Mechanisms of expanded NK cell cytotoxicity against CLL. (A) ND-XNK effectors and CLL target cells were labeled with PKH67 and CellVue Claret. CLL targets were labeled with 5 μg/mL antibody (or obinutuzumab-derived F(ab′)2) and then cocultured with NKs at 2:1 NK:CLL ratio for 30 minutes. Conjugation was measured by flow cytometry and quantified as the fraction of CLL cells bound to NK cells. Graphs show mean ± standard deviation of n = 3 to 6 experiments. (B) ND-XNKs were treated for 90 minutes with 1 μg/mL concanamycin A and then cocultured with primary CLL cells ± 5 μg/mL obinutuzumab for a 4-hour calcein release assay. Graphs show mean ± standard deviation of n = 4 to 8 experiments. (C) ND-XNKs were treated for 30 minutes with 50 μg/mL blocking antibodies against FasL, TNF-α, or TRAIL and then cocultured with primary CLL cells ± 5 μg/mL obinutuzumab for a 4-hour calcein release assay. Graphs show mean ± standard deviation of n = 5 experiments. (D) ND-XNK cells were treated as in panel C and then cocultured with primary CLL cells at a 2.5:1 effector:target ratio for 4 hours. Cytotoxicity was measured using Annexin V/Fixable Live-Dead staining flow cytometry and quantified using count beads. ∗P < .05 by ANOVA (A,D) or mixed effect modeling (B,C). ns, not significant.