Figure 1.
PD-L1 t-haNK cells are haNK cells engineered to target PD-L1-expressing tumor cells. (A) Immune-related transcriptomes of irradiated PD-L1 t-haNK and haNK cells were analyzed using the nCounter PanCancer immune profiling panel. Heatmap showing select NK-related genes with data presented as fold change values on a scale of −3 (blue) to 3 (red). (B) Representative FACS plots showing the frequencies of anti-PD-L1 CAR+, CD16+, and perforin+granzyme B+ irradiated PD-L1 t-haNK and haNK cells. Results are from two independent experiments and were gated on live cells. Blue histograms represent controls, while red histograms represent samples. (C) Immunofluorescence microscopy data showing perforin (green) and NKG2D (orange) expressions on irradiated PD-L1 t-haNK cells. (D) MDA-MB-231 tumor cell lysis by PD-L1 t-haNK and haNK cells with or without anti-PD-L1 antibody (1 µg/mL) was evaluated via 111In-release assay at 25:1 E:T ratio. Results shown are the means with SEM of triplicate measurement and representative of three independent experiments. (E) Cytolytic capacity of PD-L1 t-haNK cells on CMA and anti-CD16 treatment with MDA-MB-231 as target at 25:1 E:T ratio as evaluated in 111In-release assay. Results shown are the means with SEM of triplicate measurement. (F) PD-L1 t-haNK cell-mediated killing of MDA-MB-231 tracked in real time using live-cell imaging. CellTracker violet BMQC was used to differentiate the PD-L1 t-haNK cells (blue) from the MDA-MB-231 tumor cells (bright field), and CellEvent-caspase 3/7 was used to identify cells undergoing apoptosis. Scale bars on microscopy images (C, F) represent 10 µM. One-way analysis of variance with Tukey’s multiple comparison test was used for statistical analyses. *P<0.05, **P<0.01, ***P<0.005, ****P<0.001. CAR, chimeric antigen receptor; CMA, concanamycin A; E:T, effector to target; FACS, fluorescence-activated cell sorting; haNK, high-affinity NK; NK, natural killer; PD-L1, programmed death-ligand 1; t-haNK, targeting high-affinity natural killer.