Anti cd3 monoclonal antibody clone okt3

After receiving informed consent, blood (10–20 mL) was collected from each healthy donor in evacuated tubes containing heparin (BD Vacutainer). Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (GE Healthcare) density-gradient centrifugation and then washed three times and plated into 25 cm² flasks at a concentration of 2 × 10⁶ cells/ml in X-VIVO 15 hematopoietic medium (Lonza, 04-418Q) containing human IFN-γ (#300-02, PeproTech). On the next day, anti-CD3 monoclonal antibody (clone OKT3; 16-0037-85, eBioscience) and IL-2 (#200-02, PeproTech) were added to the medium [21 (link)]. Cells were incubated in a humidified atmosphere with 5% CO₂ at 37 °C and changed medium every 2–3 days. After culture for at least 14 days, cells were harvested and a survival rate of >95% was observed and counted via a LUNA automated cell counter (Logos Biosystems, Inc. Annandale, VA, USA). An overview of the CIK cell preparation schedule is shown in Figure 1A.

19305DP, NY-ESO-1-specific CD8 single positive T-cell clones (CD8SP) and NY-ESO-1-specific CD4⁺ T-cell clones (CD4SP) (0.5 × 10⁶) were untreated or stimulated with immobilized anti-CD3 monoclonal antibody (clone OKT3, 5 μg/ml, eBioscience) for 2 h. The cells were harvested and washed with PBS, and then the cell pellets were frozen. RNA extraction and gene expression analyses using Nanostring PanCancer Immune Panel on nCounter Analysis System version 2.6 were performed at the Genomics Shared Resource at Roswell Park Comprehensive Cancer Center. The heat map was generated by the Heatmapper tool (http://bar.utoronto.ca/ntools/cgi-bin/ntools_heatmapper.cgi) or the nSolver Analysis Software version 4.0.