Background/Purpose . The detection of anti-citrullinated protein antibodies (ACPA) aids RA diagnosis, while B cell depletion by anti-CD20 can provide clinical benefits. We therefore undertook investigations of the phenotypic and functional properties of citrulline (Cit)-specific B cells in the bloodstream of RA patients.  

Methods . RA met 2010 ACR/EULAR criteria. PBMC were cultured +/- CpG2006/IL-21/sCD40L, and ELISpots performed. CD19+ B cell subsets were FACS sorted and cultured with CD40L-cell line/CpG2006/IL-21.  Sera and supernatants were evaluated for total IgG, and for IgG1 and IgG4 subclasses by bead-based multiplex analysis, against 8 sets of paired Cit/native cyclic self-peptides, plus CCP3, CQP3 (Cit to Q), Cit/native fibrinogen (Fib), tetanus toxoid (TT) and control ligands. We also surveyed 87 CCP3-seropositive and 50 seronegative RA serum samples, plus panels of disease specific control samples (SS, OA, PsA, SLE).

Results . ELISpots documented the presence of circulating anti-Cit B cells, as seropositive RA had 41.3+/-0.5 CCP3+ IgG (mean +/-SE) but only 1.9+/-0.6 CQP+ spots/10⁽⁶⁾ PBMC, while these were undetectable in seronegative RA and healthy adults.  Anti-TT ISC were detected at 71.1+/-2.6 and 65.4+/-1.2 spots/10⁽⁶⁾PBMC in seropositive RA and controls, respectively. By multiplex assay, ACPA fine specificity reactivities in sera were compared with supernatant antibodies from stimulated PBMC. In 36 PBMC from seropositive RA, highly significant correlations by ACPA fine specificity were found in comparisons with sera ACPA by Spearman correlations. From sorted B cells, we found ACPA-secreting B cells were enriched among CD19+IgD-CD27+ switched-memory (0.019 – 0.059% of CD19+CD27+IgD-), and included highly CCP3-reactive IgG.  Yet, only rare weakly CCP3-reactive naïve B cells (IgD+CD27-) were detected, and none amongst pre-switched memory (IgD+CD27+). From subclass-specific sera multiplex assays, amongst IgG1 ACPA-expressing seropositive RA, about half showed anti-CCP3 IgG1 only, while the remainder had both anti-CCP3 IgG1 and IgG4.  Whereas reactivity levels were generally greater for IgG1 than IgG4, IgG1 responses usually displayed great Cit than native antigen-specific responses, while the IgG4 displayed greater reactivity with the native (non-Cit) form in 5/9 antigen sets.

Conclusion . In seropositive RA PBMC, IgG ACPA in vitro secretion was detected across all treatment groups tested (MTX, MTX+TNFi, MTX+abatacept), suggesting these cells persist despite therapeutic intervention.  DAS28 score correlated with neither the level nor specificity of supernatant or sera ACPA. Pilot studies also suggest ACPA-secreting B cells predominantly reside within peripheral switched memory. Our data therefore suggest that seropositive RA patients have recirculating but quiescent memory B cells with repertoires akin to serum ACPA patterns. Furthermore, while IgG1 autoantibodies in RA are predominantly Cit-specific, with subclass switch to IgG4 there may be epitope spreading with progression to include recognition of determinants on the native forms of the self-antigens. Our studies highlight defects in B cell tolerance in RA that may help guide development of better therapy.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/citrulline-specific-autoimmunity-resides-in-quiescent-circulating-memory-b-cells-in-rheumatoid-arthritis/