Background/Purpose: Ectopic lymphoid structures (ELS) have been observed in synovial tissue of rheumatoid arthritis (RA) patients but their functional relevance in the disease remains unclear. Additionally, little is known about B cell activation pathways during ectopic lymphoid neogenesis (ELN). In this study, we utilized single cell RNA sequencing coupled with B cell repertoire sequencing to characterize B cell subsets that may play a role in ELN in RA synovial tissue and support local B cell activation and development of autoreactive plasma cells.

Methods: Synovial tissue was selected based on the presence of lymphocytic infiltrates by histology (n=4 RA patients). Tissue was disaggregated using protocols established by the Accelerating Medicines Partnership (AMP) consortium. scRNA-seq was performed on sorted tissue B cells using the 10x genomic platform with poly-A selected, 5’ initiated expression and V(D)J libraries generated from each single cell. Transcriptomic clusters were compared using supervised classification techniques to AMP phase I RA synovial B cells (n=10), SLE kidney B cells, and blood B cells. Combined single cell repertoire/RNA sequencing from an additional 13 RA synovial samples and 10 matched blood B cells are currently under analysis in AMP phase II. In vitro studies were conducted for elucidation of signals promoting in situ B cell activation, with NR4A detected by qPCR and flow cytometry.

Results: Using single cell RNA sequencing analysis, we identified a unique B cell subset in the RA synovium characterized by high expression of NR4A1-3, a family of orphan nuclear receptors (NUR77, NURR1, and NOR1) that are induced by acute and chronic antigen stimulation in lymphocytes and function as ligand-independent transcription factors. The NR4A⁺ B cell cluster showed evidence of somatic hypermutation (SHM) and class-switched recombination based on repertoire analysis.  The rate of SHM was positively correlated with NR4A1 and NR4A2 gene expression and inversely correlated with IGHD gene expression. Gene Set Enrichment Analysis revealed that the NR4A⁺ cluster has a transcriptomic profile between naïve and germinal center (GC) B cells sorted from tonsil and differentially expressed genes characteristic of GC centrocytes including CD83 and GPR183. In SLE kidney, and peripheral blood B cells, NR4A⁺ B cells were reduced to 0.7% and 1.5% abundance, respectively, compared to >40% abundance in synovial tissue from AMP phase I and the 10X platform (p< 0.001 under logistic mixed models). NRA4 was upregulated at both the RNA and protein level upon activation through the B cell receptor in vitro. NR4A1 protein was expressed spontaneously in RA tissue B cells by flow cytometric and histologic analysis.

Conclusion: Our data suggest a dynamic progression of B cell activation in RA synovial ectopic lymphoid structures, with NR4A a potential read-out of chronic antigen activation and local adaptive immune responses.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/role-of-nr4a-nuclear-receptor-family-in-ra-synovial-ectopic-lymphoid-neogenesis-revealed-by-single-cell-profiling/