Harnessing arginine metabolism overcomes hyperthermia-induced metabolic dysfunction of CAR T-cells

Abstract Chimeric antigen receptor (CAR) T-cells have shown remarkable success in the treatment of hematological malignancies, but many patients still relapse. One common adverse on-target effect of CAR T-cells is cytokine release syndrome. Importantly though, the impact of fever, a hallmark of this syndrome, on CAR T-cell function is not known. We find that exposure of CAR T-cells to hyperthermia (40°C) significantly decreased their subsequent cytotoxicity against NALM6 leukemic cells, both ex-vivo and in-vivo. This was associated with reduced secretion of IL-2, IFNg, and IL-8 by CAR T-cells and high dimensional analyses revealed the induction of a terminally differentiated T cell phenotype. Gene profiling assays highlighted a negative enrichment of mTORC1, glycolysis, and oxidative phosphorylation gene sets in CAR T-cells subjected to hyperthermia and metabolome analyses unveiled significant reductions in urea cycle metabolites. Notably, pharmacological supplementation of arginine markedly enhanced the ex-vivo and in-vivo cytotoxicity of hyperthermia-exposed CAR T-cells. Moreover, in the absence of hyperthermia, short-term arginine supplementation enhanced the metabolic fitness of CAR T-cells, augmenting their long-term in-vivo persistence and cytotoxicity. We identify arginine metabolism as a critical pathway in CAR T-cells rendered dysfunctional by exposure to hyperthermia and pharmacological arginine support will inform future iterations of CAR T-cell interventions.