Metabolic profiling of CAR T-cells in patients reveals a shift toward amino acid-supported OXPHOS and informs transporter engineering.

CAR T-cell efficacy requires post-infusion expansion and persistence, yet metabolic programs supporting T-cells in patients remain poorly defined. Here, we developed a high-throughput single-cell immunometabolic profiling pipeline and applied it across pediatric leukemia trials, revealing a conserved post-infusion CAR T-cell shift from glycolysis toward amino acid-driven oxidative phosphorylation (OXPHOS). Within this remodeling, OXPHOS-dependent stem-like CAR T-cell subsets were enriched in patients achieving complete remission. Longitudinal plasma metabolomics revealed cytokine release syndrome-associated depletion of multiple amino acids, including glutamine and arginine, during CAR T-cell expansion, creating a nutrient-restricted environment. Analysis of public CAR T-cell datasets showed that responders upregulated amino-acid solute carrier transporters, whereas disrupting uptake impaired translation, OXPHOS, stemness, and cytotoxicity. Guided by these findings, we screened amino-acid transporters for CAR T-cell engineering. SLC1A5-, SLC7A1-, and SLC38A9-armored CAR T-cells emerged as the most promising, with enhanced oxidative capacity and anti-leukemic efficacy, establishing amino acid transport as a targetable metabolic checkpoint.