Protective antitumor immunity following genetic deletion of PD-1 is associated with transcriptional signatures of IFN stimulation, effector activity, and terminal exhaustion in CD8 ⁺ TILs in MC38 tumors. (A) Schematic of study design for interrogating mechanisms by which PD-1 loss promotes the development of protective immunity in CD8⁺ TILs in MC38 tumors, and for determining which changes are dependent on a cell-intrinsic loss of PD-1 versus cell-extrinsic consequences of PD-1 loss (e.g., changes in function in CD8⁺ TILs still expressing PD-1). The data in this figure compare total CD8⁺ TILs in inducible PD-1^del. mice (UBC-CreERT2⁺ PD-1^f/f) versus WT control mice (UBC-CreERT2⁻ PD-1^f/f) to define the impact of PD-1 deletion on the global CD8⁺ TIL population. (B) Clustering and UMAP visualization of CD8⁺ T cells (n = 15,729 cells) sorted from MC38 tumors from inducible PD-1^del. versus WT control mice at days 16–17 after tumor implantation. 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. (C) UMAP visualization of CD8⁺ TILs from WT (n = 4,394 cells) versus inducible PD-1^del. mice (n = 3,785 cells). (D) Stacked bar plots showing the frequency of each population belonging to each functional cluster. The legend for the colors used in the bars is shared with the legend for the clusters in B. Cluster 7 was excluded from the bar plot since it likely contained non-T cells. 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 and ϕ coefficients showing the enrichment of cells across all clusters between the WT TME and inducible PD-1^del. TME can be found in Fig. S3 B. (E) Bar plot showing the enrichment of different topics in CD8⁺ TILs from WT control versus inducible PD-1^del. mice. The functional annotations for the top five ranked topics in each direction are indicated. A full list of topics and top 50 genes by weight in each topic can be found in Table S5. (F) Heat map showing the Cohen’s D effect size of the top 5 differentially enriched topics in each direction in the MC38 dataset (up in CD8⁺ TILs from WT control mice, and up in CD8⁺ TILs from inducible PD-1^del. mice), and the enrichment of these topics in patient cohorts where scRNA-seq data were available on CD8⁺ TILs pre- and postcheckpoint blockade (either anti-PD-1 or anti-PD-1 plus anti-CTLA-4) (Bassez et al., 2021; Liu et al., 2022; Prakadan et al., 2021; Yost et al., 2019). (G) Pie chart showing the distribution of clone sizes in CD8⁺ TILs from WT control versus inducible PD-1^del. mice. Each pie represents data from an individual mouse. Also indicated is the Simpson Index (abbreviated as “SI” in the figure) for each sample. For analyses in B, shown are cells from two independent experiments (Exp 1+ Exp 2). For analyses in C–G, shown are cells from one independent experiment (Exp 2) where total CD8⁺ TILs were sorted regardless of PD-1 protein status in order to define the broad differences between CD8⁺ TILs in inducible PD-1^del. versus WT control mice, representing n = 2 mice per genotype. “WT TME” refers to CD8⁺ TILs that were isolated from WT control (UBC-CreERT2⁻ PD-1^flf) mice, and “PD-1^del. TME” refers to CD8⁺ TILs that were isolated from inducible PD-1^del. (UBC-CreERT2⁺ PD-1^flf) mice. Schematics/images in A and F were created with https://BioRender.com. Abbreviations, Supp.: Suppressed, Prog.: Progenitor-like, T_EX: T exhausted cell, Eff. function: Effector functions, LMD: leptomemingeal disease, Breast: breast cancer, NSCLC: non-small cell lung cancer, BCC: basal cell carcinoma, SCC: squamous cell carcinoma.