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Abstract
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.
Hyperthermia-exposed CAR T-cells exhibit attenuated anti-leukemic cytotoxicity(A) Schema of the experimental strategy used to expose CAR T-cells to hyperthermia. Human peripheral T cells were transduced with a CD19-41BB-CAR lentiviral construct and the resulting CAR T-cells were stimulated (S) by co-culture with CD19+ NALM6 leukemic cells (GFP+Luc+) at an effector/target ratio of 1 in physiological (37°C, CART37S) or hyperthermic (40°C, CART40S) conditions. Non-stimulated (NS) CAR T-cells (CART37NS) were maintained at 37°C as a control.(B) The cytotoxic potential of pre-stimulated CART37S and CART40S was evaluated by Incucyte technology at a 1:1 effector/target ratio at 37 °C and non-transduced (Mock) T cells and non-stimulated CART (CART37NS) were used as positive and negative controls, respectively. NALM6 cells were added at the indicated time points (arrows) and cytotoxicity is presented as a function of GFP intensity over time (n?=?3 technical replicates).
Hyperthermia-exposed CAR T-cells exhibit a dysregulated metabolic state.(A) Hyperthermia-exposed CAR T-cells were stimulated with CD19+ NALM6 cells as shown in Fig. 1A and transcriptional changes were evaluated by RNASeq. Gene Set Enrichment Analysis (GSEA) plots of significantly altered metabolic pathways in CART40S relative to CART37S are presented (n = 3 biological replicates).(B) The metabolomes of CAR T-cells pre-exposed to 37°C (CART37S, blue) and 40°C (CART40S, red) were evaluated by mass spectrometry following a 48h stimulation by NALM6 cells at 37°C as shown in Fig. 1A. The abundance of urea cycle metabolites in CART37S and CART40S are presented (n = 4 biological replicates, statistical difference assessed by a two-tailed unpaired Student’s t-test).
Short-term arginine supplementation enhances CAR T-cell function under stress conditions.(A) Human peripheral T cells were transduced with CD19-41BB-CAR lentivirus and expanded from days 4-8 in either control media (blue) or in arginine-supplemented media (10mM, red). CD19CAR T-cells were then harvested and ex-vivo cytotoxicity against NALM6 was evaluated in stress conditions, at a 1:10 effector/target (E: T) ratio in control media. Cytotoxicity was evaluated as a function of GFP intensity and NALM6 cells were added every 48h as indicated (arrows). Non-transduced (Mock) T cells, expanded in control (dark blue) or arginine-supplemented (dark red) conditions, were used as controls.(B) To evaluate in-vivo cytotoxicity of CD19CAR T-cells under stress conditions, suboptimal numbers of CAR T-cells (0.5x106), prepared as in panel A, were injected into NALM6 (GFP+Luc+)-bearing NSG mice at day 3 following engraftment. Tumor growth was monitored by bioluminescent imaging (BLI) using the Xenogen IVIS Lumina at the indicated time points and data were quantified by radiance (photons/s/cm2/sr).