Cd161 percpcy5

For multicolor flow cytofluorimetric analysis, PBMCs, LMNCs and PMNCs were stained with the following conjugated mAb as previously described: CD56-PE-Cy5 and NKp46-PE (Beckman Coulter, clone BAB281), CD16-PE-Cy7, CD19-APCH7, IFN-γ-PE, DNAM-1 PE, CD49e-PE, CD11c-PE, CCR5-PE, CD69-PE, CCR4-PE and CXCR3-PE (BD-Pharmigen), CD45-PB, CD62L-PE-Cy7, CCL5-AF647, CD161-PerCpCy5.5, CCR3-PE, CXCR2-PE and CXCR4-PerCpCy5.5 (Biolegend), CCR7-FITC and CXCR6-PE (R&D System), CX₃CR1 PE (MBL). Aqua LIVE/DEAD (Life Technologies) was used to eliminate dead cells from the analysis. Intracellular staining was performed using Cytofix/Cytoperm (Beckton Dickinson), according to the manufacturers instructions. The gating strategy used to select NK cells from both PBMC and LMNC is depicted in Supplemental Figure 1. For measurement of IFN-γ production, whole PBMC and LMNC were stimulated with 18 hours with 20ng/ml rhIL-12 (R&D Systems) and 200U/ml rhIL-2 (Peprotech). For the final 4 hours, GolgiStop was added (Beckton Dickenson). Flow cytometry data was acquired using an LSR Fortessa (Beckton Dickinson) and data was analyzed using FlowJo Software (Tree Star).

T-cell (CD3/CD4), NK-cell, and B-cell absolute numbers were analyzed by our routine central laboratory using flow cytometry and the TruCount system (Becton & Dickinson, www.bd.com). For analysis of T-regulatory cells and CD3+ CD161+ NK/T cells, whole blood (30 mL) was collected in EDTA tubes at day+30, +90, and +150 after allo-SCT. Peripheral blood mononuclear cells were isolated by density-gradient centrifugation (Pancol^®, www.pan-biotech.com). Cells were stained with the following fluorescence-labeled antibodies: CD20 Pacific Blue and CD161 PerCP-Cy5.5 (www.biolegend.com), CD4 Pacific Blue, CD25 PE-Cy7, CD127 AlexaF700, CD3 APC-H7 (www.bd.com), CD19 Alexa780 APC (www.eBioscience.com), CD45 Pacific Orange (www.lifetechnologies.com).
Flow cytometry was performed using a Gallios^® cytometer (Beckman Coulter, www.beckmancoulter.com). Data were analyzed by Kaluza^® flow cytometry software (Beckman Coulter).