Background/Purpose: Anti-citrullinated protein antibodies (ACPA) have proven highly useful as biomarkers in rheumatoid arthritis (RA) and appear to be important to disease pathogenesis. Nevertheless, the relationship between progressive B cell receptor (IGH, IGK, and IGL) gene somatic hypermutation (SHM) and citrullinated protein antigen specificity in RA B cells remains only poorly understood. In this investigation, we aimed to characterize citrullinated peptide reactivity as a function of heavy and light chain SHM events using a panel of citrullinated autoantigen–specific RA B cells.

Methods: Three subjects from the University of Minnesota ACPA⁺ RA cohort gave consent for the study of peripheral blood B cell receptor antigen specificity. Citrullinated filaggrin peptide (CFC1) and citrullinated a-enolase peptide (CEP-1) tetramer–bound B cells were subjected to flow cytometric cell sorting and single-cell IGH, IGK, and IGL gene sequencing. BCR genes demonstrating related V-(D)-J region gene usage and conserved junction structures were compared using the PHYLIP/DNApars algorithm and B cell lineage relationships (clades) were characterized. BCR gene sequences were also expressed as recombinant monoclonal antibodies (mAbs) for direct evaluation of citrullinated antigen binding by ELISA.

Results: Paired heavy and light chain Ig gene sequences were obtained at a single time point from subjects RA14 (n = 9) and RA62 (n = 10). Additionally, 86 paired nucleotide sequences were obtained from subject RA37 at three separate time points. Parsimonious clustering of related immunoglobulin gene nucleotide sequences revealed that 52 of these 105 B cells (50%) arose within 9 unique clades as a consequence of clonal expansion and progressive SHM. The frequency of clade membership by citrullinated peptide tetramer-bound B cells in each subject varied from 44% to 89% (χ² = 7.0, P-value = 0.030). The distribution of clade membership by CEP-1 tetramer-bound B cells in subject RA37 also varied significantly across time (χ² = 25.9, P-value = 0.011). In ELISA, 9 of 20 recombinant human mAbs (45%) generated from a sample of these BCR sequences proved capable of binding to at least one citrullinated antigen, and all 9 of these citrullinated peptide-binding mAbs derived from a highly mutated clade-associated B cell. Citrulline–dependent cross-reactivity (citrulline multispecificity) extending beyond the citrullinated peptides used for B cell capture was observed in all clade-derived mAbs tested.

Conclusion: Our findings suggest that the acquisition of broad ACPA specificity in RA arises from a restricted repertoire of continuously evolving citrulline–multispecific B cell clades.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/citrulline-multispecific-b-cell-receptor-clades-in-rheumatoid-arthritis/