These findings demonstrate that established RA is characterized by a persistent IgA ACPA response that exhibits ongoing affinity maturation. This observation suggests the presence of a persistent mucosal antigen that continually promotes the production of IgA plasmablasts and their affinity maturation and epitope spreading, thus leading to the generation of ACPAs that bind additional citrullinated antigens and more potently stimulate macrophage production of TNF.

Objective. Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA). While epitope spreading of the serum ACPA response is believed to contribute to RA pathogenesis, little is understood regarding how this phenomenon occurs. This study was undertaken to analyze the antibody repertoires of individuals with RA to gain insight into the mechanisms leading to epitope spreading of the serum ACPA response in RA.Methods. Plasmablasts from the blood of 6 RA patients were stained with citrullinated peptide tetramers to identify ACPA-producing B cells by flow cytometry. Plasmablasts were single-cell sorted and sequenced to obtain antibody repertoires. Sixty-nine antibodies were recombinantly expressed, and their anticitrulline reactivities were characterized using a cyclic citrullinated peptide enzyme-linked immuosorbent assay and synovial antigen arrays. Thirty-six mutated antibodies designed either to represent ancestral antibodies or to test paratope residues critical for binding, as determined from molecular modeling studies, were also tested for anticitrulline reactivities.Results. Clonally related monoclonal ACPAs and their shared ancestral antibodies each exhibited differential reactivity against citrullinated antigens. Molecular modeling identified residues within the complementarity-determining region loops and framework regions predicted to be important for citrullinated antigen binding. Affinity maturation resulted in mutations of these key residues, which conferred binding to different citrullinated epitopes and/or increased polyreactivity to citrullinated epitopes.Conclusion. These results demonstrate that the different somatic hypermutations accumulated by clonally related B cells during affinity maturation alter the antibody paratope to mediate epitope spreading and polyreactivity of the ACPA response in RA, suggesting that these may be key properties that likely contribute to the pathogenicity of ACPAs.

Lyme disease (Borrelia burgdorferi infection) is increasingly recognized as a significant source of morbidity worldwide. Here, we show that blood plasmablasts and CD27− memory B cells are elevated in untreated Lyme disease, with higher plasmablast levels associated with more rapid resolution of clinical symptoms. Stronger serum reactivity to surface proteins and peptides from B. burgdorferi was also associated with faster resolution of clinical symptoms. Through molecular identifier-enabled antibody heavy-chain sequencing of bulk B cells and single-cell paired-chain antibody sequencing of blood plasmablasts, we characterized immunoglobulin gene usage patterns specific to B. burgdorferi infection. Recombinantly expressed antibodies from expanded lineages bound B. burgdorferi antigens, confirming that these clones are driven by the infection. Furthermore, recombinant sequence-derived antibodies were functional, inhibiting growth of B. burgdorferi in vitro. Elevations and clonal expansion of blood plasmablasts were associated with rapid return to health, while poor plasmablast responses were associated with a longer duration of symptoms following treatment. Plasmablasts induced by B. burgdorferi infection showed preferential antibody gene segment usage, while bulk sequencing of total B cells revealed convergent CDR3 motifs specific to B. burgdorferi-infected patients. Our results show that robust plasmablast responses encoding Bb-static antibodies are associated with more rapid resolution of Lyme disease, and these antibodies could provide the basis for next-generation therapeutics for Lyme disease.