Generation and characterization of anti-citrullinated protein antibody-producing B-cell clones from rheumatoid arthritis patients.

Objective Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA). Aside from autoantibody production, the function of autoantigen-specific B cells remains poorly understood in the context of this disease. This study set out to elucidate autoantigen-specific B cell functions through the isolation and immortalization of unique citrullinated protein/peptide (CP)-reactive B cell clones from RA patients. Methods B cell clones from either the blood or synovial fluid of cyclic citrullinated peptide 2 (CCP2) antibody-positive RA patients were immortalized by genetic reprogramming with Bcl-6 and Bcl-xL. Enzyme-linked immunosorbent assay and flow cytometry were used to identify CCP2-reactive clones and to further characterize surface marker and cytokine expression as well as B cell receptor signaling competence. Global gene expression profiles were interrogated by RNA sequencing. Results Three unique CP-reactive memory B cell clones were generated from the blood or synovial fluid of 2 RA patients. CP-reactive memory B cells did not appear to be broadly cross-reactive, but rather had a fairly restricted epitope recognition profile. These clones were able to secrete both pro- and antiinflammatory cytokines and had a unique surface profile of costimulatory molecules and receptors, including CD40 and C5a receptor type 1, when compared to non-CP-reactive clones from the same patient. In addition, CP-reactive clones bound citrullinated protein, but not native protein, and could mobilize calcium in response to antigen binding. Conclusion CP-reactive memory B cells comprise a rare, seemingly oligoclonal population with restricted epitope specificity and distinct phenotypic and molecular characteristics suggestive of antigen-presenting cells. Cloning by genetic reprogramming opens new avenues to study the function of autoreactive memory B cells, especially in terms of antigen processing, presentation, and subsequent T cell polarization.