Distinct epigenetic features of tumor-reactive CD8+ T cells in colorectal cancer patients revealed by genome-wide DNA methylation analysis

Tumor-reactive CD8+ tumor infiltrating lymphocytes (TILs) represent a subtype of T cells that could recognize and destroy tumor specifically. Understanding the regulatory mechanism of tumor-reactive CD8+ T cells has important therapeutic implications. Yet the DNA methylation status of this T cell subtype has not been elucidated. In this study, we segregated tumor-reactive and bystander CD8+ TILs, as well as naïve and effector memory CD8+ T cell subtypes as controls from colorectal cancer (CRC) patients, to compare their transcriptome and methylome characteristics. Transcriptome profiling confirmed previous conclusion that tumor-reactive TILs have an exhausted tissue-resident memory signature. Whole-genome methylation profiling identified a distinct methylome pattern of tumor-reactive CD8+ T cells, with tumor-reactive markers (CD39 and CD103) being specifically demethylated. In addition, dynamic changes were observed during the transition of naïve T cells into tumor-reactive CD8+ T cells. Transcription factor (TF) binding motif enrichment analysis identified several immune-related TFs, including three exhaustion-related genes (NR4A1, BATF and EGR2) and VDR, that potentially play important regulatory role in tumor-reactive CD8+ T cells. Our study supports the involvement of DNA methylation in shaping tumor-reactive and bystander CD8+ TILs, and provides a valuable resource for the development of novel DNA methylation markers and future therapeutics. We used whole-genome methylome profiling and RNA-seq to investigate the characteristics of tumor-reactive and bystander CD8+ TILs, as well as naive and effector memory CD8+ T cell subsets as controls from colorectal cancer patients. Raw sequence data access provided at: China Genomic Sequence Archive (GSA) (https://bigd.big.ac.cn/gsa-human/browse/HRA000059) under accession HRA000059. ****Raw sequence data access provided at: China Genomic Sequence Archive (GSA) (https://bigd.big.ac.cn/gsa-human/browse/HRA000059) under accession HRA000059.