Background/Purpose: Rheumatoid arthritis (RA) is characterized by the presence of anti-citrullinated protein antibodies (ACPA) in the majority of patients. ACPA represent highly disease-specific biomarkers that predict disease onset in patients with arthralgia. Experimental data suggest that ACPA could be involved in disease pathogenesis. Of interest, however, depletion of CD20⁺B cells is effective in reducing disease activity, while ACPA serum titres remain rather stable. Also, interference with plasma cell development seems clinically ineffective. Therefore, we hypothesized that ACPA could be surrogate markers for a (potentially pathogenic) auto-reactive B cell response and set out to identify citrullinated antigen-specific B cells in peripheral blood and synovial fluid of patients with RA.

Methods: We generated differentially labelled streptavidin and extravidin tetramers conjugated to biotinylated CCP2 or control antigens to identify citrullinated antigen-specific B cells in peripheral blood and synovial fluid by flow cytometry. Blocking and fluorescence activated cell sorting experiments followed by in-vitro stimulation and culture were used to confirm specificity of the staining approach. Cells were further phenotypically characterized by flow cytometry.

Results: The combination of differentially labelled CCP2 and control tetramers allowed the  successful separation of ACPA-expressing B cells from non-specific background signals. Tetramer-positive B cells, but not tetramer-negative cells, produced large amounts of ACPA upon stimulation. Interestingly, phenotypic analysis revealed that citrullinated antigen-specific B cells are mainly class-switched, post-germinal centre memory B cells and plasma blasts/cells expressing IgG or IgA. Only few cells exhibited a naïve phenotype. We observed a remarkably high frequency of memory B cells (up to 1 in 500) in peripheral blood, which correlated with ACPA serum titres and spontaneous ACPA production in culture.

Conclusion: We developed a novel technology that identifies ACPA-expressing B cells in peripheral blood and synovial fluid of patients with RA, providing the basis for a detailed characterisation of this immune response on a single cell level. First analyses show the presence of a high frequency of memory B cells, which is remarkable considering the continuous presence of citrullinated antigens. These data pave the path for a detailed understanding of the development and maintenance of citrullinated antigen-reactive B cells and could lead to the identification of targets for novel therapeutic interventions.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/citrullinated-antigen-specific-b-cells-in-peripheral-blood-and-synovial-fluid-of-patients-with-rheumatoid-arthritis-identification-and-phenotypic-characterization/