ASCT2 overexpression enhances antihuman BCMA-CAR T-cell activity. (A) Schematic diagrams of lentiviral vectors used to overexpress ASCT2 in human BCMA-CAR T cells. (B) Expression levels of the CAR construct, measured by BFP fluorescence. (C) Human ASCT2 expression detected on CAR T cells by flow cytometry. (D) Proliferation of CAR T cells in response to BCMA-coated plate stimulation at different Gln concentrations, measured by [³H]-thymidine incorporation. (E) Phenotypic analysis of CD4⁺ and CD8⁺ CAR T cells at the time of manufacture. (F) Asct2 silencing in H929 MM cells using 3 different single-guide RNAs (TadCBEd). (G) Measurement of [³H]-Gln uptake in WT and ASCT2-silenced H929 MM cells. (H) Cytotoxic activity of BCMA-CAR T cells against WT and ASCT2-silenced H929 MM cells. Data are representative of 2 independent experiments. Data represent mean ± SEM and were analyzed using 2-way ANOVA with Bonferroni multiple comparisons test (∗∗∗P < .001). BFP, blue fluorescent protein; cpm, counts per million; KO, knockout; neg, negative; SSC-A, side scatter area; T_CM, T central memory; T_EM, T effector memory; T_EMRA, T effector memory recently reactivated; T_SCM, T stem cell memory; WT, wild-type.