Background/Purpose: T cells can either be activated through their T cell receptor (TCR) in an antigen-specific manner, or in response to cytokines. Identification of antigen-reactive T cells in rheumatoid arthritis (RA) would allow us to investigate arthritogenic clones and elucidate early events in disease pathogenesis. We developed a model to identify and study such T cell responses in RA using a specific marker of TCR signaling—Nur77 (Nr4a1)—and identified antigen-reactive T cells in the SKG arthritis model and in RA synovial tissue. The immediate early gene Nr4a1 (encoding the orphan nuclear hormone receptor Nur77) is upregulated in response to antigen, but notably is not induced by cytokine stimulation. Using a fluorescent reporter of Nur77 expression in SKG mice, we found that higher levels of Nur77-eGFP (GFP^hi) in SKG CD4 T cells were more arthritogenic and autoreactive and found an analogous population in RA synovial tissue. Here, we set-out to examine the transcriptional program and TCR repertoire of the more antigen-experienced SKG GFP^hi T cells that may contribute to disease.

Methods: Paired single cell transcriptional and TCR repertoire analysis was performed on sorted naïve SKG and Balb/c (WT) CD4 T cells from 2 mice based on their Nur77-eGFP levels (GFP^hi and GFP^lo) using the 5’ 10X single-cell platform. We examined our dataset for differentially expressed genes (DEG) and TCR variable genes enriched in the arthritogenic SKG ­GFP^hi T cells.

Results: After filtering, there were 99,074 cells with a mean of 12,384 cells per condition (min 9,979, max 14,899). Using leiden clustering of nearest neighbors, 12 heterogeneous cell clusters were identified from SKG and WT GFP^hi and GFP^lo naïve CD4 T cells. DEG analysis by Wilcoxon rank-sum test identifies a cluster marked by high Nr4a1 expression (cluster 7). Cells in cluster 7 are associated with a memory-like phenotype, which is enriched in the SKG GFP^hi T cells (Fig 1). Within cluster 7 there are two highly variable gene (hvg) modules that correlate with either Egr2 and Egr3 (both immediate early gene transcription factors), or Tnfrsf9 (4-1BB – the TCR inducible co-stimulatory receptor) expression using Spearman’s correlation for all cells within cluster 7. The Egr module correlates with additional immediate early genes or markers of early T cell activation, whereas the Tnfrsf9 module is associated with markers of chronic or prolonged TCR input. Furthermore, the SKG GFP^hi T cells in Nr4a1-high cluster 7 are associated with a biased TCR variable beta (Vb) repertoire. We find significantly higher expression of TCR Vb genes TRBV3 (P < 0.001) , TRBV16 (P < 0.0049), and TRBV26 (P < 0.001) in SKG GFP^hi T cells compared to SKG GFP^lo and WT GFP^hi groups (Fig 2 ).

Conclusion: This scRNA and TCR sequencing dataset highlights the surprising heterogeneity found within naïve CD4 T cells and identifies a memory-like cluster marked by high Nr4a1 expression with 2 hvg modules that likely segregate acute from chronic antigen-activated T cells. Moreover, the altered SKG GFP^hi TCR Vb repertoire may be an important contributor to their autoreactivity and arthritogenicity and suggests further peripheral repertoire pruning.

Figure 1: Nr4a1-high cluster 7 memory-like cells associated with markers of TCR signaling and enriched in SKG GFPhi CD4 T cells Transcriptional analysis of SKG and WT GFPhi and GFPlo CD4 naïve (CD4+CD25-CD62LhiCD44lo) T cells sorted based on Nur77-eGFP expression. Unbiased leiden clustering demonstrates high Nr4a1 expression in cluster 7, which is enriched with GFPhi T cells (a) and is associated with expression of immediate early genes and markers of anergy and exhaustion (b). DEG analysis by Wilcoxon rank-sum test P * < 0.05, ** < 0.01, *** < 0.001

Figure 2: Altered SKG GFPhi TCR Vb repertoire. Bar graphs of TCR beta variable gene usage that are significantly enriched in SKG GFPhi T cells in cluster 7. Graph labels: TRBV gene name (TCR Vb protein name). Significance determined by one-way ANOVA followed by Tukey’s HSD. NA – no named TCR Vb protein, ns: P > 0.05, **P < 0.01, ***P < 0.001

ACR Meeting Abstracts - https://acrabstracts.org/abstract/nr4a1-high-arthritogenic-t-cells-from-skg-mice-are-associated-with-markers-of-recent-and-chronic-antigen-stimulation-and-an-altered-t-cell-receptor-repertoire/