08.34 Ra synovial recombinant monoclonal antibodies from single b cells target citrullinated calreticulin

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Fibroblast-like synoviocytes (FLS) play a crucial role in the pathogenesis of rheumatoid arthritis (RA) directly contributing to local cartilage destruction and synovial inflammation.1 Autophagy, a mechanism whereby damaged proteins are removed from the cells, has been implicated in the pathogenesis of RA by activating the citrullination process in RA-FLS.2 The protein expression pattern of FLS has been also characterised with the identification of >200 proteins which have been involved in the normal or pathological FLS function.3 Among all the proteins identified in the RA-FLS, calreticulin (CRT) has been implicated in the pathogenesis of RA.4,5 In particular, citrullinated-(cit)-CRT has been shown to better recognise the RA ‘share epitope’ HLA domain compared to native CRT.4 Therefore, here we tested the immunoreactivity of RA synovial recombinant monoclonal antibodies (RA-syn-rmAbs) towards RA-FLS and towards cit-CRT.

Materials and methods

82 RA-syn-rmAbs were generated from single CD19+B cells FACS-sorted from fresh synovial cell suspensions following IgVH+VL genes cloning (6). RA-syn-rmAbs were tested by means of i) cell-based immunofluorescence assays with FLS of RA patients and controls (osteoarthritis (OA)/healthy donors (HD)-FLS and dermal fibroblast (DF)) and ii) immunoenzymatic tests using native/cit-CRT. Control rmAbs were also used (Sjögren’s syndrome/HD-IgG rmAbs).


Immunofluoresce on RA-FLS demonstrated reactivity of 21% of RA-syn-rmAbs (14/67 mAbs) towards cytoplasmic/nuclear components of FLS. This reactivity was not exclusively directed against RA-FLS since it was also observed for OA/HD-FLS and DF. When tested in ELISA for native vs cit-CRT, 50% (7/14 mAbs) of the FLS+ RA clones showed to be reactive towards CRT. Interestingly, 4 out of 7 rmAbs displayed an increased reactivity towards cit-CRT. Controls mAbs showed no reactivity towards FLS and CRT.


Here, we provided evidence that locally differentiated B cells within RA synovial germinal center-like structures can react towards native/cit-CRT likely expressed in RA-FLS. Importantly, this reactivity was disease-specific. The identification of additional cit-antigens such as cit-CRT may provide further insight into the RA pathogenesis. Thus, they might be used as potential new biomarkers in RA diagnosis.

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