Single cell analysis of RA synovial B cells reveals a dynamic spectrum of ectopic lymphoid B cell activation and hypermutation characterized by NR4A nuclear receptor expression

Abstract

Ectopic lymphoid structures (ELS) are present in rheumatoid arthritis (RA) synovial tissue, but the precise pathways of B cell activation and the role of in situ synovial B cell differentiation and selection in disease are not well understood. Here, we identified a B cell population in the synovium characterized by expression of NR4A1-3, a family of orphan nuclear receptors, that is highly enriched at both early and late stages of RA. NR4A B cells are rare in healthy peripheral blood, RA blood, and SLE kidney, but share markers with blood transcriptomic signatures that peak during RA disease flare. Using combined single cell transcriptomics and B cell receptor (BCR) sequencing, we demonstrate that NR4A synovial B cells have an activated transcriptomic profile that significantly overlaps with germinal center (GC) light zone (LZ) B cells and an accrual of somatic hypermutation that correlates with loss of naive B cell status. NR4A B cells uniquely co-express lymphotoxin β and IL6, supporting important functions in ELS promotion and pro-inflammatory cytokine production. The presence of shared clones in this activated B cell state, NR4A expressing synovial plasma cells (PC), and CCR6+ memory B cell (MBC) precursors further points to in situ differentiation. NR4A1 was expressed at the protein level in RA synovial B cells and PC, was high in tonsil GC B cells with a LZ-DZ intermediate phenotype, and was rapidly induced at both the RNA and protein level upon activation through the BCR. Taken together, we identified a dynamic progression of B cell activation in RA synovial ELS, with NR4A as a read-out of likely antigen activation and local adaptive immune responses.

Spatial mapping of rheumatoid arthritis synovial niches reveals specific macrophage networks associated with response to therapy

“…Of note, CD11C + HLADR + myeloid cells (IM2 cells), resembling FDC, were observed withing the ELS2 niche. In the context of active RA and before any treatment intervention, both ELS niches were also strongly connected with resident sublining fibroblasts (PDPN -CD90 + ) near vascular vessels belonging to the SL1 and SL4 niches, consistent with the described presence of activated stromal cells and HEVs, respectively (27)(28)(29). Importantly, we also highlighted the presence of different subtypes of IMs cells within the ELS2 niche.…”

“…Within the sublining compartment, notably, different subsets of IMs and lymphoid cells were shown to communicate together and form two distinct ELS niches. Such ELS organization recapitulates the architecture of secondary lymphoid organs, with (i) a distinct T cell rich zone in contact with a B cell rich area; (ii) a network of follicular dendritic cells (FDC) and activated resident stromal cells such as fibroblasts; and (iii) the presence of high endothelial venules (HEVs) that participate to T and B cells recruitment and in situ differentiation into plasma cells, that produce somatically mutated disease-specific autoreactive antibodies (27)(28)(29). Our multiplexed spatial approach supports this view and provides additional insights into ELS organization.…”