CD8+ T cell differentiation into activated/cytotoxic, exhausted (TEX) and tissue-resident memory (TRM) phenotypes is a major determinant of successful cancer immunotherapy, which relies on the ability of CD8+ T cells to detect and eliminate cancer cells. Understanding the epigenetic profile of each of these states can help determine the major epigenetic regulators that lead to exhaustion and tissue residency, paving the way for improved T cell-based immunotherapies.

An in vitro human CD8+ T cell stimulation assay using healthy donor cells was utilized to generate different T-cell phenotypes. Naïve T cells underwent CD3/CD28, TCR-independent stimulation for 9 days to promote an activated/cytotoxic (3 days) and a TEX phenotype (9 days). TGF-β exposure (days 0-9) occurred to induce a TRM phenotype. Flow cytometry with 15 cell-surface markers aimed to validate the induction of T cell states.  Genome-wide mapping for activating (H3K4me1, H3K4me3, H3K27Ac) and repressive (H3K9me3, H3K27me3, H4K20me3) histone marks utilizing CUT&Tag was conducted, together with RNA-seq. 

Flow cytometry supports the successful induction of activated, TEX and TRM phenotypes in three healthy human donors. Feasibility of conducting genome-wide mapping for activating and repressive histone marks using CUT&Tag on unstimulated CD8+ T cells has also been confirmed. CUT&Tag-generated DNA libraries from T cells derived from three healthy human donors and stimulated via our in vitro protocol are being sequenced and analyzed, along with correlations with RNA-seq data.