Search

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.

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.