Panel A shows a schematic diagram of the experimental design for single-cell RNA sequencing (scRNAseq) analysis of tdTomato subsets in tumors and peripheral tissues on days 11 and 19 following intradermal injection of Lag3iCreERT2 Rosa26LSL-tdTomato mice with 1.25 times 10 superscript 5 B16-F10 melanoma cells. Panel B shows a bar graph of T-cell receptor (TCR) clonal expansion in the indicated CD8 positive T-cell subsets on days 11 and 19, comparing highly expanded (10 or more copies) and lowly expanded (fewer than 10 copies) clones. Panel C shows circos plots illustrating the distribution of highly expanded clones and PD1 expression among tumor-infiltrating and peripheral CD8 positive T-cell subsets on days 11 and 19, highlighting clones unique to individual subsets and those shared among multiple subsets. Panel D shows a schematic diagram of the adoptive transfer strategy in which LAG3 positive tdTomato positive CD8 positive T cells isolated from day 11 B16-F10 tumors of Lag3iCreERT2 Rosa26LSL-tdTomato (Thy1.2 positive CD45.2 positive) mice were transferred intratumorally into CD45.1 positive recipient mice bearing established day 7 B16-F10 tumors. Panel E shows a representative flow cytometry plot illustrating the gating strategy used to identify and sort LAG3 positive tdTomato positive CD8 positive T cells from day 11 tumors. Panel F shows a representative flow cytometry plot confirming the purity of the sorted LAG3 positive tdTomato positive CD8 positive T-cell population. Panel G shows a bar graph summarizing the frequencies of LAG3 positive tdTomato positive and LAG3 negative tdTomato positive CD8 positive T cells quantified from the individual tumors shown in Panel H. Panel H shows representative flow cytometry plots from six individual recipient tumors on day 12 following intratumoral adoptive transfer. Panel I shows a heatmap illustrating the distribution of transcriptional clusters within each tdTomato subset on days 11 and 19, with hierarchical clustering based on cluster composition ratios. Panel J shows a bar graph illustrating changes in the proportions of naive or stem-like memory, activated or migratory, and cytotoxic effector or exhausted transcriptional programs across tdTomato subsets over time. Panel K shows RNA velocity streamlines projected onto UMAP plots on days 11 and 19, highlighting predicted trajectories between single-positive tdTomato positive subsets, including tumor-infiltrating LAG3 negative tdTomato positive cells and peripheral CD44 positive tdTomato positive cells.
Temporal and spatial cues shape the fate of tdT + cells. (A) scRNAseq analysis of tdT subsets in the tumor and periphery on d11 and d19 following i.d. injection of Lag3iCreERT2Rosa26LSL-tdT mice with 1.25 × 105 B16-F10 melanoma cells. (B) TCR clonal expansion of indicated CD8+ T cell subsets at d11 and d19, showing proportions of highly expanded (≥10 copies) versus lowly expanded (<10 copies) clones. (C) Circos plots showing the distribution of highly expanded clones and PD1 expression across tumor-infiltrating and peripheral CD8+ T cell subsets at d11 (left) and d19 (right), highlighting clones that are unique to individual subsets versus those shared across multiple subsets. (D) Schematic overview of the adoptive transfer strategy. LAG3+tdT+ CD8+ T cells were isolated from d11 B16-F10 tumors of Lag3iCreERT2Rosa26LSL-tdT (Thy1.2+ CD45.2+) mice and intratumorally transferred 3–4 × 103 cells into CD45.1+ recipient mice bearing established d7 B16-F10 tumors. Recipient tumors and DLNs were harvested at d12 for flow cytometric analysis. (E) Representative flow cytometry plot showing the gating strategy for identification and sorting of LAG3+tdT+ CD8+ T cells from d11 tumors of Lag3iCreERT2Rosa26LSL-tdT mice. (F) Representative flow cytometry plot confirming the purity of the post-sort LAG3+tdT+ CD8+ T cell population. (G) Summary bar graph showing the frequencies of LAG3+tdT+ and LAG3−tdT+ CD8+ T cells, quantified from the individual tumors shown in H. (H) Representative flow cytometry plots of six individual recipient tumors at d12 following intratumoral adoptive transfer. Results are from three independent experiments. (I) Distribution of transcriptional clusters within each tdT subset at d11 (top) and d19 (bottom). Hierarchical clustering reflects relationships among subsets based on cluster composition ratio. (J) Cell ratio shifting on temporal dynamics of naive/stem-like memory, activated/migratory, and cytotoxic effector/exhausted programs across tdT subsets. (K) RNA velocity streamlines projected onto UMAP at d11 and d19, showing predicted trajectories between single-positive tdT+ subsets, with tumor-infiltrating LAG3-tdT+ (red) and peripheral CD44+tdT+ (pink) cells highlighted. Data in B and C and I–K are representative of two independent experiments. Error bars represent mean ± SEM.