Effects of CLL targeted therapies on NK expansion. (A) ND-XNK cells were expanded as described above with the addition of 1 μM ibrutinib, 1 μM acalabrutinib, 5 μM idelalisib, or 1 μM lenalidomide in the growth media, which were refreshed with each media change (days 0, 2 or 3, 5, 7, 9, 12, 14, 17). Portions of each culture were frozen at various points during expansion, and the graph shows projected fold expansion based on the cells left in expansion culture (mean ± standard deviation of 3 to 12 samples). Doubling times were calculated based on an exponential fit of growth curves. (B) XNK cells produced in panel A were cocultured with Mec1, OSU-CLL, or HG3 CLL cell lines ± 5 μg/mL obinutuzumab and cytotoxicity measured by 4-hour calcein release assay. Graph shows mean ± standard deviation of n = 2 to 3 experiments. (C) Diagram of NK maturation sequence and markers used. KIR⁺ refers to any combination of KIR2DL1, KIR2DL2/3, and/or KIR3DL1. (D) NK phenotypes were analyzed according to the sequence described in panel C for NKs expanded in panel A (n = 3 acalabrutinib/lenalidomide, 6 ibrutinib/idelalisib). ∗P < .05 by ANOVA (A) or mixed effect modeling (B,D).