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OBJECTIVE The effector T cell and B cell cytokine networks have been implicated in the pathogenesis of systemic autoimmune diseases, but the association of these cytokine networks with the heterogeneity of clinical manifestations and immune profiles has not been carefully examined. This study was undertaken to examine whether cytokine profiles can delineate distinct groups of patients in 4 systemic autoimmune diseases (systemic lupus erythematosus, Sjogren's syndrome, rheumatoid arthritis, and systemic sclerosis). METHODS A total of 179 patients and 48 healthy volunteers were enrolled in the multicenter cross-sectional PRECISE Systemic Autoimmune Diseases (PRECISESADS) study. Multi-low-dimensional omics data (cytokines, autoantibodies, circulating immune cells) were examined. Coculture experiments were performed to test the impact of the cytokine microenvironment on T cell/B cell cross-talk. RESULTS A proinflammatory cytokine profile defined by high levels of CXCL10, interleukin-6 (IL-6), IL-2, and tumor necrosis factor characterized a distinct group of patients in the 4 systemic autoimmune diseases. In each disease, this proinflammatory cluster was associated with a specific circulating immune cell signature, more severe disease, and higher levels of autoantibodies, suggesting an uncontrolled proinflammatory Th1 immune response. We observed in vitro that B cells reinforce Th1 differentiation and naive T cell proliferation, leading to the induction of type 1 effector B cells and IgG production. This process was associated with an increase in CXCL10, IL-6, IL-2, and interferon-γ production. CONCLUSION This composite analysis brings new insights into human B cell functional heterogeneity based on T cell/B cell cross-talk, and proposes a better stratification of patients with systemic autoimmune diseases, suggesting that combined biomarkers would be of great value for the design of personalized treatments.

International audience; Antibody-secreting cells (ASC) are divided into two principal subsets, including the long-lived plasma cell (PC) subset residing in the bone marrow and the short-lived subset, also called plasmablast (PB). PB are described as a proliferating subset circulating through the blood and ending its differentiation in tissues. Due to their inherent heterogeneity, the molecular signature of PB is not fully established. The purpose of this study was to decipher a specific PB signature in humans and mice through a comprehensive meta-analysis of different data sets exploring the PB differentiation in both species and across different experimental conditions. The present study used recent analyses using whole RNA sequencing in prdm1-GFP transgenic mice to define a reliable and accurate PB signature. Next, we performed similar analysis using current data sets obtained from human PB and PC. The PB-specific signature is composed of 155 and 113 genes in mouse and human being, respectively. Although only nine genes are shared between the human and mice PB signature, the loss of B-cell identity such as the down-regulation of PAX5, MS4A1, (CD20) CD22 and IL-4R is a conserved feature across species and across the different experimental conditions. Additionally, we observed that the IRF8 and IRF4 transcription factors have a specific dynamic range of expression in human PB. We thus demonstrated that IRF4/IRF8 intranuclear staining was useful to define PB in vivo and in vitro and able to discriminate between atypical PB populations and transient states.

Immunological memory is essential for effective immune protection upon antigen rechallenge. Memory B cells encompass multiple subsets, heterogeneous in terms of phenotypes, origins and precursors, anatomical localization, and functional responses. B-cell responses are conditioned by micro-environmental signals, including cytokines. Here, we analyzed in vitro the effects of two cytokines implicated in B-cell differentiation, interferon-alpha (IFN-α) and interleukin (IL)-21, on the early functional response of four different mature B-cell subsets (IgD- CD27- naive, IgD+ CD27+ unswitched, IgD- CD27+ switched and double-negative B cells). The dual response of naive and memory B cells to IL-21 allowed us to uncover a unique IgD+ CD27- CD10- B-cell population (referred to as NARB+) characterized by the expression of marginal zone B-cell markers CD45RB and CD1c. Similar to memory B cells, NARB+ cells were in a pre-activated state, allowing them to rapidly differentiate into plasmablasts upon innate signals while maintaining their susceptibility to IL-21 activation-induced apoptosis as observed for the naive compartment. Both in-depth phenotypic analysis of circulating B cells, and identification of these cells in spleen, tonsil and gut-associated lymphoid tissues, supported that NARB+ are uncommitted precursors of human marginal zone B cells.

Immunological memory is essential for effective immune protection upon antigen rechallenge. Memory B cells encompass multiple subsets, heterogeneous in terms of phenotypes, origins and precursors, anatomical localization, and functional responses. B-cell responses are conditioned by micro-environmental signals, including cytokines. Here, we analyzed in vitro the effects of two cytokines implicated in B-cell differentiation, interferon-alpha (IFN-α) and interleukin (IL)-21, on the early functional response of four different mature B-cell subsets (IgD+ CD27- naive, IgD+ CD27+ unswitched, IgD- CD27+ switched and double-negative B cells). The dual response of naive and memory B cells to IL-21 allowed us to uncover a unique IgD+ CD27- CD10- B-cell population characterized by the expression of marginal zone B-cell markers CD45RB and CD1c (referred to as NARB+). Similar to memory B cells, NARB+ cells displayed a pre-activated state, allowing them to rapidly differentiate into plasmablasts upon innate signals. However, this population also maintained its susceptibility to IL-21 activation-induced apoptosis similarly to the naive compartment. Both in-depth phenotypic analysis of circulating B cells, and identification of these cells in spleen, tonsil, and gut-associated lymphoid tissues, suggested that NARB+ could be uncommitted precursors of different developmental pathways including human marginal zone-like B cells (MZB). In this regard, we showed that patients with primary Sjogren’s syndrome exhibited a severe reduction of IgD+CD27+ MZB-like cells associated with a diminution of the NARB+ subset suggesting a developmental relationship.

International audience; The innate B cell (IBC) population is heterogeneous and involved in the primary immune response. IBC functions include a high ability to produce natural antibodies with IgM isotype, the elimination of apoptotic cells, and a capacity to be cognate help to T cells. Among IBC subsets, B-1 cells and marginal zone B cells are the main producers of IgM, act as rapid immune responders that may relocate to follicular lymphoid and differentiate to cytokine and antibody-secreting cells shortly after infection. IBCs functions are highly dependent on their localization site and the nature of their B cell receptor repertoire, suggesting a high plasticity range of different immune responses. In this review, we will describe the nature and functions of the different innate-like B cell subsets, first in mice and then in humans. Besides this, we will emphasize the strong ability of these cells to undertake different protective functions from the first line of defense against pathogens to the regulatory role of the broader immune response.

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

Objective: This report presents translational research showing that a pathogenic cytokine network could stratify systemic lupus erythematosus (SLE) patients associated with B cell dysfunction. Methods: Forty-five patients with SLE and 48 healthy volunteers (HV) were recruited from 10 centers in Europe. Cytokines analyses on patient's sera were performed using the Luminex technology. Autoantibodies measurement was realized on analyzer IDS-iSYS chemiluminescent. Patient's phenotyping was performed on whole blood. Functional profiles were explored using flow cytometry and quantitative RT-PCR. Cytokines in supernatants were measured using a multiplex bead-based assay. Results: To better understand how the cytokine microenvironment could be associated with different disease status in patients, we profiled 12 cytokines using the Luminex technology in serum from 45 SLE patients and 48 HV. We thus documented a unique pathogenic cytokine cluster in a group of patient delineated by CXCL10, IL-6, and IFN-γ. This pathogenic network was associated with pro-inflammatory helper B cells and an increase in systemic autoreactivity. We thus designed an in vitro model to study cytokine networks involved in pro- and anti- inflammatory B-cell functions. We demonstrated that CD4+ CD45RA+ naive T cells (TRA) polarized B cells toward pro-inflammatory B cells defined by a memory-like phenotype and an exacerbated production of CXCL-10, IFN-γ, and IL-2. Conclusion: This composite analysis brings new insights on human B cell functional heterogeneity and, on the other hand, proposes a better stratification of SLE patients according to their functional cytokine microenvironment suggesting that combined biomarkers would be a great value to design new personalized treatments. Clinical Trial Number: Registered on clinicaltrials.gov NCT 02890121 and NCT 02890147. Funding Statement: The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking PRECISESADS under grant agreement n° 115565, resources of which are composed of financial contribution from the European Union's Seventh Framework Program (FP7/2007-2013) and EFPIA companies in kind contribution. Declaration of Interests: The authors declare no competing interests. Ethical Approval Statement: The study was performed in accordance with the Declaration of Helsinki and was approved by ethical committees at each site for respective sub-cohorts. All patients gave their informed consent for participating in the study, which was registered on clinicaltrials.gov NCT 02890121 and NCT 02890147.

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