Figure 3.
iDuo NK cells efficiently kill primary CLL cells and preferentially target malignant B cells. (A) iDuo NK cells and feeder-expanded PBNK cells were cocultured with CellTrace Violet-labeled CLL cells collected from five different patients in the presence or absence of 1 μg/mL rituximab for 5 hours. Percent specific killing was determined by subtracting the percentage of live CLL cells in each condition to CLL cells cultured alone. Results are from two independent experiments. Statistical significance was determined by one-way ANOVA with multiple comparisons. (B) iDuo NK cells were cocultured with IncuCyte Caspase-3/7 dye-labeled Raji cells and peripheral blood mononuclear cells (mixed at a 1:1 ratio) at the indicated E:T ratios for 4 hours. Shown is caspase 3/7 cleavage in target cells as determined by flow cytometry. Results are representative of two independent experiments. (C) iDuo NK cells and primary anti-CD19 CAR T cells were cocultured for 4 hours with CellTrace Violet-labeled CD19+ B cells isolated from five healthy donors and Nalm6 cells expressing GFP at a 1:1 ratio. Shown are the ratios of healthy B cells to Nalm6 cells in each culture condition relative to targets cultured alone. Statistical significance was determined by unpaired two-tailed Student t tests. Results are from two independent experiments. (D) iDuo NK cells and (E) primary anti-CD19 CAR T cells were cocultured for 4 hours with CellTrace Violet-labeled CD19+ B cells isolated from four healthy donors and Nalm6 cells expressing GFP at the indicated E:T ratios in the presence of 10 μg/mL mouse IgG1κ or anti-NKG2D blocking antibody. Shown are the ratios of healthy B cells to Nalm6 cells in each culture condition relative to targets cultured alone. (F) Flow cytometry plots showing surface levels of the indicated activating receptors on non-transduced iNK cells and iDuo NK cells. Results are representative of two independent experiments. Statistical significance was determined by unpaired two-tailed Student t tests. Results are from two independent experiments. Shown is mean ± SD. ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001, ∗∗∗∗P ≤ .0001.

iDuo NK cells efficiently kill primary CLL cells and preferentially target malignant B cells. (A) iDuo NK cells and feeder-expanded PBNK cells were cocultured with CellTrace Violet-labeled CLL cells collected from five different patients in the presence or absence of 1 μg/mL rituximab for 5 hours. Percent specific killing was determined by subtracting the percentage of live CLL cells in each condition to CLL cells cultured alone. Results are from two independent experiments. Statistical significance was determined by one-way ANOVA with multiple comparisons. (B) iDuo NK cells were cocultured with IncuCyte Caspase-3/7 dye-labeled Raji cells and peripheral blood mononuclear cells (mixed at a 1:1 ratio) at the indicated E:T ratios for 4 hours. Shown is caspase 3/7 cleavage in target cells as determined by flow cytometry. Results are representative of two independent experiments. (C) iDuo NK cells and primary anti-CD19 CAR T cells were cocultured for 4 hours with CellTrace Violet-labeled CD19+ B cells isolated from five healthy donors and Nalm6 cells expressing GFP at a 1:1 ratio. Shown are the ratios of healthy B cells to Nalm6 cells in each culture condition relative to targets cultured alone. Statistical significance was determined by unpaired two-tailed Student t tests. Results are from two independent experiments. (D) iDuo NK cells and (E) primary anti-CD19 CAR T cells were cocultured for 4 hours with CellTrace Violet-labeled CD19+ B cells isolated from four healthy donors and Nalm6 cells expressing GFP at the indicated E:T ratios in the presence of 10 μg/mL mouse IgG1κ or anti-NKG2D blocking antibody. Shown are the ratios of healthy B cells to Nalm6 cells in each culture condition relative to targets cultured alone. (F) Flow cytometry plots showing surface levels of the indicated activating receptors on non-transduced iNK cells and iDuo NK cells. Results are representative of two independent experiments. Statistical significance was determined by unpaired two-tailed Student t tests. Results are from two independent experiments. Shown is mean ± SD. ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001, ∗∗∗∗P ≤ .0001.

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