Evaluating NK Cell Activity Assay

The ADCC assay for measuring intracellular NK cell IFN-γ and CD107a expression was conducted and analysed with the gating strategy as previously described^{23 (link)}. Briefly, 96-well plates were coated overnight at 4 °C with A/California/04/09 HA protein (1 μg/ml) and chimeric cH9/1 HA protein (1 μg/ml). The plates were then washed with PBS and incubated with heat-inactivated sera (prediluted 1:10) for 2 h at 37 °C. Plates were then washed again with PBS and incubated with 10⁵ CD16 176 v NK-92 cells per well (mycoplasma-free, human NK cell line expressing high affinity 176 V variant CD16 receptor) (Fox Chase Cancer Center, Philadelphia, PA, USA). As a negative control, NK-92 cells lacking the expression of CD16 were added to an additional well per sample. Cells were incubated with anti-CD107a-AF488 antibody (Biolegend, San Diego, CA, USA), Brefeldin A (5 μg/ml, BD) and monensin (5 μg/ml, BD) for 16 h at 37 °C. After incubation, the cells were stained with LIVE/DEAD Fixable Aqua dead cell staining kit (Invitrogen), anti-CD3-PE CF594 (BD) and anti-CD56-APC (BD) before intracellular staining with anti-IFN-γ-BV-421 (Biolegend). The cells were acquired on BD Fortessa. Data analysis was performed using FlowJo version 10 (treeStar).