Cd16 56 pe

Cells (1×10⁶ PBMCs) were incubated for 10 min at room temperature with 5 μL of the following fluorochrome-conjugated antibodies: CD3-FITC, CD19-PE, CD8-PE, CD4-APC, CD16,56-PE, CD4-FITC and/or CD25-APC (all from BD Biosciences, USA). The cells were washed with FACS buffer (phosphate-buffered saline, 0.2% fetal bovine serum, 0.02% sodium azide) and centrifuged for 5 min at 500 ×g. For analysis of FoxP3 expression, the cells were permeabilized by 300 μL of FACS permeabilizing solution (BD Biosciences) for 10 min at room temperature in the dark, washed, and labeled with 5 μL FoxP3-PE antibody for 10 min at room temperature (eBioscience, USA). Isotype controls were used for each staining procedure as negative controls and for compensation of fluorescence. Thirty thousand events were acquired for each cell subset analysis and 100,000 events for regulatory T-cell analysis, using a FACSort flow cytometer (BD Biosciences). The CD127lo marker was not evaluated in this study.

Whole blood was drawn in EDTA tubes and the cells were incubated with fluorochrome-conjugated antibodies against human CD3-FITC, HLA-DR-FITC, CD45RA-FITC, CD8-PE, CD16+56-PE, CD69-PE, CD28-PE, CD3-PerCP, CD45-PerCP, CD45RO-PerCP-Cy5.5, CD4-PE-Cy7, CD25-PE-Cy7, CD25-APC, CD39-APC, CD62L-APC, CD8-APC-H7, CD4-APC-H7, CD3-Horizon V450, CD56-Horizon V450, all from BD Biosciences (San Jose, USA). Anti-Foxp3-PE was purchased from eBioscience (San Diego, USA) and Helios-Pacific Blue from Biolegend (San Diego, USA). Lymphocyte subpopulations from peripheral blood were measured by 4- or 7-color combinations. TruCount^™ tubes (BD Biosciences) were used for assessment of absolute numbers (cells/μl) of leukocytes (CD45), and major lymphocyte populations; T lymphocyte (CD3), T helper (CD4), T cytotoxic (CD8), B (CD19) and NK (CD16+CD56) cells, as described by the manufacturer. Absolute numbers of subpopulations were derived from the major populations. Proportions of major populations were given as % of lymphocytes and proportions of subpopulations were given as % of their parent population.

Absolute numbers and percentage of monocytes and lymphocyte subsets were determined with flow cytometry (FACS Calibur) on whole heparinized blood. Quantification beads (Trucount, BD) in combination with CD45 fluorescein isothiocyanate (FITC), CD14 phycoerythrin (PE), CD3 peridinin chlorophyll protein (PerCP) and CD19 allophycocyanin (APC) were used to measure absolute numbers of lymphocytes, monocytes, B cells and T cells according to the manufacturer’s instructions. Absolute numbers of cell subsets were calculated using the percentage of cells within a main cell type that was measured with quantification beads. For the subsets CD45 and CD3 or CD19 were always taken in multiple tubes combined with the following markers: CD8 PE, CD4 APC, CD16/56 PE, CXCR3 APC, CD80 PE and CD27 FITC (all products from BD Biosciences, San Jose, CA, USA). Flow cytometry data were analysed using FACSDIVA software version 6.1.3. Forward, sideward scatter and CD45^bright were used to select lymphocytes. CD16⁺CD56⁺CD3⁻ cells were defined as natural killer (NK) cells and CD16/CD56⁺ CD3⁺cells are expected to be predominantly CD56⁺CD16⁻. As CD56⁺CD3⁺ T cells, also described as NK T-like cells, have been described as activated effector cells [7 (link), 8 (link)] we define them here as activated T cells. Gating strategy is depicted in Additional file 1: Figure S1.