Transcriptional mechanisms governing TOX expression in exhausted CD8+ T cells 3245

Abstract Description CD8+ T cells can become exhausted during prolonged antigen exposure, such as in chronic infections and cancer. Exhausted T cells show unique transcriptional, epigenetic, and metabolic profiles that differ from typical effector or memory T cells. We previously showed that nuclear factor of activated T cells (NFAT), independent of AP-1, initiates a transcriptional program of exhaustion, in part by upregulating key drivers like TOX and NR4A. However, the transcriptional mechanisms that control these drivers still need to be better defined. In this study, we show that intermittent ionomycin stimulation of CD8+ T cells, mimicking the Ca2+ signaling from T cell receptor activation, significantly increases TOX mRNA levels, akin to those in tumor-infiltrating lymphocytes. Using H3K27 acetylation patterns and the production of bidirectional short RNA transcripts, we identified two active enhancers within the first intron of the TOX gene that overlap known NFAT binding sites. NFAT protein deletion in CD8+ T cells prevents chromatin opening at these enhancers, resulting in significantly lower TOX levels. CRISPR-Cas9 editing of these enhancers also reduces TOX induction following ionomycin stimulation, underscoring a regulatory mechanism for TOX expression. These insights into TOX regulation in CD8+ T cells suggest a pathway to engineer CAR-T cells with modulated TOX levels, presenting a promising strategy for enhancing cancer immunotherapy. Funding Sources Supported by National Institutes of Health (NIH) grants AI040127 and AI109842 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)