Antibody-dependent NK cell degranulation was conducted as previously described [83 (link),84 (link)]. Spike or RBD protein was coated on Maxisorp ELISA plate (Thermo Fisher Scientific) and then blocked with 5% BSA. Diluted serum (1:25 dilution) was then added and incubated for 2 hours at 37°C. Human NK cells were isolated from peripheral blood by negative selection using the RosetteSep Human NK cell enrichment cocktail (STEMCELL Technologies - Vancouver, British Columbia, Canada) following the manufacturer’s instructions. Human NK cells were then added to the bound antibody and incubated for 5 hours at 37°C in the presence of RMPI+10% FBS, GolgiStop (BD), Brefeldin A (Sigma - Burlington, Massachusetts, USA), and anti-human CD107a-PE-Cy5 antibody (BD Biosciences). After incubation, cells were washed, stained with CD16-APC-Cy7, CD56-PE-Cy7, and CD3-Pacific Blue (BD Biosciences) and fixed in 4% PFA for 15 minutes. Intracellular staining was performed using the FIX/PERM Cell fixation and permeabilization kit (Thermo Fisher Scientific), and cells were stained for IFNγ-APC and macrophage inflammatory protein-1β-PE (BD Biosciences). Results were reported as percentage of NK cells positive for CD107a, IFNγ, or macrophage inflammatory protein-1β. Flow cytometry was performed with an LSRII (BD), and analysis was performed using FlowJo V10.7.1.
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