Panel A shows a schematic diagram of the experimental design in which Lag3iCreERT2 Rosa26LSL-tdTomato mice receive 1.25 times 10 superscript 5 B16-F10 cells by intradermal injection, followed by tamoxifen treatment on days 8 to 10 and tissue collection on day 19. Panel B shows a correlation matrix of tdTomato positive CD8 positive T-cell subsets isolated from non-draining lymph nodes, draining lymph nodes, and tumors based on Pearson correlation analysis. The ellipses represent correlation values, with elongated ellipses indicating correlations approaching 1 and circles indicating correlations closer to 0, while the color scale indicates correlation strength from low to high. Panel C shows a Venn diagram illustrating the overlap of highly variable chromatin accessibility peaks among tdTomato positive CD8 positive T-cell subsets. Panel D shows aggregate ATAC-seq accessibility profiles and heatmaps centered on transcription start sites (TSSs) across the indicated cell subsets. Panel E shows a heatmap of representative transcription factors associated with exhaustion, effector, and memory programs, with quantile-normalized expression scores across the indicated subsets. Panel F shows a joint principal component analysis (PCA) plot of ATAC-seq data from tdTomato positive CD8 positive T-cell subsets and reference naive, effector, memory, and exhausted differentiation states. The tdTomato positive populations include LAG3 negative tdTomato positive cells from non-draining lymph nodes, LAG3 negative tdTomato positive cells from draining lymph nodes, intratumoral LAG3 negative tdTomato positive cells, and intratumoral LAG3 positive tdTomato positive cells, while reference states are shown in shades of blue and grey. Red dashed lines connect each population to its closest reference state with the corresponding Euclidean distance values. Panel G shows stacked bar graphs representing the percentages of naive (CD44 negative CD62L positive), TCM (CD44 positive CD62L positive), TEFF (CD44 positive CD62L negative CD127 negative KLRG1 positive), MPECs (CD44 positive CD62L negative CD127 positive KLRG1 negative), SLECs (CD44 positive CD62L negative CD127 negative KLRG1 positive), and other immune cell subsets in tumors and draining lymph nodes across the indicated tdTomato subsets.
Epigenetic and functional divergence of intratumoral and peripheral tdT + cells. (A) Experimental strategy for analysis of chromatin accessibility between different tdT CD8+ T cell subsets. B16-F10 tumor implanted in Lag3iCreERT2Rosa26LSL-tdT mice, treated with three tamoxifen injections (2 mg in 5% EtOH/sunflower oil) at d8–10, and assessed for chromatin accessibility on d19. Lymphocytes were isolated from NDLNs and DLNs, and intratumoral LAG3−tdT−, LAG3+tdT+, and LAG3−tdT+ CD8+ T cell subsets were flow-sorted and subjected to ATACseq. (B) Pairwise correlations among tdT+ CD8+ T cell subsets isolated from NDLNs, DLNs, and tumors, determined by Pearson correlation analysis. Shapes represent correlation values, as indicated by the corresponding numbers in the all-by-all matrix. Correlations approaching 1 are depicted as elongated lines, whereas correlations closer to zero resemble circles. The color bar indicates correlation values, with red indicating positive correlation. (C) Venn diagram showing overlap of high variable peaks among tdT+ CD8+ T cell subsets. (D) Heatmaps with aggregate profiles of chromatin accessibility centered on TSSs across indicated subsets. (E) Heatmap of representative transcription factors associated with exhaustion, effector, and memory programs, showing quantile-normalized expression scores across subsets. (F) Joint PCA of ATACseq data from tdT+ CD8+ T cell subsets and reference differentiation states (naive, effector, memory, and exhausted). Different tdT+ populations include LAG3−tdT+ of NDLNs (pink), LAG3−tdT+ of DLNs (coral), intratumoral LAG3−tdT+ (red), and intratumoral LAG3+tdT+ (purple). Reference states are shown in shades of blue/grey. Red lines indicate the closest reference state with Euclidean distance values. (G) Phylum plot represents the percentage of Tnaive (green), TCM (blue), TEFF (yellow), MPECs (magenta), SLECs (red), and other (dark blue) immune cell subsets in both tumor and DLNs across different tdT subsets. n = 5 mice, pooled from two independent experiments. Data in B–F are representative of two independent experiments. ***P < 0.001 by Pearson correlation coefficient (B). Error bars represent mean ± SEM.