Il 15

To determine activation of primary NK cells following exposure to target cells pulsed with different peptide variants, NK cell degranulation assays measuring CD107a expression were performed using freshly isolated and purified NK cells from nine healthy KIR2DL3+ individuals [38 (link)]. Primary NK cells were isolated by incubating whole blood with RosetteSep Human NK Cell Enrichment Cocktail (Stemcell Technologies) for 20 min at room temperature, followed by Histopaque-1077 (Sigma) density gradient centrifugation. NK cells were rested overnight in RPMI medium 1640 (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum (Sigma-Aldrich), 2,500 U/ml penicillin, 2,500 μg/ml streptomycin, 100 mM L-glutamine (Cellgro), and 1.0 ng/ml IL-15 (Cellgro). Subsequently, NK cells (1 × 10⁵) were co-incubated with peptide-pulsed 721.221-ICP47-C*03:04 cells (5 × 10⁵) at an effector:target ratio of 1:5 in RPMI containing anti-human CD107a-PE-Cy7 (12.5 μl/ml) and monensin (1.5 μl/ml, BD GolgiStop). Cells were incubated for 6 h at 26°C in 5% CO₂. Cells were stained with 7AAD, anti-CD3-PB, anti-CD16-BV785, anti-CD56-BV605, anti-CD14/19-BV510, anti-KIR2DL2/3-PE, and anti-KIR2DL3-APC, washed, fixed with 4% (w/v in PBS) paraformaldehyde (Affymetrix), and analyzed by multiparameter flow cytometry using a BD LSR II.