Background/Purpose: We previously showed that up to 40% of RA synovial recombinant monoclonal antibodies (RA-rmAbs) generated from germinal center-like structure (GC-LS+) RA synovium recognize citrullinated antigens contained in neutrophils extracellular traps (NETs) (1). The cellular source of other potential autoantigens targeted by the majority of locally differentiated B cells remains undefined. Recently, RA-fibroblast-like synoviocytes (RA-FLS) have been implicated in the release of citrullinated antigens (2, 3). However, whether these cells are targeted by RA-rmAbs is still unknown. Here, we aimed to define i) the RA-rmAbs immunoreactivity towards RA-FLS and ii) identify potential stromal-derived autoantigens.

Methods: 67 RA-rmAbs were generated from single CD19+ B cells FACS-sorted from fresh synovial cell suspensions following IgV_H+V_L genes cloning (1). RA-rmAbs were tested by means of i) cell-based immunofluorescence assays with FLS of RA patients and controls (osteoarthritis (OA)-FLS and RA-dermal fibroblast (RA-DF)), ii) co-localization with stromal specific markers and iii) immunoenzymatic tests with co-localizing antigens. Control rmAbs were also used (Sjögren’s syndrome/healthy donor-IgG rmAbs).

Results: Immunofluorescence on RA-FLS demonstrated reactivity of 22.4% of RA-rmAbs (15/67 rmAbs) towards FLS. Only 4 rmAbs out of 15 were binding both FLS and NETs components. For some RA-rmAbs this reactivity was not specific to RA-FLS since it was also observed for OA-FLS (3% rmAbs). Interestingly, strong co-localization was observed with calreticulin (CRT) which has been shown to bind the RA shared-epitope (SE) ligand and to increase the signalling pathway activated by the SE ligand in its citrullinated form (3). When tested in ELISA for native vs cit-CRT, 20% (14/67 rmAbs) of the FLS-reactive RA clones showed binding to CRT with 6 out of 14 RA-rmAbs displaying increased immunoreactivity towards cit-CRT. Controls rmAbs showed no reactivity to either FLS or CRT. RA-rmAbs binding to CRT was further confirmed in western blot immunoassays.

Conclusion: Here, we provide novel evidence that a subset of locally differentiated B cells within RA synovial GC-LS can react towards RA-FLS derived antigens. Preliminary data suggest that part of this reactivity is directed towards CRT. Identification of immunodominant epitopes within CRT is under investigations.

References:

(1) Corsiero et al, ARD 2015

(2) Sorice et al, Rheumatology 2016

(3) Ling et al, AR 2013

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/characterization-of-novel-stromal-derived-autoantigens-recognized-by-ra-synovial-monoclonal-antibodies/