Background/Purpose: B cells are central drivers of seropositive RA, and B cells that express anti-citrullinated protein antibodies (ACPA) may arise from past and/or ongoing breaches in immune tolerance. While ACPA levels are only modestly affected by commonly prescribed therapeutic agents (e.g, Methotrexate, TNFi), it is unclear whether these treatments significantly affect autoimmune lymphocytes and whether these cells persist after treatment induced clinical remission. We therefore assessed the frequencies and epitope reactivity patterns of ACPA-producing switched-memory B cells, with comparisons to serum ACPA levels and disease activity scores.

Methods: In a cross-sectional study of seropositive RA patients that met 2010 ACR/EULAR criteria, blood samples were biobanked, and later cryopreserved PBMC were cultured +/- sCD40L/CpG2006/IL-21 for 6 days and then ELISpots performed. Synovial fluid mononuclear cells and FACS-sorted switched-memory B cells [CD19+/CD27+/IgD-] were cultured with CpG2006/IL-21 in the presence of a CD40L-expressing feeder cell line for 12 days. Sera, synovial fluid, and culture supernatants were analyzed by multiplex bead-based assay for ACPA-IgG reactivity for 8 citrullinated and native peptide/protein pairs, as well as reactivity with the diagnostic CCP3 peptide (Inova Diagnostics) and glutamine-containing CQP3, plus other control ligands. We then performed Spearman correlations.

Results: In select patients with active disease, we found a high frequency of synovial fluid cells that spontaneously secreted ACPA in culture, suggesting these ACPA producing B cells are especially common at the site of disease, and epitope specificities were similar to those in autologous synovial fluid. For cultures of PBMC from groups of patients receiving different treatment regimens (MTX or TNFi+/-MTX), ACPA-IgG secretion was also detected, although in vitro stimulation was generally required. In the circulation of these RA patients, switched memory B cells were the predominant source of ACPA-IgG. Notably, serum anti-CCP3-IgG levels significantly correlated with frequencies of ACPA-producing switched-memory B cells (r = 0.57, p = 0.003). For individual donors, the ACPA fine-specificity patterns in serum samples were similar to those in supernatants from cultured B cells. A majority (16/24) of RA patients had wells with detectable in vitro ACPA secretion. Yet frequencies of ACPA memory B-cells did not attain significant correlation with DAS28 scores (r = 0.35, p = 0.09). In fact, a subset of RA patients, which included patients treated with methotrexate and/or TNFi, had high frequencies of recirculating ACPA memory-B cells despite being in DAS remission.

Conclusion:  We have documented that DAS score cannot be used as a predictor of reduced or absent ACPA memory B cell burden, as quiescent disease-associated anti-Cit memory B-cells commonly persist in peripheral circulation despite treatment to clinical remission. Our results help to explain why therapeutic cessation most often results in reactivation or flare. In the future, our platform may be useful for evaluating new agents and for informing treatment decisions in individual RA patients.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/disease-associated-anti-citrullinated-protein-memory-b-cells-in-rheumatoid-arthritis-persist-in-das-remission/