Panel A shows bar graphs assessing tdTomato expression in CD8 positive, Tconv (CD4 positive FOXP3 negative), and Tregs (CD4 positive FOXP3 positive) TEX cells from non-draining and draining lymph nodes under different tamoxifen treatment regimens. The x-axis represents the treatment groups, and the y-axis represents the percentage of Thy1.2 positive T cells expressing tdTomato. Panel B shows flow cytometry plots and bar graphs of tdTomato expression in CD8 positive T cells isolated from the spleen, non-draining lymph nodes, and draining lymph nodes in vehicle-treated and tamoxifen-treated mice. The x-axis represents the tissue source, and the y-axis represents the percentage of Thy1.2 positive CD8 positive T cells expressing tdTomato. Panel C shows flow cytometry plots and bar graphs assessing tdTomato expression in CD8 positive CD44 positive T cells isolated from the spleen and lymph nodes of mice injected intradermally with PBS or B16-F10 and treated with tamoxifen. The x-axis represents the treatment groups, and the y-axis represents the percentage of Thy1.2 positive CD8 positive CD44 positive T cells expressing tdTomato. Panel D shows bar graphs of PD1 expression on tdTomato positive cells from Panel C. The x-axis represents the treatment groups, and the y-axis represents the percentage of live CD8 positive CD44 positive tdTomato positive cells expressing PD1. Panel E shows flow cytometry plots and bar graphs of PD1 and LAG3 expression on tdTomato subsets of tumor-infiltrating CD8 positive T cells. The accompanying plot shows the percentages of PD1 high LAG3 high, PD1 intermediate LAG3 low, PD1 negative LAG3 negative, PD1 high LAG3 negative, and PD1 intermediate LAG3 positive CD8 positive T cells. Panel F shows flow cytometry plots of LY108 and TIM3 expression on tdTomato subsets of tumor-infiltrating CD8 positive T cells. Panel G shows bar graphs assessing LY108 and TIM3 expression on LAG3 negative tdTomato negative, LAG3 positive tdTomato negative, and LAG3 negative tdTomato positive CD8 positive T cells in draining lymph nodes on days 11 and 19. The accompanying plot shows the percentages of LY108 negative TIM3 negative, LY108 positive TIM3 negative, LY108 positive TIM3 positive, and LY108 negative TIM3 positive CD8 positive T cells within these subsets.
Validation of a tamoxifen-inducible LAG3 + cell–specific Cre line. (A) tdT expression was assessed on the CD8+, Tconv (CD4+ FOXP3−), and Treg (CD4+ FOXP3+) TEX cells from NDLNs (left panel) and DLNs (right panel) to evaluate labeling efficiency in respective T cell compartment with different tamoxifen treatment regimen. Gated on Thy1.2+ T cells. (B)Lag3iCreERT2Rosa26LSL-tdT mice were implanted with 1.25 × 105 B16-F10 cells i.d. and treated with three intraperitoneal tamoxifen injections (2 mg in 5% EtOH/sunflower oil) and oil control on d8–10. Harvested on d19. tdT expression was assessed on the CD8+ T cells isolated from spleen, NDLNs, and DLNs in the vehicle control versus tamoxifen-treated mice. (C) tdT expression was assessed on the CD8+ CD44+ T cells isolated from spleen and LNs in the mice injected with 1× PBS versus B16-F10 i.d. and treated with three intraperitoneal tamoxifen injections (2 mg in 5% EtOH/sunflower oil) d8–10. Harvested on d18. (D) PD1 expression was assessed on tdT+ cells from C. (E) PD1 and LAG3 expression on tdT subsets of tumor-infiltrating CD8+ T cells. Phylum plot represents the percentage of PD1hiLAG3hi (green), PD1−LAG3lo (blue), PD1−LAG3− (yellow), PD1hiLAG3− (red), and PD1intLAG3− (purple) CD8+ T cells. (F) Flow cytometry plots showing LY108 and TIM3 expression on tdT subsets of tumor-infiltrating CD8+ T cells. (G) Ly108 and TIM3 expression was assessed on LAG3−tdT−, LAG3+tdT−, and LAG3− tdT+ CD8+ T cells in DLN on d11 and d19 from the Lag3iCreERT2Rosa26LSL-tdT mice. Phylum plot represents the percentage of Ly108−TIM3− (blue), Ly108+TIM3− (purple), Ly108+TIM3+ (pink), and Ly108−TIM3+ (green) CD8+ T cells within those subsets. Data in A, B, E, and F are from n = 5–9 mice per group, pooled from three independent experiments. Data in C and D are representative of two independent experiments with n = 6–7 mice per group. *P < 0.05; **P < 0.01; ns, not significant by (A–D) unpaired t test. Error bars represent the mean ± SEM.