CHARACTERIZATION OF HSP60, A STROMAL-DERIVED AUTOANTIGEN, RECOGNIZED BY RA SYNOVIAL RECOMBINANT MONOCLONAL ANTIBODIES

Background Up to 50% of rheumatoid arthritis (RA) patients display synovial ectopic lymphoid structures (ELS) supporting B-cell autoreactivity toward locally generated citrullinated and other translationally modified antigens. Recently, screening a large number of recombinant monoclonal antibodies (rmAbs, n=71) which we derived from locally differentiated B-cells from RA ELS+ synovium [1], we identified a subset of antibodies which specifically recognise fibroblast-like-synoviocytes (FLS) (10 out of 71), suggesting FLS as a cellular source of autoantigens fuelling the local autoimmune response. We reported that calreticulin is one of the antigenic targets of these anti-FLS rmAbs, while the nature of other FLS-derived autoantigens is still unclear [2]. Objectives Here we aimed to define other stromal-derived autoantigens from RA-FLS targeted by RA-rmAbs. Methods Western blotting/mass-spectrometry were used to identify potential autoantigens from RA-FLS protein extracts. Putative candidates were validated using colocalization immunofluorescence confocal microscopy/ELISA/immunoprecipitation assay. Finally, both serum and synovial fluid (SF) from RA patients (OA patients used as control) were tested for immunoreactivity towards the putative antigen. Results Following immunoprecipitation and mass-spectrometry analysis, among the anti-FLS antibodies we identified a subset of RA-rmAbs which display strong reactivity towards heat shock protein 60 (HSP60). Three RA-rmAbs confirmed a clear immunoreactivity towards HSP60 in ELISA assay in a dose-dependent manner. Confocal microscopy did not show co-localization between anti-HSP60 RA-rmAbs and HSP60, suggesting that HSP60 act as autoantigen when released from the RA-FLS in stress condition. Finally, anti-HSP60 Abs were preferentially detected in RA-SF versus OA-SF, with an accumulation of HSP60 in RA-SF versus RA sera. Conclusion Here, we identified synovial B cell-derived RA-rmAbs locally differentiated within the ELS+ RA synovium reacting toward HSP60, suggesting that FLS-derived HSP60 may contribute to fuel the local autoimmune response. Elucidating the mechanisms involved in RA-FLS activation in vitro/in vivo will be important to clarify the anti-FLS rmAbs functional role in modulating inflammation. References [1]Corsiero et al , ARD 2016; [2] Corsiero et al , JI 2018. Acknowledgements This work was supported by a research grant from Versus Arthritis (Grant 22440 to E. Corsiero). Disclosure of Interests None declared.