Fig 1: IL-7Rhi CD8+ cells have a distinct poised epigenetic landscape regulated by DNA methylation that imparts superior antitumor immunity. (A) Methylation-specific PCR targeting CpG loci in Tcf7 promoter region in nonmethylated DNA standard (Ctrl, leftmost lane), IL-7Rlo and IL-7Rhi CD8+ T cells from tumor-draining lymph nodes of functional memory mice. PCR amplification of Tcf7 locus in IL-7Rlo and IL-7Rhi genomic DNA (loading control, regular primers). (B) Relative quantification of Tcf7 methylation IL-7Rlo and IL-7Rhi CD8+ T cells (C) Relative quantification of genomic DNA/loading control in the IL-7Rlo and IL-7Rhi conditions. (D and E) Quantification of Il7r (D) and Tcf7 (E) expression by RT-PCR in murine T cells upon treatment with vehicle control (black) or hypomethylating agent RG-108 (red). (F) Visualization of chromatin accessibility at or near transcriptional start sites (TSS) of indicated genes. Peaks from exhausted phenotype shown in black, functional memory shown in red. See also SI Appendix, Fig. S4A. (G) Diagram of tumor rechallenge experiment in functional memory mice shown in H and I. (H) Bar graphs showing comparison of IFN-g, IL-2, IL-12, and IL-1ß cytokine levels in functional memory (red) and tumor rechallenged conditions (purple). See also SI Appendix, Fig. S4 B and C. (I) Quantification of IFN-g, TNF-a and IL-2 production upon stimulation of IL-7Rlo (black) and IL-7Rhi (red) CD8+ T cells from tumor-draining lymph nodes of functional memory mice. (J) Kaplan–Meier survival curves (J, Left) for C57BL/6 mice grafted with melanoma cells after receiving untreated IL-7Rlo (dark blue) population, untreated IL-7Rhi population (crimson), RG-108-treated IL-7Rlo population (light blue), or RG-108-treated IL-7Rhi population (red) of CD8 T cells from tdLN of functional memory mice by adoptive transfer (red). Pie charts (J, Right) showing percent survival in these same groups. (K) Scatter plot of TCF7 methylation (beta value) and gene expression (RSEM log2) in cohort of human melanoma samples (72). Each dot represents an individual sample (Pearson’s r = 0.67, P = 2.51 × 10−62). See also SI Appendix, Fig. S4D. (L) Bar graph of Tcf7 promoter methylation in cohort of human melanoma samples stratified into an IL-7Rhi (red) and IL-7Rlow (black) group. (M) Heatmap showing DNA methylation of IL7Rhi-defining genes in a large cohort of melanoma patients stratified by IL-7R expression. Hypomethylation shown in blue, hypermethylation shown in red. Student’s t test, Log-rank or ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001. Bar graphs shown as mean +/− SEM, based on three biological replicates. Violin plots show the median (dashed line) and quartiles (dotted lines). Cytokines measured by ELISA using recombinant protein standard curve. Cell count based on luminescence of CellTiterGLO v2 assay. Results based on three biological replicates.