Prolonged TGF-β locks NK cells in a dysfunctional state through persistent epigenetic remodeling of IRF, T-bet and EOMES binding sites

Transforming growth factor beta (TGF-β) is a central suppressor of natural killer (NK) cell function in tissues and tumor microenvironments, yet it remains unclear whether NK cell suppression requires continuous TGF-β signaling or can become stably imprinted. Here, we dissect the outcome of TGF-β signaling on NK cell transcriptional, epigenetic, and functional programs in short-term vs. long-term stimulation. We show that short-term TGF-β exposure induces limited and largely reversible transcriptional and chromatin accessibility changes, resulting in transient impairment of NK cell effector function. In contrast, prolonged TGF-β signalling drives durable epigenetic remodelling accompanied by sustained suppression of cytotoxicity and cytokine production that persists even after signal withdrawal, indicating that NK cell dysfunction becomes independent of continued TGF-β exposure. Prolonged TGF-β exposure is associated with loss of chromatin accessibility at regulatory elements controlling NK cell effector programs. These persistently less accessible regions are enriched for binding motifs of interferon regulatory factors (IRFs) as well as the lineage-defining transcription factors EOMES and T-BET, providing a mechanistic basis for impaired cytokine responsiveness. Reduced H3K4me3 occupancy at selected effector gene promoters further supports a repressive chromatin state. In contrast to the lasting repression of effector genes, genes associated with tissue residency exhibit largely reversible transcriptional and epigenetic regulation, revealing distinct regulatory modes governing effector function versus tissue adaptation. NK cells from patients with hepatocellular carcinoma display impaired effector function and epigenetic alterations overlapping with features induced by prolonged TGF-β exposure, consistent with chronic signaling in vivo . Together, our findings reveal persistent TGF-β-driven epigenetic remodelling as a central mechanism of NK cell dysfunction in cancer and identify signal duration as a key determinant of NK cell fate, with implications for therapeutic strategies aimed at restoring NK cell function.