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:
The authors are grateful to Erik Peterson, Jerry Molitor, and Emily Gillespie for assistance with University of Minnesota ACPA-positive RA cohort development, Patt Carlson for acting as the study coordinator for patient recruitment, Joseph Wilson for processing samples for the study, Johanna Steen and Ragnhild St?lesen for helping produce monoclonal antibodies, and Marc Jenkins and Yoji Shimizu for reading the manuscript and providing critical feedback.
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).
© 2018, American College of Rheumatology