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BACKGROUND Little is known about the autoreactive B cells in antineutrophil cytoplasmic antibody–associated (ANCA-associated) vasculitis (AAV). We aimed to investigate tolerance checkpoints of circulating antigen-specific proteinase 3–reactive (PR3+) B cells. METHODS Multicolor flow cytometry in combination with bioinformatics and functional in vitro studies were performed on baseline samples of PBMCs from 154 well-characterized participants of the RAVE trial (NCT00104299) with severely active PR3-AAV and myeloperoxidase-AAV (MPO-AAV) and 27 healthy controls (HCs). Clinical data and outcomes from the trial were correlated with PR3+ B cells (total and subsets). RESULTS The frequency of PR3+ B cells among circulating B cells was higher in participants with PR3-AAV (4.77% median [IQR, 3.98%–6.01%]) than in participants with MPO-AAV (3.16% median [IQR, 2.51%–5.22%]) and participants with AAV compared with HCs (1.67% median [IQR, 1.27%–2.16%], P < 0.001 for all comparisons), implying a defective central tolerance checkpoint in patients with AAV. Only PBMCs from participants with PR3-AAV contained PR3+ B cells capable of secreting PR3-ANCA IgG in vitro, proving they were functionally distinct from those of participants with MPO-AAV and HCs. Unsupervised clustering identified subtle subsets of atypical autoreactive PR3+ memory B cells accumulating through the maturation process in patients with PR3-AAV. PR3+ B cells were enriched in the memory B cell compartment of participants with PR3-AAV and were associated with higher serum CXCL13 levels, suggesting an increased germinal center activity. PR3+ B cells correlated with systemic inflammation (C-reactive protein and erythrocyte sedimentation rate, P < 0.05) and complete remission (P < 0.001). CONCLUSION This study suggests the presence of defective central antigen-independent and peripheral antigen-dependent checkpoints in patients with PR3-AAV, elucidating the selection process of autoreactive B cells. Trial registration ClinicalTrials.gov NCT00104299. Funding The Vasculitis Foundation, the National Institute of Allergy and Infectious Diseases of the NIH, and the Mayo Foundation for Education and Research.

Background: Sjogren’s syndrome (SS) is a chronic systemic autoimmune disease, characterized by mouth and eye dryness, due to irreversible destruction of glandular tissue by infiltrated lymphocytes. It is now well established that B cells play a key role in the physiopathology of SS. Indeed, B cells exhibit various signs of hyperactivity and produce excessive amounts of pro-inflammatory cytokines and immunoglobulins (Ig), especially IgG type-antibodies (Abs) (Kroese et al.,2014). Currently, it is suggested that the interleukin (IL)-21 (Wang et al., 2018) and IFNα (Yao et al., 2013), promote the generation of autoreactive IgG-producing plasma cells (PC). On the other side, some studies highlight the protective role of IgM producing cells in autoimmunity through enhanced efferocytosis and immune-regulatory properties (Ehrenstein and Notley, 2010). Objectives: This study aims to understand the influence of IL21 and IFNα on the B cell differentiation and the subsequent functional responses of the cells according to their maturation stage. Moreover, this will also enable a further understand of the contribution of these cytokines to the pathogenesis of SS. Methods: We established in vitro models to study the differentiation of different B cell subsets in a T-independent (TI) and T-dependent (TD) manner, under different cytokine stimulations. Naive, switched memory, unswitched memory and CD27-negative memory B cells were isolated from peripheral blood of healthy controls (HC) and SS patients and thereby cultured in those different conditions. After three days of culture, the expression of surface markers and transcription factors was analysed by flow cytometry and molecular identity was further determined by transcriptional analysis. Additional functional assay was also performed to measure Abs and cytokine secretion. Results: First results on HC suggest that switched memory B cells differentiate into IgG and IgA-secreting plasmablasts (PB), when stimulated in a TI manner. This process is highly increased in presence of IFNα. Unswitched memory B cells mostly become PB able to secrete IgM and IL10. IL21 and IFNα oppose distinct effector functions. IL21 promote activation of B cells with an upregulation of the surface marker CD11c and T-bet transcription factor. Transcriptomic studies are still in progress to further define the molecular profile of those distinct effector B cells. Preliminary results on SS patients suggest that B cell differentiation is altered with a biased ratio between activated cells and PB. Conclusion: This work will allow us a better understanding of the heterogeneity of the distinct effector B cells and their involvement in SS pathogenesis. References [1] Ehrenstein, M. R. & Notley, C. A. The importance of natural IgM: scavenger, protector and regulator. Nature Reviews Immunology10, 778–786 (2010). [2] Kroese, F. G. M. et al. B-cell hyperactivity in primary Sjogren’s syndrome. Expert Rev Clin Immunol10, 483–499 (2014). [3] Wang, S. et al. IL-21 drives expansion and plasma cell differentiation of autoreactive CD11chiT-bet+ B cells in SLE. Nat Commun9, 1758 (2018). [4] Yao, Y., Liu, Z., Jallal, B., Shen, N. & Ronnblom, L. Type I interferons in Sjogren’s syndrome. Autoimmun Rev12, 558–566 (2013). Disclosure of Interests: None declared

Background:B cells play a crucial role in the pathogenesis of systemic autoimmunity through various effector functions, including auto-antibody production, secretion of pro-inflammatory cytokines and antigen presentation to T cells. Interferon alpha (IFNα), mainly produced by innate cells (Menon et al., 2016), and interleukin (IL)-21 which is secreted by follicular helper T cells (Berglund et al., 2013), promote the generation of auto-reactive IgG-secreting plasma cells. However, it is suggested that IFNα participate also to the generation of regulatory B cells.Objectives:To further understand the disturbing microenvironmental signals leading to autoimmune diseases, we aim to define a coherent framework integrating the B-cell subsets having different ability to respond to microenvironmental signals, and the signalling pathways driving the differentiation and the functional fate of B cells.Methods:Naïve and several populations of memory B cells were isolated from peripheral blood of healthy donors and differentiatedin vitroin the presence of IL21 or IFNα. The phenotype and the expression of transcription factors were analysed by flow cytometry and molecular identity of these cells was further determined by transcriptomic approaches. Functional analyses were performed to assess the effector functions of B cells and their potential regulatory effects on T cells.Results:IFNα, in synergy with CpG, promote the generation of CD27highCD38highplasmablasts (PB), mainly arising from memory subsets, but not IL21. However, IFNα and IL21 induce the transcriptional program of B-cell differentiation by up-regulating IRF4 and Blimp1 expression. Unlike IFNα, IL21 drive the expansion of CD11c+T-bet+cells only from switched memory subsets, suggesting T-bet may be a footprint of long-lived memory B cells.Even though subtle differences were observed in the antibody production between IFNα and IL21-stimulated memory B cells, naïve cells secreted less amount of IgM and IL10 when stimulated with IL21.Transcriptomic studies are still in progress to further define the molecular profile of those distinct effector B cells.Conclusion:Taken together, these findings suggest that IFNα promote a rapid differentiation of B cells into IL10 and Ig-secreting PB whereas IL21 contributes to the generation of T-bet+atypical pre-PB that may have a role in chronic autoimmune disorders.References:[1]Berglund, L.J., Avery, D.T., Ma, C.S., Moens, L., Deenick, E.K., Bustamante, J., Boisson-Dupuis, S., Wong, M., Adelstein, S., Arkwright, P.D., et al. (2013). IL-21 signalling via STAT3 primes human naive B cells to respond to IL-2 to enhance their differentiation into plasmablasts. Blood122, 3940–3950.[2]Menon, M., Blair, P.A., Isenberg, D.A., and Mauri, C. (2016). A Regulatory Feedback between Plasmacytoid Dendritic Cells and Regulatory B Cells Is Aberrant in Systemic Lupus Erythematosus. Immunity44, 683–697.Disclosure of Interests:None declared

Background:B cells play a central role in many autoimmune diseases (AIDs) including ANCA-associated vasculitis (AAV) and primary Sjögren’s syndrome (pSS). Most of the research that has been conducted on AID has focused on the production, secretion, and pathogenicity of auto-antibodies, but little is known on the characteristics of autoreactive B cells in humans.Objectives:This study aims at characterizing circulating autoantigen (PR3 ou SSA)-specific B-cells in patients with AAV and pSS compared to healthy subjects to better understand their role in thenatural and pathological autoimmunityand define the mechanisms leading to the breakdown of self-tolerance in patients with AID.Methods:First, we developed a new flow-cytometry method to detect circulating auto-reactive B cell based on the specificity of their B-cell receptor (BCR). To study surface phenotype of specific B cells by flow cytometry, blood samples were collected from patient with PR3-ANCA AAV, pSS and from healthy subjects. Functional analysis of antigen-specific B cells was also elicited by in vitro analysis of their capacity to secrete immunoglobulins against SSA or PR3 antigens by ELISPOTResults:Phenotype analysis showed that antigen-specific B cells in patients have a memory phenotype compared with healthy controls (5 to 9% are IgG-expressing memory B cells). It suggests that in AID, theses auto-reactive cells are able to differentiate into IgG isotype-switched cells and escape peripheral tolerance checkpoint but not in healthy subjects. Interestingly, Naturalauto-reactive Bcells are able to secrete only IgM isotype autoantibodies uponin vitrostimulation but not IgG class switched antibodies. In order to better understand what differentiates auto-reactive B cells and the mechanisms leading to pathological autoimmunity, agenomic analysisof the antibody repertoire as well as atranscriptional profilingof these cells by single-cell RNA seq is ongoing to understand further the differences of these autoreactive B cells between healthy subjects and patients with AIDs.Conclusion:We developed a technology to identify and isolate antigen-specific B cells from the peripheral blood of patients with AID. Our results suggest that autoreactive B cells escape peripheral tolerance checkpoint and are able to differentiate into IgG isotype-switched cells in patients with AIDs but not in healthy subjects.References:[1]D. Cornec, A. Berti, A. Hummel, T. Peikert, J.-O. Pers, et U. Specks, « Identification and phenotyping of circulating autoreactive proteinase 3-specific B cells in patients with PR3-ANCA associated vasculitis and healthy controls »,J. Autoimmun., vol. 84, p. 122–131, 2017[2]P. F. Kerkman et al., « Identification and characterisation of citrullinated antigen-specific B cells in peripheral blood of patients with rheumatoid arthritis », Ann. Rheum. Dis., vol. 75, no 6, p. 1170‑1176, juin 2016, doi: 10.1136/annrheumdis-2014-207182.Acknowledgments:with support of vasculitis foundation, CSL Berhing, SFRDisclosure of Interests:None declared