Cd158a percp cy5

Flow cytometric analysis was performed using cryopreserved PBMC. Viability was ascertained by trypan blue exclusion. Cells were washed, and then stained on ice for 20 min with the following fluorescent Abs in various combinations: CD3 (PerCP-Cy5.5 or allophycocyanin) or FITC, anti–TCR-αβ-1-FITC, and anti–TCR-γδ-1-PE (all BD Oncomark); Vα24 TCR-FITC and Vβ11 TCR-PE (Immunotech); CD56-PE, CD107a-PE, CD94-allophycocyanin, CD158b-PE, and CD16–PerCP-Cy5.5 (all from BD Pharmingen); and CD158a–PerCP-Cy5.5 (eBiosciences). After washing, stained cells were fixed in PBS/2% FCS/1.6% paraformaldehyde and acquired on a FACSCalibur flow cytometer (BD Biosciences). For intracellular staining, cells were first surface stained, followed by washing and incubation for 30 min on ice with Fix/Perm buffer (eBioscience). After washing in permeabilization buffer, cells were incubated for 30 min on ice with perforin-PE (Perforin reagent set; BD Pharmingen 556437) or IFN-γ allophycocyanin. The cells were washed again, fixed in PBS/2% FCS/1.6% paraformaldehyde, and acquired. Data were analyzed using Flowjo software (Tree Star, Ashland, OR).

Flow cytometry was performed on cryopreserved cells. As per the gating strategy in figure 1A, NK cells were identified using a combination of Fixable blue dead cell stain (Life technologies), CD3-Pacific Blue, CD56-PE-Cy7 and CD16- APC-Cy7 (BD Biosciences). NK cell receptor expression was assessed using combinations of CD158a-PerCP-Cy5.5 (eBioscience), CD158b-FITC, KIR3DL1-Alexafluor700 (both Biolegend), KIR2DL3-PE, KIR2DL1-APC (both R&D Systems), NKG2A-PE (Beckman Coulter), NKG2D-APC, CD94-FITC, 2B4-FITC, NKp46-PE, NKp30-APC (all BD Biosciences), TRAIL-PE (Biolegend), CD161-FITC (Miltenyi) and CD160-Alexafluor647 (Biolegend). For intracellular staining, cells were fixed and permeabilized (PermA/B solution, Caltag) according to the manufacturers' instructions, prior to incubation with Perforin-PerCP-Cy5.5 (eBioscience). At least 1500 NK cells were acquired for all samples on either a five laser BD LSRFortessa or a four laser BD LSRII system, equipped with FACSDiva Version 8.8.3 (BD biosciences). Rainbow beads ensured a consistent, comparable level of fluorescence across all samples on different days of acquisition. Gates were set using fluorescence minus one or unstimulated samples where appropriate. The data were analysed using FlowJo version 9.5.3 (Treestar, OR, USA).

Cryopreserved peripheral blood mononuclear cells (PBMC) were stained with combinations of antibodies that comprehensively interrogate the breadth of NK-cell receptors including the killer immunoglobulin receptors (KIRs), the c-type lectin receptors (NKG2), and the natural cytotoxicity receptors (NCRs). These included CD158a-PerCP-Cy5.5 (eBioscience), KIR3DL1-Alexafluor700 (Biolegend), NKG2A-PE (Beckman Coulter), NKG2D-APC, CD94-FITC, 2B4-FITC, NKp46-PE, NKp30-APC, CD158b-FITC (all BD Biosciences), TRAIL-PE (Biolegend), CD161-FITC (Miltenyi) and CD160-Alexafluor647 (Biolegend). Dead cells were excluded using the Fixable Blue Dead Cell Stain (Life Technologies) prior to surface staining and fixation. For perforin staining, samples were then permeabilized (Perm A/B, Caltag) and stained with anti-Perforin-PerCP-Cy5.5 (eBioscience). NK cells were identified as CD3 negative lymphocytes expressing CD56 and/or CD16. At least 1500 NK cells were acquired per sample. Fluorescence minus one was used to set gates. The data were analyzed with Flowjo v9.5.4 (Treestar).