Background/Purpose: Citrulline–modified proteins arising from the post-translational modification of arginine residues are recognized as primary rheumatoid arthritis (RA) autoantigen targets based on the strong association of anti-citrullinated protein antibodies (ACPA) with RA disease development. Nevertheless, the repertoire of citrullinated protein–specific B cell antigen receptors (BCRs) has not previously been directly assessed.

Methods: 89 subjects from the IRB-approved University of Minnesota ACPA⁺ RA cohort who met the 2010 ACR/EULAR criteria for RA provided blood samples and clinical data for use in this study. Citrullinated filaggrin peptide CFC1 and citrullinated a-enolase peptide CEP-1 were used in the construction of tetramer sets designed to specifically capture and characterize autoreactive citrullinated protein–specific B cells in the unaltered, polyclonal repertoire of RA patients. Citrullinated peptide tetramer–bound B cells were subjected to flow cytometric cell sorting and single cell IGH, IGK, and IGL gene sequencing for B cell lineage determinations. BCR gene sequences were also expressed as recombinant human monoclonal antibodies, and tested for their ability to bind to citrullinated peptides and proteins. Finally, select human V-(D)-J sequences were expressed as recombinant mouse monoclonal antibodies to test their ability to prolong endotoxin-induced arthritis.

Results: Tetramer–binding CFC1– and CEP-1–specific IgD^– CD27⁺ switched-memory B cells were found in increased numbers in the blood of RA subjects who also demonstrated high titers of anti-CFC1 and/or –CEP-1 serum antibodies, respectively (5.7-fold increase for CFC1, Pvalue < 0.01; 5.3-fold increase for CEP-1; Pvalue = 0.01). The frequency of CFC1–specific switched-memory B cells was also positively associated with the duration of disease (p= 0.02), the presence of subcutaneous nodules (p = 0.02), and the DAS28-ESR disease activity index (p = 0.01). Citrullinated peptide tetramer–specific BCRs had highly mutated immunoglobulin (Ig) heavy and light chain complementarity determining region (CDR) sequences, biased V-region gene usage, and conserved CDR3 junction lengths. Parsimonious clustering of related IGH, IGK, and IGL nucleotide sequences demonstrated that the clonal expansion of rare individual B cell lineages occurs in association with progressive amino acid sequence divergence. Recombinant human monoclonal antibodies generated from citrullinated peptide tetramer–sorted B cells within extended clades confirmed target peptide antigen–binding for most clones, yet citrulline–dependent cross-reactivity to a broad set of distinct citrullinated peptides and proteins implicated in RA was also observed. Finally, a pair of citrullinated protein–specific recombinant monoclonal antibodies with cross-reactive autoantigen–binding profiles promoted arthritis in mice.

Conclusion: Broad anti-citrullinated protein antibody specificities in RA may arise from a restricted repertoire of B cell clades with evolving and divergent citrulline–polyspecific BCRs.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/citrulline-polyspecific-b-cell-antigen-receptors-arising-from-somatic-hypermutation-within-clades-demonstrate-pathogenicity-in-rheumatoid-arthritis/