Panel A shows a schematic diagram of the experimental design for the YUMM1.7 melanoma model, illustrating the timeline for tamoxifen treatment and tissue collection. Panel B shows flow cytometry plots and a line graph of tdTomato and LAG3 expression in CD8 positive T cells on days 11, 15, and 19. The flow cytometry plots have LAG3 on the x-axis and tdTomato on the y-axis, while the line graph has days on the x-axis and the percentage of Thy1.2 positive CD8 positive T cells on the y-axis. Panel C shows a schematic diagram of the experimental design for the YUMMER1.7 melanoma model, including the tamoxifen treatment and tissue collection timeline. Panel D shows flow cytometry plots and a line graph of tdTomato and LAG3 expression in CD8 positive T cells on days 11, 15, and 19, with LAG3 on the x-axis and tdTomato on the y-axis in the flow cytometry plots, and days on the x-axis and the percentage of Thy1.2 positive CD8 positive T cells on the y-axis in the line graph. Panel E shows a schematic diagram of the experimental design for the MC38 colon adenocarcinoma model, illustrating the tamoxifen treatment and tissue collection timeline. Panel F shows flow cytometry plots and a line graph of tdTomato and LAG3 expression in CD8 positive T cells on days 11, 15, and 19, with LAG3 on the x-axis and tdTomato on the y-axis in the flow cytometry plots, and days on the x-axis and the percentage of Thy1.2 positive CD8 positive T cells on the y-axis in the line graph. Panel G shows a schematic diagram of adoptive transfer of pMEL cells followed by dual tumor inoculation with B16-gp100 and MC38 cells. Panel H shows flow cytometry plots and a bar graph of tdTomato positive CD8 positive T cells in tumors. The flow cytometry plots have LAG3 on the x-axis and tdTomato on the y-axis, while the bar graph has tumor groups on the x-axis and the percentage of live CD8 positive T cells on the y-axis. Panel I shows flow cytometry plots and a graph of tdTomato positive CD8 positive T cells in draining and non-draining lymph nodes. The flow cytometry plots have LAG3 on the x-axis and tdTomato on the y-axis, while the graph has lymph node groups on the x-axis and the percentage of live CD8 positive T cells on the y-axis.
Population kinetics of fate-mapped tdT+ CD8+ T cells across multiple tumor models. (A) Scheme for evaluation of population kinetics of tdT+ cells in tumor models. 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. (A–D) LAG3 and tdT expression was assessed on CD8+ T cells isolated from (A and B) YUMM1.7 and (C and D) YUMMER1.7, gated on Thy1.2+ CD8+ T cells. (E) Scheme for evaluation of population kinetics of tdT+ cells in the MC38 colon adenocarcinoma model. Lag3iCreERT2Rosa26LSL-tdT mice were s.c. implanted with MC38 cells (5.0 × 105), treated with three tamoxifen injections (2 mg in 5% EtOH/sunflower oil) at d8–10, and harvested on d11, d15, and d19. (F) LAG3 and tdT expression was assessed on CD8+ T cells isolated from E, gated on Thy1.2+ CD8+ T cells. (G) C57BL/6 mice received adoptive transfer of 50,000 Lag3iCreERT2Rosa26LSL-tdT pMEL cells (i.v.) followed by dual tumor inoculation with B16gp100 (i.d.) and antigen-irrelevant MC38 (s.c.). Tamoxifen (2 mg in 5% EtOH/sunflower oil) was administered on d8–10. (H and I) LAG3 and tdT expression were then assessed in (H) tumors, (I) DLNs, and non-NDLNs. Data in B, D, and F are from n = 5–6 mice per group, pooled from three independent experiments. Data in H and I are representative of two independent experiments with n = 14 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; ***P < 0.001; ****P < 0.0001; ##P < 0.01; ###P < 0.001; ns, not significant, by two-way ANOVA (B, D, and F) and paired t test (H and I).