Objective A hallmark of rheumatoid arthritis (RA) is the production of autoantibodies, including anti-citrullinated protein antibodies (ACPAs). Nevertheless, the specific targets of these autoantibodies remain incompletely defined. During an immune response, B cells specific for the inciting antigen(s) are activated and differentiate into “plasmablasts”, which are released into the blood. In this study we sequence the plasmablast antibody repertoire to define the targets of the active immune response in RA. Methods We developed a novel DNA barcoding method to sequence the cognate heavy- and light-chain pairs of antibodies expressed by individual blood plasmablasts in RA. The method uses a universal 5’ adapter that enables full-length sequencing of the antibodies’ variable regions and recombinant expression of the paired antibody chains. The sequence datasets were bioinformatically analyzed to generate phylogenetic trees that identify clonal families of antibodies sharing heavy- and light-chain VJ sequences. Representative antibodies were expressed, and their binding properties characterized using CCP2 ELISA and antigen microarrays. Results We used our sequencing method to generate phylogenetic trees representing the antibody repertoires of peripheral blood plasmablasts of 4 individuals with anti-CCP+ RA, and recombinantly expressed 14 antibodies that were either “singleton” antibodies or representative of clonal antibody families. CCP2 ELISA identified four ACPAs, and antigen microarray analysis identified ACPAs that differentially targeted epitopes on α-enolase, citrullinated fibrinogen, and citrullinated histone 2B. Conclusions Our data provide evidence that autoantibodies targeting α-enolase, citrullinated fibrinogen, and citrullinated histone 2B are produced by the ongoing activated B cell response in, and thus may contribute to the pathogenesis of, RA.

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Objectives The co-occurrence of rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA) is well described in rheumatoid arthritis (RA). However, the mechanisms underlying the potential interaction between these two distinct autoantibodies has not been well defined. We sought to evaluate the epidemiologic and molecular interaction of ACPA and RF with both disease activity as well as measures of RA-associated inflammation. Methods In a cohort of 1,488 Veterans with RA, measures of disease activity and levels of serum cytokines and multiplex ACPA were compared between the double-negative (aCCP-/RF-), ACPA-positive and RF-negative (aCCP+/RF-), ACPA-negative and RF-positive (aCCP-/RF+), or double-positive (aCCP+/RF+) subgroups. Studies were additionally performed using an in vitro immune complex (IC) stimulation assay in which macrophages were incubated with ACPA-ICs in the presence or absence of monoclonal IgM RF, and TNFα production measured as readout of macrophage activation. Results Compared with the double negative (as well each single positive) subgroup, the aCCP+/RF+ subgroup exhibited higher disease activity, serum CRP, and inflammatory cytokines (all P<0.001). In vitro stimulation of macrophages by ACPA-ICs induced increased cytokine production with the addition of monoclonal IgM RF as compared to ACPA-ICs alone (P=0.003) Conclusions The combined presence of ACPA and IgM RF mediates increased proinflammatory cytokine production in vitro, and is associated with increased systemic inflammation and disease activity in RA. Our data suggest that IgM RF enhances the capacity of ACPA-ICs to stimulate macrophage cytokine production, thereby providing a mechanistic link by which RF enhances the pathogenicity of ACPA-ICs in RA.