Nkg2a apc

Whole blood and intratumoural immune cells were stained with CD56-FITC-Viobright, NKG2A-APC, CCR5-FITC, CCR1-APC, CCR3-PE (Miltenyi Biotec), CD3-APC-Cy7, NKp30-BV421, NKp46-PE-Cy7, NKG2D-PE-Cy5, PD-1-PE-Cy7, TIGIT-PE-Cy5, CD69-BV510 (BioLegend). Red blood cells were lysed using BD Lysing Solution (BD Biosciences) as per manufacturer’s instructions. NK cells were quantified as CD56⁺CD3⁻ cells within the lymphocyte gate. Cells were acquired using the CANTO II (BD Biosciences) flow cytometer and analysed using FlowJo software (Tree Star).

NK cells were enriched from freshly isolated PBMCs using an EasySep Human NK Cell Enrichment Kit (Stemcell, Vancouver, Canada) as per the manufacturer's guidelines and sorted using a BD FACS Aria-Fusion. NK cells were labeled with CD3-AF700, CD14-APC-Cy7, CD19-APC-Cy7, NKG2A-APC (Miltenyi Biotec, Bergisch Gladbach, Germany–REA110), LIVE/DEAD fixable near-IR dye and a combination of KIR2DL1/KIR2DS5-PE, KIR2DL2/S2/L3-PE (BioLegend–DX27), and KIR3DL1-PE (BioLegend–DX9) depending on specific donor genotype for 20 min at 4°C in the dark. Cells were sorted to select for NKG2A-educated NK cells and uneducated NK cells while excluding KIR-educated NK cells and were subsequently incubated overnight in RPMI containing 10% (v/v) FBS with 1 ng/mL IL-15. NK cells were washed then resuspended in calcium buffer (HBSS + 1 mM CaCl2 + 0.5 mM MgCl2 + 10 mM HEPES + 0.1% FBS) and stained with 4 μM Fura2 (Life Technologies, CA, USA) for 30 min at 37°C. Cells were resuspended in calcium buffer then observed under a fluorescent microscope. Biotinylated anti-NKp46 (BioLegend–9E2) and anti-2B4 (BioLegend–C1.7) antibodies were added after 3 min then streptavidin (BioLegend) after a further 2 min. Thapsigargin (Merck Millipore, MA, USA) was added at 17 min. The 340/380 ratio of Fura2 was recorded to measure intracellular calcium release by cells over time at 37°C using an incubator chamber.

Peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy donors by density gradient centrifugation and resuspended in RPMI supplemented with 10% FBS and 1% penicillin–streptomycin (complete RPMI (cRPMI), Gibco). PBMC were seeded at a density of 500,000 cells per 500 µL of cRPMI and treated for 24 h with 5 μM or 10 μM of Olaparib. Following treatment, the cells were surface stained for flow cytometric analysis using CD56-FITC (BioLegend), CD3-APC-Cyanine7 (BioLegend), and the following antibodies for activating NKRs, NKp46-PE-Cyanine7 (BioLegend), DNAM-1-PE (BioLegend), NKp30-BV421 (BioLegend), NKG2D-PE-Cyanine5 (BioLegend), CD16-PE-Cyanine7 (BioLegend), inhibitory receptors NKG2A-APC (Miltenyi Biotec), PD-1-PE-Cyanine7 (BioLegend), and TIGIT-PerCP-Cyanine5.5 (BioLegend), death receptor ligands TRAIL-APC (BioLegend) and FasL-BV421 (BioLegend), and phenotypic markers, CD57-PE (BioLegend), CD69-BV510 (BioLegend), and CD27-Pacific blue (BioLegend). All samples were acquired using the BD FACS CANTO II (BD Biosciences) flow cytometer and analysed using FlowJo v10 software (BD Biosciences). NK cells were defined as CD56⁺ CD3⁻ in the lymphocyte gate. The gating strategies are available in Supplementary Figure S1.