T Cell-Dependent Affinity Maturation and Innate Immune Pathways Differentially Drive Autoreactive B Cell Responses in Rheumatoid Arthritis

Our findings suggest that ACPA+ and RF+ B cells are imprinted with distinct transcriptional programs, which suggests that these autoantibodies associated with increased inflammation in RA arise from 2 different molecular mechanisms.

We developed antigen-tetramers to isolate ACPA+ and RF+ B cells and performed single-cell RNA sequencing on 2,349 B cells from 6 RA patients and 1 healthy donor to analyze their immunoglobulin repertoires and transcriptional programs. Prominent immunoglobulins were expressed as monoclonal antibodies and tested for autoantigen reactivity.

Rheumatoid arthritis (RA) is characterized by the activation of B cells that produce anti-citrullinated protein antibodies (ACPAs) and rheumatoid factors (RFs), but the mechanisms by which tolerance is broken in these B cells remain incompletely understood. We undertook this study to investigate whether ACPA+ and RF+ B cells break tolerance through distinct molecular mechanisms.

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 used more class-switched isotypes and exhibited more somatic hypermutations relative to RF+ B cells, and these differences were accompanied by down-regulation of CD72 and up-regulation 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.