Regnase-1 KO CAR-T cells acquire memory-, effector-, and exhaustion-associated epigenetic programs. CD8⁺ WT and KO CAR-T cells were cotransferred 1:1 into tumor-bearing mice. CD8⁺Ly108⁺ cells were sorted from spleens 21 days later for whole-genome DNA methylation profiling. (A) Number of DMRs in Ly108⁺ WT and KO CAR–T-cell genomes relative to T_N, T_CM, effector memory T cell (T_EM), and T_EX genomes. The number of demethylated and methylated regions were calculated based on ≥30% and ≤30% methylation difference between the 2 populations, respectively. (B) Normalized CpG methylation plots at sites surrounding and within DMRs of memory-associated genes, (C) effector-associated genes, and (D) exhaustion-associated genes obtained by whole genome bisulfite sequencing (WGBS) analysis. Red and blue lines depict methylated and unmethylated CpG sites, respectively. DMRs are indicated by green boxes. Data represent 10 biological replicates pooled for triplicate analysis.