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

CD8+ 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 TEX progenitors towards terminally differentiated and effector (TEFF)-like TEX. In mouse T-cells, TET2 -deficient terminally differentiated TEX retained aspects of TEX progenitor 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 TEFF and 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 for TET2- targeted CAR T-cells, using CRISPR/Cas9 editing and site-specific adeno-associated virus transduction to simultaneously knock-in a CAR at the TRAC locus and a functional safety switch within TET2 . Disruption of TET2 with this safety switch in CAR T-cells restrained terminal TEX differentiation in vitro and enhanced anti-tumor responses in vivo . Thus, TET2 regulates pivotal fate transitions in TEX differentiation and can be targeted with a safety mechanism in CAR T-cells for improved tumor control and risk mitigation. One Sentence Summary Modulation of exhausted CD8+ T-cell differentiation by targeting TET2 improves therapeutic potential of CAR T-cells in cancer. ### Competing Interest Statement R.O. has contributed to patents licensed to Novartis in Biomedical Research and possesses equity interests in Nucleus Biologics and Stoic Bio, additionally serving as a scientific advisor to Nucleus Biologics. J.J.M. discloses receiving fees from IASO Biotherapeutics and Poseida Therapeutics, as well as Kite Pharma, unrelated to this work. He holds patents related to enhancing immune cell efficacy and predicting chimeric antigen responsiveness, issued to Novartis. S.L.M. has obtained clinical trial support and advisory roles with Novartis and Wugen and holds a Novartis-pending patent. S.R.R. and D.L.P. have disclosed involvement with various pharmaceutical companies and hold positions that include receiving research funding, holding equity, and serving in advisory capacities. D.L.P. also benefits from patents and royalties with Tmunity and Wiley and Sons Publishing. B.L.L. maintains consultancy and advisory positions with several biotech companies, including Terumo and GSK, and has equity in Tmunity Therapeutics and Capstan Therapeutics. S.S-M. and N.V.F. are engaged with Sana Biotechnology. N.V.F. has consultancy roles with Novartis and Syndax Pharmaceuticals, besides obtaining funding from Kite Pharma. S.G. has disclosed receiving support and serving in advisory capacities for multiple entities within the pharmaceutical sector, including Novartis and Servier. M.H.P. is involved with Graphite Bio and Allogene on their Board of Directors and Scientific Advisory Board, respectively, and is an advisor to Versant Ventures, holding equity in CRISPR Tx and Kamau Therapeutics. E.W.W. is a consultant to and holds equity in Lyell Immunopharma and consults for Umoja Immunopharma. E.J.W. is an advisor for Arsenal Biosciences, Coherus, Danger Bio, IpiNovyx, New Limit, Marengo, Pluto Immunotherapeutics, Prox Bio, Related Sciences, Santa Ana Bio, and Synthekine. E.J.W. is a founder of and/or holds stock in Coherus, Danger Bio, Prox Bio and Arsenal Bioscience. J.A.F. has patents and holds intellectual property in T-cell-based cancer immunotherapy, from which he has received royalties. He also receives funding from Tmunity Therapeutics and Danaher Corporation, consults for Retro Biosciences, and is a member of the Scientific Advisory Boards for Cartography Bio, Shennon Biotechnologies Inc., CellFe Biotech, OverT Bio, Inc., and Tceleron Therapeutics, Inc. The remaining authors declare no competing interests.