Decoding Histone-DNA Methylation Crosstalk in Exhausted T Cells to Enhance Immunotherapy 2253691

Abstract Introduction Epigenetic scarring restricts the long-term function of exhausted CD8 T cells (TEX), impairing their ability to control chronic infections and tumors, or to respond effectively to immunotherapy. While our prior work established that de novo DNA methylation reinforces terminal exhaustion, how upstream histone modifications influence these methylation programs remains largely unknown. Defining these molecular mechanisms is essential for reversing exhaustion and enhancing the durability of T cell immunotherapies. Methods We employed a novel in vitro model of human CD8 T cell dysfunction alongside preclinical murine models of T cell exhaustion. Using integrative epigenomic approaches, we profiled H3K4 methylation states (H3K4me1/3) and DNA methylation signatures across distinct TEX subsets. To investigate functional relevance, we performed CRISPR/Cas9-gene editing, retroviral transduction, and pharmacological inhibition of histone demethylases to assess their impact on TEX functions, stemness, and response to immune checkpoint blockade (ICB). Results While distinct histone and DNA methylation landscapes defined TEX subsets in both human and murine models, H3K4me1/3–histone marks that inhibit Dnmt3a-mediated DNA methylation–were enriched at effector/memory-associated genes in cytolytic/progenitor TEX but diminished in terminally exhausted cells. Genetic or therapeutic inhibition of specific H3K4 demethylases (KDM5A/B) improved effector function and cytotoxicity in dysfunctional human CD8 T cells. In vivo, KDM5A/B targeting enhanced TEX fitness and responsiveness to anti-PD-L1 therapy during chronic viral infection and cancer. Conclusion Our findings uncover a central histone—DNA methylation circuit, regulated by KDM5A/B and DNMT3A, that drives epigenetic scarring and terminal exhaustion in CD8 T cells. Therapeutic targeting of this circuit offers a novel approach to epigenetically reprogram TEX cells and enhance the efficacy of cancer immunotherapy. Funding Source R01AI170926 (NIH, NIAID) Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)