Genome-wide DNA methylation analysis of senescence in repetitively infected memory cytotoxic T lymphocytes.

The immune system including antigen-specific CD8 T cells, which are cytotoxic T lymphocytes (CTLs), can acquire the potential for more effective elimination of the pathogen at re-infection. As memory CTLs could exert protective immunity after the next response, we aimed to elucidate the substantial change of repetitively infected memory CTLs. Currently, DNA methylation status in repetitively infected memory CTLs is unknown, so we performed next-generation sequencing to evaluate methylation status and transcriptional regulation of naive, primary and secondary memory CD8 T cells on the basis of transcription start sites (TSS). Notably, total CpG sites in the entire regions of all genes were significantly unmethylated in primary memory CTLs (young memory CTLs) and even more unmethylated in secondary memory CTLs (old memory CTLs). However, total proximal regions from TSS, which cover transcriptional promoters, were steadily methylated with repeated infections. In contrast, distal regions from TSS, which are the majority of entire regions and include transcriptional enhancers, were extensively unmethylated by infections. In association between transcriptional and methylation changes, accompanied by genes characteristic of the immune response, natural killer cell signature genes, known to be expressed in senescent CD8 T cells, were transcriptionally up-regulated and unmethylated in young memory CTLs, and more so in old memory CTLs, whereas ribosomal proteins were transcriptionally down-regulated and methylated in proximal region from the TSS by infections. Our results suggest that epigenetically augmented enhancers and suppressed promoters, which could consequently lead to global decline of transcription and translation, could represent the senescence of memory CTLs.