Fig. 4: scRNA-seq analysis reveals unique signaling pathways involved in the BCAT1-mediated regulation of IL-17A production.

Multiplex scRNA-seq analysis of human CD4⁺ memory T cells from three different groups was performed using Seurat in R software (version 4.3.0): no TCR stimulation (TCR-), TCR stimulation for 72 h without Bi2 (TCR+Bi2-), and TCR stimulation for 72 h with Bi2 (10 μM) (TCR+Bi2+), a Individual cells (28,651 cells) were color-coded based on the cluster (n = 31) in a t-distributed stochastic neighbor-joining (t-SNE) plot generated by unsupervised Seurat clustering. b Major CD4⁺ memory T-cell subsets were identified by canonical cell type marker expression. c t-SNE plots segregated on the basis of major CD4⁺ memory T-cell subsets (top, TCR-, n = 10,109 cells; TCR+Bi2-, n = 9506 cells; TCR+Bi2 +, n = 9036 cells). Dotted regions highlight Th17 cluster changes after TCR stimulation with Bi2 treatment. Pie charts showing relative Th subset abundances under different conditions (bottom). Activated T cells were annotated by the average expression of activation markers listed in Fig. S4A. d Pathway enrichment analysis of differentially expressed genes (DEGs) in activated Th17 cells between the TCR+Bi2- and TCR+Bi2+ groups. Representative genes in each pathway are shown. e Volcano plot of DEGs in activated Th17 cells between the TCR⁺Bi2^- and TCR⁺Bi2⁺ groups. Volcano plots were generated using the EnhancedVolcano package (version 1.16.0). f Expression of HIF1A in activated Th17 cells was projected onto the t-SNE plot (top) shown as feature plots (top). Violin plots showing the distribution of HIF1A expression levels, with dots representing individual cells (bottom). g Gene set enrichment analysis (GSEA) revealed 15 pathways enriched in 23,457 DEGs in the activated Th17 cluster with an FDR < 0.25. Red bars represent gene sets with a nominal p value < 0.1. h GSEA was used to examine the significantly enriched pathways. GSEA enrichment plot (left) and heatmap of downregulated DEGs (right) in the PI3K-AKT-mTOR signaling pathway in Bi2-treated cells. All transcripts within annotated genes were uploaded to a locally installed GSEA tool and compared with the hallmark gene sets.