Fig. 4

Unsupervised clustering of T-cell subsets according to the level of intracellular gene expression. Single-cell RNA sequencing of five fresh tumor specimens. A UMAP displaying clusters defined based on their gene expression profile. Created with Cerebro (R-studio^©). The list of genes expressed by each cluster is provided in Supplementary Data 11. B Stacked violin plot displaying the expression distribution of selected cell markers in the T cell clusters. C Pie chart showing the relative abundance (mean) of each cluster in the whole cohort. D Stacked bar chart showing the relative abundance of each cluster in each tumor specimen. E Volcano plot displaying differential gene expression between CD4⁺FOXP3⁻ vs CD8⁺ T-cells (upper left panel); CD4⁺FOXP3⁺ vs CD4⁺FOXP3⁻T cells (bottom panel) and CD4⁺FOXP3⁺ vs CD8.⁺ T cells (upper right panel). Genes are plotted as log2 fold change versus the − log10 of the adjusted p value. Genes in red are significantly differentially expressed with a fold change > 1.5 compared to the reference population. F. Stacked violin plot displaying the expression distribution of ICPs in the T cell clusters. ICPs: immune checkpoints: (CD25, CD28, CD39, 4-1BB, CTLA-4, ICOS, OX40, PD-1, PD-L1, and TIGIT)