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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.)

Dysfunctional T cells in the tumour microenvironment have abnormally high expression of PD-1 and antibody inhibitors against PD-1 or its ligand (PD-L1) have become commonly used drugs to treat various types of cancer 1-4 . The clinical success of these inhibitors highlights the need to study the mechanisms by which PD-1 is regulated. Here we report a mechanism of PD-1 degradation and the importance of this mechanism in anti-tumour immunity in preclinical models. We show that surface PD-1 undergoes internalization, subsequent ubiquitination and proteasome degradation in activated T cells. FBXO38 is an E3 ligase of PD-1 that mediates Lys48-linked poly-ubiquitination and subsequent proteasome degradation. Conditional knockout of Fbxo38 in T cells did not affect T cell receptor and CD28 signalling, but led to faster tumour progression in mice owing to higher levels of PD-1 in tumour-infiltrating T cells. Anti-PD-1 therapy normalized the effect of FBXO38 deficiency on tumour growth in mice, which suggests that PD-1 is the primary target of FBXO38 in T cells. In human tumour tissues and a mouse cancer model, transcriptional levels of FBXO38 and Fbxo38, respectively, were downregulated in tumour-infiltrating T cells. However, IL-2 therapy rescued Fbxo38 transcription and therefore downregulated PD-1 levels in PD-1 + T cells in mice. These data indicate that FBXO38 regulates PD-1 expression and highlight an alternative method to block the PD-1 pathway.

CD28 provides an essential costimulatory signal for T cell activation, and its function is critical in antitumor immunity. However, the molecular mechanism of CD28 transmembrane signaling remains elusive. Here we show that the conformation and signaling of CD28 are regulated by two counteractive charged factors, acidic phospholipids and Ca 2+ ions. NMR spectroscopy analyses showed that acidic phospholipids can sequester CD28 signaling motifs within the membrane, thereby limiting CD28 basal signaling. T cell receptor (TCR) activation induced an increase in the local Ca 2+ concentration around CD28, and Ca 2+ directly disrupted CD28-lipid interaction, leading to opening and signaling of CD28. We observed that the TCR, Ca 2+ , and CD28 together form a dual-positive-feedback circuit that substantially amplifies T cell signaling and thus increases antigen sensitivity. This work unravels a new regulatory mechanism for CD28 signaling and thus contributes to the understanding of the dependence of costimulation signaling on TCR signaling and the high sensitivity of T cells.