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Background  Tumor-reactive CD8+ tumor-infiltrating lymphocytes (TILs) represent a subtype of T cells that can 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.    Results  In this study, we segregate tumor-reactive and bystander CD8+ TILs, as well as naïve and effector memory CD8+ T cell subtypes as controls from colorectal cancer patients, to compare their transcriptome and methylome characteristics. Transcriptome profiling confirms previous conclusions that tumor-reactive TILs have an exhausted tissue-resident memory signature. Whole-genome methylation profiling identifies a distinct methylome pattern of tumor-reactive CD8+ T cells, with tumor-reactive markers  CD39  and  CD103  being specifically demethylated. In addition, dynamic changes are observed during the transition of naïve T cells into tumor-reactive CD8+ T cells. Transcription factor binding motif enrichment analysis identifies several immune-related transcription factors, including three exhaustion-related genes ( NR4A1 ,  BATF , and  EGR2 ) and  VDR , which potentially play an important regulatory role in tumor-reactive CD8+ T cells.    Conclusion  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.

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