(A) Schematic of experimental design. (B) Principal component analysis of approximately 70,000 differentially methylated cytosines (DMCs) identified from a generalized linear model and ANOVA-like testing with FDR q value < 0.05. Ellipses represent normal contour lines with 1 standard deviation probability. n = 3 mice/group (young — naive, aged — naive, young — influenza, and aged — influenza). (C) Venn diagram partitioning into differentially expressed genes or DEGs (yellow, 1,813 genes), gene promoters containing DMCs (dark orange, 5,535 genes), and genes that are both DEGs and have gene promoters containing DMCs (light orange intersection, 1,319 genes). Promoters were defined as 1 kb surrounding the transcription start site. A hypergeometric P value is shown. K-means clustering of 1,319 genes with an FDR q value < 0.05. Fold change–fold change plot for young Treg cell response to influenza versus aged Treg cell response to influenza infection highlighting methylation-regulated DEGs. GSEA results showing the top 5 positively enriched gene sets with an FDR q value < 0.25. Genes were ordered by log₂(fold change) and ranked by the young Treg cell phenotype. n = 5 mice/group (young — naive, aged — naive, young — influenza, and aged — influenza).