PD-1 expressed by CD8 ⁺ T cells is largely sufficient for protective antitumor immunity against subcutaneous MC38. (A) Schematic for germline deletion of PD-1 selectively on CD8⁺ T cells (using the E8i-Cre^POS PD-1^f/f mouse) or on all cells (using the global PD-1^KO mouse). E8i-Cre^NEG PD-1^f/f mice were included as a WT control. Schematic created using https://BioRender.com. (B) Representative flow cytometry contour plots showing PD-1 (y axis) versus CD8α (x axis) expression. Plots are gated on CD45⁺ CD3⁺ TILs at day 11 after implantation of MC38 tumor cells into WT (E8i-Cre^NEG PD-1^f/f), CD8 conditional PD-1 KO (E8i-Cre^POS PD-1^f/f), and PD-1 germline KO (global PD-1^KO). (C) Quantification of the flow cytometry in B, showing the frequency PD-1⁺ of CD8⁺ T cells, CD4⁺ FoxP3⁻ T cells (conventional CD4⁺ T cells), and CD4⁺ Foxp3⁺ (Tregs) from MC38 tumors at day 11 after tumor cell implantation. Data shown are from one representative experiment with n = 4–8 mice per group. Data are representative of two independent experiments with at least three mice per group comparing all three mouse strains (CD8-Cre^NEG PD-1^f/f, CD8-Cre^POS PD-1^f/f, and global PD-1^KO), and an additional four independent experiments comparing CD8-Cre^NEG PD-1^f/f and CD8-Cre^POS PD-1^f/f mice. A two-way ANOVA was performed with Tukey’s multiple comparisons test. A P value for all comparisons is marked with “***,” P < 0.0001. For comparisons labeled as not significant (ns): CD8⁺ T cells, P = 0.9906; CD4⁺ FoxP3⁻ T cells, P = 0.6966; CD4⁺ FoxP3⁺ T cells, P = 0.2088. (D) Tumor growth curves in WT control (E8i-Cre^NEG PD-1^f/f), CD8 conditional PD-1 KO (E8i-Cre^POS PD-1^f/f), and PD-1 germline KO (global PD-1^KO) mice, showing tumor volume (mm³) over time (days) after implantation of MC38 tumor cells subcutaneously in the flank. Data shown are from one representative experiment with n = 5–10 mice per group. Data are representative of three independent experiments with at least four mice per group. A one-way ANOVA (Kruskal–Wallis test) with Dunn’s multiple comparisons was performed for the day 23 time point (endpoint for CD8-Cre^NEG PD-1^f/f). CD8-Cre^NEG PD-1^f/f versus CD8-Cre^POS PD-1^f/f, P = 0.0136*; CD8-Cre^POS PD-1^f/f versus global PD-1^KO, P = 0.8391; CD8-Cre^NEG PD-1^f/f versus global PD-1^KO, P = 0.0021**. (E–J) Analysis of TIL from MC38 tumors at days 11–13 after tumor cell implantation in CD8-Cre^NEG PD-1^f/f and CD8-Cre^POS PD-1^f/f mice using flow cytometry. (E) Frequency of CD8⁺ T cells (left, P < 0.0001***) and Treg cells (right, P = 0.0027**) of total CD45⁺ cells. (F) Number of CD8⁺ T cells (left, not significant, P = 0.0630) and Treg cells (right, not significant, P = 0.0514) per milligram of tumor. (G) Ratio of CD8⁺ T cells to Treg cells in the tumor, P < 0.0001***. (H) Frequency (left, P = 0.0190*) and number per milligram (right, P = 0.0089**) of CD8⁺ T cells expressing granzyme B protein (abbreviated “GzmB”). (I) Frequency of IFNγ-producing CD8⁺ T cells following ex vivo stimulation with PMA and ionomycin, P = 0.0006***. (J) Frequency (left, not significant, P = 0.2351) and number per milligram (right, not significant, P = 0.6842) of CD8⁺ T cells expressing Ki-67 protein. Data shown in E–J are from two experiments combined for a total of n = 10 CD8-Cre^NEG PD-1^f/f and n = 10 CD8-Cre^POS PD-1^f/f. For E–J, significance was determined using an unpaired t test if both groups were normally distributed, or the Mann–Whitney test if both groups were not normally distributed. Representative of 2–7 total experiments with at least three mice per group per experiment.