NK Cell Cytotoxicity Evaluation

NK cell function was assessed by IFNγ production and degranulation (CD107a) as an indirect marker of cytotoxicity [17 (link)]. NK cells were stimulated with K562 cells by co-culture of 10⁶ PBMC at a 10:1 effector-to-target ratio as previously described [18 (link)]. Following 30 min. of pre-incubation, cells were incubated during 4 hours in the presence of Brefeldin A (Biolegend) at 0.5 μg/mL, Monensin (Golgi-Stop^TM, BD Biosciences) at 0.3 μg/mL and anti-CD107a-APC (H4A3) (BD Bioscience). Cells were then surface-stained using anti-CD3 PerCP and anti-CD56 PE followed by intracellular staining for IFNγ using Cytofix/Cytoperm^TM (BD Biosciences) and anti-IFNγ-FITC (4S.B3) (Biolegend). For experiments testing the impact of stress hormones on NKG2D expression in vitro, cells were incubated for 3 h in serum-free RPMI 1640 (Life Technologies) in the presence of cortisol (Sigma) at 280 ng/mL, epinephrine (Sintetica) at 50 pg/mL, or a combination of both and NKG2D mean fluorescence intensity (MFI) was assessed.