Figure 6. Cell-intrinsic loss of PD-1... | Rockefeller University Press

$Cell-intrinsic loss of PD-1 drives increased clonal expansion of PD-1KOCD8+TILs compared with PD-1HiCD8+TILs. (A) Schematic of the experimental design for interrogating the CD8+ T cell–intrinsic changes following PD-1 deletion. PD-1KO versus PD-1Hi CD8+ TILs in the inducible PD-1del. TME are compared head to head to define the cell-intrinsic effect of PD-1 deletion. The schematic was created with https://BioRender.com. (B) Differentially expressed (DE) genes between PD-1Hi and PD-1KO CD8+ TILs in the inducible PD-1del. TME. Individual genes of interest are indicated on the plot. The entire list of differentially expressed genes can be found in Table S11. (C) Quantification of the number of cells within each clonotype, separated by CD8+ TILs in the WT TME (left) and inducible PD-1del. TME (right). Cells with a shared TCR sequence are shown as a dot, with lines connecting the dots containing the cells with the same TCR sequence between groups. CDES refers to Cohen’s D effect size. Significance was determined used a paired t test. Asterisks (***) indicate P < 0.0001. Exact P values are as follows: WT TME PD-1Lo versus PD-1Hi, P = 4.1 × 10−5; CDES = −0.93. PD-1KO TME PD-1KO versus PD-1Hi, P = 8.9 × 10−9; CDES = 0.59. (D) Scatterplot showing the correlation between the fraction of TILs in each clone within the inducible PD-1del. mice that are PD-1KO (y axis) versus the size of the clone (x axis). Asterisks (***) indicate P < 0.0001. Exact P values are as follows: KT correlation = 0.41, P = 1.52 × 10−7. P value was calculated using the Kendall tau test. (E) Frequency of TCF-1+ TIM-3− (progenitor TEX) and TIM-3+ TCF-1− (terminal TEX) subpopulations within the PD-1Hi or PD-1Lo CD8+ TIL populations in MC38 tumors from WT versus inducible PD-1del. mice assessed using flow cytometry. Full gating strategy was size, singlets, live, CD45+, CD8α+, and either PD-1Hi or PD-1Lo. Significance was assessed using the Kruskal–Wallis one-way ANOVA with Dunn’s multiple comparisons test. Progenitor TEX (left graph): WT TME PD-1Lo versus WT TME PD-1Hi, P < 0.0001***; WT TME PD-1Lo versus PD-1del. TME PD-1Lo P = 0.1150 ns; WT TME PD-1Lo versus PD-1del. TME PD-1Hi, P < 0.0001***; WT TME PD-1Hi versus PD-1del. TME PD-1Lo, P = 0.0104*; WT TME PD-1Hi versus PD-1del. TME PD-1Hi, P > 0.9999, ns; PD-1del. TME PD-1Lo versus PD-1del. TME PD-1Hi, P = 0.0886, ns. Terminal TEX (right graph): WT TME PD-1Lo versus WT TME PD-1Hi, P < 0.0001***; WT TME PD-1Lo versus PD-1del. TME PD-1Lo, P = 0.0008***; WT TME PD-1Lo versus PD-1del. TME PD-1Hi, P = 0.0003***; WT TME PD-1Hi versus PD-1del. TME PD-1Lo, P > 0.9999, ns; WT TME PD-1Hi versus PD-1del. TME PD-1Hi, P > 0.9999, ns; PD-1del. TME PD-1Lo versus PD-1del. TME PD-1Hi, P > 0.9999, ns. Data shown in E are combined from two representative experiments for a total of n = 14 WT control and n = 12 inducible PD-1del. mice. (F) Heat map showing average Z score for select signatures from the literature in cells separated by genotype (WT versus inducible PD-1del.) and PD-1 status (PD-1Lo or PD-1Hi) in the WT TME, or PD-1KO versus PD-1Hi in the inducible PD-1del. TME. Signatures obtained or derived from Beltra et al. (2020), Hudson et al. (2019), Im et al. (2016), Kurtulus et al. (2019), Miller et al. (2019), Pauken et al. (2016). List of gene signatures can be found in Table S2. Analyses in B include cells from two independent experiments (Exp 1 + Exp 2), representing n = 4 mice per genotype. Analyses in C and D include cells from one independent experiment (Exp 2 only), representing n = 2 mice per genotype. WT TME refers to CD8+ TILs taken from UBC-CreERT2− PD-1f/f mice, and PD-1del. TME refers to CD8+ TILs taken from UBC-CreERT2+ PD-1f/f mice. PD-1KO and PD-1Hi labels on CD8+ TILs from the inducible PD-1del. mice were determined as described in Fig. 4 and Materials and methods. All mice received tamoxifen daily from days 7 to 11 i.p. https://BioRender.com.$

Figure 6.

Cell-intrinsic loss of PD-1 drives increased clonal expansion of PD-1 ^KO CD8 ⁺ TILs compared with PD-1 ^Hi CD8 ⁺ TILs. (A) Schematic of the experimental design for interrogating the CD8⁺ T cell–intrinsic changes following PD-1 deletion. PD-1^KO versus PD-1^Hi CD8⁺ TILs in the inducible PD-1^del. TME are compared head to head to define the cell-intrinsic effect of PD-1 deletion. The schematic was created with https://BioRender.com. (B) Differentially expressed (DE) genes between PD-1^Hi and PD-1^KO 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 S11. (C) Quantification of the number of cells within each clonotype, separated by CD8⁺ TILs in the WT TME (left) and inducible PD-1^del. TME (right). Cells with a shared TCR sequence are shown as a dot, with lines connecting the dots containing the cells with the same TCR sequence between groups. CDES refers to Cohen’s D effect size. Significance was determined used a paired t test. Asterisks (***) indicate P < 0.0001. Exact P values are as follows: WT TME PD-1^Lo versus PD-1^Hi, P = 4.1 × 10⁻⁵; CDES = −0.93. PD-1^KO TME PD-1^KO versus PD-1^Hi, P = 8.9 × 10⁻⁹; CDES = 0.59. (D) Scatterplot showing the correlation between the fraction of TILs in each clone within the inducible PD-1^del. mice that are PD-1^KO (y axis) versus the size of the clone (x axis). Asterisks (***) indicate P < 0.0001. Exact P values are as follows: KT correlation = 0.41, P = 1.52 × 10⁻⁷. P value was calculated using the Kendall tau test. (E) Frequency of TCF-1⁺ TIM-3⁻ (progenitor T_EX) and TIM-3⁺ TCF-1⁻ (terminal T_EX) subpopulations within the PD-1^Hi or PD-1^Lo CD8⁺ TIL populations in MC38 tumors from WT versus inducible PD-1^del. mice assessed using flow cytometry. Full gating strategy was size, singlets, live, CD45⁺, CD8α⁺, and either PD-1^Hi or PD-1^Lo. Significance was assessed using the Kruskal–Wallis one-way ANOVA with Dunn’s multiple comparisons test. Progenitor T_EX (left graph): WT TME PD-1^Lo versus WT TME PD-1^Hi, P < 0.0001***; WT TME PD-1^Lo versus PD-1^del. TME PD-1^Lo P = 0.1150 ns; WT TME PD-1^Lo versus PD-1^del. TME PD-1^Hi, P < 0.0001***; WT TME PD-1^Hi versus PD-1^del. TME PD-1^Lo, P = 0.0104*; WT TME PD-1^Hi versus PD-1^del. TME PD-1^Hi, P > 0.9999, ns; PD-1^del. TME PD-1^Lo versus PD-1^del. TME PD-1^Hi, P = 0.0886, ns. Terminal T_EX (right graph): WT TME PD-1^Lo versus WT TME PD-1^Hi, P < 0.0001***; WT TME PD-1^Lo versus PD-1^del. TME PD-1^Lo, P = 0.0008***; WT TME PD-1^Lo versus PD-1^del. TME PD-1^Hi, P = 0.0003***; WT TME PD-1^Hi versus PD-1^del. TME PD-1^Lo, P > 0.9999, ns; WT TME PD-1^Hi versus PD-1^del. TME PD-1^Hi, P > 0.9999, ns; PD-1^del. TME PD-1^Lo versus PD-1^del. TME PD-1^Hi, P > 0.9999, ns. Data shown in E are combined from two representative experiments for a total of n = 14 WT control and n = 12 inducible PD-1^del. mice. (F) Heat map showing average Z score for select signatures from the literature in cells separated by genotype (WT versus inducible PD-1^del.) and PD-1 status (PD-1^Lo or PD-1^Hi) in the WT TME, or PD-1^KO versus PD-1^Hi in the inducible PD-1^del. TME. Signatures obtained or derived from Beltra et al. (2020), Hudson et al. (2019), Im et al. (2016), Kurtulus et al. (2019), Miller et al. (2019), Pauken et al. (2016). List of gene signatures can be found in Table S2. Analyses in B include cells from two independent experiments (Exp 1 + Exp 2), representing n = 4 mice per genotype. Analyses in C and D include cells from one independent experiment (Exp 2 only), representing n = 2 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 from the inducible PD-1^del. mice were determined as described in Fig. 4 and Materials and methods. All mice received tamoxifen daily from days 7 to 11 i.p. https://BioRender.com.

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