Nk 92

HEK293T, Jurkat, and NK-92 cell lines were purchased from ATCC. YT cell line was a kind gift of Dr. A.V. Filatov. Primary human T cells were isolated from peripheral blood of a healthy donor who provided written informed consent in accordance with the approval of the Ethics Committee on Animal and Human Research of the Institute. T cells and cell lines were grown in IMDM (ThermoFisher) supplemented with 10% FCS (15% for NK-92), 100 μg/ml penicillin and 100 μg/ml streptomycin, at 37 °С in a humidified atmosphere of 5% CO2. IL-2 (200 μg/ml) was included into growth media for NK-92 cell cultivation.

DC4 + human primary AML samples and normal peripheral blood mononuclear cells (PBMCs) were obtained from residual samples using a protocol approved by the Institutional Review Board of Stony Brook University. THP-1, U937, TALL104, and NK-92 cell lines were obtained from ATCC (Manassas, VA, USA). MOLM-13 was obtained from AddexBio (San Diego, CA, USA) T cells were cultured in filtered T cell media, defined as 50% AIM V, 40% RPMI 1640 and 10%FBS, with 1% Pen/Strep (all Gibco, Waltham, MA, USA) and supplemented with IL-2 (300 IU/mL; Peprotech, Rocky Hill, NJ, USA), unless otherwise specified. NK-92 cells were cultured in filtered NK cell media, defined as alpha-MEM without ribonucleosides and deoxyribonucleosides with 2mM L-glutamine, 1.5 g/L sodium bicarbonate (Gibco), 12.5% heat-inactivated horse serum (Gibco), 12.5% heat-inactivated FBS (Atlanta Biologicals, Atlanta GA, USA), 1% Pen/Strep (Gibco), 0.2% inositol (Sigma), 0.02% folic acid (Fisher), and 50 uM beta-mercaptoethanol (Fisher), supplemented with IL-2 (300 IU/mL), unless otherwise specified. THP-1, U937, and MOLM-13 cell lines were cultured in RPMI, 10% FBS, 1% Pen/Strep (Gibco). TALL104 cells were cultured in IMDM adding 300 IU/ml recombinant human IL-2, 2.5 mg/ml human albumin, 0.5 mg/ml D-mannitol, and 20% FBS.

The human MM cell lines RPMI8226 and MM.1S, and the human natural killer cell line NK-92 were obtained from ATCC (Manassas, VA, USA). RPMI8226 and MM.1S cells were cultured in RPMI-1640 medium (GIBCO/BRL, Grand Island, NY, USA) supplemented with 100 U/ml penicillin-streptomycin and 10% fetal bovine serum (FBS). The RPMI8226-Luc cell line was constructed by luciferase-encoding lentiviral infection. NK-92 cells were cultured in MEM-alpha medium (GIBCO/BRL, Grand Island, NY, USA) supplemented with 100 U/ml rhIL-2, 100 U/ml penicillin-streptomycin, 12.5% FBS, 12.5% horse serum, and 0.1 mM β-mercaptoethanol. The human NK cell line NKL was a gift from Professor Jin BQ (Department of Immunology, Fourth Military Medical University, Xi’an, PR China). NKL cells were cultured in complete RPMI-1640 medium containing (100 U/ml) rhIL-2. All cells were incubated at 37°C in a humidified incubator with 5% CO₂. Cells in the logarithmic growth phase were selected for subsequent experiments.

Natural Killer Cell line (NK-92) were originally obtained from ATCC (ATCC, Manassas, USA). Cells were cultured in the Alpha Minimum Essential Medium without ribonucleosides and deoxyribonucleosides but with sodium bicarbonate (MEM, Corning, New York, USA) supplemented with 2mM L-Glutamine (Thermo Fisher Scientific, Waltham, USA), 0.2mM myo-Inositol (Sigma-Aldrich, Taufkirchen, Germany), 50mM β-mercaptoethanol (Sigma-Aldrich, Taufkirchen, Germany), 0.02mM Folic Acid (Sigma-Aldrich, Taufkirchen, Germany), 150U/ml recombinant human Il-2 (Thermo Fisher Scientific, Waltham, USA), 12.5% Horse Serum (Thermo Fisher Scientific, Waltham, USA) and 12.5% foetal bovine serum (EURx, Gdansk, Poland). Cells were passaged 24h before all experiments using standard protocol to a 24-well plate.

MDA-MB-231 (HTB-26™), a human tiple-negative (ER⁻/PR⁻/HER2⁻) breast cancer cell line, and PC-3 (CRL-1435™), a human androgen-independent prostate adenocarcinoma cell line, were used to assess immune cell killing of tumor cells in vivo. Both cell lines were purchased from the American Type Culture Collection (ATCC). MDA-MB-231 cells were cultured in RPMI-1640 medium (HyClone) supplemented with 10% heat-inactivated FBS (Biowest) and 1% antibiotic–antimycotic solution (Gibco Life Technologies). PC-3 cells were grown in Ham’s F-12 K medium (HyClone) supplemented with 10% heat-inactivated FBS and 1% antibiotic–antimycotic solution. Cell lines were maintained at 37 °C in 5% CO₂ and detached with trypsin (HyClone) for passaging and further culture. Breast cancer line MDA-MB-468 (HTB-132™) was kindly gifted by Dr. Feng Lin from Cleveland Clinic. This cell line was also purchased from ATCC and grown in Leibovitz’s medium in the presence of 10% FBS. For the RNA sequencing experiments, we purchased NK-92 cells from ATCC. NK-92 cells were cultured in MEM-α medium (HyClone) supplemented with 12.5% FBS, 12.5% horse serum (Gibco Life Technologies), 100 IU/ml IL-2 (R&D Systems, Minneapolis, MN, USA), 0.2 mM inositol (Sigma-Aldrich, St. Louis, MO, USA), 0.02 mM folic acid (Sigma-Aldrich) and 0.1 mM mercaptoethanol (Gibco Life Technologies).