Fig. 1: Epigenetic profiles of physiologically aged and ML functional memory T cells.

a, Schema for generating ML memory CD8⁺ T cells. Naive mice were infected with VSV generating antigen-specific memory CD8⁺ T cells. Heterologous VSV infection and boosting of the memory T cells was performed after 30 versus 60 days post previous infection. After three infections, the antigen-specific memory CD8⁺ T cells were transferred into congenically distinct naive mice, and the boosting strategy was repeated over a time frame that yielded memory T cells with age ranging between 0.5 and ~4 mouse lifetime (LT) equivalents. For young memory, n = 6; 0.5× LT, n = 3; 2–3× LT, n = 5; and 4× LT, n = 3. b, Representative FACS analysis of cell trace violet (CTV) dilution from 3° (CD45.2) and 51° (CD45.1) memory T cells transferred into recipient CD45.1/2 mice. CTV label was measured 10 days after VSV-Indiana challenge of the chimeric mice. c, PCA plot of 3,000 most variable CpG sites comparing ML and ‘young’ memory T cells. Plot showing principal components 1 and 2 (PC1 and PC2). d, Violin plots showing average genome-wide CpG methylation for naive, young memory and ML CD8⁺ T cells. The red triangles represent the median values. e, Heatmap showing top ML gain of methylation DMRs. f, GSEA using cell cycle regulators and gain versus loss of methylation DMRs for 4× LT memory CD8⁺ T cells relative to young memory CD8⁺ T cells. The P value is based on the weighted Kolmogorov–Smirnov statistic with no adjustment. g, Genomic and CpG island annotation of top loss and gain of methylation ML DMRs. h, Summary graph showing mean methylation across top ML gain of methylation DMRs relative to boosting and age of memory T cells. Different lengths of resting period also result in significant changes of mean methylation levels. The P value is based on the two-sided Student’s t-test.