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

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.

Different Hierarchies of Anti–Modified Protein Autoantibody Reactivities in Rheumatoid Arthritis

“…It is intriguing to hypothesize that ACPA subsets, based on reactivity fingerprints and consensus motifs, may be associated with different pathogenic properties. Polyclonal ACPAs have been observed to modulate both osteoclast and fibroblast functionality, have proinflammatory properties, and mediate pain behavior (22,24–26,48–50). Our previous studies demonstrated that the ACPA clone 1325:04C03 (with the Gly–Cit motif) have osteoclastogenic properties and have no effect on fibroblasts (22,23,51).…”

“…The included ACPA mAb have been described previously and have been verified to have citrulline reactivity down to a concentration of at least 100 ng/ml (22,24–26). All recombinant ACPA mAb were expressed as human IgG1 in Expi 293 cells (Life Technologies), and the mAb were purified with protein G resin (GE Life Sciences).…”

Variable domain N‐linked glycosylation and negative surface charge are key features of monoclonal ACPA: Implications for B‐cell selection

“…The transduced cells were sorted into wells with flow cytometry and cultured in the presence of CD40L and IL‐21. Supernatants were screened for citrullinated peptide reactivity by ELISA and the ACPA clone 1003:10A01 was identified . RA blood memory derived ACPA positive clone obtained by EBV immortalization : The ACPA positive blood memory derived clone BVCA1 was isolated from RA PBMC using methods previously described . Briefly, CD22 + cells were enriched using MACS microbeads (Miltenyi) and subsequently sorted for IgG + cells.…”

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

“…In combination with cell sorting, it also allows subsequent analysis, such as transcriptomic profiling by single cell‐based (“next generation”) sequencing techniques. Furthermore, it is possible to analyze antigen‐specific B cell receptor (BCR) repertoires, to obtain full‐length BCR sequences for mAb generation, and to perform functional studies of isolated single B cells or B cell populations, which includes the generation of immortalized, antigen‐specific B cell clones . This wealth of possibilities permits unprecedented insights into human B cell biology; it requires, however, particular care and adherence to relevant and tedious control steps to ensure that the antigen‐specific B cell populations identified by FCM, which are frequently very rare, indeed represent the antigen‐specific B cell population of interest.…”