Figure 1.

iDuo NK cells target malignant B cells through anti-CD19 CAR and hnCD16. (A) Representative flow cytometry plots showing surface expression of hnCD16, anti-CD19 CAR, and membrane-bound IL-15RF on non-transduced iNK cells and iDuo NK cells. (B) Non-transduced iNK cells, iDuo NK cells, and PBNK cells activated with 10 ng/mL IL-15 overnight were used as effectors against NLR-transduced Daudi, JeKo-1, Raji, and AHR-77 cells at the indicated E:T ratios in 72-hour IncuCyte assays. Shown are the percentages of live target cells over time normalized to tumor cells alone. Results are representative of two independent experiments. (C) Non-transduced iNK cells and iDuo NK cells were cocultured with NLR-transduced AHR-77 cells labeled with IncuCyte Caspase-3/7 dye at the indicated E:T ratios. Statistical significance was determined by two-way ANOVA. Results are representative of three independent experiments. (D) Non-transduced iNK cells and iDuo NK cells were cocultured with NLR-transduced CD19 knockout AHR-77 cells labeled with IncuCyte Caspase-3/7 dye with or without 1 μg/mL rituximab at the indicated E:T ratios. Statistical significance was determined by two-way ANOVA. Non-transduced iNK cells, anti-CD19 CAR iNK cells, and anti-CD19 CAR/IL-15RF iNK cells were cocultured (E) wild-type Nalm6 cells and (F) CD19 knockout Nalm6 cells labeled with IncuCyte Caspase-3/7 dye at the indicated E:T ratios. Statistical significance was determined by two-way ANOVA. Non-transduced iNK cells and iDuo NK cells were used as effectors and cocultured with Raji cells or Raji cells + 1 μg/mL rituximab at a 2:1 E:T ratio for 5 hours. NK cells were then assessed by flow cytometry for (G) surface CD107a levels and (H) intracellular IFN-γ levels. Results are from three independent experiments. Shown is mean ± standard deviation. Statistical significance was determined by paired two-tailed Student t tests. ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001, ∗∗∗∗P ≤ .0001.