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P047 The plasma cell bone marrowniche in ACPA+ ra patients contain citrulline specific cells

Career situation of first and presenting author Post-doctoral fellow Introduction Anti-citrullinated antibodies (ACPA) in RA have been postulated to contribute to disease pathogenesis. This has been supported by studies of monoclonal APCA generated by cloning from single cell sorting of different B cell subsets from peripheral blood and synovial fluid. It has been debated if the bone marrow (BM) compartment of RA patients hosts long lived plasma cells producing ACPA or if ACPA are primarily coming from plasmablasts at the sites of inflammation, e.g. the synovium. Objectives The main objectives were to study primary lymphoid tissue from RA patients, the BM plasma cell IgG repertoire and further characterize recombinantly expressed antibodies from selected sequences to explore if ACPA producing long lived plasma cells reside in this niche. Methods For this study we collected bone marrow from proximal or distal femur of RA patients undergoing hip replacement surgery due to secondary osteoarthritis. BM samples were processed from four ACPA+RF+ and one ACPA-RF+ RA patients. Mononuclear cells were obtained by Ficoll separation and 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. Selected sequences were cloned and expressed as IgG in Expi293 cells. Results Overall 465 paired IgG BM plasma cell sequences were obtained, 97 from the ACPA- patient and 368 (range 20–194) from ACPA+ patients. We observed statistically significant changes in heavy chain variable gene usage with lower VH-1 and higher VH-3 frequency in the ACPA+ sequences compared to ACPA- seqences. We also found statistically significant increase in VH N-glycosylation in ACPA+sequences (22.6% ACPA+ vs 12.4% ACPA-; p=0.03). There was however no difference in mutation numbers. From these, 34 clones were selected, based on mutation numbers and presence of Fab N-glycosylation sites, for subsequent mAb-expression. Among the 34 clones we found two CCP2+ and cit-peptide positive clones and one malondialdehyde-acetaldehyde (MAA) adduct reactive clone, originating from different APCA+ patients. Conclusions We could identify BM plasma cells producing autoantibodies to malondialdehyde-modified proteins and citrullinated peptides, isolated from RA patients. Hence, we show for the first time, that not just at the site of inflammation but also long-lived plasma cells in bone marrow do produce RA specific autoantibodies. Acknowledgements Orthopedic staff at Karolinska University Hospital. Danika Shepis, Louise Berg and Christina Gerstner for sorting help. Disclosure of Interest None declared