Fig. 6: Single-cell transcriptomic analysis highlighted the enhanced cytotoxicity of CDK9⁺CD8⁺ T cells.

a Left: The scRNA-seq (GEO: GSE151530) UMAP projection of the 16,462 T cells from 37 patients with liver cancer. Each color represents a T-cell cluster. Cell types and marker genes are assigned to each cluster. Right: Expression levels of marker genes identified for clusters. b Violin plot showing the expression profiles of genes related to T-cell activation (top), cytotoxicity (middle), and exhaustion (bottom). A Wilcoxon Rank Sum test was performed to determine the P values (two-tailed test). c Pathway enrichment of top 200 marker genes enriched in CDK9⁺CD8⁺ T cells as compared to CDK9^-CD8⁺ T cells using Metascape analysis. Significance shown by the Bonferroni-corrected P values (two-tailed test).  d The gene expression of PRF1, LAYN, CDK9 overlaid on the cell trajectory projection. The size and color represent the gene expression levels. e Activated mouse CD8⁺ T cells were treated with 250 nM NVP-2 for 6 h or 24 h, and apoptotic cells were detected by flow cytometry. f Activated mouse CD8⁺ T cells were treated with 250 nM NVP-2 for 6 h or 24 h, and senescent cells were detected by flow cytometry. β-Gal positive cells are shown as senescent cells. g Activated mouse CD8⁺ T cells were treated with 250 nM NVP-2 for 24 h, and then the indicated signal was detected by flow cytometry. h Activated mouse CD8⁺ T cells were treated with 100 nM NVP-2 for indicated days, then the cytotoxicity- and exhaustion-related genes were determined by qRT-PCR. The heat map showed the fold changes when compared to the DMSO group each day. i Flow cytometry quantification of the percentage of PD-1⁺CD8⁺T and LAG3⁺ CD8⁺T in tumor tissues from orthotopically xenografted mice (n = 9 for each group). Error bars represent mean ± SEM. Statistical analyses were determined by a two-tailed Student’s t-test. Source data are provided as a Source Data file.