MFI 10.6±3.1 vs 7.3±2.5, P<0.05). While treatment with LP and P did not affect CD68(+) macrophages after damage, LP vs P-, EL- and S- treatment led to more anti-inflammatory CD163(+) macrophages (2.8±1.5 vs 0.9±1.9%, 0.3±0.1% and 0.6±0.6%, p<0.05). In addition, MCP-1 mRNA was reduced from 8.7±2.4% to 3.5±2.0 and 4.8±2.9 (p<0.05) after S vs LP and P treatment. Conclusion: LP treatment of renal IRI results in local uptake in the kidney and a shift of macrophages towards an anti-inflammatory state. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 64 2224 Miche Rombouts Silencing RNA as a tool to overcome the TH1-polarizing capacity of mature dendritic cells 1 2 2 2 2 1 3 M Rombouts , AH Nuyts , J Derdelinckx , K Peeters , K Van Camp , GRY De Meyer , H Goossens , ZN 2 1,2 1 2 Berneman , I Van Brussel , DM Schrijvers and N Cools 1 2 Laboratory of Physiopharmacology; Laboratory of Experimental Hematology, Vaccine & Infectious 3 Disease Institute (Vaxinfectio); Laboratory of Medical Microbiology, Vaccine & Infectious Disease Institute (Vaxinfectio); University of Antwerp, Wilrijk, Belgium Considering the established role of dendritic cells (DC) in maintaining the balance between immunity and tolerance, it has been suggested that DC-based strategies could be instrumental in correcting autoreactive immune responses. We aimed to confirm the key role of hallmark molecules required for Th1 polarization using neutralizing antibodies. Next, we investigated if modifying their expression using silencing RNA could modulate Th1-mediated immune responses. We demonstrate that a pro-inflammatory cytokine cocktail supplemented with resiquimod and IFN-γ induces highly potent human mature DC (CRImDC) compared with DC stimulated with the “gold standard” cytokine cocktail. Moreover, CRImDC are strong inducers of IFN-γ secretion by naive T cells in an IL-12dependent, but CD80 and CD70-independent, manner. By modifying the expression of IL-12 using silencing RNA we could modulate the Th1/Th2-polarizing capacity of both murine and human DC. However, since this strategy focuses on only one target, it is likely that IL-12-silenced DC can still convert towards a more immune-stimulatory phenotype following in vivo activation. Simultaneous targeting of multiple key effector genes might offer a solution. Ultimately, this can improve the safety and efficacy of DC-based therapies to restore the immunological balance in autoimmune diseases. 2225 Irina Mitrofanova Chitinase-like protein YKL-39 stimulates monocyte migration and reversely correlates with hematogenous and lymphatic metastasis in human breast cancer 1 2 1,4 1,4 1,4 Irina Mitrofanova , Tengfei Liu , Michail Buldakov , Marina Zavjalova , Nikolai Litviakov , Nadezhda 1,4 1,2,3 Cherdyntseva , Julia Kzhyshkowska 1 Laboratory for Translational Cellular and Molecular Biomedicine, Tomsk State University, Tomsk, Russia Medical Faculty Mannheim, Heidelberg University, Institute of Transfusion Medicine and Immunology, 3 Mannheim, Germany German Red Cross Blood Service Baden-Württemberg – Hessen, Mannheim, 4 Germany Tomsk Cancer Research Institute, Tomsk, Russia 2 Human chitinase-like proteins are considered as а novel biomarker of cancer, inflammation and tissue remodeling. The role of YKL-39 and its associations with tumor progression has not been studied until now. YKL-39 gene expression was measured by RT-PCR. We found that it was strongly up-regulated with IL4/TGF-beta in human macrophages on 6 and 12 days. With using of IF staining and confocal microscopy the intracellular distribution of YKL-39 was checked. YKL-39 was detected in the TGN, p62lck positive late endosomes, Lamp-1 positive lysosomes and CD63 positive secretory lysosomes. Monocyte migration was performed with a trans-well system. Recombinant YKL-39 significantly enhanced the migration of monocytes by 1.9 fold in 1 hour, and reached to 4.9 fold in 3 hours. The correlations of YKL-39 with metastasis in human breast cancer were verified by RT-PCR, IHC and IF staining. In patients with breast cancer, low levels of YKL39 correlated with a higher frequency of lymphatic metastasis (p=0.036, n=74) and hematogenous metastasis (p=0.0337, n=74). The pre-dominant TAM phenotypes were YKL39+/stabilin-1+. Thus YKL-39 is secreted by alternatively activated macrophages via lysosomal pathway, stimulates migration of monocytes and demonstrates a reverse correlation with hematogenous and lymphatic metastasis in human breast cancer. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 65 2226 Ilka Knippertz Transcriptional targeting of mature DCs by the newly characterized human CD83 promoter-prospects for new in vivo DC vaccination strategies 1 1 2 1 3 Ilka Knippertz , Marcello F. Stein , Thomas H. Winkler , Andrea Deinzer , Dirk M. Nettelbeck , Michael 4 5 6 1 Stürzl , Robert K. Slany , Thomas Werner , and Alexander Steinkasserer 1 Department of Immune Modulation, University Hospital Erlangen, 91052 Erlangen, Germany; 2 Department of Biology, Nikolaus-Fiebiger-Zentrum für Molekulare Medizin, Friedrich-AlexanderUniversität Erlangen-Nürnberg, 91054 Erlangen, Germany; 3 German Cancer Consortium, DKFZ, 69120 Heidelberg, Germany; 4 Division of Molecular and Experimental Surgery, University Hospital Erlangen, 91054 Erlangen, Germany; 5 Department of Biology, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91058 Erlangen, Germany; 6 Genomatix Software GmbH, 80335 Munich, Germany One of the biggest challenges facing cancer therapy is the development of new treatment strategies, including DC-based cancer vaccines. Since the CD83 molecule is only expressed by immunogenic mature DCs (mDCs) but not by immature, tolerogenic DCs (iDCs), the CD83 promoter represents an ideal tool for transcriptional in vivo targeting of mDCs directly in tumor patients. In this regard, ChIP-on-chip-, biocomputational-, gene reporter-, and EMSA-analyses revealed a transcriptional activation complex formed by a 3D-folding process involving three distinct DNA regions. Herein, we identified a complex framework of IRF- and NFκB-transcription factor binding sites involved in the exact arrangement of the tripartite structure, with NFkB-family members to synergize with interferon regulatory factors. Noteworthy, this complex is specifically active in mDCs, but not in iDCs, nor in subsets of activated B- and T cells. Addressing the therapeutic potential of this promoter-complex for its use to transcriptionally target mDCs we generated different adenoviral vectors encoding therapeutic molecules such as MelanA and IL-12 under the control of this CD83 promoter. Transduction of human mDCs demonstrated the CD83 promoter to efficiently and specifically drive expression of both therapeutic molecules simultaneously only in mDCs. Therefore, this newly identified DC- and stadium-specific CD83 promoter complex allows for the development of new in vivo-targeting strategies for next generation DC-vaccination directly in patients suffering from malignant disorders. 2227 Stefano Bonelli TARGETING NEUROPILIN-1 THROUGH SINGLE-DOMAIN ANTIBODIES: A NOVEL STRATEGY TO TACKLE TUMOR-ASSOCIATED MACROPHAGES 1,2 1,2 1,2 1,2 Bonelli Stefano , Laoui Damya , Keirsse Jiri , Schoonooghe Steve , Van Ginderachter Jo 1 1,2 2 Myeloid Cell Immunology Lab, VIB Inflammation Research Center, Ghent, Belgium Lab of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium. Neuropilin-1 (Nrp-1) is a transmembrane receptor which binds several ligands including VEGF-A, class 3 semaphorin proteins, and PlGF. Recently, Nrp1 expression in the tumor environment has recently sparked the interest of scientists as a potential target for cancer therapy. In particular, Nrp1 expression in infiltrating immune cells from the host has drawn the attention of immunologists. Nrp1 has been shown to be needed by tumor-associated macrophages to migrate to hypoxic regions of the tumor, where they can establish an immunosuppressive and pro-angiogenic environment and mediate tumor progression. Hence, blocking Nrp1 by means of antibodies or antibody-like molecules holds great therapeutic promise. Given these premises, we have generated a panel of single-domain antibody fragments (also referred to as ‘nanobodies’) with high affinity for human and mouse Nrp1 which have shown to bind to Nrp1expressing cell cultures and ex vivo primary cells. Because of their small size and high affinity, we are evaluating the nanobodies’ ability to penetrate the tumor tissue and to target tumor-associated 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 66 macrophages. All together, these studies will establish a novel nanobody-based strategy to tackle the immunosuppressive function of tumor-associated macrophages and, ultimately, will provide a new tool for cancer therapy. 2228 Lenneke A.M. Cornelissen Tumor sialylation negatively instructs T cell mediated anti-tumor responses while promoting tumorassociated Tregs 1 1 1 1 Lenneke Cornelissen , Maurizio Perdicchio , Ingeborg Streng-Ouwehand , Steef Engels , Marleen I. 1 2 1 3 1 Verstege , Dirk Geerts , Sandra J. van Vliet , Wendy W.J. Unger * and Yvette van Kooyk *. *Shared authorship 1 Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The 2 Netherlands. Department of Pediatric Oncology/Hematology, Erasmus University Medical Center 3 Rotterdam, The Netherlands. Present address: Department of Pediatrics, div. Infectious Diseases, ErasmusMC-Sophia Children’s Hospital, Rotterdam, The Netherlands A tolerogenic microenvironment, which allows escape from tumor-specific effector cells and an altered glycosylation pattern are two important hallmarks of cancer. Overexpression of carbohydrate structures containing Sialic acids (Sia) is frequently found within the tumor-associated glycan repertoire. Therefore, we hypothesized that Sia may play a crucial role in inducing the tolerogenic microenvironment. Indeed, a genetically engineered glycovariant of the B16 melanoma harboring low Sia grew slower than wild-type high Sia B16 tumors [1]. Moreover, higher frequencies of IFNγ-producing effector T-cells were detected in low high the tumor-microenvironment of Sia compared to the Sia tumors. This was paralleled by a 50% + + low reduction in FoxP3 CD4 regulatory T-cell (Treg) frequencies in Sia tumors [1]. Since antigen presenting cells (APC), and in particular dendritic cells, are instrumental for induction of effective adaptive anti-tumor immune responses, we are currently investigating the effect of tumor-associated Sia on APC phenotype and function. Sia can be recognized by APCs through Siglec receptors. Most Siglec receptors contain an intracellular ITIM motif, suggesting that Sia might be able to induce a tolerogenic APC phenotype via their high low interaction with Siglecs, thus explaining the observed in vivo characteristics of the B16 Sia vs B16 Sia tumors. Reference 1. Perdicchio, M., et al., Tumor sialylation impedes T cell mediated anti-tumor responses while promoting tumor associated-regulatory T cells. Oncotarget, 2016. Epub ahead of print. 2229 Athanasios A. Blanas Type II Lewis Antigens in Colorectal Cancer (CRC): Evaluation of their immune modulatory capacity on antigen presenting cells 1 1 Athanasios Blanas , Yvette van Kooyk and Sandra J. van Vliet 1 1 Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands Increased fucosylation arises as a new hallmark of Colorectal Cancer (CRC). Specifically, high expression of type II Lewis antigens on the surface of colon cancer cells has been strongly associated with poor prognosis, disease progression and metastatic potential. Recognition of tumor Lewis epitopes by innate antigen presenting cells bearing glycan-specific receptors (such as C-type Lectins) can lead to a complex interaction between these types of cells. The influence of Lewis determinant overexpression on antigen presenting cells and their ability to shape subsequent anti-tumor immune responses have not been fully described so far. In order to study the immunological aspects of differential tumor glycosylation, we generated isogenic Lewis antigen positive or negative glycovariants of the murine CRC cell line MC38. Specific fucosyltransferases that account for increased Lewis-X/Y antigen formation in tumors were overexpressed in MC38, following gene activation using the CRISPR-Cas9 technology. Our aim is to 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 67 investigate the effect of differentially fucosylated MC38 on dendritic cells and macrophage phenotype and + + function as well as their ability to induce effector or regulatory CD4 /CD8 T cell responses. 2230 Sophie A. Dusoswa Repurposing glioblastoma exosomes as personalized multi-antigenic anti-tumor vaccine 1 1 1 2 2,3 Sophie A. Dusoswa , Sophie K. Horrevorts , Sjoerd T. Schetters Jordi Berenguer , Thomas Würdinger , 1 1 Yvette van Kooyk , Juan J. Garcia-Vallejo 1 Department of Molecular Biology and Immunology, VU University Medical Center, Cancer Center 2 Amsterdam, Amsterdam, The Netherlands Department of Neurosurgery, VU University Medical Center, 3 Cancer Center Amsterdam, Amsterdam, The Netherlands Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, USA. Introduction: The composition of glial tumor derived exosomes (TEX) reflects plasma membrane and cytosol-derived proteins of the donor cell and represents, therefore, interesting candidates for antiglioma vaccination. However, recent literature reports potent immunosuppressive characteristics of TEX. TEX surface glycosylation may alter C-type lectin receptor triggering and/or cargo internalization thereby modifying the immunogenic/tolerogenic balance of the cellular immune response. The aim of this study is to investigate whether we can repurpose TEX from tolerogenic vesicles towards an immunogenic vehicle for multi-antigenic vaccination by de novo glycosylation of their surface. Methods: Prior to their chemo-enzymatic modification, TEX were carefully characterized by ELISA and electron microscopy. TEX uptake and downstream routing was assessed by imaging flow cytometry analysis. Results: The surface glycan profile of TEX was dominated by sialic acid-capped N-glycans, high mannose glycans and truncated Tn-bearing O-glycans, with low affinity for DC-SIGN. In contrast, glycan-modified TEX, which had been introduced with a high affinity ligand for DC-SIGN, were efficiently taken up by human dendritic cells and fused with the endosomal pathway. Conclusion: Glioma-derived exosomes exhibit glycans that are typically associated with tolerogenic responses, indicating a possible role in glioma immune escape. Repurposing through de novo glycanmodification could provide a strategy for multi-antigenic anti-glioma vaccination. 2231 Inès Dufait Granzyme B expression in myeloid-derived suppressor cells: a current unidentified mechanism? 1,2 3 2 Inès Dufait , David Escors , Mark De Ridder and Karine Breckpot 1 1 2 Vrije Universiteit Brussel, Laboratory for Molecular and Cellular Technology, Jette, Belgium UZ-Brussel, 3 Vrije Universiteit Brussel, Department of Radiotherapy, Jette, Belgium Navarrabiomed-Fundacion Miguel Servet. Immunomodulation group. Pamplona. Navarra, Spain For a long time, only NK cells and cytotoxic T lymphocytes (CTLs) were considered to express granzyme B (GzmB). However, certain publications reported the expression of GzmB in other cell types. Several myeloid-derived suppressor cell (MDSC) -mediated immune suppressive functions are already described. However there could still remain some unrecognized mechanisms. In our culture system that exists of in vitro- generated MDSCs, we have found a high GzmB expression and secretion, but a distinct lack of perforin. Perforin together with GzmB is a key component of the lytic machinery of CTLs, leading to activation of caspase-3, which results in apoptosis. However evidence of perforin-independent functions of GzmB also exist, such as involvement in inflammation and anoikis. Similar observations, as found in the in vitro cultures, were made in MDSCs isolated from tumor-bearing mice. In both tumor and spleenderived MDSCs (in breast, lung and colon cancer models) we could detect GzmB, but no perforin. In vitro functional assays did not yet reveal any contributions of GzmB to MDSCs immunosuppressive function, possibly because of the lack of environmental perforin. However, we believe that we might have 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 68 discovered a new therapeutic target to dampen MDSC responses and thereby facilitate the introduction of immunotherapy. 2232 Sophie K. Horrevorts Glycan modified apoptotic vesicles as a multi-antigenic source for anti-tumor vaccination. 1 1 1 2 3 Horrevorts S.K. , Stolk D.A. , van Vliet S.J. , de Gruijl T.D. , van de Loosdrecht A.A. and van Kooyk Y. 1 1 Dept. of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, the 2 Netherlands Dept. of Medical Oncology, VU University Medical Center, Amsterdam, the Netherlands 3 Dept. of Hematology, VU University Medical Center, Amsterdam, the Netherlands To counteract tumorderived immune suppression, effective immunotherapies should boost existing or elicit de novo, tumorspecific immune responses. Vaccines for the induction of these responses are not yet realized, because of knowledge gaps concerning the choice of suitable antigens and targeting of antigens to dendritic cells (DCs), cells that play a vital role in the presentation of antigens to the T cells. We investigated a promising new approach which encompasses vaccination with apoptotic vesicles derived from the tumor. Using this strategy, DCs are not only targeted with a broad variety of known tumor antigens, but also with (neo)antigens that are unknown. To enhance the uptake of apoptotic vesicles and processing of vesiclecontained antigens, we modified their glycocalyx, which resulted in the expression of high mannose glycan structures. High mannose is the natural ligand of the DC specific C-type lectin receptors DC-SIGN and Langerin, expressed on skin dermal DCs (dDCs) and Langerhans cells (LCs), respectively. By specifically targeting apoptotic vesicles to dDCs and LCs we are able to increase vesicle uptake, antigen presentation and even maturation of the DCs. The latter possibly because of the expression of damage-associated molecular patterns (DAMPs) on the apoptotic vesicles. Our current research is focused on unraveling the effectiveness of glycan modification on vesicles to both target skin resident DCs and LCs using ex vivo skin explants as a model for intradermal vaccination in humans. 2233 Yannick De Vlaeminck Dissecting the biology of tumor-associated macrophages 1 1 1 1 Yannick De Vlaeminck , Cleo Goyvaerts , Carlo Heirman , Kris Thielemans , Jo Van Ginderachter 1 Karine Breckpot 2,3 and (1) Laboratory of Molecular and Cellular Therapy, Department of Biomedical Sciences and (2) Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium, (3) VIB Department of Molecular and Cellular Interactions, Brussels, Belgium Tumors are infiltrated with type 2 tumor-associated macrophages (M2) that play a key role in tumor progression. Several strategies are investigated to modulate them yet these are often not selective leading to toxicity. Since M2 are characterized by expression of macrophage mannose receptor (MMR), we propose to use MMR as a bull’s eye. We applied the nanobody (Nb) display technology to generate lentiviral vectors (LVs) coated with an MMR-specific Nb (MMR-LVs). Transduction of splenocytes in vitro KO or hepatocytes in vivo from wild type (WT) or MMR mice with Thy1.1 or Firefly luciferase encoding LVs, + + showed selective transduction of MMR CD11b cells by MMR-LVs using flow cytometry or bioluminescence imaging respectively. However, using flow cytometry we failed to show transduction of + + MMR CD11b cells upon intratumoral injection. As we observed that a considerable percentage of tumor cells were transduced with VSV.G-LVs, we propose to use VSV.G-LVs encoding Nb MMR fused to a protein of interest to transform tumor cells into factories producing Nb-fusion proteins. We successful showed specific binding of Nb MMR fusion proteins on M2 using flow cytometry and fluorescence microscopy. These results warrant further investigation into the use of LVs and Nb MMR as a tool to specifically target M2 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 69 2234 Rens Braster Peri-operative targeting of circulating tumour cells to prevent colorectal liver metastasis formation 1# 2# 1 2 2 Rens Braster , , Marije Overdijk , , Rianne Korthouwer , Sandra Verploegen , Paul Parren , and Marijn 1,§ 1,3,§ Bögels , Marjolein van Egmond 1 Department of Molecular cell biology and immunology, VU university medical centre, Amsterdam, the 2 3 Netherlands; Genmab, Utrecht, the Netherlands; Department of Surgery, VU university medical centre, Amsterdam, the Netherlands; #,§ Authors contributed equally to this research Colorectal cancer (CRC) patients with a restricted primary tumour are in principle undergoing curative surgery. However, resecting the tumour inflicts trauma, which is associated with increased risk of metastasis development, especially in the liver. Metastases can develop from circulating tumour cells (CTCs). In animal models development of liver metastasis was successfully prevented when monoclonal antibodies (mAbs) targeting CTCs were administered around surgery. The macrophages in the liver were the primary effector cells, and removed CTCs via antibody-dependent phagocytosis (ADCP). Therefore we assessed whether this strategy can be used to treat human patients with CRC. Currently, patients with metastases that have wildtype K-RAS are treated with mAbs blocking the epidermal growth factor receptor (EGFR). We demonstrate that anti-EGFR mAbs induced ADCP by human macrophages at low concentrations independent of K-RAS status. Furthermore, anti-EGFR mAbs did not interfere with migration and proliferation, which are two important processes in wound healing. This data suggest that CRC patients can be treated with anti-EGFR antibodies peri-operative to induce ADCP and prevent metastasis formation, without hampering wound healing after surgery. We anticipate that this may significantly improve patient outcome. 2235 Dorian A Stolk Lipo-based vaccines against melanoma: an in situ approach to target skin resident immune cells and invariant natural killer T cells 1 2 2 2 Dorian A. Stolk , Jana Vree , Hans J. van der Vliet , Tanja D. de Gruijl , Yvette van Kooyk 1 1 Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam Medical Oncology, VU University Medical Center, Amsterdam 2 Effective vaccination strategies for the treatment of cancer require strong activation of the innate and adaptive immune system which is driven by antigen presenting cells (APC). Therefore, skin resident APCs such as dermal dendritic cells (dDCs) and Langerhans cells (LCs) are an attractive target for in situ antitumor vaccination. In an ex vivo skin explant model we have shown that glycan modification of melanoma tumor antigens enhances endosomal routing and cross-presentation by targeting C-type lectin receptors. dDCs can also activate invariant natural killer T (iNKT) cells, which operate at the boundary of innate and adaptive immunity, through presentation of α-galactosylceramide (αGalCer) in a CD1d dependent manner. Indeed, we have shown before that skin APCs take up injected αGalCer which results in activation of iNKT while simultaneously enhancing the efficacy of a tumor vaccine. In this study, we developed a lipo-based vaccine containing palmitoylated synthetic long peptides and αGalCer to specifically target both innate and adaptive players of the immune system. Combination of melanoma specific antigens and αGalCer in one lipo-formulation resulted in strong CD8+ T cell activation and increased IFNγ secretion by iNKT. Addition of lewis Y to the liposomes for C-type lectin targeting further enhanced this effect. Funding: KWF 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 70 2236 Natasa Prokopi Loss of skin dendritic cells and their role in a spontaneous melanoma mouse model 1 1 1 1 1 Natasa Prokopi , David G. Mairhofer , Christoph H. Tripp , Daniela Ortner , Kerstin Komenda , Suzie 2 3 1 Chen , Björn E. Clausen and Patrizia Stoitzner 1 Department of Dermatology, Venereology and Allergology, Innsbruck Medical University, Innsbruck, 2 Austria; Susan Lehman Cullman Laboratory for Cancer Research, Rutgers University, Piscataway, New 3 Jersey, USA; Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg University, Mainz, Germany Ectopic overexpression of the metabotropic glutamate receptor 1 (Grm1) in melanocytes confers them an anti-apoptotic and hyperproliferative phenotype. This alteration, that has been detected in 40% of melanoma patient samples, leads to spontaneous melanoma development with 100% penetrance in the tg(Grm1)EPv mouse model. We have characterized the immune cell network changes that occur in the skin and the tumor draining lymph nodes in regards to skin dendritic cells (DC) as well as effector cells. Our results show a clear decrease in the skin DC, with this event already occurring very early in tumor development. Furthermore, we investigated the capacity of skin DC to prime CD8+ T cells and our results show that all skin DC subsets are able to induce priming irrespective of the stage of tumor development. Understanding the processes underlying these effects will offer useful information in regards to the role + of the different skin DC subsets in tumor antigen presentation and activation of CD8 T cells. 2237 Nathalie van Leeuwen CD141 expressing monocytes show an inflammatory profile and are associated with low-risk inflammatory features in myelodysplastic syndromes 1 1 2 3 Nathalie van Leeuwen-Kerkhoff , Theresia M. Westers , Shahram Kordasti , Tanja D. de Gruijl and Arjan 1 A. van de Loosdrecht 1 2 Department of hematology, VU university medical center, Amsterdam, The Netherlands; Department of haematological medicine King's College London and King's College Hospital, London, United Kingdom; 3 Department of medical oncology, VU university medical center, Amsterdam, The Netherlands Myelodysplastic syndromes (MDS) are clonal disorders of the hematopoietic stem cell. Low-risk disease is Preliminary findings showed that MDS-derived monocytes often have elevated expression levels of the dendritic cell marker BDCA3/CD141 compared to healthy monocytes. In this study, the role of these monocytes in immune responses in MDS was investigated. The percentage of CD141+ monocytes was examined in 22 normal bone marrow (NBM) and 143 MDS BM samples. Furthermore, CD141 expression was assessed in different disease stages. Sorted CD141+/monocytes were tested for functional capacities. The percentage of CD141+ monocytes was highly increased in MDS BM compared to NBM (36% vs 12%, p<0.0001). This increase was more evident in low-risk groups and correlated with low blast counts. The median fluorescent intensity (MFI) of HLA-DR was elevated on CD141+ monocytes compared to total monocytes (1.5 fold, p<0.001). Functional analyses showed a trend towards higher production of IL-1β, IL6 and TNF and a reduction in FoxP3+ T regulatory cell skewing. In conclusion, CD141+ monocytes are found in MDS BM and their presence correlates with a proinflammatory profile. Further research is warranted to investigate the role of CD141+ monocytes in immune responses. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 71 2238 Kevan Hosseini The carcinogen diethylnitrosamine (DEN) induces leukocyte recruitment and lipid deposition in mouse livers Kevan Hosseini, Sonja M. Kessler, Jessica Hoppstädter, Stephan Laggai, Katja Gemperlein, Rolf Müller, Alexandra K. Kiemer The excessive lipid accumulation in hepatic steatosis can lead to inflammatory processes, which drive the progression to cirrhosis and hepatocellular carcinoma. The carcinogen diethylnitrosamine (DEN) induces hepatocarcinogenesis (Naugler et al., 2007) after long-term exposure and short-term (48 h) DEN treatment is suggested to mimic a carcinogenic inflammatory process. However, the effects of short-term DEN on hepatic lipids and leukocyte recruitment are rather ill characterized. Therefore, we analysed changes in hepatic lipid and leukocyte composition in the short-term DEN mouse model. DEN-treated animals lost body and liver weight and the transaminases AST and ALT were elevated, indicating severe liver injury. The absence of caspase-3 activity suggested a minor involvement of apoptotic events. The total leukocyte amount was higher in DEN-treated animals: monocytes, macrophages, neutrophils, natural killer, and dendritic cells were increased while eosinophils were decreased. Triglycerides, saturated as well as polyunsaturated fatty acids were elevated in the livers of DEN-exposed animals. In serum, triglycerides were lowered, whereas cholesterol levels were increased. In summary, our data characterize the metaflammatory and tumor-promoting environment induced in the short-term DEN mouse model. 2239 Tamara Tyrinova The impairments in TNFα expression underlie the defect of monocyte-derived dendritic cell cytotoxicity in Hodgkin’s lymphoma patients T. Tyrinova, O. Leplina, M. Tikhonova, D. Batorova, G. Ushakova, V. Sergeevicheva, S. Sizikova, I. Kryuchkova, A.Ostanin, E. Chernykh Institute of Fundamental and Clinical Immunology, Novosibirsk, Russia Dendritic cells (DCs) possess direct antitumor cytotoxic activity. DC cytotoxicity can be stimulated greatly by I IFN. Here we investigated the association between cytotoxicity of monocyte-derived IFNα-induced DCs (IFN-DCs) and TNFα expression in IFN-DCs of malignant lymphoma patients. Patient IFN-DCs showed low cytotoxicity against TNFα-sensitive tumor HEp-2 cells which was caused by low membrane TNFα (mTNFα) expression on IFN-DCs. Low cytotoxicity and mTNFα expression were observed mainly in IFN-DCs of patients with Hodgkin’s lymphoma (HL). Decreased mTNFα expression was not associated with the changing of the active form of NFkB level in HL patient IFN-DCs. IFN-DCs of patients with non-Hodgkin lymphoma efficiently killed HEp-2 cells; mTNFα molecule expression was similar to that in donor IFN-DCs. Blocking TNFα-converting enzyme with TAPI-0 enhanced the expression of mTNFα on HL patient IFN-DCs and significantly increased their cytotoxic activity. rhIL-2 and dsDNA increased mTNFα expression on HL patient IFN-DCs and enhanced DC cytotoxicity against HEp-2 cells. The level of NF-kB active form in rhIL-2or dsDNA-stimulated IFN-DCs does not differ from intact IFN-DCs. The current study shows an important role of mTNFα as mediator of DC tumoricidal activity and as molecular targets for the regulation of DC cytotoxicity 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 72 Session VIII Host –microbe crosstalk 2244 Claudia Kitzmueller No title Claudia Kitzmüller, Martina Schuschnig, and Barbara Bohle Department of Molecular Biology, University of Salzburg, Christian Doppler Laboratory for Immunomodulation, Department of Pathophysiology and Allergy Research, Medical University Vienna, Background: Epithelial cells (EC) of the oral cavity have important barrier functions and might be involved in the development of oral tolerance. Little is known about their role in the modulation of immune responses. Aim: To analyse whether oral EC react to pathogen-associated molecular patterns (PAMPs) and upregulate components of the antigen-processing and -presentation machinery under inflammatory or infectious conditions. Methods: We analysed the expression of Toll-like receptors, NOD-like receptors and C-type lectin receptors by the buccal EC line HO-1-N-1, the sublingual EC line HO-1-u-1 and by primary human oral keratinocytes (HOK) using PCR. We studied the functional response of receptor stimulation by IL-8 ELISA and the up-regulation of Cathepsin S and MHC class I and II in response to direct stimulation with IFN- and indirect stimulation by PAMPs by Western Blot and flow cytometry. Results: HO-1-N-1 expressed mRNA of TLR 1, 2, 4, 6 and NOD1 and increased IL-8 production after stimulation of TLR 2, 3, 4 and NOD1. HO-1-u-1 expressed mRNA of TLR 1, 3, 4, 5, 6, and NOD1 and secreted elevated levels of IL-8 after stimulation of TLR 3 and 5. HOK expressed mRNA of all the TLR and NLR analysed and reacted strongly to TLR 3 stimulation. Direct stimulation with IFN- led to an upregulation of Cathepsin S and MHC class I and II on HO-1-N-1 and HOK. HOK increased the levels of MHC class I molecules also after stimulation of TLR 3. Conclusion: Oral EC can be stimulated by TLR ligands. Under inflammatory and infectious conditions they could have antigen-presenting potential. 2245 Aurélie Detavernier Regulation of interleukin-27 expression in the context of infection. 1 2 1 3 1 Aurélie Detavernier , Guillaume Oldenhove , Laurye Van Maele , David Svec , David Torres , Muriel 1 1 1 Nguyen , Séverine Thomas , Stanislas Goriely . 1 2 : Institute for Medical Immunology, Université Libre de Bruxelles, Brussels, Belgium : Department of 3 Immunobiology, Université Libre de Bruxelles, Brussels, Belgium : Laboratory of Gene Expression, Institute of Biotechnology CAS, Vestec u Prahy, Czech Republic. The inflammatory response, initiated by pathogens or danger signals, is required for the establishment of the immune response. This process has to be tightly regulated, as it may lead to tissular damages and chronic inflammatory states. Among the Interleukin(IL)-12 family members, IL-27 plays a central role in the control of inflammation. However, little is known about the in vivo cellular sources of IL-27. Using IL12-YFP mice, we confirmed that conventional dendritic cells produce primarily IL-12 upon Toxoplasma gondii or Listeria monocytogenes infection. In sharp contrast, using a novel IL27-GFP reporter mouse, we + + + identified CD11b CD64 LY6C inflammatory monocytes as the main source of IL-27. This result suggests that these populations fulfill different functions in the course of infection. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 73 By studying the expression profile of inflammatory genes within inflammatory monocytes by single cellqPCR, we showed that among the most correlated genes with IL27p28, many are associated with IFN (type I/II) signaling. Further work will aim at identifying the epigenetic regulation of IL-27 that may lead to this functional specialization. 2246 Martijn Nolte Bone marrow harbors a unique population of resident DCs with anti-fungal properties 1 1 1 1 2 Marieke Goedhart , Fernanda Pascutti , Sulima Geerman , Giso Brasser , Timo Rademakers , Carlijn 1 2 Vo Department of Hematopoiesis and Department of Molecular Cell Biology; Sanquin Research and Landsteiner Laboratory; Academic Medical Centre; University of Amsterdam; Amsterdam, the 1 1 Netherlands ermans , Martijn Nolte Systemic yeast and fungal infections are a major cause of morbidity and mortality after hematopoietic stem cell transplantations and in patients with bone marrow (BM) failure. As BM is enriched for memory T cells recognizing fungal antigens, we hypothesize that BM plays an important role in anti-fungal immunity. To investigate this, we examined the anti-fungal properties of DCs residing in the BM. We found that the majority of DCs present in murine BM belong to the IRF4-regulated subtype, indicating Th2/Th17 inducing potential. These BM-resident DCs can efficiently cross-present OVA-protein and thereby activate naïve + OVA-specific CD8 T cells. Interestingly, we discovered that most DCs in BM express the pattern recognition receptor Dectin-1, which can bind beta-glucans expressed on fungi and yeast. Indeed, BMresident DCs were much more efficient in phagocytosis of yeast-derived zymosan-particles than their splenic counterparts. Importantly, also DCs present in human BM were able to efficiently take up zymosan. In summary, BM contains a unique population of resident DCs that is capable of priming naïve T cells, potentially driving Th2 and Th17 immune responses. Our findings signify an important role for the BM in general and local DCs in particular in anti-fungal immunity, which we are currently investigating. Funding: PPOc Project grant #13-030 2247 Mariangela Cavarelli Intestinal Dendritic Cells and Macrophages Differentially Affect HIV-1 Transmission Across the Intestinal Mucosa. 1 2 3 1 1 4 Mariangela Cavarelli , Tilo Schorn , Chiara Foglieni , Antonio Cosma , Nathalie Bosquet , Ugo Elmore , 2 1 Gabriella Scarlatti , Roger le Grand 1 CEA, Immunology of Viral Infection and Auto-immune Diseases (IMVA) UMER1184, IDMIT infrastructure, 2 iMETI/DRF, Fontenay-aux-Roses, Université Paris-Sud 11, Orsay, France. San Raffaele Scientific Institute, 3 Viral Evolution and Transmission Unit, Milan, Italy San Raffaele Scientific Institute, Cardiovascular 4 Research Area, IRCCS, Milan, Italy San Raffaele Scientific Institute, Gastrointestinal Surgery Unit and Vita Salute San Raffaele University, Milan, Italy. Human colonic lamina propria CD11c+DCSIGN+CD68- cells sample luminal R5 HIV-1 in an ex vivo model, a mechanism mediated by CCR5 and exploited by HIV to bypass the intact epithelial barrier (Cavarelli 2013). We used multicolor flow cytometry, immunofluorescence and ex vivo explant culture of colorectal mucosa to define mononuclear phagocytes (MP) distribution and their susceptibility to HIV/SIV infection in humans and Cynomolgus macaques. Three subset of DC (CD11c+CD64-) were identified on the basis of CD103 and CX3CR1 expression. The totality of colonic Mf (CD11c+CD64+) was CX3CR1+ while about 50% expressed the CD163. Interestingly, CCR5 was preferentially expressed by CD11c+CX3CR1+ cells, which support their involvement in active sampling of HIV and in transmission of infection in situ. In support of this, CD11c+ CX3CR1+ but not CD103+ cells penetrated the intestinal epithelium following ex vivo R5 HIV-1 stimulation. The same effect was observed when macaque tissues were treated with SIVmac but not with SIVagm. Moreover, cells emigrated from the macaque mucosa productively transferred SIV infection to co-cultured target cells. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 74 We outlined new findings concerning the phenotype and function of intestinal MP and discuss the relative contribution of different subsets of MP in the early events of HIV transmission at mucosal sites. Funding: ANRS, Marie Curie Actions 2248 Stefanie Dichtl Dopamine regulates iron homeostasis and innate immune responses of macrophages to Salmonella infection 1 1 1 2 1 1 Stefanie Dichtl , David Haschka , Egon Demetz , Oliver M. D. Lutz , Manfred Nairz , Malte Aßhoff , Markus 1 1 3 1 Seifert , Sylvia Berger , Christian Huck , Günter Weiss 1 2 Department of Internal Medicine VI, Medical University of Innsbruck, Austria Austrian Drug Screening 3 Institute, Innsbruck, AUSTRIA University of Innsbruck, AUSTRIA Backround: Siderophores are catechol based compounds which can bind iron. Iron is an essential growth factor for mammalian cells and microbes. Based on previous observations, showing increased bacterial growth in the presence of catechols, we asked whether this may be referred to hormone mediated alterations of iron homeostasis. Methods: We studied the effects of the catecholamine dopamine on the regulation of iron in BMDM obtained from C57Bl/6wt. The in vivo effects of dopamine were studied in wt mice infected with the Gram negative bacteria Salmonella typhimurium (S.tm.). Results: Administration of dopamine to macrophages resulted in a dose dependent increase of heme oxygenase-1 and ferroportin expression. This effect could also be reproduced upon infection of macrophages with S.tm.. The in vivo administration of dopamine to mice infected with S.tm. resulted in an increased bacterial burden in liver and spleen as compared mice receiving solvent. This is linked to an increased delivery of iron to bacteria in the presence of dopamine along with an impaired proinflammatory immune response of macrophages. Conclusion: Our data demonstrate that dopamine may deteriorate the course of infection by promoting bacterial growth which can be a major concern from the treatment of patients with bacterial sepsis receiving catecholamines 2249 Stephanie Obermeyer Pathogen control in Leishmania infections is regulated by the iron metabolism 1 1 Stephanie Obermeyer , Ulrike Schleicher and Christian Bogdan 1 1 Mikrobiologisches Institut – Klinische Mikrobiologie, Immunologie, und Hygiene, Universitätsklinikum Erlangen and Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg, Erlangen, Germany Iron-dependent survival of Leishmania within phagosomes is counteracted by inducible nitric oxide (NO) synthase (NOS2). Here, we analyzed whether the antimicrobial function of NOS2-derived NO and cellular iron metabolism crossregulate each other during Leishmania infection. Addition of FeCl3 or FeSO4 reversed the killing of intracellular Leishmania in macrophages induced by endogenous NO or an exogenous NO donor raising the possibility that NO causes iron deprivation of the parasite. However, NOS2-derived NO failed to upregulate the iron-exporter ferroportin-1 (Fpn-1) on mRNA and protein level in infected macrophages. In a reverse approach we analyzed the course of Leishmania infection in ironloaded mice. Iron overload exacerbated cutaneous leishmaniasis (CL, L. major), but ameliorated visceral disease (L. infantum). Aggravated CL was associated with an enhanced influx of Ly6C+Ly6G+ cells into infected lesions and spleens, with decreased IFNγ and increased arginase 1 mRNA-levels in lesions. In contrast, expression of NOS2 and production of NO in the tissue was not affected. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 75 These data indicate that iron withdrawal or destruction of FeS clusters in enzymes might be an indirect mechanism of NOS2-dependent Leishmania control. The mechanism behind the impaired pathogen control in CL in iron-loaded mice requires further studies. 2250 Ger van Zandbergen Human macrophages are superior to dendritic cells as host cell for Leishmania major parasites 1 1 1 1 3 Peter Crauwels , Laura Roßmann , Abel Martínez-Rodrigo , Rebecca Bohn , Cees van Kooten , Ger van 1,2 Zandbergen 1 Immunology Division Paul-Ehrlich Institute, Federal Institute for Vaccines and Biomedicines, Langen, 2 Germany Institute of Immunology, University Medical Center of the Johannes Gutenberg University of 3 Mainz, Mainz, Germany Department of Nephrology, Leids Universitair Medisch Centrum, Leiden, the Netherlands An appropriate T-cell response to Leishmania infection is critical for an effective immune response. Human macrophages (hMDMs) as well as dendritic cells (DCs) can internalize Leishmania parasites and serve as antigen presenting cells, leading to T cell activation. In this study we compare hMDMs and DCs for their suitability as host cell for Leishmania major (L.major) by analyzing infection rate, parasite transformation and their ability to induce T cell proliferation, having an effect on parasite survival. + + + - L.major parasites were able to infect both pro-inflammatory hMDM1 (CD14 CD206 CD209 CD163 + + + + + MHCII ) and anti-inflammatory hMDM2 (CD14 CD206 CD209 CD163 MHCII ) as well as DCs (CD14 + + + + CD206 CD1a CD209 MCHII CD163 ). After 24 hours, hMDM2 ((45.5 % ± 5.9) shows to be most susceptible for L.major infection, followed by hMDM1 (32.5 % ± 2.5) and DCs (12.4% ± 1.8). Remarkably, high over a period of 7 days parasite developed is the strongest in DCs. The intracellular promastigotes (ABC low low high and GP63 ) transformed into the multiplying amastigote form (ABC and GP63 ), analyzed by RT1 PCR . Upon coculturing infected cells with autologous CFSE labeled PBMCs, proliferation, detected as low CFSE cells, was induced the strongest by DCs (49 % ± 2.4) followed by hMDM1 (44.1 % ± 2.8) and hMDM2 (36.6 % ± 4.2). As a consequence of proliferation, L.major infection rates were dramatically reduced about 20% in hMDM1, 40% in hMDM2 and almost 60% in DCs. Interestingly, preliminary data showed the presence of nitric oxide to be increased in DCs, which may contribute to the strong parasite elimination. When hMDMs encounter virulent L.major promastigotes, the autophagy machinery got 2 activated, leading to a significantly reduction of T cell proliferation . In contrast, induction of the autophagy machinery upon L.major infection in DCs is absent. So far, we could demonstrate that both human macrophage phenotypes as well as dendritic cells can be infected with L.major promastigotes, which develop, in all 3 cell types, into amastigotes. Moreover, apoptotic L.major misuse the autophagy machinery in macrophages – but not in dendritic cells - to lower T cell proliferation, securing intracellular survival, making human macrophages superior to dendritic cells as host cell for L.major. 1. Wenzel UA, Bank E, Florian C, et al. Leishmania major parasite stage-dependent host cell invasion and immune evasion. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2012;26(1):29-39. 2. Crauwels P, Bohn R, Thomas M, et al. Apoptotic-like Leishmania exploit the host´s autophagy machinery to reduce T-cell-mediated parasite elimination. Autophagy. 2015;11(2):285-297. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 76 2251 Dagmara Wiatrek Zinc homeostasis during dendritic cell maturation 1 1 2 1 Dagmara Wiatrek , Swati Arya , Catherine H. Botting , Simon J. Powis , Alan J. Stewart 1 1 2 School of Medicine, University of St Andrews, St Andrews KY16 9TF, UK Biomedical Sciences Research Complex, University of St Andrews, St Andrews KY16 9ST, UK. Correspondence should be addressed to D.W. ([email protected]). The importance of zinc for cellular immunity is well described; however the exact mechanisms are still not clear. Here we show that LPS stimulation of Toll-like receptor 4 (TLR4) changes intracellular free zinc levels in the key antigen presenting cell type, dendritic cells (DCs). Those changes occur via alteration of zinc transporters and are involved in DC maturation. Using real time PCR, western blot, immunofluorescence, flow cytometry and SWATH proteomics, we have found significant changes in the expression of both zinc importers (Zip transporters) and exporters (ZnT transporters). The biggest alterations occur between 6 to 24h post-stimulation, where the relative levels of ZnT1, ZnT7, Zip7 and Zip6 transporters decrease. Moreover, SWATH proteomic data reveal 3-7 fold increases in the expression of histocompatibility complex proteins, involved in the antigen presentation. Our data suggest that zinc homeostatic control is an important part of antigen presentation process. Collectively, our findings help to build a fuller picture of the behaviour of Zip and ZnT transporters and other proteomic changes that occur during this crucial time period for DCs. 2252 LISBETH GONÇALVES ROLE OF FAST (Fas-activated serine threonine phosphoprotein) IN PHAGOCYTOSIS OF BACTERIA BY MACROPHAGES LISBETH GONÇALVES DE FREITAS, MARÍA SIMARRO GRANDE, ANTONIO ORDUÑA DOMINGO, MIGUEL ANGEL BRATOS PÉREZ, MIGUEL ANGEL DE LA FUENTE No affiliation In recent years remarkable progress has been made towards the understanding of the basic molecular and cellular functions of FAST and its homologs. The purpose was to further explore the effect of FAST deficiency in innate immune system, particularly in some macrophage functions such as phagocytosis and intracellular killing of gram positive and gram negative bacteria. Compared with wild-type macrophages, -/FAST macrophages exhibited increased phagocytosis of Escherichia coli both in vitro and in vivo. Evaluation of the expression of the receptors TLR2 and TLR4 and maturation markers revealed no -/significant differences between wild-type and FAST macrophages. Both cell types also showed a similar ability to kill bacteria and to produce reactive oxygen species. Bacterial counts at the early time points in the gentamicin protection assay correlated well with the phagocytic indexes. Consistent with the findings -/in FAST macrophages, human THP-1 macrophages with silenced FAST expression showed increased -/phagocytosis of Escherichia coli compared with control cells. We have demonstrated, that FAST macrophages have a decrease in mitochondrial membrane potential caused by a specific deficiency of complex I of the respiratory chain (40%). In conclusion, our findings reveal a novel role for FAST in the regulation of phagocytosis. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 77 2253 Riem Gawish Myeloid interferon signaling protects from murine CMV infection and promotes extramedullary hematopoiesis *1 *1 1,2 3 1 1 Riem Gawish , Mario Biaggo , Caroline Lassnig , Zsuzsanna Bago-Horvath , Rita Rom , Lena Amenitsch , 1 1 4 4 1 1,2 Juliana Kornhoff , Marion Bokor , Astrid Krmpotic , Stipan Jonjic , Birgit Strobl and Mathias Müller 1 2 Department of Biomedical Science, University of Veterinary Medicine Vienna, Austria Biomodels Austria, 3 University of Veterinary Medicine, Vienna Department of Pathology, Medical University of Vienna, 4 Vienna, Austria Department of Histology and Embryology, University of Rijeka, Rijeka, Croatia * Authors contributed equally Cytomegalovirus (CMV) infection causes congenital birth defects, can be fatal for immune-compromised patients and represents a major health care problem. While systemic interferon (IFN) responses clearly protect against various infections, including CMV, the specific contribution of myeloid IFN-induced antiviral immunity remains still elusive. Using mice deficient for the signal transducer and activator of ΔMonoMac transcription 1 (STAT1) in myeloid cells (Stat1 ), i.e. harboring monocytes and macrophages that are unresponsive to all types of IFN, we studied the impact of myeloid STAT1 signaling during CMV infection as well as during CpG-induced sterile inflammation. Myeloid STAT1 suppressed CMV replication in the spleen and profoundly protected from splenic macrophage death and immunopathology. ΔMonoMac Surprisingly, despite increased tissue damage during infection, Stat1 mice developed less severe splenomegaly than littermate controls. This was at first counterintuitive but turned out to be the result of an impaired induction of extramedullary hematopoiesis (EMH). To address, whether this was due an altered inflammatory milieu or due to CMV-induced macrophage death in the absence of STAT1, we induced sterile inflammation using CpG, a well-established trigger of EMH. Even though no macrophage death occurred in this model, EMH was still dependent on myeloid STAT1. Taken together our data show that STAT1 signaling in macrophages on the one hand protects from CMV infection and on the other hand crucially mediates inflammation-induced EMH. Funding: Austrian Science FWF Infect-ERA project eDEVILLI (I2187) 2254 Ernesto Rodriguez Glycans from Fasciola hepatica modulate antigen presenting cells through the 1,2 1 1 1 3 Ernesto Rodríguez , Paula Carasi , Natalie Brossard , Verónica Noya , Cecilia Giacomini , Sandra van 2 2 2 1 Vliet , Yvette van Kooyk , Juan J. García Vallejo , Teresa Freire 1 2 Immunobiology Department, School of Medicine, UdelaR, Montevideo Uruguay; Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, Netherlands; 3 Biochemistry Department, School of Chemestry, Montevideo, Uruguay. The infection by Fasciola hepatica is an important parasitic disease of livestock that causes significant economic losses worldwide and consider as an emerging zoonosis with an increasing number of human infections globally. Like other helminth parasites, F. hepatica can modulate the immune response of the host during the infection, inducing a strong Th2/Treg immune response. We recently describe the important role of glycans structures in the immunomodulation induced by F. hepatica, although the specific receptors and glycan structures involved are yet to be determinated. In this work, we identify the Macrophage Galactose Lectin (MGL) as a major receptor for F. hepatica antigens. Our results suggest that the Tn antigen expressed in the surface of F. hepatica can modulate the TLR2-induced maturation of human dendritic cells by interaction with MGL, upregulating the production of IL10 and TNFα. Furthermore, we show that there is an important recruitment of mMGL2+ CD11c+ cells to the peritoneum of infected-mice, which express the regulatory cytokines IL10, TNFα and TGFβ and a variety of regulatory markers. Interestingly, these cells can modulate CD4+ T cells allogeneic responses by dowregulation of INFγ and upregulation of IL10. Together, our data suggest a main role of MGL in the immunomodulation induced by F. hepatica. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 78 2255 Lenka Kavanova Alveolar macrophages vs. Monocyte derived macrophages: different macrophages with different immune response to respiratory pathogens. 1,2 1 1 1 Kavanova Lenka , Matiaskova Katarina , Nedbalcova Katerina , Martin Faldyna , Jiri Salat 1 1 2 Veterinary Research Institute, Hudcova 296/70, 62100 Brno, Czech Republic Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlarska 267/2, 61137 Brno, Czech Republic Alveolar macrophages as resident cells provide one of the first lines of defence against microbes invading the lung tissue. On the other hand, monocyte derived macrophages (MDMFs) as inflammatory macrophages are naive cells accumulating in site of inflammation. Macrophages serve as the target of replication of viruses like porcine reproductive and respiratory syndrome (PRRS) which is an important swine disease causing severe economic losses worldwide and can predispose pigs to infection by bacteria such as Haemophilus parasuis. Concurrent infection of porcine alveolar macrophages (PAMs) or MDMFs with PRRS virus and H. parasuis was analysed in vitro and difference dependent on macrophage type were observed. New infiltrated MDMFs were more sensitive to PRRSV infection resulting in higher mortality of cells and higher production of IFN-α compared to fully differentiated PAMs. Elevated level of IFN-α decreased expression of pro-inflammatory cytokines (IL-1β and IL-8) which was confirmed by experimental addition of IFN-α to MDMFs followed by infection with H. parasuis. Furthermore, infection of MDMFs with H. parasuis caused cell fusions and formation of multinucleated giant cells. The work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (project LO1218) and the Ministry of Agriculture of the Czech Republic (QJ1210120). Funding The work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (project LO1218) and the Ministry of Agriculture of the Czech Republic (QJ1210120). 2256 Julie Deckers Titel: Epicutaneous sensitization to house dust mite allergen via intact skin depends on IRF4-dependent dermal dendritic cells 1,2,3 1,2 1,2,4 1,2 Julie Deckers , Dorine Sichien , Maud Plantinga , Justine Van Moorleghem , Manon 1,2 5 1,2 3 Vanheerswynghels , Bernard Malissen , Martin Guilliams , Karolien De Bosscher , Bart N. 1,2,4* 1,2* Lambrecht , and Hamida Hammad 1 2 VIB Inflammation Research Center, a VIB-UGhent department, Ghent, Belgium Laboratory of 3 Immunoregulation and Mucosal Immunology, Ghent University, Ghent, Belgium Receptor Research Laboratories, Nuclear Receptor Lab, Medical Biotechnology Center, a VIB-UGhent department, Ghent, 4 Belgium Department of Pulmonary Medicine, Erasmus University Medical Centre Rotterdam, the 5 Netherlands Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université UM2, Inserm Exposure to allergens like house dust mite (HDM) via the skin has been described to often precede the development of allergic airway inflammation. In most instances, Th2 sensitization via the skin happens when this barrier has been damaged or disrupted. Whether allergic sensitization to HDM can also happen through an intact skin barrier, and the exact cell types involved in this process are currently unknown. Allergic sensitization to HDM was induced by the epicutaneous application of HDM on the intact unmanipulated ear skin. To study the role of the different DC subsets in epicutaneous sensitization to HDM, mice strains lacking different DC populations were used. Finally, the intrinsic capacity of several skin-derived DC subsets to induce Th2 sensitization via the skin was determined by adoptive transfer into naïve recipient mice. We found that epicutaneous application of HDM on intact skin barrier led to features of allergic airway inflammation upon intranasal challenge with HDM. This process did not require skin damage or the + enzymatic activity of HDM. Primary proliferation of CD4 T cells transgenic for Derp1-specific TCR occurred 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 79 only in the lymph node draining the site of HDM application. Finally, epicutaneous sensitization was driven by two variants of IRF4-dependent dermal conventional dendritic cell (cDC2) subsets, and not by epidermal Langerhans cells. These findings identify skin cDC2 as crucial players in Th2 sensitization to common inhaled allergens that enter the body through an intact skin, and can provoke features of allergic asthma. 2257 Katarína Matiašková Molecular-biological detection of the immune response of porcine alveolar macrophages to in vitro infection with different serovars of Haemophilus parasuis 1 1 1 1 1 Kateřina Nedbalcová , Monika Vícenová1, Jiří Salát , Lenka Kavanová , Vladimír Babák , Ján Matiašovic , 1 Martin Faldyna 1 Veterinary Research Institute, Brno, Czech Republic2University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic Porcine alveolar macrophages (PAMs) are essential defence mechanism in the lung of pigs against aerogenous bacterial infection such as Haemophilus parasuis. H. parasuis is a part of the normal flora of the respiratory tract of pigs, but it can also induce severe diseases. The aim of this study was to compare the immune responses of PAMs to in vitro infection with various H. parasuis serovars of different virulence. Stimulation of PAMs with H. parasuis up-regulated the expression of all selected genes (IL-1β, TNFα, IL-8, IL-10 and IL-1Ra) at 4 and 24 hours post infection (PI). Up-regulation was higher after 24 hours PI except for TNF-alpha. Despite the fact that strains of serovars with assumed different virulence were used in this study, no significant differences among serovars in the expression of mRNA for the investigated genes were detected under in vitro conditions. Expression of these genes was strongly dependent on the individual variability of pigs. On the other hand, virulent strains were able to suppress production of reactive oxygen species in PAMs more than avirulent strains, indicating the presence of defence mechanism against respiratory burst which is one of the most important antibacterial mechanism of PAMs. The study was supported by Ministry of Agriculture of the Czech Republic (QJ1210120) and MEYS LO1218. Funding: Ministry of Agriculture of the Czech Republic (QJ1210120) and MEYS LO1218 2258 Rui Martins Heme drives hemolysis-induced susceptibility to infection and sepsis via disruption of phagocyte cytoskeletal dynamics 1,2 1,2 1,2 1 1,2 1,2 Rui Martins , Julia Maier , Anna-Dorothea Gorki , Kilian V. M. Huber , Omar harif , Simona Saluzzo , 1,2 1,2 1,2 2 3 Philipp Starkl , Federica Quattrone , Riem Gawish , Karin Lakovits , Michael C. Aichinger , Branka 1 1 4 5 5 Radic-Sarikas , Charles-Hugues Lardeau , Markus Brown , Kari Vaahtomeri , Michelle Duggan , Dontscho 4 6 1 7 3 Kerjaschki , Harald Esterbauer , Jacques Colinge , Stephanie C. Eisenbarth , Thomas Decker , Keiryn L. 1 1 5 1,8 1,2 Bennett , Stefan Kubicek , Michael Sixt , Giulio uperti-Furga , and Sylvia Knapp 1 2 CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria; 3 Department of Medicine I, Laboratory of Infection Biology, Medical University of Vienna, Vienna, Austria; 4 Max F. Perutz Laboratories, University of Vienna, Vienna, Austria; Department of Pathology, Medical 5 University of Vienna, Vienna, Austria; IST Austria (Institute of Science and Technology Austria), 6 Klosterneuburg, Austria; Clinical Department of Medical and Chemical Laboratory Diagnostics, Medical 7 University of Vienna, Vienna, Austria; Department of Laboratory Medicine and Department of Immunobiology, Yale University School of Medicine, New Haven, USA Hemolysis is a serious complication of systemic inflammation and an independent predictor of poor outcome from sepsis. Moreover, people suffering from hemolytic disorders like malaria or sickle cell disease exhibit a considerably increased susceptibility to bacterial infections. How hemolysis intersects with infection susceptibility and outcome from sepsis is not completely understood, yet often considered a consequence of heme-iron serving as an essential nutrient for bacteria. Here, we show that elevated heme levels impair the control of bacterial 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 80 proliferation independent of heme-iron acquisition by pathogens. Instead, we demonstrate that heme strongly inhibits phagocytosis and migration of phagocytes by disrupting actin cytoskeleton dynamics. This heme-induced interference with cytoskeletal dynamics is conserved between human and mouse phagocytes and is mediated via DOCK8-triggered CDC42 activation. Furthermore, a chemical screening approach revealed that quinine effectively prevents the effects of heme on the cytoskeleton and thereby restores phagocytosis and survival from sepsis. These mechanistic insights on how hemolysis exhibits immunosuppressive effects expose potential therapeutic targets to help patients with sepsis and protect those with hemolytic disorders from bacterial infections.; 8 Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria Funding: Science Fund of the Austrian National Bank (14107) and the Austrian Science Fund FWF (I1620B22) within the Infect-ERA framework. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 81 Participants Name First name Ackermann Jochen Insertion Institute Residence Country E-mail Universitätsklinikum Erlangen Erlangen Germany [email protected] [email protected] [email protected] Affandi Alsya UMC Utrecht Utrecht The Netherlands Agod Zsófia University of Debrecen Debrecen Hungary Alem Carla Barbaria van The Netherlands The Netherlands United Kingdom Leiden University Medical Centre Leiden Arnaud VUMC Amsterdam Baru Abdul GlaxoSmithKline Stevenage Bhushan Sudhanshu Justus-Liebig University Giessen Blanas Anthanasios VUMC Amsterdam Bloemendaal Felicia AMC Amsterdam Blomgran Robert Linköping University Linköping Sweden [email protected] Bogdan Christian Universitätsklinikum Erlangen Erlangen Germany [email protected] [email protected] Germany The Netherlands The Netherlands [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] Boltjes Arjan UMC Utrecht Utrecht The Netherlands Bonelli Stefano Vrij Universiteit Brussel Brussels Belgium [email protected] ACTA Amsterdam The Netherlands [email protected] VIB Ugent Gent Belgium [email protected] Bontkes Hetty Borght Katrien Braster Rens VUMC Amsterdam Breedveld Annelot VUMC Amsterdam Brinke Anja ten Sanquin Research Amsterdam Broek Lenie van den VUMC Amsterdam Bruggeman Christine Sanquin Research Amsterdam Brunner Julia Medical University of Vienna Vienna van der The Netherlands The Netherlands The Netherlands The Netherlands The Netherlands Austria The Netherlands The Netherlands The Netherlands The Netherlands [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] Burg Sjoerd van der LUMC Leiden [email protected] Burgsteden Johan van AMC Amsterdam Cardoso Sandra UMC Utrecht Utrecht Carvalheiro Tiago UMC Utrecht Utrecht Cattepoel Susann CSL Behring AG Bern Switzerland [email protected] France [email protected] [email protected] [email protected] [email protected] Cavarelli Mariangela CEA Fontenayaux-Roses Clausen Björn University Medical Center Mainz Mainz Germany [email protected] Clement François Namur University Namur Belgium [email protected] Amsterdam The Netherlands [email protected] New Haven USA [email protected] Cornelissen Lenneke VUMC Cresswell Peter Yale University Medicine Crommentuijn Matheus VUMC Amsterdam The Netherlands [email protected] Culemann Stephan Universitätsklinikum Erlangen Erlangen Germany [email protected] Deckers Julie VIB Ugent Ghent Belgium [email protected] Delputte Peter University Antwerp Antwerpen Belgium peter.delputte@uantwerpen/be Detavernier Aurelie Institute for medical immunology Gosselies Belgium [email protected] Dichtl Stefanie Medical University of Innsbruck Innsbruck Austria [email protected] Dinther Dieke Drewniak Dudziak van School of VUMC Amsterdam Agata AMC Amsterdam Diana University Hospital Erlangen Erlangen The Netherlands The Netherlands Germany [email protected] [email protected] [email protected] 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 82 Dufait Inès Dunnen Jeroen Dusoswa Sophie Egmond Marjolein Eikmans Vrij Universiteit Brussel den Brussels Belgium The Netherlands The Netherlands The Netherlands The Netherlands The Netherlands The Netherlands The Netherlands [email protected] AMC Amsterdam VUMC Amsterdam VUMC Amsterdam Michael Leiden University Medical Centre Leiden Eisden Tracy-Jane AMC Amsterdam Essen Mieke Leiden University Medical Centre Leiden Everts Bart LUMC Leiden Faas Maria Universitätsklinikum Erlangen Erlangen Germany [email protected] Florczyk Urszula Jagiellonian University Kraków Poland [email protected] [email protected] [email protected] van van Forss Cecilia University of Manchester Manchester United Kingdom Gawish Riem Institute of Animal Breeding and Genetics Vienna Austria The Netherlands The Netherlands [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] Ghiboub Mohammed AMC Amsterdam Gollnast Doron UMC Utrecht Utrecht Gonçalves Lisbeth Universidad de Valladolid Valladolid Spain [email protected] Gouwy Mieke Rega Institute Leuven Belgium [email protected] Govers Coen Wageningen Universiteit Wageningen The Netherlands [email protected] Goverse Gera KU Leuven Leuven Belgium [email protected] Goyvaerts Cleo Vrij Universiteit Brussel Brussels Belgium [email protected] Graczyk Damian Institute of Biochemistry and Biophysics Warsaw Poland [email protected] Grau Veronika Justus-Liebig University Giessen Germany [email protected] Grein Susanne UMC Utrecht Utrecht Groot Kormelink Tom UMC Utrecht Utrecht Gruijl Tanja de VUMC Amsterdam Haan Joke den VUMC Amsterdam Ham Marieke van Sanquin Research Amsterdam Hampton-O'Neil Lea University of Bristol Bristol Haniffa Muzlifah Newcastle University Newcastle Hansen Ivo AMC Amsterdam Harris Robert Karolinska Institutet Stockholm Sweden [email protected] Haschka David Medical University of Innsbruck Innsbruck Austria [email protected] van der Heemskerk Niels VUMC Amsterdam Heideveld Esther Sanquin Research Amsterdam Heinsbroek Sigrid AMC Amsterdam Herdoíza-Padilla Estefania University of Ulm Ulm The Netherlands The Netherlands The Netherlands The Netherlands The Netherlands United Kingdom United Kingdom The Netherlands The Netherlands The Netherlands The Netherlands Germany The Netherlands The Netherlands [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] Hertoghs Nina AMC Amsterdam [email protected] Hoepel Willianne AMC Amsterdam Hoppstädter Jessica Saarland University Saarbrücken Germany [email protected] [email protected] [email protected] Horrevorts Sophie VUMC Amsterdam The Netherlands Hosseini Kevan Saarland University Saarbrücken Germany [email protected] Houseman Maja University of Berne Bern Switzerland [email protected] Irvine Katharine University of Queensland Brisbane Australia [email protected] 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 83 Jantsch Jonathan Institute of Clinical Microbiology and Hygiene Regensburg Jenkins Stephen University of Edinburgh Edinburgh Jong Esther AMC Amsterdam Karia Rashmi ISA Pharmaceuticals BV Leiden Kavanova Lenka Veterinary Research Institute Brno Kelly Aoife University of Manchester Manchester Kitzmueller Claudia University of Salzburg Salzburg Austria [email protected] Knippertz Ilka Universitätsklinikum Erlangen Erlangen Germany [email protected] VUMC Amsterdam The Netherlands [email protected] de van Germany United Kingdom The Netherlands The Netherlands Czech Republic United Kingdom [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] Kooyk Yvette Krljanac Branislav Universitätsklinikum Erlangen Erlangen Germany [email protected] Krönke Gerhard University Hospital Erlangen Erlangen Germany [email protected] Kubes Paul University of Calgary Calgary Canada [email protected] Kuttke Mario Medical University of Vienna Vienna Austria [email protected] Lammers Aart Wageningen Universiteit Wageningen The Netherlands [email protected] Lányi Arpád University of Debrecen Debrecen Hungary [email protected] The Netherlands The Netherlands The Netherlands Lebre Maria AMC Amsterdam Leenen Pieter Erasmus MC Rotterdam Leeuwen Nathalie VUMC Amsterdam Lemmens Stefanie BIOMED Diepenbeek Belgium [email protected] Radboudumc Nijmegen The Netherlands [email protected] VUMC Amsterdam Nederland [email protected] [email protected] Lent Peter Li Eveline van van [email protected] [email protected] [email protected] Lindenbergh Marthe Utrecht University Utrecht The Netherlands Linke Monika Medical University of Vienna Vienna Austria [email protected] Lippuner Christoph University of Bern Bern Switzerland [email protected] Loosdrecht Arjan Lubbers van de The Netherlands The Netherlands The Netherlands United Kingdom VUMC Amsterdam Joyce VUMC Amsterdam Maas-Krebber Merle UMC Utrecht Utrecht MacDonald Andrew University of Manchester Manchester Marsland Benjamin University of Lausanne Lausanne Switzerland [email protected] Martins Rui CeMM Vienna Austria [email protected] The Netherlands Czech Republic [email protected] [email protected] [email protected] [email protected] Marut Wioleta UMC Utrecht Utrecht [email protected] Matiaskova Katarina Veterinary Research Institute Brno Germany [email protected] [email protected] Melandri Emiliano Miltenyi Biotec Bergisch Gladbach Mitrofanova Irina National Research Tomsk State University Tomsk Russia [email protected] Molhoek Anoushka VUMC Amsterdam The Netherlands [email protected] Miltenyi Biotec Bergisch Gladbach Germany [email protected] Vlaardingen The Netherlands [email protected] Madrid Spain [email protected] Morrissey-Wettey Frank Motta Alexandre Unilever R&D Municio Cristina Inst. de Invest. Gregorio Maranon Netea Mihai Radboudumc Nijmegen Newling Melissa AMC Amsterdam Nijmeijer Bernadien AMC Amsterdam Nolte Martijn Sanquin Research Amsterdam Sanitaria The Netherlands The Netherlands The Netherlands The Netherlands [email protected] [email protected] [email protected],nl [email protected] 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 84 Obermeyer Stephanie Universitätsklinikum Erlangen Erlangen Germany Czech Republic The Netherlands Czech Republic [email protected] Oreskovic Zrinka Veterinary Research Institute Brno [email protected] Ottria Andrea UMC Utrecht Utrecht Palová Jelínková Lenka Sotio a.s. Prague Parsa Roham Karolinska Institutet Stockholm Sweden [email protected] Pázmándi Kitti Linda University of Debrecen Debrecen Hungary [email protected] Pearce Edward University of Pennsylvania Philadelphia USA [email protected] [email protected] [email protected] [email protected] Pelgrom Leonard Leiden University Medical Centre Leiden The Netherlands Penny Xianne Singapore Immunology Network Singapore Singapore [email protected] Petit Jules Wageningen Universiteit Wageningen The Netherlands [email protected] Petzer Verena Medical University of Innsbruck Innsbruck Austria [email protected] Pieber Melanie Karolinska Institutet Stockholm Sweden [email protected] Piket Eliane Karolinska Institutet Stevenage Sweden [email protected] [email protected] Pokorna Katerina Sotio a.s. Prague Chech Republic Popović Jelena Technical University of Dresden Dresden Germany [email protected] [email protected] Prins Marileen AMC Amsterdam The Netherlands Prokopi Natasa Medical University of Innsbruck Innsbruck Austria [email protected] [email protected] Raaijmakers Marc Erasmus MC Rotterdam The Netherlands Raes Geert Vrij Universiteit Brussel Brussels Belgium [email protected] Raggi Federica G.Gaslini Institute Genova Italy [email protected] [email protected] Reginato Eleonora GlaxoSmithKline Stevenage United Kingdom Reinecke Julio Orthogen Dusseldorf Germany [email protected] Amsterdam The Netherlands [email protected] St. Johns Canada [email protected] Ulm Germany [email protected] Ribeiro Carla AMC Richardson Vernon Memorial University Newfoundland Riedel Christian University of Ulm of The Netherlands The Netherlands Rodrigues Neves Charlotte VUMC Amsterdam Rodriguez Ernesto VUMC Amsterdam Rombouts Miche University Antwerp Antwerpen Belgium [email protected] Saferding Victoria Medical University of Vienna Vienna Austria [email protected] Sagara Masaki Takeda pharmaceutical Kanagawa Japan [email protected] The Netherlands The Netherlands [email protected] [email protected] Sahasrabudhe Neha VUMC Amsterdam [email protected] Salvagno Camilla NKI Amsterdam Sanchez Selien BIOMED Diepenbeek Belgium [email protected] Saris Anno Sanquin Research Amsterdam The Netherlands [email protected] Schabbauer Gernot Medical University of Vienna Vienna Austria [email protected] [email protected] [email protected] Schetters Sjoerd VUMC Amsterdam The Netherlands Schleicher Ulrike Universitätsklinikum Erlangen Erlangen Germany [email protected] Schryver Majorie University Antwerp Wilrijk Belgium [email protected] Schuijs Martijn VIB Ugent Gent Belgium [email protected] Schultze Joachim University of Bonn Bonn Germany Sethi Mohammad Hannover Medical School Hannover Germany [email protected] Sichien Dorine VIB Ugent Zwijnaarde Belgium [email protected] [email protected] [email protected] de Sprokholt Joris AMC Amsterdam The Netherlands Steinkasserer Alexander Universitätsklinikum Erlangen Erlangen Germany 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 85 Stolk Dorian Teijlingen Nienke Thielemans Tomczyk The Netherlands The Netherlands VUMC Amsterdam AMC Amsterdam Kris Vrij Universiteit Brussel Brussels Belgium [email protected] Mateusz Jagiellonian University Kraków Poland [email protected] [email protected] van [email protected] [email protected] Annelies Sanquin Research Amsterdam The Netherlands Tyrinova Tamara Institute of Fundamental and Clinical Immunolgy Novosibirsk Russia [email protected] Varesio Luigi Instituto Giannina Gaslini Genoa Italy [email protected] Turksma Ven Rieneke Verdoes van de The Netherlands The Netherlands The Netherlands The Netherlands VUMC Amsterdam Martijn Radboudumc Nijmegen Versnel Marjan Erasmus MC Rotterdam Verwoolde Michel Wageningen Universiteit Wageningen Vieten Gertrud Hannover Medical School Hannover Germany [email protected] Vlaeminck Yannick Vrij Universiteit Brussel Brussels Belgium [email protected] VUMC Amsterdam The Netherlands [email protected] de van [email protected] [email protected] [email protected] [email protected] Vliet Sandra Vogel Andrea Medical University of Vienna Vienna Austria [email protected] Wehner Rebekka Medical Faculty TU Dresden Dresden Germany [email protected] Weichhart Thomas Medical University of Vienna Vienna Austria [email protected] Weiss Guenther Medical University of Innsbruck Innsbruck Austria [email protected] The Netherlands United Kingdom The Netherlands The Netherlands The Netherlands The Netherlands The Netherlands Wentzel Annelieke Wageningen Universiteit Wageningen Wiatrek Dagmara University of St. Andrews St. Andrews Wichers Harry Wageningen Universiteit Wageningen Wijngaard Rene AMC Amsterdam Wildenberg Manon AMC Amsterdam Winkel Beatrice Leiden University Medical Centre Leiden Winther Menno AMC Amsterdan Yildirim Ceren Medical University of Vienna Vienna Austria [email protected] Zaal Anouk Sanquin Research Amsterdam The Netherlands [email protected] Zakrzewicz Anna Justus-Liebig University Giessen Germany [email protected] Zandbergen Ger Paul-Ehrlich-Institut Langen Germany [email protected] Zhang Xing-Mei Karolinska Institutet Stockholm Sweden [email protected] Ziegler-Heitbroek Loems Monocytomics Herrsching Germany [email protected] Zinser Elisabeth Universitätsklinikum Erlangen Erlangen Germany [email protected] Leiden The Netherlands [email protected] Zom Gijs van den de van ISA Pharmaceuticals BV [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 86 Poster walks Wednesday September 21st. Guided poster 11.30-12.30 walk Presented by Institute Country I Carla Ms Ribeiro AMC NL Control of APC function Gera Goverse KU LEUVEN BE Balcony I Anno Saris Sanquin NL Microbiology and Biotechnology Ulm University Germany Alsya J Affandi UMC Utrecht NL Anouk Zaal Sanquin NL Jelena Popović Medical School, MTZ, Dresden University of Technology Germany II Roham Parsa Karolinska Institute Sweden Chronic inflammatory Arjan Boltjes UMC Utrecht NL disorders Elisabeth Zinser Universitätsklinikum Erlangen Germany Balcony I Anoushka K. Molhoek VUmc NL Ivo S Hansen AMC NL Victoria Saferding Medical University of Vienna Austria Eliane S Piket Karolinska Institute Sweden Katharine M Irvine University of Queensland Austalia Marjan A. Versnel Erasmus MC R'dam NL Annelot C Breedveld VUmc NL III Monika Linke Medical University of Vienna Austria Chronic inflammatory Manon E. Wildenberg AMC NL disorders Peter L Delputte University of Antwerpen BE Balcony I Felicia Bloemendaal AMC NL Felicia Bloemendaal AMC NL Maria Faas UK Erlangen Germany UMC Utrecht NL Instituto de Investigacion Sanitaria Gregorio Maranon Spain Frederica Raggi G.Gaslini Institute Italy Frederica Raggi G.Gaslini Institute Italy Leonard R Pelgrom LUMC NL Hweixian Penny Singapore Immunology Network Estefania Padilla Herdoiza Wioleta Marut Cristina Rueda IV Metabolism Signaling Balcony II and Municio Leong Maria Christina Lebre AMC NL Jonathan Jantsch Institute of Clinical Microbiology and Hygiene FAU, Erlangen Germany Mieke Gouwy Rego Institute, Univ. Leuven BE Melanie Pieber Karolinska Institut Sweden R.J. Eveline Li MCBI VUmc NL Lea A O'Neil University of Bristol UK Hampoton- 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands poster number (11:00) 2016092101 (11:00) 2016092102 (11:00) 2016092103 (11:00) 2016092104 (11:00) 2016092105 (11:00) 2016092106 (11:00) 2016092107 (11:00) 2016092108 (11:00) 2016092109 (11:00) 2016092112 (11:00) 2016092113 (11:00) 2016092114 (11:00) 2016092115 (11:00) 2016092116 (11:00) 2016092117 (11:00) 2016092118 (11:00) 2016092119 (11:00) 2016092110 (11:00) 2016092111 (11:00) 2016092120 (11:00) 2016092121 (11:00) 2016092122 (11:00) 2016092123 (11:00) 2016092124 (11:00) 2016092125 (11:00) 2016092126 (11:00) 2016092127 (11:00) 2016092128 (11:00) 2016092129 (11:00) 2016092131 (11:00) 2016092132 (11:00) 2016092133 (11:00) 2016092134 (11:00) 2016092135 (11:00) 2016092136 87 Veronika Grau Dep of General and Thoracic Surgery, Justus-Liebig-University, Giessen Germany Manon E Wildenberg AMC NL Sandra C.S. Cardoso UMC Utrecht NL Zrinka Oreskovic Veterinary Research Institute, Brno Czech Republic Anna Zakrzewicz Medical Univ Giessen Germany Tiago F Carvalheiro UMC Utrecht NL Zsofia Agod Dep of Immunology, Faculty of Medicine, University of Debrecen Hungary Mateus Tomczyk Jagiellonian Univ, Krakow Poland VI Geert Raes VIB-VUB BE/China Macrophage function in Stephen J Jenkins University of Edinburgh UK health and disease Esther Heideveld Sanquin NL Balcony II David Haschka Medical Univ Innsbruck Austria Sudhanshu Bhushan Institute for Anatomy and Cell Biology, Giessen, Germany Mohammed Ghiboub AMC/Tytgat NL Jules Petit Wageningen University NL VII Aoife Kelly University of Manchester UK Macrophage function in Pieter JM Leenen Erasmus MC R'dam NL health and disease Annelieke S. Wentzel Dep of Animal Sciences, Cell Biology and Imm Group, Wageningen University NL Balcony II Christine Bruggeman Sanquin NL Branislav Krljanac FAU Germany Gertrud Vieten Hannover Medical School Germany Selien Sanches Hasselt University-BIOMED BE V Metabolism Signaling and Balcony II W. 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands (11:00) 2016092130 (11:00) 2016092140 (11:00) 2016092137 (11:00) 2016092138 (11:00) 2016092139 (11:00) 2016092141 (11:00) 2016092142 (11:00) 2016092143 (11:00) 2016092144 (11:00) 2016092147 (11:00) 2016092146 (11:00) 2016092149 (11:00) 2016092150 (11:00) 2016092151 (11:00) 2016092152 (11:00) 2016092145 (11:00) 2016092148 (11:00) 2016092153 (11:00) 2016092154 (11:00) 2016092155 (11:00) 2016092156 (11:00) 2016092157 88 Poster walks Thursday September 22nd. Guided poster walk 11.00-12.00 Presented by Institute Country poster number VIII Andrew S MacDonals University of Manchester UK antigen processing and presentation Martijn Verdoes RadboudUMC, Nijmegen NL Katrien Van der Borght Dieke van Dinther VIB, Ghent University BE VUmc NL Marjorie De Schryver University Antwerpen BE Cecilia I Forss University of Manchester UK Anno Saris Sanquin Research A'dam NL Christoph Lippuner Switzerland Robert Blomgran Department of Anaesthesiology and Pain Therapy, University Hospital Bern Linköping University IX Rieneke van de Ven VUmc - CCA NL Functional heterogeneity of APCs Anouk Zaal Sanquin NL Dorine Sichien VIB IR Un.GENT BE Balcony II Julia S Brunner Medical University Vienna Austria Urszula Florczyk Jagiellonian University, Krakow Poland Nathalie van Leeuwen VUmc NL Alsya J Affandi UMC Utrecht NL Susanne van der Grein Emiliano Melandri Faculty of Veterinary Medicine Utrecht University NL Miltenyi Biotec GmbH NL X Mario Kuttke Med Univ of Vienna Austria Clinical applications and tumor immunology Camilla Salvagno NKI NL Carla M.A. van Alem LUMC NL Balcony I Miche Rombouts University of Antwerp BE Irina Mitrofanova National Research tomsk State University Russia Ilka Knippertz Univ. Erlangen Germany Stefano Bonelli VU Brussel BE Lenneke AM Cornelissen Athanasios A Blanas MCBI VUmc NL MCBI VUmc NL Sophi A Dusoswa MCBI VUmc NL Ines Sufait VU Brussel BE XI Rebekka Wehner Inst of Imm Medical Fac. TU Dresden Germany Clinical applications and tumor immunology Niels Heemskerk MCBI VUmc NL Sophie K Horrevorts MCBI VUmc NL Balcony I Yannick De Vlaeminck VU Brussel BE Rens Braster MCBI VUmc NL Dorian A Stolk MCBI VUmc NL Natasa Prokopi Med Univ of Innsbruck Germany (10:30) 2016092202 (10:30) 2016092204 (10:30) 2016092201 (10:30) 2016092203 (10:30) 2016092205 (10:30) 2016092206 (10:30) 2016092207 (10:30) 2016092208 (10:30) 2016092209 (10:30) 2016092210 (10:30) 2016092211 (10:30) 2016092212 (10:30) 2016092213 (10:30) 2016092214 (10:30) 2016092215 (10:30) 2016092216 (10:30) 2016092217 (10:30) 2016092218 (10:30) 2016092219 (10:30) 2016092220 (10:30) 2016092223 (10:30) 2016092224 (10:30) 2016092225 (10:30) 2016092226 (10:30) 2016092227 (10:30) 2016092228 (10:30) 2016092229 (10:30) 2016092230 (10:30) 2016092231 (10:30) 2016092221 (10:30) 2016092222 (10:30) 2016092232 (10:30) 2016092233 (10:30) 2016092234 (10:30) 2016092235 (10:30) 2016092236 Balcony I Sweden 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 89 Nathalie van Leeuwen VUmc NL Kevan Hosseini Saarland University Germany Tamara Tyrinova Insitute of Fundamental and Clinical Immunology Russia XII Ulrike Schleicher University Hospital Erlangen Germany Host-microbe crosstalk Balcony II Joris K. Sprokholt AMC NL Claudia Kitzmueller University of Salzburg Austria Aurélie Detavernier Int. Medical Immunology,Univ. De Bruxelles BE Martijn Nolte Sanquin NL Mariangela Cavarelli Universite Paris-Sud (CEA) France/IT Stefanie Dichtl Medical Unviersity of Innsbruck Austria Stephanie Obermeyer Microbiology Institute, Erlangen, Germany Ger van Zandbergen Paul-Ehrlich Institut Germany Dagmara Wiatrek University of St Andrews UK XIII Melissa Newling AMC NL Host-microbe crosstalk Balcony II Diana Dudziak University Hospital Erlangen Germany Lisbeth Goncalves ? Brasil? Riem Gawish University of Veterinary Medicine Vienna Autstria Ernesto Rodriguez MCBI VUmc NL Lenka Kavanova Verterinary Res. Institute Brno Julie Deckers University Gent Czech Republic BE Katarína Matiašková Veterinary Research Institute,v.v.i., Brno, Czech Republic Rui Martins CeMM- Research Center for Molecular Medicine of the Austrian Academy of Sciences Czech Republic Austria 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands (10:30) 2016092237 (10:30) 2016092238 (10:30) 2016092239 (10:30) 2016092240 (10:30) 2016092242 (10:30) 2016092244 (10:30) 2016092245 (10:30) 2016092246 (10:30) 2016092247 (10:30) 2016092248 (10:30) 2016092249 (10:30) 2016092250 (10:30) 2016092251 (10:30) 2016092241 (10:30) 2016092243 (10:30) 2016092252 (10:30) 2016092253 (10:30) 2016092254 (10:30) 2016092255 (10:30) 2016092256 (10:30) 2016092257 (10:30) 2016092258 90 NOTES 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 91 NOTES 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 92 NOTES 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 93 NOTES 30th Annual Conference of the EMDS, September 21st-23rd, Amsterdam, The Netherlands 94
