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Although tumor-infiltrating lymphocytes (TILs) maintain their ability to proliferate, persist, and eradicate tumors, they are frequently dysfunctional in situ. By performing both whole-genome CRISPR and metabolic inhibitor screens, we identify that nicotinamide phosphoribosyltransferase (NAMPT) is required for T cell activation. NAMPT is low in TILs, and its expression is controlled by the transcriptional factor Tubby (TUB), whose activity depends on the T cell receptor-phospholipase C gamma (TCR-PLCγ) signaling axis. The intracellular level of NAD + , whose synthesis is dependent on the NAMPT-mediated salvage pathway, is also decreased in TILs. Liquid chromatography-mass spectrometry (LC-MS) and isotopic labeling studies confirm that NAD + depletion led to suppressed glycolysis, disrupted mitochondrial function, and dampened ATP synthesis. Excitingly, both adoptive CAR-T and anti-PD1 immune checkpoint blockade mouse models demonstrate that NAD + supplementation enhanced the tumor-killing efficacy of T cells. Collectively, this study reveals that an impaired TCR-TUB-NAMPT-NAD + axis leads to T cell dysfunction in the tumor microenvironment, and an over-the-counter nutrient supplement of NAD + could boost T-cell-based immunotherapy., Competing Interests: Declaration of interests Authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Clinical evidence suggests that poor persistence of chimeric antigen receptor-T cells (CAR-T) in patients limits therapeutic efficacy. Here, we designed a CAR with recyclable capability to promote in vivo persistence and to sustain antitumor activity. We showed that the engagement of tumor antigens induced rapid ubiquitination of CARs, causing CAR downmodulation followed by lysosomal degradation. Blocking CAR ubiquitination by mutating all lysines in the CAR cytoplasmic domain (CAR KR ) markedly repressed CAR downmodulation by inhibiting lysosomal degradation while enhancing recycling of internalized CARs back to the cell surface. Upon encountering tumor antigens, CAR KR -T cells ameliorated the loss of surface CARs, which promoted their long-term killing capacity. Moreover, CAR KR -T cells containing 4-1BB signaling domains displayed elevated endosomal 4-1BB signaling that enhanced oxidative phosphorylation and promoted memory T cell differentiation, leading to superior persistence in vivo. Collectively, our study provides a straightforward strategy to optimize CAR-T antitumor efficacy by redirecting CAR trafficking., Competing Interests: Declaration of Interests A patent application has been submitted based on results presented in this manuscript. H. Wang, P.W., W.L., and S.Q. are listed as the inventors. The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)