Functional ADCC Assay

For analysis of NK cell mediated ADCC responses, RAJI cells (1–2 × 10⁶ cells/ml) were coated with anti-CD20 or murine immunoglobulin G (IgG) (InvivoGen) at 2.5 μg/ml for 30 min. Subsequently RAJI cells were washed and then mixed with PBMCs in a V bottom 96-well plate at a 10:1 E:T ratio and incubated for 6 h 37C in the presence of CD107a-APC-H7 antibody (BD Biosciences, Cowley, U.K.). GolgiStop (containing Monensin, 1/1,500 concentration, BD Biosciences) and GolgiPlug (containing brefeldin A, 1/1,000 final concentration, BD Biosciences) were added for the last 5 h of culture. Following incubation cells were washed and stained for extracellular receptors prior to permeabilization and intracellular staining for IFN-γ. Boolean gating analysis was used to analyse CD107a and IFN-γ production in CD56^dim NK cell subpopulations expressing CD57, NKG2A, and KIRs and combinations thereof.