Rosettesep human nk cell enrichment cocktail

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).

Tonsil-derived single cell suspensions were pelleted by centrifugation at 524 × g for 10 min at RT. The cell pellets were then resuspended in 2 mL RT FBS, 1 mL leukocyte-depleted RBC, and 100 μL RosetteSep “Human NK cell Enrichment Cocktail” (StemCell Technologies) per 1E8 cells. The RosetteSep “Human NK cell Enrichment Cocktail” contains bivalent antibodies to tether cells expressing CD3, CD4, CD14, CD19, CD36, CD66b, and CD123 to human RBC. Where indicated, we also used the human CD8+ T cell and monocyte enrichment cocktails (StemCell Technologies). The tonsil cell/FBS/RBC/RosetteSep mixtures were incubated at RT for 30 min on a nutator. After incubation, the mixtures were diluted 2-fold in RT PBS and then layered over Ficoll (25 mL of cell mixture over 15 mL Ficoll in each 50 cc tube) and centrifuged at 754 × g for 30 min at RT with the brake off. Mononuclear cell layers at the Ficoll/supernatant interface were collected, and then residual RBC were lysed by resuspending the mononuclear cell preparations in 10 mL 0.8% NH₄Cl buffer (StemCell Technologies) per donor for 5–10 min at RT. Enriched cells were then washed with PBS, enumerated as above, and used for flow cytometry immunophenotyping and/or in vitro stimulation for cytokine production (see below).

Burkitt's lymphoma Raji cells (ATCC® CCL-86™) and SK-BR-3 breast tumor cell lines (ATCC® HTB-30™) were obtained from American Type Culture Collection. Raji cells were cultured in complete RPMI with 10% fetal bovine serum (FBS) and SK-BR-3 cells were cultured in complete DMEM with 10% FBS. The collections of CHO cells expressing FLAG-tagged hFcγRs were described previously (14 (link), 40 (link), 45 (link)).
Human PBMCs and neutrophils were purified from anonymous healthy volunteers using Histopaque density gradient centrifugation (Sigma-Aldrich). Neutrophils were activated with 50 ng/mL hIFNγ for 24 h. NK cells were isolated by negative immunodensity isolation using the RosetteSep Human NK Cell Enrichment Cocktail (StemCell Technologies). Human GM-CSF differentiated macrophage cells were differentiated from CD14⁺ monocytes with 50 ng/mL GM-CSF (BioLegend®) for 7 days. Ectodomains of hFcγRI, hFcγRIIa, hFcγRIIb, and hFcγRIIIa, were produced in Expi293 cells (Invitrogen). All primers were synthesized by Integrated DNA Technologies.

Human peripheral blood mononuclear cells (PBMC) were obtained from the Shanghai Blood Center under a research protocol approved by the Department of Shanghai Blood Administration. Human NK cells were purified from PBMC using the RosetteSep Human NK Cell Enrichment Cocktail (StemCell Technologies, Vancouver, BC, Canada) as described previously30 (link). Murine splenocytes were obtained from wild-type (WT) mice or mice in which floxed STAT3 was deleted in hematopoietic cells by Cre expression under the Tie2 promoter31 (link). Murine NK cells were isolated from splenocytes using the EasySep Mouse NK Cell Enrichment Kit (Stemcell Technologies Vancouver, BC, Canada). All experiments involving animals were conducted in accordance with Institutional Animal Care and Use Committee approval at University of Texas MD Anderson Cancer Center.