Isolation and Expansion of NK Cells from AML Patients

NK cells from AML patients and controls from normal donors were isolated by density-gradient purification followed by flow-based sorting. NK cells from both healthy donors and untreated AML patients were sorted into two subpopulations based on NK cell development (CD94^+/-/NKp80⁺/CD16⁺/CD57^- and CD94^+/-/NKp80⁺/CD16⁺/CD57⁺ (stage 5 and 6, respectively)) as previously described [10 (link),11 (link),12 (link),13 (link),14 (link),15 (link)]. NK cells for in vitro gene editing studies were isolated by negative selection using RosetteSep™ Human NK Cell Enrichment Cocktail (Stem Cell Technologies, 15065, Vancouver, BC, Canada). Purified NK cells (depleted of T cells, B cells, and monocytes and typically > 95% CD16/56+) were stimulated weekly for two weeks with irradiated CSTX002 feeder cells in AIM-V expansion medium supplemented with ICSR (CTS™AIMV™SFM/CTS^TM Immune Cell SR, Thermo Fisher Scientific) [16 (link)] and 50 IU of human recombinant IL-2 (rIL-2) (Novartis). NK cell purity of WT and KO NK cells was determined after expansion and was uniformly > 98% (Supplementary Figure S1).