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08.34 Ra synovial recombinant monoclonal antibodies from single b cells target citrullinated calreticulin
  1. Elisa Corsiero,
  2. Lucas Jagemann,
  3. Edoardo Prediletto,
  4. Costantino Pitzalis,
  5. Michele Bombardieri
  1. Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, UK

Abstract

Background 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).

Results 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.

Conclusions 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.

References

  1. I McInnes and G Schett. N Engl J Med, 2011.

  2. M Sorice, et al. Rheumatology, 2016.

  3. K Dasuri, et al. Arthritis Res Ther, 2004.

  4. J Holoshitz, et al. Ann N Y Acad Sci, 2010.

  5. M Ni, et al. J Clin Immunol, 2013.

  6. E Corsiero, et al. Ann Rheum Disease, 2015.

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