TET2 regulates early and late transitions in exhausted CD8+T-cell differentiation and limits CAR T-cell function

Abstract

AbstractCD8+T-cell exhaustion hampers disease control in cancer and chronic infections and limits efficacy of T-cell−based therapies, such as CAR T-cells. Epigenetic reprogramming of CAR T-cells by targeting TET2, a methylcytosine dioxygenase that mediates active DNA demethylation, has shown therapeutic potential; however, the role of TET2 in exhausted T-cell (TEX) development is unclear. In CAR T-cell exhaustion models and chronic LCMV infection, TET2 drove the conversion from stem cell-like, self-renewing TEXprogenitors towards terminally differentiated and effector (TEFF)-like TEX. In mouse T-cells,TET2-deficient terminally differentiated TEXretained aspects of TEXprogenitor biology, alongside decreased expression of the transcription factor TOX, suggesting that TET2 potentiates terminal exhaustion. TET2 also enforced a TEFF-like terminally differentiated CD8+T-cell state in the early bifurcation between TEFFand TEX, indicating a broad role for TET2 in mediating the acquisition of an effector biology program that could be exploited therapeutically. Finally, we developed a clinically actionable strategy forTET2-targeted CAR T-cells, using CRISPR/Cas9 editing and site-specific adeno-associated virus transduction to simultaneously knock-in a CAR at theTRAClocus and a functional safety switch withinTET2. Disruption ofTET2with this safety switch in CAR T-cells restrained terminal TEXdifferentiationin vitroand enhanced anti-tumor responsesin vivo. Thus, TET2 regulates pivotal fate transitions in TEXdifferentiation and can be targeted with a safety mechanism in CAR T-cells for improved tumor control and risk mitigation.One Sentence SummaryModulation of exhausted CD8+T-cell differentiation by targeting TET2 improves therapeutic potential of CAR T-cells in cancer.