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OP0175 Characterization of novel stromal-derived autoantigens recognized by ra synovial monoclonal antibodies
  1. E Corsiero,
  2. L Jagemann,
  3. E Prediletto,
  4. C Pitzalis,
  5. M Bombardieri
  1. Centre for Experimental Medicine & Rheumatology, William Harvey Research Institute, London, United Kingdom

Abstract

Background 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 (NET) (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.

Objectives Here, we aimed to define the RA-rmAbs immunoreactivity towards i) 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 IgVH+VL 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 21% of RA-rmAbs (14/67 rmAbs) towards cytoplasmic components of FLS. Only 4 rmAbs out of 14 were binding both FLS and NET components. For some rmAbs this reactivity was not specific to RA-FLS since it was also observed for OA-FLS and RA-DF. Interestingly, strong co-localization was observed with calreticulin (CRT) which in its citrullinated (cit-CRT) form has been previously shown to recognize the RA “shared epitope” HLA domain sequence (3). When tested in ELISA for native vs cit-CRT, 57% (8/14 rmAbs) of the FLS-reactive RA clones showed binding to CRT with 4 out of 8 rmAbs displaying increased immunoreactivity towards cit-CRT. Controls rmAbs showed no reactivity to either FLS or CRT. Preliminary data suggest that RA patient serum preferentially recognize the lectin-like N-terminal domain of CRT (4).

Conclusions 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.

  4. Hong et al, JI 2010.

References

Disclosure of Interest None declared

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