Tonic signaling of chimeric antigen receptor (CAR), i.e., the spontaneous CAR activation in the absence of tumor antigen stimulation, is considered to be a pivotal event controlling CAR-T efficacy. However, the molecular mechanism underlying the spontaneous CAR signals remains elusive. Here, we unveil that positively charged patches (PCPs) on the surface of the CAR antigen-binding domain mediate CAR clustering and result in CAR tonic signaling. For CARs with high tonic signaling (e.g., GD2.CAR and CSPG4.CAR), reducing PCPs on CARs or boosting ionic strength in the culture medium during ex vivo CAR-T cell expansion minimizes spontaneous CAR activation and alleviates CAR-T cell exhaustion. In contrast, introducing PCPs into the CAR with weak tonic signaling, such as CD19.CAR, results in improved in vivo persistence and superior antitumor function. These results demonstrate that CAR tonic signaling is induced and maintained by PCP-mediated CAR clustering. Notably, the mutations we generated to alter the PCPs maintain the antigen-binding affinity and specificity of the CAR. Therefore, our findings suggest that the rational tuning of PCPs to optimize tonic signaling and in vivo fitness of CAR-T cells is a promising design strategy for the next-generation CAR.

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Insulin resistance is associated with a greatly increased risk of type 2 diabetes. Administration of fibroblast growth factor-1 (FGF-1) resulted in a marked improvement in insulin sensitivity. However, the underlying molecular mechanism whereby FGF-1 represses insulin resistance remains largely unknown. Here, we sought to delineate the role of FGF-1 in insulin resistance with respect to its anti-inflammatory capability. In this study, we found that FGF-1 had positive effects on glucose intolerance, hepatic lipid accumulation, and insulin resistance, while it markedly repressed cytokine secretion (TNF-α and IL-6) in serum and reduced liver inflammation in diet-induced obesity (DIO) mice. Further, FGF-1 treatment significantly represses TNF-α-induced insulin resistance in vitro and in vivo. These results indicate that FGF-1 likely ameliorates insulin resistance via a mechanism that is independent of its glucose-lowering activity. Subsequent experiments demonstrated that FGF-1 ameliorated insulin resistance, and inflammation was accompanied by decreased c-Jun N-terminal kinase (JNK) signaling. In addition, it is likely that FGF-1 impedes JNK phosphorylation via blocking the transforming growth factor-β activated kinase 1 (TAK1) and TAK1 binding protein 1 (TAB1) interaction. These findings reveal that FGF-1 regulates insulin sensitivity and may represent an attractive therapeutic target for preventing the development of insulin resistance.

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