Antibody-dependent natural killer cell activation
FRANKLIN
MaxiSorp enzyme-linked immunosorbent assay (ELISA) plates (Thermo Fisher) were coated with RSV preF antigen for 2 hours at room temperature and blocked with 5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA). Diluted (1:500) serum samples (100 µL) were added to the wells and incubated overnight at 4°C. Natural killer (NK) cells were isolated from buffy coats from healthy donors using the RosetteSep Human NK Cell Enrichment Cocktail (STEMCELL Technologies, Cambridge, MA, USA) and stimulated with recombinant human interleukin-15 (1 ng/mL; STEMCELL Technologies) at 37°C overnight. NK cells were added to the washed ELISA plate and incubated together with anti-human CD107a (BD Biosciences, Franklin Lake, NJ, USA; clone: H4A3; PE-Cy5; 1:40 dilution), brefeldin A (Sigma-Aldrich), and monensin (BD Biosciences) for 5 hours at 37°C. Cells were surface stained for CD56 (BD Biosciences; clone: B159; PE-Cy7; 1:200 dilution), CD16 (BD Biosciences; clone: 3G8; APC-Cy7; 1:200 dilution), and CD3 (BD Biosciences; clone: UCHT1; Pacific Blue; 1:800 dilution). Cells were fixed and permeabilized using the FIX & PERM™ Cell Permeabilization Kit (Thermo Fisher) and stained for intracellular macrophage inflammatory protein-1β (MIP-1β; BD Biosciences; clone: D21-1351; PE; 1:50 dilution) and interferon-γ (IFN-γ; BD Biosciences; clone: B27; FITC; 1:17 dilution). NK cells were defined as CD3