2.6. NK Cell Degranulation Assay

The cytotoxic degranulation of NK cells was determined by measuring the cell surface expression of CD107a, as previously described [35]. Briefly, IL-2-stimulated PBMCs or primary expanded NK cells (1 × 10⁵ cells) were mixed with an equal number of KCL-22 M cells in 96-well V-bottom culture plates (Corning Costar, NY, USA) and incubated for 2 h at 37 °C. For the blockade of NK activating receptors, Fc receptors on IL-2-stimulated PBMCs or primary expanded NK cells were blocked with human Fc Receptor Binding Inhibitor (eBioscience, San Diego, CA, USA) and then incubated with 20 μg/mL control IgG1 or Abs to the indicated NK activating receptors for 30 min at 4 °C prior to mixing with target cells. The cell pellets were resuspended in flow cytometry buffer (phosphate-buffered saline [PBS] with 1% FBS) and stained with anti-human CD3-PerCP, anti-human CD56-PE, and anti-human CD107a-FITC Abs for 35 min in the dark at 4 °C. Lymphocytes were gated on forward and side scatter characteristics, and the CD107a expression on CD3⁻CD56⁺ NK cells was analyzed by flow cytometry using a FACS Accuri C6 (BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo software (ver.10, Treestar, Ashland, OR, USA).