Fig. 5

Immune checkpoints are more expressed by expanded clonotypes of intratumoral CD4⁺Foxp3⁺ T-cells. TCR repertoire analysis from the single-cell RNA sequencing dataset of five fresh tumor specimen. A TCR diversity showing the number of clonotypes per patient in each T cell cluster. Dunn’s multiple comparison test, *p value ≤ 0.05; **p value ≤ 0.01. B Stacked bar chart displaying the distribution of clonotype frequency in each cluster. C Sankey diagram showing clonotype sharing between clusters and according to clonality (LC ≤ 2 cells; HC > 2 cells). D Heatmap displaying differential ICP expression (median Log2 fold-change) between LC and HC T cells in each CD4⁺ (left panel) and CD8⁺ (right panel) clusters. E Stacked bar chart showing the distribution of T-cells from CD4⁺ clusters according to the level of ICP expression (above the median expression level (HE) or below the median expression level (LE)) and the expansion status (LC or HC). Median expression level was calculated independently for each sample and for each ICP. F Graph displaying the average number with standard deviation of ICP expressed per cells in LC and HC T cells for each cluster; Mann–Whitney test, *p value ≤ 0.05; **p value ≤ 0.01; ***p value ≤ 0.001; ****p value ≤ 0.0001.TCR: T cell receptor; LC: low clonality; HC: high clonality. ICPs: immune checkpoints: (CD25, CD28, CD39, 4-1BB, CTLA-4, ICOS, OX40, PD-1, PD-L1, and TIGIT)