Fig 1: Combination treatment mitigates tumor-infiltrating CD8+ T cell exhaustion and promotes T cell activation, proliferation, and function.(A to C) scRNA-seq analysis of T cells from tumors (n = 3 per group). (A) UMAP and (B) RNA expression of marker genes used for T cell classification. CD8+ Teff, CD8+ Teff; CD8+ Tmem, CD8+ T memory; prolif T, proliferating T cell. (C) Gene expression in CD8+ T cells. * indicates any statistical comparisons between the CT26LIGHT + anti–CTLA-4 group and any of the other groups. (D) Heatmap of protein expression in CD8+ T cells from CyTOF analysis (n = 3 per group). (E) Percentage of Ki-67+ of CD3+ T cells via flow cytometry analysis (n = 6–8 per group). (F) Percentage of Granzyme B+ of CD8+ cells in the CRLMs from flow cytometry analysis (n = 6 to 8 per group). (G) Percentage of CD44+CD62L− cell (Teff memory) and CD44+CD62L+ cell (T central memory) out of CD8+ T cells via flow cytometry analysis (n = 6 to 8 per group). (H) Percentage of Perforin+ and Granzyme B+ CD8+ T cells after in vitro stimulation with LIGHT and anti–CTLA-4 via flow cytometry analysis. For (E) to (H), means ± SEM is shown on plots, and a two-tailed unpaired t test was performed between groups. (I) Exhaustion score is plotted for each sample in the TCGA dataset (n = 396 cases) stratified by CTLA4 and TNFSF14 expression (n = 148 for LIGHThighCTLA4high, n = 50 for LIGHThighCTLA4low, n = 50 for LIGHTlowCTLA4high, and n = 148 for LIGHTlowCTLA4low). Median score and interquartile range are shown. *P < 0.05; ***P < 0.005. (J) UMAP plot of low, moderate, and highly clonal T cells and plot of the percentage of high clonality T cells out of T cells (moderate clonality = 2 unique T cells with the same TCR; high clonality refers to >2 T cells with the same TCR). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0005.