Search

B cell abnormalities are common in systemic lupus erythematosus (SLE), and include expansion of double negative (DN) and age-associated-like B cells (ABC-like). We aimed to investigate rituximab (RTX) effects on DN and ABC-like B-cell subsets and, when possible, also secondary effects on T cells. Fifteen SLE patients, fulfilling the ACR 1982 criteria, starting RTX and followed longitudinally up to two years, were analyzed for B- and T- lymphocyte subsets using multicolor flow cytometry. DN were defined as IgD-CD27- and ABC-like as CD11c+CD21- within the DN gate. Additional phenotyping was performed adding CXCR5 in the B-cell panel. Cellular changes were further analyzed in the context of the generation of anti-drug antibodies (ADA) against RTX and clinical information. The SLE patients were mainly females (86.6%), of median age 36.7 (29.8-49.4) years and disease duration of 6.1 (1.6-11.8) years. Within the DN subset, ABC-like (IgD-CD27-CD11c+CD21-) B cell frequency reduced from baseline median level of 20.4% to 11.3% (p=0.03), at early follow-up. The DN B cells were further subdivided based on CXCR5 expression. Significant shifts were observed at the early follow-up in the DN2 sub-cluster (CD11c+CXCR5-), which reduced significantly (-15.4 percentage points, p=0.02) and in the recently described DN3 (CD11c-CXCR5-) which increased (+13 percentage points, p=0.03). SLE patients treated with RTX are at high risk of developing ADA. In our cohort, the presence of ADA at 6 months was associated with lower frequencies of DN cells and to a more pronounced expansion of plasmablasts at early follow-up. The frequency of follicular helper T cells (T

Based on the epidemiological link between periodontitis and rheumatoid arthritis (RA), and the unique feature of the periodontal bacterium

Background Systemic lupus erythematosus (SLE) is a systemic autoimmune disease, which exhibits multiple B cell abnormalities including expanded populations of memory B cells and elevated levels of autoantibodies. Belimumab is a monoclonal antibody targeting the B cell cytokine BAFF (a.k.a. BLyS), approved for the treatment of SLE. Methods In this prospective cohort study, B cells from peripheral blood of 23 SLE patients initiating belimumab treatment and followed longitudinally for up to three years, were assessed using mass cytometry. Findings B cells decreased during the study period, with a rapid decrease of both naive and CD11c+CD21− B cells at the first follow-up visit, followed by a continuous reduction at subsequent follow-ups. In contrast, plasma cells and switched memory B cells remained stable throughout the study. The observed immunological changes correlated with early, but not late, clinical improvements. Moreover, high baseline B cell counts were predictive of failure to attain low disease activity. In summary, our data unveiled both rapid and gradual later therapy-associated alterations of both known and unforeseen B cell phenotypes. Interpretation Our results suggest that evaluation of B cell counts might prove useful prior to initiation of belimumab treatment and that early treatment evaluation and discontinuation might underestimate delayed clinical improvements resultant of late B cell changes.

Arthritis is a common clinical feature of systemic lupus erythematosus (SLE) and is usually non-erosive as opposed to rheumatoid arthritis (RA). While RA synovial pathology has been extensively studied, little is known about the pathophysiology of lupus arthritis. Here, we aimed to explore the cytokine and cellular compartments in synovial fluids of SLE patients with arthritic manifestations. Acellular synovial fluid and paired serum samples from SLE patients (n=17) were analyzed with cytokine bead array for T helper associated cytokines. From two SLE patients, synovial fluid mononuclear cells (SFMC) were analyzed by multiparameter flow cytometry to dissect T cell, B cell, monocyte and dendritic cell phenotypes. SLE-derived SFMC were further stimulated in vitro to measure their capacity for producing IFN and IL-17A. All patients fulfilled the ACR 1982 classification criteria for SLE. Clinical records were reviewed to exclude the presence of comorbidities such as osteoarthritis or overlap with RA. IL-17A and IL-6 levels were high in SLE synovial fluid. A clear subset of the synovial CD4+ T cells expressed CCR6+, a marker associated with Th17 cells. IL-17-production was validated amongst CD4+CCR6+ T cells following in vitro stimulation. Furthermore, a strong IFN production was observed in both CD4+ and CD8+ cells. Our study shows high IL-17A and IL-6 levels in synovial fluids of patients with lupus arthritis. The Th17 pathway have been implicated in several aspects of SLE disease pathogenesis and our data points to Th17 involvement also for lupus arthritis.

Summary Arthritis is a common clinical feature of systemic lupus erythematosus (SLE) and is usually non‐erosive, as opposed to rheumatoid arthritis (RA). While RA synovial pathology has been extensively studied, little is known about the pathophysiology of lupus arthritis. Here, we aimed to explore the cytokine and cellular compartments in synovial fluids of SLE patients with arthritic manifestations. Acellular synovial fluid and paired serum samples from SLE patients (n = 17) were analyzed with cytokine bead array for T helper‐associated cytokines. From two SLE patients, synovial fluid mononuclear cells (SFMC) could also be captured and were analyzed by multiparameter flow cytometry to dissect T cell, B cell, monocyte and dendritic cell phenotypes. SLE‐derived SFMC were further stimulated in vitro to measure their capacity for producing interferon (IFN)‐γ and interleukin (IL)‐17A. All patients fulfilled the ACR 1982 classification criteria for SLE. Clinical records were reviewed to exclude the presence of co‐morbidities such as osteoarthritis or overlap with RA. IL‐17A and IL‐6 levels were high in SLE synovial fluid. A clear subset of the synovial CD4+ T cells expressed CCR6+, a marker associated with T helper type 17 (Th17) cells. IL‐17A‐production was validated among CD4+CCR6+ T cells following in‐vitro stimulation. Furthermore, a strong IFN‐γ production was observed in both CD4+ and CD8+ cells. Our study shows high IL‐17A and IL‐6 levels in synovial fluids of patients with lupus arthritis. The Th17 pathway has been implicated in several aspects of SLE disease pathogenesis and our data also point to Th17 involvement for lupus arthritis., IL‐17A and IL‐6 are elevated in synovial fluid of SLE arthritis patients. The Th17 pathway have been implicated in several aspects of SLE disease pathogenesis and our data points to Th17 involvement also for lupus arthritis.

Objective Anti-citrullinated protein antibodies (ACPAs) have proven highly useful as biomarkers for rheumatoid arthritis (RA). However, composition and functionality of the associated autoreactive B cell repertoire have not been directly assessed. We aimed to selectively investigate citrullinated autoantigen-specific B cell receptors (BCRs) involved in RA and initiate studies on their pathogenicity. Methods Blood samples were obtained from patients in a University of Minnesota cohort with ACPA-positive RA (n = 89). Tetramer sets bearing citrullinated filaggrin peptide cfc1 or citrullinated α-enolase peptide were constructed to specifically capture autoreactive B cells from the unaltered, polyclonal repertoire in RA patients. Citrullinated peptide tetramer-bound B cells were subjected to flow cytometric cell sorting and single-cell IGH, IGK, and IGL gene sequencing for B cell lineage determinations. BCR gene sequences were also expressed as recombinant monoclonal antibodies (mAb) for direct evaluation of citrullinated autoantigen binding and effector functionality. Results Using citrullinated peptide tetramer enrichment to investigate single autoreactive blood B cells, we identified biased V-region gene usage and conserved junction arrangements in BCRs from RA patients. Parsimonious clustering of related immunoglobulin gene nucleotide sequences revealed clonal expansions of rare individual B cell clades, in parallel with divergent sequence mutations. Correspondingly, recombinant mAb generated from such BCR lineages demonstrated citrulline-dependent cross-reactivity extending beyond the citrullinated peptides used for B cell capture. A pair of citrullinated autoantigen-specific mAb with cross-reactive binding profiles also promoted arthritis in mice. Conclusion Our findings suggest that broad ACPA specificities in RA arise from a restricted repertoire of evolving citrulline-multispecific B cell clades with pathogenic potential.

Background: Antibodies targeting citrullinated proteins (ACPA) are highly specific for rheumatoid arthritis (RA). However, the etiology and molecular basis for ACPA production is still unclear. Based on an epidemiological association between RA and periodontitis (PD), and the unique ability of the oral pathogen P.gingivalis (Pg) to express a PAD enzyme that can citrullinate both bacterial and human proteins, it has been hypothesised that the ACPA response may be triggered in the gum mucosa in response to Pg. Objectives: The main purpose of this study was to investigate if citrulline-reactive B cells reside in inflamed gingival tissue, and to examine ACPA cross-reactivity between citrullinated bacterial and human epitopes on a monoclonal level. Methods: Using a single-cell antibody cloning approach, 55 recombinant monoclonal antibodies (mAbs) were generated from gingival tissue (GT) CD19+ B cells (n=1 ACPA+ RA/PD patient). Citrulline reactivity was determined using the anti-CCP2 kit (EuroDiagnostica AB), and in-house peptide ELISAs (including a citrullinated peptide derived from Pg PAD (CPP3) and citrullinated peptides derived from human α-enolase, fibrinogen, vimentin, fillaggrin and histone 4). Reactivity against CCP2 and CPP3 was also investigated in: 19 mAbs from bronchoalveolar lavage (BAL) CD19+ B cells (n=2 ACPA+ RA patients); 29 mAbs from bone marrow (BM) plasma cells (n=4 ACPA+ RA patients); 142 mAbs from synovial fluid (SF) plasma cells (n=5 ACPA+ RA patients); and 36 mAbs from peripheral blood memory/plasma cells (n=4 ACPA+ RA patients). Predicted germline versions were produced for two of the mAbs. Results: Among 55 GT mAbs, 14 unique clones (25%) were reactive to the bacterial CPP3-peptide. We also detected CPP3-reactive mAbs from BAL (n=9/19), BM (n=3/29), SF (n=1/142) and peripheral blood (n=1/36). Interestingly, 11 out of 28 (39%) CPP3-reactive clones also bound citrullinated peptides derived from human proteins. Notably, three of these clones were positive in the clinical anti-CCP2 test, and when converted back to the predicted germline sequence, these clones became CCP2 negative, while maintaining reactivity against the bacterial CPP3 peptide. Conclusion: Our data show that B cells reactive with a citrullinated peptide derived from Pg PAD are present in gingival tissue, lungs, bone marrow, blood and the inflamed joint of ACPA+ RA patients. Moreover, the finding that a number of these clones are cross-reactive with citrullinated peptides derived from human proteins as well as the gold standard CCP2 test suggests mechanisms of molecular mimicry in the generation of ACPA. Importantly, the germline versions of these ACPA were Pg-reactive but not autoreactive, supporting the hypothesis of a bacterial origin for this ACPA response. Disclosure of Interests: Natalia Sherina: None declared, Vijay Joshua: None declared, Radha Thyagarajan: None declared, Natalie Sippl: None declared, Lena Israelsson: None declared, Heidi Wahamaa: None declared, Ragnhild Stalesen: None declared, Kaja Eriksson: None declared, Tulay Yucel-Lindberg: None declared, Aase Hensvold: None declared, Caroline Gronwall: None declared, Anca Catrina Grant/research support from: Yes, but not for the presented study., Vivianne Malmstrom: None declared, Antonio Lanzavecchia: None declared, Luca Piccoli: None declared, Khaled Amara: None declared, Karin Lundberg: None declared

Background: Musculoskeletal involvement is a common clinical feature of systemic lupus erythematosus (SLE), that can be present either at the onset or in later disease course. SLE related arthritis is usually non-erosive and non-deforming as opposed to rheumatoid arthritis (RA). While RA synovial pathology has been extensively studied, little is known about the pathophysiology of arthritis in SLE. Objectives: to explore the cellular compartments in synovial fluid of SLE patients with arthritic manifestations. Methods: paired synovial fluid (SF) samples from large joint aspiration and peripheral blood samples (PBMC) obtained at the same time point from five SLE patients were analyzed by multicolor flow cytometry. The patients fulfilled the ACR 1982 classification criteria for SLE [1]. Clinical records were reviewed in order to exclude the presence of comorbidities such osteoarthritis or overlap with RA. Three different lineage-specific panels for B cells, T cells (cytotoxic and helper) were developed. Results: The overall frequency of CD4+ and CD8+ T cells was similar across the SF and PBMC samples. Among the CD4+ T cells, those co-expressing CCR4, showed a much higher frequency in the SF compared to the peripheral blood in 4 out of 5 patients (mean percentage 8.9±7.0% vs 2.1±1.6%, p=ns). In addition, in 4 out of 5 patients we could identify an increased frequency of CD4+ expressing CCR6+, a marker for Th17 cells in SF as compared to PBMC (mean percentage 35±16.6% vs 12.7±8.9%, respectively, p=ns). In all patients, a higher frequency of EOMES+ Granzyme A + CD4+ T cells was observed in SF when compared to PBMC (9.2±2.5% vs 4.5±2.5%, p=0.03). Moreover, in all patients, we could observe a higher proportion of regulatory T cells (FOXP3+/CD25+) in the SF (21.5±15.4% vs 8.4±7.8%, p=ns). No relevant differences were observed in the Th1 compartment (CXCR3+). CD19+ cells (B-lymphocytes) were scarcely present in the SF of SLE patients as opposed to the peripheral blood. Conclusion: Although SLE is usually considered to be a B cell driven disease, its common clinical features like arthritis could be driven in situ by T cells, namely subtypes of CD4+ (helper) cells such as TH17 cells and CD4+ T cells with cytotoxic profile. Further confirmation of the present findings is needed. Reference [1] Tan, E.M., et al., The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum, 1982. 25(11 Disclosure of Interests: Francesca Faustini Consultant for: I have participated to an advisoryborad for Pfizer Sweden in one occasion during 2018, Speakers bureau: I have been speaker for an internal education meeting for Novartis Sweden in one occasion during 2018, Natalie Sippl: None declared, Karine Chemin: None declared, Iva Gunnarsson: None declared, Vivianne Malmstrom: None declared

Career situation of first and presenting author Student for a master or a PhD. Introduction The presence of anti-citrullinated protein antibodies (ACPAs) is a hallmark of rheumatoid arthritis (RA). ACPAs specifically recognize citrullinated epitopes, a result of a post-translational modification catalyzed by peptidyl arginine deiminases (PAD). Based on the unique feature of the periodontal bacteria Porphyromonas gingivalis (P.gingivalis) to express P.PAD it has been suggested that ACPA-positive RA may be precipitated in the gum mucosa. Objectives To address this hypothesis our aims were to investigate the antibody response against a citrullinated P.PAD peptide (CPP3) in patients with RA, chronic periodontitis (PD) and in controls. In addition, we generated monoclonal antibodies (mAbs) from gingival tissue B cells of RA patient aiming to investigate whether citrulline-specific B cells may reside in the gingiva. Methods Gingival tissue-derived single CD19+ B cells from an ACPA-positive RA patient with PD were sorted by flow cytometry. Immunoglobulin variable region genes were sequenced and expressed to generate recombinant mAbs. CPP3-reactivity was analysed by ELISA in serum samples from 66 PD patients, 63 periodontally healthy controls (non-PD), 200 RA patients, and 120 non-RA controls, as well as in 55 mAbs. Differences in antibody levels were examined using Mann-Whitney U test for independent groups. Results Anti-CPP3 antibody levels were low in non-PD controls, while 65% of PD patients showed elevated levels (p Conclusions This study shows that a substantial proportion of systemically healthy individuals possess ACPAs directed against P.gingivalis, and these ACPAs also bind epitopes on human proteins. Based on our data, we propose that the ACPA response may be triggered by P.gingivalis via an antibody response against CPP3, which cross-reacts with human citrullinated proteins by mechanisms of molecular mimicry. Disclosure of Interest None declared.

Background:Epidemiologic studies have demonstrated a link between periodontitis (PD) and rheumatoid arthritis (RA), specifically RA characterized by anti-citrullinated protein antibodies (ACPA). The keystone pathogen driving PD, Porphyromonas gingivalis (Pg), is the only pathogen known to express peptidylarginine deiminase (PAD), a citrullinating enzyme. Hence, Pg has been proposed to be involved in triggering the ACPA response, by generating citrullinated antigens in an inflammatory milieu(1). Another major virulence factor of Pg is arginine gingipain B (RgpB), a proteinase which cleaves proteins so that P.PAD can access the site where citrullination takes place. We have previously shown elevated anti-RgpB IgG levels in ACPA+ RA patients, even before clinical onset(2, 3), and we hypothesize that anti-RgpB IgG could serve as a serological marker to identify PD patients with increased risk of developing ACPA+ RA.Objectives:Based on this hypothesis, we set out to investigate whether anti-RgpB IgG was associated with PD, PD severity, autoimmunity in general, and the ACPA response in particular.Methods:Anti-RgpB IgG, as well as RA- and systemic lupus erythematosus (SLE)-related autoantibodies targeting cyclic citrullinated peptide(s) (CCP2), rheumatoid factor (RF), dsDNA, cardiolipin, and β2 glycoprotein, were measured by ELISA in serum samples from the ParoKrank study, which is a well-characterized cohort of 805 patients with a first myocardial infarction and 805 matched controls, where periodontal status has been determined by dentists(4). In this study, individuals with PD (n=941) were compared to individuals without PD (n=557).Results:We detected significantly elevated (pConclusion:Our data demonstrates a specific association between severe PD, elevated anti-RgpB IgG levels and RA-related autoantibodies, supporting a role for Pg in linking PD to ACPA+ RA. Further investigation will be needed to confirm whether anti-RgpB IgG can be used as a serological marker to identify PD patients with increased risk of developing ACPA+ RA.References:[1]Rosenstein ED, Greenwald RA, Kushner LJ, Weissmann G. Hypothesis: the humoral immune response to oral bacteria provides a stimulus for the development of rheumatoid arthritis. Inflammation. 2004;28(6):311-8.[2]Kharlamova N, Jiang X, Sherina N, Potempa B, Israelsson L, Quirke AM, et al. Antibodies to Porphyromonas gingivalis Indicate Interaction Between Oral Infection, Smoking, and Risk Genes in Rheumatoid Arthritis Etiology. Arthritis Rheumatol. 2016;68(3):604-13.[3]Johansson L, Sherina N, Kharlamova N, Potempa B, Larsson B, Israelsson L, et al. Concentration of antibodies against Porphyromonas gingivalis is increased before the onset of symptoms of rheumatoid arthritis. Arthritis Res Ther. 2016;18(1):201.[4]Rydén L, Buhlin K, Ekstrand E, Faire Ud, Gustafsson A, Holmer J, et al. Periodontitis Increases the Risk of a First Myocardial Infarction. Circulation. 2016;133(6):576-83.Disclosure of Interests:None declared

Background:Immune system’s abnormalities in SLE involve several subsets of the B-cell compartment, including double negative B-cells (DN) and CD11c+CD21- B cells (also referred to as ABC-age associated B cells), which are expanded in the disease. ABC cells are also known to interact with T helper cells, T follicular and peripheral helper cells (1). Rituximab, a chimeric anti- CD20 antibody, depleting B cells, is commonly used off-label as treatment for SLE patients, especially in lupus nephritis. Little is known on the impact of B-cell depletion on such B-cell subsets and on B-T-cell interactions.Objectives:to investigate the effects of rituximab (RTX) on the frequencies of double negative B-cell subsets and CD11c+CD21- ABC cells and as well as T follicular helper (TFH, CXCR5+ PD-1+) and T peripheral helper (TPH, PD-1high) CD4+ T-cell subsets.Methods:15 SLE patients, starting RTX and followed longitudinally up to two years, were analyzed for lymphocyte subsets using multicolor flow cytometry. Cryopreserved PBMC were thawed and stained at the same time together with one buffy coat. Around 1 x 106 PBMC for each panel were labeled and further stained with fluorescent antibodies for B and T-cell markers. For the B-cell panel, PBMC were stained with anti-CD3, CD14, CD16, CD19, IgD, CD27, CD38, CD11c, CD21 and in some samples with anti-CXCR5 antibodies. For the T-cell panel, PBMC were labeled with anti-CD16, CD14, CD19 and CD3, CD4, CD8, PD-1, CCR7, CXCR5, CD45RA antibodies. All patients fulfilled the ACR 1982 classification criteria for SLE. Cellular changes were analyzed in the context of clinical information.Results:in the present cohort, the SLE patients were mainly female (86.6%) and of median age of 36.7 (29.8-49.4) with a disease duration of 6.1(1.6-11.8) years, and active disease with SLEDAI-2K at baseline 12.0 (8.0-16.0). The frequency of age-associated B cells (ABCs; CD27-IgD-CD11c+ CD21-) decreased by 13% (p=0.03) in the first two to four months after rituximab start, while globally the DN (IgD-CD27-) B cells transiently increased by around 3% (p=0.15) at the first follow-up. This increase could not be attributed to the DN1 (CXCR5+CD11c-) or DN2 (CXCR5-CD11c+) subsets but to the CD11c-CXCR5- DN (DN3) B cells (increase= 6.7%, p=0.03). In parallel, T effector cells (CCR7- CD45RA+) and TEMRA (CD45RA+ CCR7-) frequencies increased after first follow up in both CD4+ and CD8+ T cells. The frequency of TFH (CXCR5+ PD-1+) cells did not change after rituximab, however a decrease of PD-1high CD4+ cells was observed in most patients, although not significant, after 2-4 month of treatment. In most patients the frequency of PD-1high CD4+ cells either reduce or stay the same after RTX treatment (reduction= 0.53, p=0.28). After 11-15 months of RTX treatment the frequency of PD-1high CD4+ T cell reduces by a -0.5% in comparison to 2-4 months (p=0.039). The SLEDAI at baseline did not correlate with the frequency of PD-1high CD4+ T cells (r=0.03, p=0.9).Conclusion:the importance of T cell - B cell interactions in SLE pathogenesis was recently strengthened by the identification of the lymphocyte subsets TFH/TPH and ABCs respectively. Here, in the context of rituximab treated SLE, we could detect a reduction in the frequencies of both ABCs and PD-1high T cells after treatment with rituximab, while the DN3 and effector memory T cells frequencies increased. Our data suggests that anti-CD20 mediated B-cell depletion affects both B-cell and T-cell subsets frequencies, and that monitoring these specific cell subsets may be clinically relevant.References:[1]Bocharnikov AV, Keegan J, Wacleche VS, Cao Y, Fonseka CY, Wang G, et al. PD-1hiCXCR5- T peripheral helper cells promote B cell responses in lupus via MAF and IL-21. JCI insight. 2019;4(20)Disclosure of Interests:Francesca Faustini Speakers bureau: More than two years ago and not in relation to any aspect of the present research, Natalie Sippl: None declared, Ragnhild Stålesen: None declared, Karine Chemin: None declared, Iva Gunnarsson: None declared, Vivianne Malmström: None declared.

Background: Antiphospholipid syndrome (APS) is defined by arterial, venous, or microvascular thrombosis or obstetric morbidity together with confirmed positive test results, at least 12 weeks apart ...

Introduction Increasing evidence points to the autoimmune process of rheumatoid arthritis (RA) originating at mucosal surfaces. Previous work from our group described a subset of B cells in the synovium and synovial fluid (SF) of RA patients distinguishable by their expression of Fc-like receptor 4 (FcRL4) and elevated expression of RANKL, indicating a unique pathogenic function.1,2 B cells expressing FcRL4 were originally described as a distinct memory B cell subset in human tonsils where they are localised in the epithelium.3 We have recently shown in RA that they are enriched in clones recognising citrullinated autoantigens.3 Recent in vitro work suggested that FcRL4 is a low affinity receptor for aggregated IgA.4 Objectives Investigate the interactions between IgA and FcRL4 and FcRL4+ B cells using tonsil and SF B cells, and transfected cell lines. Examine the distribution of Ig classes by flow cytometry and PCR. Methods Mononuclear cells were isolated from SF (n=10) and tonsil, labelled for CD19, FcRL4, IgA, and IgG and analysed by flow cytometry. In experiments identifying BCRs and relative loads of surface bound IgA; SFMCs were washed in an acidic buffer to remove receptor-bound proteins before staining. Heat-aggregated purified human IgA was added to assess IgA binding to FcRL4 +B cells. Single B cells were sorted from SF of RA patients and their constant region genes probed for identification of their Ig subclass by PCR. Results We show that ex vivo SF FcRL4+ B cells have a significantly higher load of IgA bound to their surface (p=0.0001) than FcRL4- B cells. Following in vitro removal of surface bound IgA, FcRL4 +B cells bind heat-aggregated IgA (p=0.03 vs control). We also demonstrate that a significantly higher proportion of FcRL4+ B cells use IgA BCRs (p=0.0061) by flow cytometry, and by probing constant region genes by PCR an enrichment for Ig genes for coding the IgA1 isotype (p=0.009) was found. Furthermore, three out of eight of the antibodies recognising citrullinated peptides had been cloned from FcRL4+ IgA+ B cells. Conclusions In conclusion, their specificity for citrullinated antigens, ability to capture IgA immune complexes through via FcRL4 and enrichment in IgA1 subclass expression, suggest a role for FcRL4 +B cells in the mucosal origin of joint inflammation. References . Yeo L, et al. Ann Rheum Dis2011;70:2022–2028. doi:10.1136/ard.2011.153312 . Yeo L, et al. Ann Rheum Dis2015;74:928–935. doi:10.1136/annrheumdis-2013-204116 . Amara K, et al. J. Autoimmun2017. doi:10.1016/j.jaut.2017.03.004 . Wilson TJ, et al. J Immunol2012;188;4741–4745. doi:10.4049/jimmunol.1102651 Acknowledgements This work was supported by the Medical Research Council UK, Rheumatoid Arthritis Pathogenesis Centre of Excellence, and Karolinska Institutet Foundations for Rheumatology Research. Disclosure of interest None declared

Background Increasing evidence points to the autoimmune process of rheumatoid arthritis (RA) originating at mucosal surfaces. Previous work from our group described a subset of B cells in the synovium and synovial fluid (SF) of RA patients which can be distinguished by their expression of the Fc-like receptor 4 (FcRL4) and elevated expression of RANKL, indicating a unique pathogenic function1,2. B cells expressing FcRL4 were originally described as a distinct memory B cell subset in human tonsils where they accumulate in the epithelium3. We have recently shown that they are enriched in cells recognizing citrullinated autoantigens (Amara, K. et al. under revision). Recent in vitro work suggested that FcRL4 is a low affinity receptor for aggregated IgA4. Objectives 1) To investigate the interaction of RA synovial fluid FCRL4+ B cells with IgA 2) To examine the distribution of Ig classes by flow cytometry and PCR Methods SF mononuclear cells were isolated, labelled for FcRL4, IgA and CD19 and analysed by flow cytometry. In experiments identifying IgA B cell receptors, SF mononuclear cells were briefly incubated in an acidic buffer to remove surface receptor bound antibodies before staining. Heat-aggregated purified human IgA was added to assess IgA binding to FcRL4+ B cells. Single B cells were sorted from SF of RA patients and their constant region genes probed for identification of their Ig subclass by PCR. Results Ex vivo, FcRL4+ B cells in SF of RA patients have a higher load of IgA bound to their surface compared to their FcRL4- counterparts. After in vitro removal of surface bound IgA, they can bind heat-aggregated IgA (p=0.0313). We also demonstrate that a significantly higher proportion of FcRL4+ B cells use IgA BCRs (p=0.0061) by flow cytometry and further more by probing constant region genes by PCR an enrichment for Ig genes for coding the IgA1 isotype (p=0.009) was found. Conclusions Both their ability to capture IgA immune complexes through binding to FcRL4 and their enrichment in IgA Ig gene expression suggest a potential role for synovial fluid FcRL4+ B cells in the mucosal origin of joint inflammation. References Yeo, L. et al. Cytokine mRNA profiling identifies B cells as a major source of RANKL in rheumatoid arthritis. Ann Rheum Dis 70, 2022–2028, doi:10.1136/ard.2011.153312 (2011). Yeo, L. et al. Expression of FcRL4 defines a pro-inflammatory, RANKL-producing B cell subset in rheumatoid arthritis. Ann Rheum Dis 74, 928–935, doi:10.1136/annrheumdis-2013–204116 (2015). Falini, B. et al. Expression of the IRTA1 receptor identifies intraepithelial and subepithelial marginal zone B cells of the mucosa-associated lymphoid tissue (MALT). Blood 102, 3684–3692, doi:10.1182/blood-2003–03–0750 (2003). Wilson, T. J., Fuchs, A. & Colonna, M. Cutting edge: human FcRL4 and FcRL5 are receptors for IgA and IgG. J Immunol 188, 4741–4745, doi:10.4049/jimmunol.1102651 (2012). Disclosure of Interest None declared

Background Rheumatoid arthritis (RA) is characterised by autoantibodies to citrullinated proteins (ACPA). Smoking and specific HLA alleles are well-established risk factors for ACPA+RA, and more recently Porphyromonas gingivalis, a major cause of periodontitis (PD), has been linked to ACPA+RA. Our ambition with this study is to clone ACPA-specific B-cells from gingival tissue of patients suffering from both PD and RA, in order to demonstrate that citrulline-specific B-cells, previously only detected in RA joints and circulation, may also reside in gingival tissue. Materials and methods Gingival tissue-derived single CD19+ B-cells from an ACPA+RA patient with PD were sorted by flow cytometry. Immunoglobulin (Ig) variable region genes were sequenced and expressed to generate recombinant monoclonal antibodies (mAbs). These mAbs will subsequently be screened for reactivity towards citrullinated antigens by ELISA and an antigen multiplex assay. Results We successfully isolated 480 CD19+ B-cells from the gingival tissue, and to this date, we have analysed 110 variable heavy chain Ig genes (IGHV). Ig gene sequences analysis demonstrated that the B-cell repertoire was predominantly polyclonal, although two clonally related B-cell populations (approximately 2%) were detected. Compared to healthy controls, RA/PD B-cells showed decrease in VH3 and JH5 genes, and increase in VH4, JH4 and JH6. By individual VH gene segments, IGHV4–31 was overrepresented compared to controls (p Alignment of VH sequences to their closest germline counterparts revealed that RA/PD B-cells exhibited extensive mutations in the IGH CDR regions and higher levels of somatic mutations in the V gene segments compared to controls (p Conclusions We have been able to successfully isolate and clone a number of gingival-tissue-derived B-cells. Based on the hypothesis that the ACPA-response may be initiated at mucosal surfaces such as gingival tissue, we now have the tools available to more directly address this etiological question.

The clinical efficacy of B cell targeting therapies highlights the pathogenic potential of B cells in inflammatory diseases. Expression of Fc Receptor like 4 (FcRL4) identifies a memory B cell subset, which is enriched in the joints of patients with rheumatoid arthritis (RA) and in mucosa-associated lymphoid tissue. The high level of RANKL production by this B cell subset indicates a unique pathogenic role. In addition, recent work has identified a role for FcRL4 as an IgA receptor, suggesting a potential function in mucosal immunity. Here, the contribution of FcRL4+ B cells to the specific autoimmune response in the joints of patients with RA was investigated. Single FcRL4+ and FcRL4- B cells were sorted from synovial fluid and tissue from RA patients and their immunoglobulin genes characterized. Levels of hypermutation in the variable regions in both populations were largely consistent with memory B cells selected by an antigen- and T cell-dependent process. Recombinant antibodies were generated based on the IgH and IgL variable region sequences and investigated for antigen specificity. A significantly larger proportion of the recombinant antibodies generated from individual synovial FcRL4+ B cells showed reactivity towards citrullinated autoantigens. Furthermore, both in analyses based on heavy chain sequences and flow cytometric detection, FcRL4+ B cells have significantly increased usage of the IgA isotype. Their low level of expression of immunoglobulin and plasma cell differentiation genes does not suggest current antibody secretion. We conclude that these activated B cells are a component of the local autoimmune response, and through their RANKL expression, can contribute to joint destruction. Furthermore, their expression of FcRL4 and their enrichment in the IgA isotype points towards a potential role for these cells in the link between mucosal and joint inflammation., Highlights • Memory B cells expressing the IgA receptor FcRL4 are found in the joints of patients with RA. • B cell receptors expressed on synovial FcRL4+ B cells more frequently belong to the IgA class. • Among recombinant antibodies cloned from FcRL4+ B cells there is more reactivity with citrullinated proteins. • The gene transcription profile of FcRL4+ B cells shows a low level of differentiation to plasma cells. • These cells may be involved in the link between mucosal and joint autoimmunity.

Scavenger receptor CD36 has been shown to mediate uptake of modified self-antigens such as apoptotic cells and oxidized LDL in macrophages and dendritic cells. Interestingly, CD36 was discovered to be preferentially expressed also on an innate B cell subtype, marginal zone B cells (MZB). Here we investigate the role of CD36 in B cell activation in the context of apoptotic cell clearance and break of tolerance to self. We found that formation of germinal center B cells and autoantibody production in response to an increased load of apoptotic cells coincide with downregulation of CD36 on MZB. We could also in connection to this show that systemic lupus erythematosus (SLE) patients have lower levels of peripheral MZB compared to healthy individuals. B cells lacking CD36 proved more easily activated in response to apoptotic cells, as was shown in bone marrow (BM) chimeras reconstituted with wild type and CD36-deficient BM. In steady-state, we found that CD36 co-localizes with the B cell receptor and the tyrosine kinase Lyn. Interestingly, upon cross-linking of the inhibitory Fc receptor, FcgRIIb, CD36 instead co-localizes with this receptor. This suggests that CD36 interacts with FcgRIIb to activate a negative signaling cascade in B cells. Our data implicate CD36 in the early regulation of B cell responses towards apoptotic cells and autoantibody production. In line with this we find that CD36-deficient B cells are more resistant to FcgRIIb-induced cell death. Thus, CD36 exerts its regulatory function by associating with a known negative signaling pathway in B cells involving Lyn and FcgRIIb. This finding could have implications for autoimmune diseases involving apoptotic cell-derived self-antigens, such as SLE.

Background and objectives Expression of Fc Receptor like 4 (FcRL4) identifies a memory B cell subset, which is enriched in the joints of patients with rheumatoid arthritis (RA) and in mucosa associated lymphoid tissue. Their high level of RANKL production indicates a unique pathogenic role. Here, B cell receptor characteristics, antigen specificity and transcriptomic profile of FcRL4 + B cells sorted from the joints of RA patients were investigated. Materials and methods FcRL4 + and FcRL4 - B cells were sorted from synovial fluid (SF) and tissue of patients with active RA. Their immunoglobulin variable region (IgVH) genes were sequenced and expressed to generate recombinant monoclonal antibodies (mAbs). Reactivity of 63 mAbs towards native and citrullinated peptides derived from α-enolase, fibrinogen, vimentin, histones 3 and 4 was determined by ELISA and Phadia’s ImmunoCAP ISAC microarray system. RNA sequencing was carried out on sorted SF FcRL4 + and FcRL4 - B cells. After preamplification using the SMARTer amplification kit, library preparation was carried out using Illumina’s TruSeq Stranded prep and sequenced on the Illumina platform. Data analysis was carried out using the programs rna-star, DESeq2 and ToppGene. Results Analysis of IgVH genes usage in FcRL4 + and FcRL4 - B cells indicated that the Ig repertoire was highly diverse in both B cell subsets, however VH1–69 gene segment was significantly over-represented in the FcRL4 + B cell population (P = 0.013). Out of the 63 mAbs tested, eight antibodies displayed high level of binding to citrullinated peptides. These were exclusively derived from the FcRL4 + subset (p = 0.018). Another eleven antibodies showed low levels of binding to citrullinated peptides. These were derived from both FcRL4 + and FcRL4 - B cells. Global transcriptome analysis showed clear differences between the two B cell subsets, including 64 genes which were significantly over or under-expressed in FcRL4 + B cells. Among the genes underexpressed in FcRL4+ B cells, the majority were associated with antibody production and plasma cell differentiation. Conclusions Reactivity of FcRL4 + B cells towards citrullinated autoantigens suggests that they are a component of the citrulline-specific autoimmune response, however, their low level of expression of immunoglobulin and plasma cell differentiation genes suggests functions alternative to antibody production.

Background We have recently identified a subset of pro-inflammatory memory B cells in RA synovial fluid and tissue, characterised by the expression of Fc receptor-like 4 (FcRL4)1-3. These cells produce RANKL, and express high levels of TNFa mRNA, indicating a destructive, pro-inflammatory role. Their immunophenoptype suggest that they are activated B cells; however, they show no signs of differentiation towards plasmablasts, suggesting that their functional role does not involve antibody production. Objectives The aim of this project was to investigate the characteristics of the immunoglobulin genes expressed in FcRL4+ B cells isolated from RA synovial fluid and tissue and to determine whether their B cell receptors recognise known autoantigens. Methods Single FcRL4-positive and -negative B cells were sorted from synovial tissue (n=2) and synovial fluid (n=5) of patients with active RA. Their Ig variable region genes were sequenced and subsequently expressed to generate recombinant monoclonal antibodies. Antigen specificities of the generated monoclonal antibodies were determined by ELISA. Results In total, we have cloned and sequenced B cell receptors from 332 individual B-cells (171 from FcRL4+ and 160 from FcRL4- cells). Ig gene sequence analysis demonstrated that the Ig repertoire was highly diverse with no major differences in the IGH and IGK or IGL gene segment usage or IGH CDR3 features between FcRL4+ and FcRL4- memory B cells. The prevalence of mutations, suggesting somatic hypermutation and selection in germinal centres, was equivalent in the FcRL4+ and FcRL4- populations. From the sequenced clones we have generated recombinant monoclonal antibodies from both FcRL4+ (n=29) and FcRL4– B cells (n=26) and have determined their specificity for citrullinated candidate autoantigens. A similar proportion of recombinant antibodies derived from the FcRL4+ and FcRL4- B cell populations (23% and 24% respectively) reacted with citrullinated antigens (including a-enolase, fibrinogen, vimentin and histones) Conclusions We conclude that memory B cells from both the FcRL4+ and FcRL4- populations are post-germinal centre hypermutated B cell subsets with similar Ig gene features. Their antigen specificity suggests that these functionally distinct B subpopulations both emerge from the autoantigen driven immune response in the inflamed joint. References 1. Yeo L, Toellner KM, Salmon M, et al. Cytokine mRNA profiling identifies B cells as a major source of RANKL in rheumatoid arthritis. Ann Rheum Dis. Nov 2011;70(11):2022-2028. 2. Yeo L, Lom H, Juarez M, et al. Expression of FcRL4 defines a pro-inflammatory, RANKL-producing B cell subset in rheumatoid arthritis. Ann Rheum Dis. Jan 15 2014. 3. Ehrhardt GR, Hsu JT, Gartland L, et al. Expression of the immunoregulatory molecule FcRH4 defines a distinctive tissue-based population of memory B cells. J Exp Med. Sep 19 2005;202(6):783-791. Disclosure of Interest None declared