Deletion of PD-1 induces a potent antitumor state and improves functionality in both PD-1 ^KO and PD-1 ^Hi CD8 ⁺ TILs. (A) Schematic of the experimental design for interrogating the CD8⁺ T cell–intrinsic versus T cell–extrinsic changes following PD-1 deletion. In this figure, a combination of PD-1 protein expression, transcript information, and logistic regression (see Materials and methods) was used to determine whether a CD8⁺ TIL is PD-1^KO (based on predicted deletion of the Pdcd1 locus and a lack of PD-1 protein) or PD-1^Hi (based on the presence of an intact Pdcd1 locus and the presence of PD-1 protein) in the inducible PD-1^del. TME. PD-1^KO versus PD-1^Hi CD8⁺ TILs in the inducible PD-1^del. TME are compared to define the cell-intrinsic effect of PD-1 deletion, and PD-1^Hi CD8⁺ TILs in the inducible PD-1^del. TME are compared with PD-1^Hi CD8⁺ TILs in the WT TME to define the cell-extrinsic effect. The schematic was created with https://BioRender.com. (B) UMAP visualization of CD8⁺ T cells (n = 15,729) sorted from MC38 tumors of WT control versus inducible PD-1^del. mice at days 16–17 after tumor implantation. In each UMAP, the color indicates the cells of the given sample (for WT control: PD-1 protein status, and for inducible PD-1^del.: both PD-1 protein status and transcript-based prediction for being non-KO or KO) and the cells in the remaining three samples are shown in gray. (C) Violin plots showing the PC1 scores for cells from B. (D) Stacked bar plots showing the frequency of each sample as indicated in the figure belonging to each transcriptional cluster shown in Fig. 3 B. Colors denote transcriptional clusters, labeled with functional annotations (see Materials and methods). A full list of upregulated genes per cluster can be found in Table S1. Clusters are indicated in order from left to right, with Cluster 0 being the left-most cluster, and Cluster 8 being the right-most cluster. P values generated using Fisher’s exact test for all groups across all clusters can be found in Fig. S5 D, and the associated ϕ coefficients can be found in Fig. S5 E. (E) Differentially expressed (DE) genes between PD-1^Hi CD8⁺ TILs in the WT TME and PD-1^Hi CD8⁺ TILs in the inducible PD-1^del. TME. Individual genes of interest are indicated on the plot. The entire list of differentially expressed genes can be found in Table S10. Analyses in B–E include all cells combined from two independent experiments, representing n = 4 mice per genotype. WT TME refers to CD8⁺ TILs taken from UBC-CreERT2⁻ PD-1^f/f mice, and PD-1^del. TME refers to CD8⁺ TILs taken from UBC-CreERT2⁺ PD-1^f/f mice. PD-1^KO and PD-1^Hi labels on CD8⁺ TILs were determined as described in Fig. 4 and Materials and methods. (F) Frequency of granzyme B⁺ PD-1^Hi and PD-1^Lo CD8⁺ T cells in MC38 tumors from WT and inducible PD-1^del. mice at day 17 after tumor cell implantation assessed using flow cytometry. Data in F are from two experiments combined with n = 15 WT control and n = 17 inducible PD-1^del. mice. Data are representative of at least four independent experiments with at least five mice per group. Normality was determined using both the D’Agostino–Pearson test and Shapiro–Wilk tests, and all groups were considered normally distributed. Significance was assessed using a one-way ANOVA with Tukey’s multiple comparisons tests. WT TME PD-1^Lo versus WT TME PD-1^Hi, P = 0.0044**; WT TME PD-1^Lo versus PD-1^KO TME PD-1^Lo, P < 0.0001***; WT TME PD-1^Lo versus PD-1^KO TME PD-1^Hi, P < 0.0001***; WT TME PD-1^Hi versus PD-1^KO TME PD-1^Lo, P = 0.0182*; WT TME PD-1^Hi versus PD-1^KO TME PD-1^Hi, P = 0.0153*; PD-1^KO TME PD-1^Lo versus PD-1^KO TME PD-1^Hi, P > 0.9999, ns. All mice received tamoxifen daily from days 7 to 11 i.p. https://BioRender.com.