Cd16 pe

Peripheral blood was collected in BD Vacutainer CPT tubes containing sodium heparin (8 ml; Becton Dickinson and Company, Franklin Lakes, NJ, USA). Within 2 h after blood collection, PBMCs were collected by centrifugation (Sorvall Legend XFR; Thermo Fisher Scientific, Waltham, MA, USA) at 1,700 × g for 20 min at room temperature. The PBMCs were washed in 30 ml phosphate-buffered saline (PBS) containing 2 mM EDTA and then centrifuged at 250 × g for 10 min at room temperature to remove any contaminating platelets and plasma.
The PBMCs were incubated with CD14-FITC (catalogue #: 2228020), CD16-PE (catalogue #: 2110040), CD3-PE/Cy7 (catalogue #: 2102100) and CD4-APC (catalogue #: 2323070) antibodies (5 µl antibody/500 µl of cell suspension) (Sony Biotechnology Inc., Tokyo, Japan) for 20 min at 4°C. After washing with 5 ml of PBS, CD14⁺⁺/CD16⁻ monocytes and CD3⁺/CD4⁺ T cells were immediately sorted using an SH800 Cell Sorter (Sony Biotechnology) from the monocyte-containing or lymphocyte-containing gate determined from light-scatter density plots (Supplementary Fig. 6). The purity of all FACS-sorted populations was analysed by flow cytometry using the SH800 Cell Sorter.
Genomic DNA and RNA were extracted from the sorted cells using the AllPrep DNA/RNA Micro Kit (Qiagen, Venlo, The Netherlands), according to the manufacturer’s instructions.

Antibodies against CD5-PC7, CD19-BV421, CD14-PC7, CD16-PE, CD64-PE, CD71-APC-Cy7, CD86-BV421, CD163-FITC, CD169-APC, CD206-BV421, CD209-FITC, and relevant isotype controls were purchased from Sony Biotechnology Europe. 7-AAD and Annexin-V-FITC viability kit were purchased from Miltenyi Biotec, France. Data were acquired on an LSRII flow cytometer (BD Biosciences, France). The obtained results were further analyzed by Flow Logic 700.2A (Inivai Technologies, Australia).

The following anti-human monoclonal antibodies (mAbs) were used for flow cytometry: CD3- PE-Cy7 (Beckman Coulter, USA, clone UCHT1), CD56-APC (Beckman Coulter, USA, clone N901), CD56-Brilliant Violet 421 (Sony, USA, clone HCD56), CD56-PE (Beckman Coulter, USA, clone N901 (HLDA6)), CD57-PE (eBioscience, USA, clone TB01), CD57-FITC (Miltenyi Biotech, Germany, clone TB03), CD57-APC (Miltenyi Biotech, Germany, TB03), CD16-PE (Sony, USA) anti-NKG2A-PE (R&D Systems, USA, clone 131411), anti-KIR2DL2/DL3-PE (Miltenyi Biotech, Germany, clone DX27), anti-HLA-DR-FITC (Beckman Coulter, USA, clone B8.12.2). Cells were incubated in PBS containing 0.5% BSA (bovine serum albumin) and 0.01% sodium azide (labeling buffer) with mAbs for 30 min on ice, then washed twice with labeling buffer and centrifugation. Samples were subsequently analyzed using FACSCalibur flow cytometer (BD Biosciences, San Jose CA, USA) equipped with 488 and 640 nm lasers. At least 30000 events were recorded in lymphocyte gate for total NK cell population and 5000 events for NK cell clones. Acquired data was analyzed using Flowing Software version 2.5.1 (PerttuTerho, Turku Centre for Biotechnology, Finland) and FlowJo software version 7.6 (FlowJo LLC, Ashland, OR, USA). For fluorescence-activated cell sorting, cells were labeled with mAbs in PBS containing 0.5% BSA and 2 mM EDTA.