Panel A shows a schematic diagram of the experimental design used to evaluate the differentiation potential of LAG3 negative tdTomato positive CD8 positive T cells in circulation following tumor re-challenge. Panel B shows bar graphs of tdTomato expression in Thy1.2 positive CD8 positive T cells from non-draining lymph nodes, draining lymph nodes, and tumors in SHAM control and tumor-bearing mice. Panel C shows flow cytometry plots and a bar graph of LAG3 and tdTomato expression in CD8 positive T cells isolated from the indicated tissues. Panel D shows SPICE plots illustrating the co-expression patterns of inhibitory receptors in tdTomato subsets. Panel E shows flow cytometry plots of LY108 and TIM3 expression in tdTomato subsets. Panel F shows bar graphs of the percentages of LY108 and TIM3 expressing cells within the indicated tdTomato subsets. Panel G shows a line graph of functional enrichment scores across 15 clusters of CD44 positive tdTomato positive cells identified by single-cell RNA sequencing (scRNAseq) analysis. Panel H shows a schematic diagram outlining the experimental design used to assess tumor growth following depletion of peripheral LAG3 negative tdTomato positive CD8 positive T cells. Panel I shows line graphs of tumor growth curves for five different groups of mice, with the x-axis representing time and the y-axis representing tumor size.
Lag3 — tdT + CD8 + T EX stem cell–like progenitor cells persist in circulation and contribute to anti-tumor immunity. (A) Scheme to evaluate differentiation potential of LAG3−tdT+ CD8+ T cells in circulation upon tumor rechallenge. Lag3iCreERT2Rosa26LSL-tdT mice were i.d. injected with B16-F10 cells (1.25 × 105), treated with three tamoxifen injections (2 mg in 5% EtOH/sunflower oil) at d8–10, and tumor was resected on d12. After a 30-day resting phase, mice were rechallenged on d42 with i.d. injection of B16-F10 cells (1.25 × 105), and tumors were harvested 18 days after rechallenge. SHAM controls were injected with 1× PBS on d0 but followed a similar regime. (B) tdT expression was assessed on Thy1.2+ CD8+ T cells in NDLNs, DLNs, and tumor isolated from A for SHAM control (grey) and tumor-bearing (blue) mice. (C) LAG3 and tdT expression on CD8+ T cells isolated from A. Phylum plot represents the percentage of tdT subsets in SHAM, NDLNs, DLNs, and tumor. (D) Spice plot visualization of IR co-expression (n = 4) on tdT subsets from A. (E) LY108 and TIM3 expression assessed on tdT subsets from A. (F) Phylum plot represents the percentage of LY108−TIM3− (blue), LY108+TIM3− (purple), LY108+TIM3+ (pink), and LY108−TIM3+ (green) in tdT subsets. (G) CD44+tdT+ cells from scRNAseq on d11, d19, and d60 were analyzed for functional enrichment of 15 clusters (C1–C15). (H) Scheme to assess tumor growth after depletion of peripheral LAG3−tdT+ CD8+ T cells persisting in the system. 300 ng of diphtherial toxin was treated on d39–41. (I) Tumor growth curves across five different groups. Yellow: Lag3iCreERT2Rosa26LSL-tdT mice received PBS instead of primary tumor on d0 and were treat with DTX on d39–41 (n = 10); red: Lag3iCreERT2Rosa26LSL-tdT mice received primary tumor and were treat with PBS instead of DTX on d39–41 (n = 10); purple: Lag3iCreERT2Rosa26LSL-tdT mice received primary tumor and were treat with DTX on d39–41 (n = 11); blue: Lag3iCreERT2Rosa26LSL-tdTRosa26LSL-DTR mice received primary tumor and were treat with PBS instead of DTX on d39–41 (n = 6); green: Lag3iCreERT2Rosa26LSL-tdTRosa26LSL-DTR mice received primary tumor and were treat with DTX on d39–41 (n = 10). Results in I are representative of three independent experiments, with the indicated n per group. Data in B–F are pooled from three independent experiments with n = 8 mice per group. Data in G are representative of two independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant, by unpaired t test (B), two-way ANOVA (C and F), or mixed-effect analysis (I). Error bars represent mean ± SEM.