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

Abstract

TGF-{beta} signaling is a major regulator of immune cell differentiation and function, yet whether prolonged signaling can durably imprint dysfunctional immune states independently of continued pathway engagement remains unclear. Here, we identify signal duration as a critical determinant of human natural killer (NK) cell fate: short-term TGF-{beta} exposure induces largely reversible transcriptional, chromatin and functional changes, whereas prolonged exposure establishes persistent effector dysfunction independent of continued signaling. Mechanistically, prolonged TGF-{beta} drives durable loss of chromatin accessibility at effector-associated regulatory elements, particularly those enriched for IRF, EOMES, and T-bet binding motifs, resulting in stable restriction of NK cell effector programs despite reversibility of active histone marks. In contrast, tissue residency-associated programs remain largely reversible, revealing that distinct NK cell fate programs differ fundamentally in their susceptibility to epigenetic fixation downstream of the same cytokine signal. SMAD4 CUT&RUN and knockout experiments further demonstrate that canonical TGF-{beta} signaling acts primarily through rewiring of upstream transcriptional regulatory networks rather than direct targeting of effector loci. Finally, NK cells from patients with hepatocellular carcinoma recapitulate key functional and epigenetic features of the persistent TGF-{beta}-associated state defined in vitro. Together, these findings identify signal duration as a critical determinant of TGF-{beta}-driven cell fate and demonstrate that prolonged TGF-{beta} exposure can induce epigenetically stabilized dysfunctional states that persist after signal withdrawal, with important implications for therapeutic strategies aimed at reversing chronic TGF-{beta}-mediated dysfunction.