Transcriptional differences between total CD8 ⁺ TILs in WT versus inducible PD-1 ^del. mice and comparison with changes in anti-PD-1–treated mice. (A) Joint plot showing the first two principal components of gene expression and distribution of CD8⁺ TILs in the WT versus inducible PD-1^del. TME. Effect size (Cohen’s D, referred to as CDES) between groups is indicated. PC1 CDES = 0.082; PC2 CDES = 1.691. (B) Heat map showing the proportion of cells in each cluster between CD8⁺ TILs in WT control and inducible PD-1^del. mice. Indicated are the raw cell counts (in the box), P values, and phi (ϕ) coefficient (effect sizes) (next to the box). P values were calculated using Fisher’s exact test. The color indicates the proportion of cells in each cluster, normalized by columns. On the left is a plot of all cells from experiment (Exp) 2 grouped based on genotype (WT versus inducible PD-1^del. mice, two mice per group). On the right is a plot of the cells from Exp 2 broken up by individual mouse, showing that the individual mice reproduce the pattern seen in the combined dataset. (C) GSEA of HALLMARK gene sets comparing CD8⁺ TILs in WT control and inducible PD-1^del. mice. Shown are only the top 10 significantly different gene sets between groups. Full names of each pathway, enrichment scores, and P values can be found in Table S4. (D) UMAPs of the topic weights for each topic. Full list of top 50 genes per topic can be found in Table S5. (E) Violin plots showing module scores in the scRNA-seq data generated from CD8⁺ TILs from WT control and inducible PD-1^del. mice. Modules shown are signatures of upregulated and downregulated genes generated from scRNA-seq from CD8⁺ TILs from mice that received anti-PD-1 treatment. Modules derived from Kumar et al. (2022). (Top) Modules shown are differentially expressed genes from CD8⁺ TILs from mice with MC38 tumors treated with anti-PD-1 versus isotype control–treated mice (harvested at day 13). The left plot shows the module of genes downregulated in anti-PD-1–treated compared with isotype control (P = 7.22 × 10⁻¹⁰⁵; CDES = 0.48), and the right plot shows the module of genes upregulated in anti-PD-1–treated compared with isotype control (P = 6.99 × 10⁻²⁸²; CDES = −0.89). (Bottom) Modules shown are differentially expressed genes from CD8⁺ TILs from mice with CT26 tumors treated with anti-PD-1 versus isotype control–treated mice (harvested at day 17). The left plot shows the module of genes downregulated in anti-PD-1–treated mice compared with isotype control (P = 6.06 × 10⁻²³⁸; CDES = 0.76), and the right plot shows the module of genes upregulated in anti-PD-1–treated mice compared with isotype control (P = 0; CDES = -1.42). P values were calculated using a two-sided Mann–Whitney U test. Plots in A–C and E include cells from one independent experiment, representing n = 2 mice per genotype. Plots in D include cells from two independent experiments, representing n = 4 mice per genotype. Asterisks (***) indicate P < 0.0001. Exact P values listed in each panel.