T cell-dependent affinity maturation and innate immune pathways differentially drive autoreactive B cell responses in rheumatoid arthritis

Rheumatoid arthritis (RA) is characterized by the activation of B cells that produce anticitrullinated protein antibodies (ACPA) and rheumatoid factors (RF), but the mechanisms by which tolerance is broken in these B cells remain incompletely understood. Here we investigate whether ACPA and RF B cells break tolerance through distinct molecular mechanisms. We developed antigen-tetramers to isolate ACPA- and RF-producing B cells and performed single-cell RNA-sequencing on 2,349 B cells from six RA patients to analyze their immunoglobulin repertoires and transcriptional programs. Prominent immunoglobulins were expressed as monoclonal antibodies and tested for autoantigen reactivity. ACPA and RF B cells were enriched in the peripheral blood of RA patients relative to healthy controls. Characterization of patient-derived monoclonal antibodies confirmed ACPA and RF targeting of tetramer-specific B cells at both antigen-inexperienced and affinity-matured B-cell stages. ACPA B cells utilized more class-switched isotypes and exhibited more somatic hypermutations relative to RF B cells, and these differences were accompanied by the downregulation of CD72 and upregulation of genes that promote class-switching and T cell dependent responses. In contrast, RF B cells expressed transcriptional programs that stimulate rapid memory reactivation through multiple innate immune pathways. Coexpression analysis revealed that ACPA- and RF- B cell enriched genes belong to distinct transcriptional regulatory networks. Our findings suggest that ACPA and RF B cells are imprinted with distinct transcriptional programs, suggesting that these autoantibodies associated with increased inflammation in RA arise from two different molecular mechanisms.