De-identified human peripheral blood buffy coats were provided by the Department of Transfusion Medicine, NIH Clinical Center. The NIH Office of Human Subjects Research Protections determined that use of this material was excepted from the requirements of institutional review board (IRB) review. Primary NK cells were isolated using RosetteSep human NK cell enrichment cocktail (STEMCELL Technologies, BC, Canada) with lymphocyte separation medium (MP Biomedicals, Irvine, CA, USA) or by sequential CD3 depletion and CD56 selection using magnetic-activated cell sorting (MACS) beads (Miltenyi Biotec, Bergisch Gladbach, Germany). NK cells were cultured in X-VIVO 20 medium (Lonza, Basel, Switzerland) supplemented with 10% heat-inactivated human AB serum (Millipore-Sigma, MA, USA), 2 mM GlutaMAX (Thermo Fisher Scientific, Waltham, MA, USA), and 500 IU/ml IL-2 (teceleukin, Roche, Basel, Switzerland), refreshing at least half the medium every 1–5 days (usually every 2–3 days). LCL cells or K562 4-1BBL IL-21 cells were irradiated (100 Gy) and added to culture as indicated. Cell numbers were enumerated by trypan blue staining and microscopic counting or using a Luna-FL fluorescence cell counter (Logos Biosystems, Anyang, South Korea) with acridine orange and propidium iodide stains. Statistical comparisons of proliferation were assessed using GraphPad Prism 8.4.1 comparing log10(fold change) values.

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