Insertion of SLC7A5 or SLC7A11 amino acid transporters upregulates arginase expression in CAR T cells. (A) The proliferation of T cells is significantly impaired by tryptophan or cystine-free culture conditions, in vitro as measured by CSFE dilution using flow cytometry after 72 hours. (B) The proliferation of anti–CD33-CAR T cells in response to CD33⁺ THP1 leukemia cells is significantly reduced by tryptophan or cystine-free conditions in vitro as measured by flow cytometry after 72 hours. Anti–CD33-CAR T cells produced from 3 individual human donors are shown. (C) Expression of SLC7A5 or SLC7A11 has no detrimental effect on the cytotoxicity of CAR T cells against target CD33⁺ THP1 in vitro. The percentage of dead THP1 cells measured by flow cytometry after 72 hours. (D) Activation–induced IFN-γ release remains unchanged by the expression of SLC7A5 or SLC7A11 after CAR T-cell culture with target CD33⁺ THP1 in vitro. IFN-γ concentration was measured by bead immunoassay in cell culture supernatants after 72 hours. (E) SLC7A5 or SLC7A11–modified anti-CD33 CAR T cells have enhanced proliferation under tryptophan- or cystine-low (75% free) culture conditions respectively, compared with unmodified CAR T cells, in vitro. CAR T-cell proliferation was measured by flow cytometry after 72 hours. (F) Schematic showing NOD-SCID mice engrafted with CD33⁺ HL60 AML. Following confirmed engraftment mice were injected with modified or control CAR T cells from 3 human donors. (G) The proliferation of unmodified or SLC7A5- or SLC7A11–modified anti-CD33 CAR T cells in NOD-SCID mice with established engraftment of CD33⁺ AML blasts (HL60). Data from the day of euthanization. CAR T-cell frequency was determined by qPCR. Pooled data from 3 human donors. (H) SLC7A5- or SLC7A11–modified anti-CD33 CAR T cells reduce the expansion of CD33⁺ HL60 in the bone marrow of murine xenografts, compared with those treated with unmodified CAR T cells. Fold change in AML on the day of euthanization from the day of CAR T administration. Pooled data from 3 human donors. (I) Heatmap of the differential gene expression analysis comparing SLC7A5 or SLC7A11–modified anti-CD33 CAR T cells with unmodified anti-CD33 CAR T cells from n = 5 human donors. Top 500 genes shown. The red arrow indicates the ARG2 gene. (J) Intracellular Arg1 enzyme expression is significantly increased in SLC7A5- or SLC7A11–modified anti-CD33 CAR T cells compared with unmodified CAR T cells cultured with CD33⁺ THP1, under R10%, tryptophan-low (75% free), or cystine-low (75% free) conditions. Intracellular ARG1 staining as measured by flow cytometry in CAR T cells from n = 3 human donors. (K) Intracellular Arg2 enzyme expression is significantly increased in SLC7A5- or SLC7A11–modified anti-CD33 CAR T cells compared with unmodified CAR T cells cultured with CD33⁺ THP1, under tryptophan-low (75% free) or cystine-low (75% free) conditions. Intracellular Arg2 staining as measured by flow cytometry in CAR T cells from n = 3 human donors. (L) Arginase enzyme activity, measured by catabolism of arginine into ornithine and urea, is increased in SLC7A5- or SLC7A11–modified anti-CD33 CAR T cells compared with unmodified CAR T cells. CAR T cells were cultured with CD33⁺ THP1 for 48 hours, before sorting and enzyme activity analysis. Data from n = 4 human donors is shown. CFSE, carboxyfluorescein succinimidyl ester; qPCR, quantitative polymerase chain reaction.