Panel A shows a schematic diagram outlining the experimental design used to evaluate the population kinetics of tdTomato positive cells in tumor-bearing hosts. Panel B shows flow cytometry plots and a paired scatter plot of LAG3 and tdTomato expression in CD8 positive T cells isolated from B16-F10 tumors on days 11, 15, and 19. The x-axis represents the days, and the y-axis represents the percentage of Thy1.2 positive CD8 positive T cells. Panel C shows line graphs of the percentage of tdTomato positive cells in draining lymph nodes and non-draining lymph nodes on days 11, 15, and 19. The x-axis represents the days, and the y-axis represents the percentage of Thy1.2 positive CD8 positive T cells. Panel D shows bar graphs of CD8 positive T-cell subsets based on LY108 and TIM3 expression on days 11 and 19, including Non-TEX (LY108 negative TIM3 negative), pTEX (LY108 positive TIM3 negative), intTEX (LY108 positive TIM3 positive), and tTEX (LY108 negative TIM3 positive). Panel E shows a flow cytometry plot and a bar graph of gp100 positive cells among CD8 positive T cells isolated from tumors on day 18. The x-axis represents gp100 expression levels, and the y-axis represents the percentage of live Thy1.2 positive CD8 positive T cells. Panel F shows bar graphs of LAG3 and tdTomato expression in gp100 high and gp100 low cell populations. Panel G shows a schematic diagram outlining the experimental design for evaluating LAG3 and tdTomato expression in pMEL-T cells transferred into C57BL/6 mice. Panel H shows flow cytometry plots and a line graph of LAG3 and tdTomato expression in CD8 positive T cells isolated from B16gp100 tumors on days 11, 15, and 19. The x-axis represents the days, and the y-axis represents the percentage of Thy1.1 positive CD8 positive T cells. Panel I shows line graphs of the percentage of tdTomato positive cells in draining lymph nodes and non-draining lymph nodes on days 11, 15, and 19. The x-axis represents the days, and the y-axis represents the percentage of Thy1.1 positive CD8 positive T cells. Panel J shows bar graphs of CD8 positive T-cell subsets based on LY108 and TIM3 expression on days 11 and 19, including Non-TEX (LY108 negative TIM3 negative), pTEX (LY108 positive TIM3 negative), intTEX (LY108 positive TIM3 positive), and tTEX (LY108 negative TIM3 positive).
The LAG3 − tdT + CD8 + T EX cells are found in periphery compared with the LAG3 + tdT + CD8 + T EX cells, which are more terminally T EX and restricted to tumor compartment. (A) Scheme for evaluation of population kinetics of tdT+ cells in tumor-bearing host. Lag3iCreERT2Rosa26LSL-tdT mice were i.d. implanted with melanoma cells (1.25 × 105), treated with three tamoxifen injections (2 mg in 5% EtOH/sunflower oil) at d8–10, and harvested on d11, d15, and d19. (B and C) LAG3 and tdT expression was assessed on CD8+ T cells isolated from (B) B16-F10 tumors and (C) DLNs and NDLNs, gated on Thy1.2+ CD8+ T cells. (D) LY108 and TIM3 expression on different tdT subsets. Phylum plot represents the percentage of LY108−TIM3− (blue), LY108+TIM3− (purple), LY108+TIM3+ (pink), and LY108−TIM3+ (green) CD8+ T cells. (E) gp100+ cells were assessed on CD8+ T cells isolated from tumor on d18 of Lag3iCreERT2Rosa26LSL-tdT mice implanted with B16gp100 and treated with three tamoxifen injections (2 mg in 5% EtOH/sunflower oil) d8–10. (F) LAG3 and tdT expression were evaluated in both gp100 high and gp100 low populations. (G) Schematic representation of Lag3iCreERT2Rosa26LSL-tdT pMEL-T cells (50,000 Thy1.1+CD8+ T cells) injected i.v. into C57BL/6 mice on d−1 followed by i.d. injection of B16gp100 tumor cells (1.25 × 105) on d0, treated with three tamoxifen injections (2 mg in 5% EtOH/sunflower oil) at d8–10, and harvested on d11, d15, and d19. (H and I) LAG3 and tdT expression was assessed on Thy1.1+ CD8+ T cells isolated from B16gp100 tumors (H), DLNs and NDLNs (I), gated on Thy1.1+ CD8+ T cells. (J) LY108 and TIM3 expression on different tdT subsets. Data in B–D and H–J are from n = 6–10 mice per group, pooled from three independent experiments. Data in E and F are representative of two independent experiments with n = 8 mice. Statistical analysis of LAG3+tdT+ and LAG3−tdT+ CD8+ T cells from d11–15 and d15–19 are represented by * and #, respectively. #P < 0.05; ns, not significant, by two-way ANOVA (B, C, H, and I) or unpaired t test (F). Error bars represent mean ± SEM.