Pevonedistat can enhance the cytotoxicities of PD-L1 CAR T cells in ATLL cells. (A) Schematic design of the durvalumab-based anti–PD-L1 CAR construct used in the study. (B) Representative plots showing the transduction efficiency of mock-control T cells and PD-L1 CAR T cells analyzed by flow cytometry. (C) Cell surface expression of PD-L1 in KK1 cells transduced with sgAAVS1 or sgPD-L1 was measured via flow cytometry. PD-L1 knockout KK1 cells were established by single-cell cloning from bulk sgPD-L1–transduced cells. (D) Values of specific lysis of KK1 cells transduced with sgAAVS1 or sgPD-L1 under cocultivation with PD-L1 CAR T cells. Data were obtained as shown in Figure 6A. (E) Values of specific lysis of unmanipulated KK1 (wild-type), KK1 PD-L1-SV#7, and KK1 PD-L1-SV#15-12 cells under cocultivation with mock T cells and PD-L1 CAR T cells. Data were obtained as shown in Figure 6A. (F-H) Values of specific lysis of unmanipulated KK1 (wild-type) (F), KK1 PD-L1 SV#7 (F), KK1 PD-L1 SV#15-12 (F), unmanipulated ST1 (G), and unmanipulated TL-Om1 cells (H) under cocultivation with the effector PD-L1 CAR T cells. The target cells were pretreated with pevonedistat (1250 nM) or DMSO for 24 hours, washed twice, stained with CellBrite red cytoplasmic membrane dye, and cocultured with the effector PD-L1 CAR T cells. Data were obtained as shown in Figure 6A. Error bars represent the mean with SEM of replicates. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; Welch 2-sample t test. All experiments were repeated at least 2 times.