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

OBJECTIVE: Anti-citrullinated protein antibodies (ACPA) are a hallmark for rheumatoid arthritis (RA). Autoantigen-specific B-cell function aside from autoantibody production remains poorly understood in the context of this disease. We set out to elucidate this function through the isolation and immortalization of unique citrullinated protein or peptide (CP)-reactive B-cell clones from RA patients.

METHODS: B-cell clones from either blood or synovial fluid of cyclic-citrullinated peptide 2 (CCP2)+ RA patients were immortalized by genetic reprogramming with Bcl6 and Bcl-xL. ELISA 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 (BCR) signaling competence. Global gene expression profiles were interrogated by RNA sequencing.

RESULTS: We generated three unique CP-reactive memory B-cell clones from two 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 can secrete both pro- and anti-inflammatory cytokines and have a unique surface profile of costimulatory molecules and receptors, including CD40 and C5aRI, when compared to non-CP-reactive clones from the same patient. Additionally, CP-reactive clones bind citrullinated but not native protein and can mobilize calcium in response to antigen binding.

CONCLUSIONS: 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. This article is protected by copyright. All rights reserved.