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Background Rheumatoid arthritis (RA) is characterized by the formation of autoantibodies against citrullinated antigens (CA). Intriguingly, depletion of CD20+ B cells leads to significant clinical improvement in the majority of patients, despite the persistence of high titer autoantibodies. This indicates that the CD20+ B cell compartment, potentially more than the autoantibodies themselves, contributes to the pathogenesis and/or chronicity of RA. Auto-reactive B cells within this compartment could be the main drivers of this effect, but evidence for this hypothesis is lacking. We recently developed the technology to visualize and characterize CA-specific B cells in patients and demonstrated that a large proportion has a CD20+ memory phenotype 1 . Objectives To study the phenotype of CA-specific B cells in comparison to tetanus-toxoid (TT)-specific B cells in peripheral blood of individual patients. Methods Differentially labeled CA and their arginine control variants were used as tetramers to identify CA-specific B cells in peripheral blood of patients with RA. TT-specific B cells were identified in the same samples using differentially labeled TT. Both antigen-specific cell populations were enumerated and phenotypically characterized by flow cytometry. Results CA-specific B cells and TT-specific B cells were detected in peripheral blood in comparable frequency (median 0.012% vs. 0.014%). Both CA- and TT-specific B cells displayed mainly markers of class-switched memory B cells. Intriguingly, CA-specific memory B cells showed remarkable overexpression of co-stimulatory molecules and markers of active proliferation. Conclusions This is the first phenotypic analysis of auto-reactive, CA-specific memory B cells in comparison to recall antigen-specific memory B cells in individual patients with RA. CA-specific memory B cells overexpress co-stimulatory molecules and proliferation markers, indicative of an active immune response against CA in these patients. These data support the hypothesis that CA-specific memory B cells contribute to RA pathogenesis and fuel efforts for their antigen-specific depletion. References Kerkman PF et al. Identification and characterisation of citrullinated antigen-specific B cells in peripheral blood of patients with rheumatoid arthritis. Ann Rheum Dis. 2015 Jun 1. doi: 10.1136/annrheumdis-2014-207182. Acknowledgement Supported by grants from the Dutch Arthritis Foundation, The Netherlands Organization for Scientific Research and the Innovative Medicines Initiative funded project BeTheCure. Disclosure of Interest H. Kristyanto: None declared, P. Kerkman: None declared, E. van der Voort: None declared, H. Spits Employee of: AIMM Therapeutics, D. Baeten: None declared, T. Huizinga: None declared, R. Toes: None declared, H. Scherer: None declared

Background Spondyloarthritis (SpA) is a major form of chronic inflammatory arthritis characterized by inflammation of axial and peripheral joints and by pathologic new bone formation leading to ankylosis. Consistent genetic, experimental, and clinical evidence indicates that IL-23/IL-17 immune axis plays a pivotal role in the pathophysiology SpA. It remains, however, unknown which IL-23 responsive cells are the major cellular source of IL-17 in SpA. Innate lymphoid cells (ILCs) are an emerging family of innate immune cells that produce various cytokines, including IL-17 and IL-22, and play critical roles in regulation of inflammation and tissue remodeling. Objectives In this study we investigated the presence and phenotype of ILCs in the peripheral blood and inflamed peripheral joints of patients with SpA. Methods Paired peripheral blood (PB), synovial fluid (SF) and synovial tissue (ST) were obtained from SpA patients with actively inflamed knee joints. ILCs (lineage negative, CD45 + CD127 + CD161 + ) were analysed by flow cytometry. Results ILCs were present in all three compartments of patients with SpA. Analysis of ST revealed a significantly increased frequency of total ILCs in the joint compared with PB ((median (IQR) 0.37 (0.12-1.12)% of the lymphocyte population in ST versus 0.06 (0.04-0.09) % in PB, p=0.016). Immunophenotyping of ILC subsets showed a statistically significant increase in the frequency of ILC1 (CRTH2 - NKp44 - ckit - ) in ST (37.8%; 74.43-20.47%) versus SF (7.27%; 0.6-25.1%, p=0.008) and PB (3.45%; 1.45-9.25%, p=0.004). The second most prominent ILCs in the joint were NCR-negative ILC3 (CRTH2 - NKp44 - ckit + ), composing 33.45% (9.54-50.64%) of the total ILCs. NCR-positive ILC3 (CRTH2 - NKp44 + ckit + ) and ILC2 (CRTH2 + ) populations were present in synovium at lower frequencies. Conclusions We observed an absolute and relative enrichment of both ILC1 and NCR-negative ILC3 in the inflamed ST of patient with spondyloarthritis as compared to PB and SF. As studies in other tissues such as gut and tonsil revealed that these IL-23 responsive ILC subsets can be an important source of IL-17 and /or IL-22 (1, 2), we will further investigate the cytokine production by these synovial ILCs. References Geremia et al. J Exp Med. 2011 208(6):1127-1133 Bernink et al. Nature Immunology 14(3):221-229 Disclosure of Interest None declared

Background Innate lymphoid cells (ILCs) represent a novel family of effector and regulatory cells in innate immunity and tissue remodelling [1]. The family comprises several phenotypically and functionally distinct subsets that produce various cytokines such as IL-22, IL-17, IFNγ, TNF, IL-13 and IL-5, of which many have been shown to be important in arthritis pathogenesis. Objectives The IL-17 and IL-22 producing ILCs are of major interest as they are implicated in chronic gut inflammation [2]. Based on the broad clinical overlap between inflammatory bowel disease and spondyloarthritis (SpA) and the clinical importance of IL-17 in SpA we hypothesize that IL-17 and IL-22 producing ILCs contribute to inflammation and remodelling in SpA synovitis. As these cells have never been described in the joint we aimed at characterising ILCs in chronic inflammatory arthritis. Methods ILCs (lineage negative, CD45 + CD127 + ) were analysed and sorted by flow cytometry from synovial tissue and fluid from rheumatoid arthritis (RA) and SpA patients as well as in blood from SpA patients and healthy donors. mRNA expression of sorted and expanded cells was analysed by qPCR. Results ILCs were identified in blood as well as in synovial tissue and fluid from both RA and SpA patients. The frequency of ILCs was higher in the inflamed joint (0,5-3,3% of the lymphocyte population) than in the peripheral blood compartment (0,1%). In blood, there was no marked difference in the frequency of the different ILC subset between healthy controls (n=10) and SpA patients (n=5). In the inflamed joint, the ILC3 (CRTH2 - NKp44 + ckit + ) and ILC1 (CRTH2 - NKp44 - ckit - ) populations, previously shown to express IL-22 and IFNγ respectively in other tissues, were present in all samples whereas the Th2 cytokine expressing ILC2s (CRTH2+) were found in very low frequencies. Frequencies of ILC subpopulations varied considerably between patients and no differences could be detected between RA and SpA patients. qPCR analysis of expanded cells revealed that ILC1s expressed TBX21 whereas ILC3s expressed RORC . Accordingly, stimulated ILC3s expressed transcripts for both IL-23R and IL-22 but not IL-17. Conclusions ILC1s and ILC3s are present in the chronically inflamed joint and express the key transcription factors associated with specific cytokine profiles. These data indicate that ILCs could contribute to local cytokine-driven immune alterations in SpA and RA. References Spits H, Cupedo T. Innate lymphoid cells: emerging insights in development, lineage relationships, and function. Annu Rev Immunol. 2012;30:647-75. Geremia, A., Arancibia-Carcamo, C. V., Fleming, M. P., Rust, N., Singh, B., Mortensen, N. J., Travis, S. P. & Powrie, F. IL-23-responsive innate lymphoid cells are increased in inflammatory bowel disease. J Exp Med 2011;208:1127-1133. Disclosure of Interest H. Hreggvidsdottir: None Declared, J. Mjosberg: None Declared, J. Bernink: None Declared, D. Baeten: None Declared, H. Spits Employee of: AIMM Therapeutics

Background Rheumatoid arthritis (RA) is a prototypic autoimmune disease characterised by a prominent humoral autoimmunity. Of particular relevance is the local production of autoantibodies such as rheumatoid factor and anti-citrullinated protein antibodies in the inflamed synovial tissue. The mechanisms underlying break of B cell tolerance and local autoantibody production remains poorly understood. Objectives To identify cellular and molecular pathways implicated in RA-specific humoral autoimmunity. Methods Synovial tissue samples were obtained by arthroscopy from untreated individuals with RA (n = 33) and inflammation matched SpA controls (n = 58). Gene expression profiling was performed on tissue samples of patients with established arthritis using 44K. Whole Genome Human microarrays (Agilent). Top differentially expressed genes were validated on three independent cohorts by Taqman based RT-qPCR and immunohistochemistry. Collagen-induced arthritis (CIA) and Experimental autoimmune encephalomyelitis (EAE) experiments were conducted using Bob1 knockout mice and their littermate controls. Results Microarray screening for genes differentially expressed in the inflamed synovium, the key target of the disease process in RA, revealed a prominent and disease-specific B cell/plasma cell signature with the B cell-specific transcriptional co-activator Bob1 and its transcriptional target BCMA among the most upregulated genes. Validation by RT-qPCR on two independent cohorts representing early and established arthritis confirmed microarray data and demonstrated elevated expression of Bob1 and BCMA not only in established RA, but also at the early phase of the disease. Quantitative evaluation of immunohistochemical stainings of synovial tissue with monoclonal antibody for Bob1 revealed significant increase in Bob1 positive cells in RA synovium (p − / − mice were fully resistant to CIA induction compared to their wild-type littermates. This remarkable protection from CIA is explained by decreased antigen-presentation/costimulatory capacity of B cells and by failure to produce pathogenic anti-collagen autoantibodies in the absence of Bob1. Conclusions The specific increase in Bob1 expressing cells in RA synovitis and the resistance of Bob1-deficient mice to development of CIA indicate that Bob1/BCMA axis may contribute to humoral autoimmunity in RA. The relationship between an aberrant Bob1 expression and the break of peripheral tolerance in RA is currently under investigation.