Cd39 apc

For phenotypic analysis of the Tregs, the following anti-human monoclonal antibody were used: CD4/PerCP (BD Biosciences, Heidelberg, Germany); CD25/FITC (BD Biosciences); CD39/APC (eBioscience; San Diego, CA, USA); FOXP3/PE (BD Biosciences); Helios/Horizon (BioLegend, San Diego, CA, USA); CD127/PeCy7 (BD Biosciences). For analysis of surface markers, cells were incubated at 4 °C for 20 min with a monoclonal antibody. For the staining of the intracellular molecules Fixation/Permeabilization buffers (eBioscience) were used. For T-cell subsets, the fresh isolated PBMCs were washed with phosphate-buffered saline (Sigma, München, Germany)/fetal calf serum 10% (Biochrom) at room temperature. For the surface antigens staining to discriminate the T-cell subsets, CD3/PerCp (BD Bioscience), CD4/Amycian (BioLegend, Fell, Germany), CD8/Pacblue (BD Bioscience), CD45RA/FITC (BD Bioscience) and CD45R0/PE (BD Bioscience) antibodies were incubated at 4 °C for 20 min and evaluated by a fluorescence-activated cell sorter (FACS). Activation markers were analysed after 24 h stimulation with the antibody blinatumomab and target cells as described in the topic proliferation assay. Cells were analysed by the BD FACSCANTOII (BD Biosciences, Heidelberg, Germany) and FlowJo Software (Tree Star Inc., Ashland, OR, USA).