Background 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 (CRS), due to the triggering of a systemic inflammatory response. Importantly though, the impact of CRS, and especially the fever that is a hallmark of this syndrome, on CAR T-cell function is not known.
Methods T cells were generated to express an anti-CD19 (FMC63) CAR construct harboring the 4-1BB costimulatory domain. CD19CAR T-cells were exposed to hyperthermia (40°C) and their cytotoxic activity against CD19+ NALM6 leukemic cells was evaluated in both ex-vivo (Incucyte) and in-vivo (NSG mouse) models. Hyperthermia-induced changes in CAR T-cells were evaluated by cytokine secretion profiles, CyTOF, RNASeq, and metabolomic analyses. Arginine supplementation was performed as described.
Results Exposure of CD19CAR T-cells to hyperthermia significantly decreased their subsequent cytotoxicity against CD19+ leukemic cells at 37°C, both ex-vivo and in-vivo (figure 1). This was associated with reduced secretion of IL-2, IFNg, and IL-8 by CD19CAR T-cells and high dimensional analyses revealed the induction of a terminally differentiated CD25HiCD39HiCD44HiTIM3Hi CAR T cell. Mechanistically, gene profiling assays highlighted a negative enrichment of mTORC1, glycolysis, and oxidative phosphorylation gene sets in CAR T-cells subjected to hyperthermia (figure 2A), and these data were confirmed by functional metabolic assays. Furthermore, the metabolome of hyperthermia-exposed CAR T-cells unveiled significant reductions in arginine and urea cycle metabolites (figure 2B). 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 to CAR T-cells during the expansion process enhanced metabolic fitness, promoting the potential of these CAR T-cells to respond to repetitive ex-vivo NALM6 stimulations (E/T=0.1) and enhancing in-vivo anti-leukemic activity under stress conditions (figure 3).
Conclusions Exposure of CAR T-cells to hyperthermia results in a metabolic reprogramming associated with attenuated cytotoxic function and the induction of a terminally differentiated state. We identify arginine metabolism as a critical pathway in CAR T-cells rendered dysfunctional by exposure to hyperthermia. On the basis of these data, we assessed the impact of short-term arginine supplementation on long-term CAR T-cell function, and our data highlight a significantly augmented CAR T persistence and cytotoxicity in stress conditions. Pharmacological arginine support will inform future iterations of CAR T-cell interventions.
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