Objective: Anti–citrullinated protein antibodies (ACPAs) have proven highly useful as biomarkers for rheumatoid arthritis (RA). However, composition and functionality of the associated autoreactive B cell repertoire have not been directly assessed. We aimed to selectively investigate citrullinated autoantigen–specific B cell receptors (BCRs) involved in RA and initiate studies on their pathogenicity. Methods: Blood samples were obtained from patients in a University of Minnesota cohort with ACPA-positive RA (n = 89). Tetramer sets bearing citrullinated filaggrin peptide cfc1 or citrullinated α-enolase peptide were constructed to specifically capture autoreactive B cells from the unaltered, polyclonal repertoire in 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 monoclonal antibodies (mAb) for direct evaluation of citrullinated autoantigen binding and effector functionality. Results: Using citrullinated peptide tetramer enrichment to investigate single autoreactive blood B cells, we identified biased V-region gene usage and conserved junction arrangements in BCRs from RA patients. Parsimonious clustering of related immunoglobulin gene nucleotide sequences revealed clonal expansions of rare individual B cell clades, in parallel with divergent sequence mutations. Correspondingly, recombinant mAb generated from such BCR lineages demonstrated citrulline-dependent cross-reactivity extending beyond the citrullinated peptides used for B cell capture. A pair of citrullinated autoantigen–specific mAb with cross-reactive binding profiles also promoted arthritis in mice. Conclusion: Our findings suggest that broad ACPA specificities in RA arise from a restricted repertoire of evolving citrulline-multispecific B cell clades with pathogenic potential.
Bibliographical noteFunding Information:
Supported by the NIH (National Institute of General Medical Sciences grants MSTP T32 and GM008244 to Mr. Titcombe). Dr. Svensson’s work was supported by the Swedish Research Council (grant 542-2013-8373), the Swedish Foundation for Strategic Research (grant FFL09-0011), the Knut and Alice Wallenberg Foundation, and the Ragnar So€derberg Foundation. Dr. Mueller’s work was supported by the Rheumatology Research Foundation (Disease Targeted Innovative Research Grant and Within Our Reach Award).