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SAT0047 PRIMARY LYMPHOID TISSUE FROM RHEUMATOID ARTHRITIS PATIENTS HARBOUR CITRULLINE SPECIFIC PLASMA CELLS

Background: Anti-citrullinated protein antibodies (ACPA) are specific markers with pathological effects in rheumatoid arthritis (RA). ACPA-specific B-cells have been identified in synovial joint fluid and peripheral blood. An increased concentration of ACPA is found in synovial fluid and lung tissue compared to blood suggest local production, partly confirmed by detection ACPA synovial plasma blasts, but primary lymphoid tissue have not previously been examined. Objectives: In this study we investigated the plasma cell repertoire in the bone marrow and occurrence of autoantibody producing plasma cells. Methods: For this study we developed a method to collected and process bone marrow samples from proximal femur in RA patients undergoing hip joint replacement. Bone marrow samples were processed and mononuclear cells were obtained by Ficoll separation. CD138+ plasma cells were single cell sorted by flow cytometry. Paired heavy and light chains were PCR amplified, sequenced, and analyzed by V-Quest and IgBLAST towards the IMGT database to annotate variable gene usage. To enrich for ACPA, sequences were selected based on high somatic hypermutations number and Fab N-glycosylation sites. Selected sequences were cloned and expressed as IgG in Expi293 cells. Monoclonal antibody reactivity to citrullinated and arginine form of vimentin, enolase, fibrinogen, histone, tenascin C peptides; carbamylated and malondialdehyde-acetaldehyde bovine serum albumin, and tetanus toxoid antigens were examined by ELISA. Included patients were clinical examined and donating peripheral blood samples. Results: After method development and quality analysis we were able to include five bone marrow samples in this study. The five included RA patients, median age 74 (range 66-82) years, had in median 20 (range 4-44) years of destructive disease, all were RF positive, four were females and anti-CCP2-positive, and all except one were treated with anti-rheumatic drugs. Overall, from the processed bone marrow, 465 paired heavy-light IgG plasma cell sequences were obtained, in total 368 (range 20-194) from ACPA positive patients and 97 from the ACPA negative patient. We observed statistically significant changes in heavy chain variable gene usage with lower VH-1 and higher VH-3 frequency in sequences from ACPA positive RA patients compared to sequences from the ACPA negative RA patient. We also found statistically significant increase in VH N-glycosylation in sequences from the ACPA positive RA patients (22.6% ACPA positive vs 12.4% ACPA negative; p=0.03) but no difference in mutation numbers. From obtained sequences, 34 interesting clones were selected for monoclonal antibody-expression. Among the 34 clones we found two citrulline specific reactive clones (reactivity towards CCP2, citrullinated vimentin and fibrinogen peptides) and one malondialdehyde-acetaldehyde (MAA) reactive clone, originating from three different APCA positive patients with the most longstanding disease. None of these clones had reactivity towards control antigens such as arginine versions of peptides. Conclusion: Plasma cells producing ACPA are present in primary lymphoid tissue from RA patients. Further studies of difference in characteristics, IgG gene usage and N-glycosylation, between ACPA-positive and ACPA-negative patients are needed. Disclosure of Interests: Radha Thyagarajan: None declared, Aase Hensvold: None declared, Lena Israelsson: None declared, Johanna Steen: None declared, Heidi Wähämaa: None declared, Annika van Vollenhoven: None declared, Khaled Amara: None declared, Anca Catrina Grant/research support from: Yes, but not for the presented study., Vivianne Malmström: None declared, Caroline Grönwall: None declared