(19d) Sequencing the Evolving Functional Antibody Repertoire in Rheumatoid Arthritis

Combining large-scale antibody repertoire sequencing data with a comprehensive analysis of antibody function can provide a more complete understanding of the role of an antibody response in disease pathology. For example, a hallmark of rheumatoid arthritis (RA) is the generation of an adaptive autoimmune B cell response against citrullinated antigens that result from a post-translational modification of arginine. However, the precise specificity and functional properties of the anti-citrullinated protein antibodies (ACPAs) in RA remain poorly understood. To address this, we applied a high-throughput single-cell barcoding methodology to sequence the entire variable region of paired heavy and light chain antibody genes expressed by individual plasmablasts (activated B cells). Bioinformatics analysis of these sequencing results precisely quantifies the degree of clonal expansion and identifies the key affinity matured, clonally expanded antibodies generated in an immune response. Importantly, with the paired heavy and light chain data, we can express these antibodies in vitro, thereby enabling the direct linkage of a patient-derived antibodyâ??s sequence with its function (e.g. antigen specificity). Furthermore, we evaluated the antibody repertoire over time to characterize the evolving adaptive autoimmune response, elucidate the key autoantibodies, and provide insight into the pathogenic mechanisms underlying RA. We sequenced single plasmablasts from peripheral blood sampled at serial time points from 8 individuals with RA and used a tetrameric citrullinated peptide-based sort reagent to isolate B cells that produce ACPAs. We bioinformatically identified lineages that persisted across the serial time points and characterized the expansion and contraction of clonal lineages, some of which expressed multiple antibody isotypes, in relation to clinical metrics (e.g. swollen and tender joints). We selected antibodies representative of the identified B cell clonal families for recombinant expression and defined their antigen targets with planar and bead-based antigen arrays. By combining detailed sequence information with antibody function, this broadly applicable approach facilitates a comprehensive investigation of the evolution of the antibody response in disease, which can reveal novel diagnostic markers and therapeutic targets.