The clusters 1 and 2 accounted for around 80% and 15% of the total cells, respectively, whereas the clusters 3C5 accounted for very few cells (Fig.?6aCc). and concomitant autoantibody production. CD8+ Tfh cells share comparable gene signatures with CD4+ Tfh, and require CD40L/CD40 and TCR/MHCI interactions to deliver help to B cells. Our study thus highlights the diversity of follicular T cell subsets that contribute to the breakdown of B-cell tolerance. and and and and and that encode Roqin-1 and Roqin-2, respectively49,50 (Fig.?5a). In contrast, CXCR5+PD-1?CD8+ T cells expressed high levels of Cytotoxic T lymphocyte (CTL) effector genes, including and (Fig.?5a). Therefore, CXCR5+PD-1+CD8+ T cells Oglufanide are a unique sublineage of CD8+ T cells that share comparable gene signatures with CD4+ Tfh cells, and might function as helper T cells. Open in a separate windows Fig. 5 Tfh-like gene expression by Stat5 deficiency in CXCR5+PD-1+CD8+ T cells. Splenic CXCR5?PD-1?CD8+, CXCR5+PD-1?CD8+, CXCR5+PD-1+CD8+ T cells were sorted from LCMV-infected Stat5+/+CD8Cre and Stat5fl/?CD8Cre mice (a, b, d, e) or from Stat5+/+CD8CreIgHELsHEL and Stat5fl/?CD8CreIgHELsHEL mice Oglufanide (c), and subjected to RNA-seq analysis. a Differentially expressed genes in wild-type CXCR5+PD-1+CD8+ T cells relative to the indicated control subsets of wild-type CD8+ T cells. b Differentially expressed Tfh-specific genes in Stat5-deficient CXCR5+PD-1+CD8+ T cells relative to the indicated control subsets of Stat5-deficient CD8+ T cells. c Differentially expressed Tfh-specific genes in Stat5-deficient CXCR5+PD-1+CD8+ T cells relative to the indicated control subsets of Stat5-deficient CD8+ T cells derived from IgHELsHEL transgenic mice. d Volcano plot of differentially expressed genes in Stat5-deficient CXCR5+PD-1+CD8+ T cells relative to wild-type CXCR5+PD-1+CD8+ T cells. e Differentially expressed Tfh-specific and effector-related genes in Stat5-deficient relative to wild-type CXCR5+PD-1+CD8+ T cells. Data shown are obtained from three mice of each experimental group. f Comparative GSEA of the Tfh, Th1 and CD8 effector Oglufanide cell signatures in wild-type and Stat5-deficient CXCR5+PD-1+CD8+ T cells As expected, the Tfh-specific genes were highly expressed in CXCR5+PD-1+CD8+ T cells derived from LCMV-infected Stat5fl/?CD8Cre/YFP mice as well as non-infected Stat5fl/?CD8Cre/YFPIgHELsHEL mice (Fig.?5b, c). The expression level of and and were highly expressed in CXCR5+PD-1+CD8+ T cells, and further upregulated in Stat5-deficient cells; whereas, the expression of memory receptor and inhibitory receptor (encoded by havcr2) was downregulated in CXCR5+PD-1+CD8+ T cells (Supplementary Figs.?10, 11). However, the protein levels of TCF1, one of the crucial CD4+ Tfh-related transcriptional factors, were comparable between Stat5-deficient and control CXCR5+PD-1+CD8+ cells (Supplementary Fig.?11). The FACS analysis also confirmed that this antigen-induced IFN production was downregulated in Stat5-deficient relative to control CD8+ T cells (Supplementary Fig.?12). Real-time quantitative PCR (RT-qPCR) confirmed the upregulated expression of and in Stat5-deficient CXCR5+PD-1+CD8+ T cells (Supplementary Fig.?13). Thus, Stat5 negatively regulates the expression of Tfh-specific genes, but positively regulates the expression of effector T-cell-related genes, in CXCR5+PD-1+CD8+ T cells. To further characterize CXCR5+PD-1+CD8+ T cells, we performed high-depth single-cell RNA-seq (scRNA-seq) analysis of these cells. CXCR5+PD-1+CD8+ T cells were sorted from acute LCMV-infected Stat5+/+CD8Cre/YFP or Stat5fl/?CD8Cre/YFP Rabbit polyclonal to ATP5B mice, and subjected to scRNA-seq analysis. Following quality control (QC), 500 cells from LCMV-infected Stat5+/+CD8Cre/YFP mice with median of 2160 genes per cell, and 723 cells from LCMV-infected Stat5fl/?CD8Cre/YFP mice with median of 2397 genes per cell were utilized for further analysis. The unbiased t-distributed stochastic neighbor embedding (t-SNE) analysis generated five cell clusters (clusters 1C5) (Fig.?6a). The clusters 1 and 2 accounted for around 80% and 15% of the total Oglufanide cells, respectively, whereas the clusters 3C5 accounted for very few cells (Fig.?6aCc). The quantitative transcriptional comparisons among the clusters revealed that each cluster differently expressed specific genes (Supplementary Figs.?14, 15). Of notice, cluster 1 expressed.