PD-1 ^Hi and PD-1 ^Lo CD8 ⁺ TILs are phenotypically, functionally, and transcriptionally distinct in MC38 tumors in WT control mice. (A) Representative flow cytometry contour plots gated on CD8⁺ T cells in MC38 tumors in WT C57BL/6 mice at day 21 after tumor cell implantation. Full gating strategy is size, singlets, live, CD45⁺, CD8⁺ cells. PD-1 is shown on the x axis, and each individual marker shown on the y axis is indicated above the plot. (B) Quantification of the flow cytometry data shown in A, showing the percentage of PD-1^Hi or PD-1^Lo CD8⁺ T cells expressing each indicated marker. Data are shown from three experiments combined for all markers except granzyme B, which is from two experiments combined. For all markers except granzyme B, n = 17 mice. For granzyme B, n = 7 mice. Significance was assessed using a paired t test. PD-1^Lo versus PD-1^Hi: CD62L, P < 0.0001***; TCF-1, P < 0.0001***; Ki-67, P < 0.0001***; granzyme B, P = 0.0015**; TIM-3, P < 0.0001***. (C) (Top) Quantification of IR transcript co-expression separated by sample (showing PD-1^Hi and PD-1^Lo cells in WT control [UBC-CreERT2⁻ PD-1^f/f] mice), showing the frequency of CD8⁺ TILs co-expressing the select number of IR transcripts (ranging from 0 to 5 IRs expressed/co-expressed). IR transcripts included in the analysis were Havcr2 (encoding TIM-3), Lag3 (encoding LAG-3), Tigit (encoding TIGIT), Cd160 (encoding CD160), and Ctla4 (encoding CTLA-4). (Bottom) Pie charts of protein data (using flow cytometry) showing the frequency of PD-1^Hi or PD-1^Lo CD8⁺ T cells co-expressing IR proteins from MC38 tumors in WT C57BL/6 mice. Flow cytometry data shown are from two experiments with n = 10 mice in total. IR proteins included were TIM-3, LAG-3, TIGIT, and CD160. (D) Volcano plot showing differentially expressed genes between PD-1^Hi and PD-1^Lo CD8⁺ TILs in WT control (UBC-CreERT2⁻ PD-1^f/f) mice. Individual genes of interest are indicated on the plot. The entire list of differentially expressed genes can be found in Table S8. For the scRNA-seq plots in C and D, shown are all cells combined from two independent experiments, representing n = 4 mice per genotype.