Trail apc

PBMC were isolated from non-cancer controls by density gradient centrifugation and seeded at a density of 1 × 10⁶ cells/mL cRPMI. Cells were treated with 30 ng/mL of recombinant fractalkine (Biolegend) and/or 100 ng/mL of recombinant IL-15 (Immunotools, Friesoythe, Germany) for 2 or 24 h. Cells were stained with CD56-FITC-Viobright (Miltenyi Biotec), L-selectin-BV510, CD3-APC-Cy7, Integrin β7-Pe-Cy5, α4-Pe-Cy7, TRAIL-APC, FasL-BV421 (Biolegend). Samples were acquired using the CANTO II (BD Biosciences) flow cytometer and analysed using FlowJo software (BD Biosciences).

Whole blood, SVF from omentum and intratumoural immune cells were stained with CD56-FITC-Viobright (Miltenyi Biotec, Bergisch Gladbach, Germany), L-selectin-BV510, CD3-APC-Cy7, Integrin β7-Pe-Cy5, α4-Pe-Cy7, TRAIL-APC, FasL-BV421 (Biolegend, San Diego, CA, USA). Red blood cells were lysed using BD Lysing Solution (BD Biosciences, Franklin Lakes, NJ, USA) 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 (BD Biosciences).

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.