Cd27 pacificblue

After Zombie™ dye and Fc‐block (both BioLegend, San Diego, CA, USA) pre‐incubation, PBMC were stained with anti‐human CD14‐FITC, CD19‐PerCP‐Cy5.5, CD40‐PE‐Dazzle, CD69‐FITC, CD86‐BV421, CD95‐PE (all BD Bioscience, NJ, USA), CD4‐PE‐Cy7, CD8‐PE, CD14‐PE‐CF594, CD19‐FITC, CD20‐APC‐Cy7, CD24‐PerCp‐Cy5.5, CD25‐BV605, CD27‐PacificBlue, CD38‐FITC, CD80‐PE‐Cy7 and major histocompatibility complex (MHC)‐II‐APC (all BioLegend, CA, USA). Cytokines were evaluated after adding 1‐µL Golgi‐Plug (BioLegend) to CpG‐stimulated cells for 4 h; 500 ng/mL ionomycin and 20 ng/mL phorbol 12‐myristate 13‐acetate (PMA; Sigma Aldrich, MO) were added after 2 h. Cells were stained with anti‐human interleukin‐ (IL‐)6‐FITC, tumor necrosis factor (TNF)‐A700 and IL‐10‐PE‐CF594 (all BioLegend).

Flow cytometry antibodies used for surface marker staining are listed as follows. Antibodies from BD Biosciences include: CD3‐BUV395 (Clone UCHT1; cat #563 546), CD4‐BV786 (Clone SK3; cat #563 877) and CD8‐PE‐Cy7 (Clone SK1; cat #335 787). Antibodies from Biolegend include: CD3‐FITC (Clone UCHT1; cat #300 406), CCR7‐PE (Clone G043H7; cat #353 204), CD45RA‐APC (Clone HI100; cat #304 112), CD45RO‐Pacific Blue (Clone UCHL1; cat #304 215),CD27‐Pacific Blue (Clone M‐T271; cat #356 413), CD28‐FITC (Clone CD28.2; cat #302 906), CD28‐PE (Clone CD28.2; cat #302 907), CD95‐FITC (Clone DX2; cat #305 605), CD127‐Brilliant Violet 785 (Clone A019D5; cat #351 329), TIM‐3‐Pacific Blue (Clone F38‐2E2; cat #345 041), CD57‐FITC (Clone HNK‐1; cat #359 603), LAG‐3‐Brilliant Violet 785 (Clone 11C3C65; cat #369 321) and human TruStain FcX reagent (Cat #422 302). Antibodies for intracellular staining include granzyme B‐PE (BD Biosciences, GB11; cat #561 142), IFN‐γ‐FITC (Miltenyi Biotec, cat #130‐090‐433), IL‐2‐PE (Miltenyi Biotec, cat #130‐090‐487) and TNF‐α‐APC (Miltenyi Biotec, cat #130‐091‐267).

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.