Development of an inducible PD-1 deletion model to study PD-1–sufficient and PD-1–deleted CD8 ⁺ T cells in the same TME. (A) Schematic for inducible PD-1 deletion. Mice containing loxP-flanked Pdcd1 alleles (PD-1^f/f) and expressing either one copy of UBC-CreERT2 (UBC-CreERT2⁺ PD-1^f/f, referred to as “inducible PD-1^del.” mouse) or no copies of UBC-CreERT2 (UBC-CreERT2⁻ PD-1^f/f, referred to as “WT control” mouse) received daily tamoxifen (1 mg/mouse/day) i.p. from days 7 to 11 (unless otherwise indicated) after MC38 tumor cell implantation to delete exons 2–4 of Pdcd1. The schematic was created with https://BioRender.com. (B) Percentage of splenic CD8⁺ T cells expressing PD-1 protein following 24 h in vitro stimulation with anti-CD3 and anti-CD28. Data shown are one experiment with n = 11 WT control and n = 7 inducible PD-1^del. mice. Data are representative of three independent experiments with at least n = 3 WT control and at least n = 5 inducible PD-1^del. per group. An unpaired t test was performed not assuming normal distribution (Mann–Whitney test), P < 0.0001***. (C) Representative flow cytometry contour plots showing PD-1 (x axis) by CD8α expression (y axis) on CD8⁺ T cells in MC38 tumors harvested at day 17. Full gating strategy was based on size, singlets, live, CD45⁺, CD8⁺ cells. (D) Quantification of the frequency of CD8⁺ T cells, CD4⁺ Foxp3⁻ T cells, and CD4⁺ Foxp3⁺ T cells expressing PD-1 in MC38 tumors in WT control mice versus inducible PD-1^del. mice shown in C. Data shown are one representative experiment with n = 6 WT control mice and n = 10 inducible PD-1^del. mice. Data are representative of five experiments with at least five mice per group. Normality was determined using the Shapiro–Wilk test, and significance was assessed using a one-way ANOVA with Tukey’s multiple comparisons tests. Asterisks (***) indicate P < 0.0001 for each pairwise comparison indicated in this figure. (E) Quantification of the levels of PD-1 phosphorylation in CD8⁺ TILs in MC38 tumors at day 17 after tumor cell implantation. Phospho-PD-1 levels were determined using an antibody that specifically detects the phosphorylated ITSM motif of PD-1 by flow cytometry as described previously (Bu et al., 2021). Data shown are one experiment with n = 5 mice per group and representative of two independent experiments with three to five mice per group. A one-way ANOVA was performed with Tukey’s multiple comparisons test. WT control PD-1^Lo versus PD-1^Hi, P < 0.0001***; inducible PD-1^KO PD-1^Lo versus PD-1^Hi, P = 0.0045**. (F–H) Tumor growth curves showing tumor volume (mm³) over time (days) after subcutaneous injection of MC38 tumor cells. Arrows indicate the timing of tamoxifen administration to induce PD-1 deletion. Tamoxifen was administered (F) prior to tumor cell injection (n = 9 WT and n = 11 inducible PD-1^del.), (G) at days 0–5 after tumor cell injection (n = 6 WT and n = 9 inducible PD-1^del.), or (H) days 7–11 after tumor cell injection (n = 12 per genotype). Data in F–H are from one representative experiment each. F and G are each representative of two independent experiments with at least n = 6 per group. H is representative of three experiments with at least n = 3 WT control and at least n = 5 inducible PD-1^del. mice per group. Significance was assessed using the Mann–Whitney unpaired t test at the endpoint for the WT control. (F) Day 27, P = 0.0019**. (G) Day 32, P = 0.0182*. (H) Day 31, P = 0.0011**. (I) CD8/Treg ratio in MC38 tumors at day 17 after tumor cell implantation. Data shown are from two experiments combined with n = 15 (WT control) and n = 17 (inducible PD-1^del.). Data are representative of three independent experiments with at least five mice per group. Significance was assessed using an unpaired t test, P = 0.0020**. In A–I, WT control refers to UBC-CreERT2⁻ PD-1^f/f mice, and inducible PD-1^del. refers to UBC-CreERT2⁺ PD-1^f/f mice.