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. 2019 Mar;71(3):340-350.
doi: 10.1002/art.40739. Epub 2019 Feb 2.

Generation and Characterization of Anti-Citrullinated Protein Antibody-Producing B Cell Clones From Rheumatoid Arthritis Patients

Kristine Germar  1 , Cynthia M Fehres  1 , Hans U Scherer  2 , Nathalie van Uden  1 , Sabrina Pollastro  1 , Nataliya Yeremenko  1 , Monika Hansson  3 , Priscilla F Kerkman  2 , Ellen I H van der Voort  2 , Evan Reed  3 , Hanna Maassen  2 , Mark J Kwakkenbos  4 , Arjen Q Bakker  4 , Lars Klareskog  3 , Vivianne Malmström  3 , Niek de Vries  1 , René E M Toes  2 , Karin Lundberg  3 , Hergen Spits  4 , Dominique L Baeten  5
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Generation and Characterization of Anti-Citrullinated Protein Antibody-Producing B Cell Clones From Rheumatoid Arthritis Patients

Kristine Germar et al. Arthritis Rheumatol. 2019 Mar.

Abstract

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.

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