Anti human cd3 okt3

The anti-Fas (IgM) antibody, anti-phospho-LAT-Tyr226, and rabbit polyclonal anti-human LAT were from Merck-Millipore; anti-PLC-γ1 mAb, anti-Lck, and anti-Erk were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies binding phospho-Erk, phospho-PLC-γ1-Tyr783, phospho-ZAP70-Tyr319, phospho-MEK-Ser221, phospho-LAT-Tyr191, and anti-MEK were from Cell Signaling Technology; anti-6His-HRP, anti-Grb2, and anti-phospho-LAT-Tyr132 antibodies were from Abcam (Cambridge, MA, USA); and anti-CD69-APC (allophycocyanin) and anti-β-actin monoclonal antibodies were from Biolegend. The protein synthesis inhibitor cicloheximide was purchased from Merck-Millipore. Stimulations were performed with anti-human CD3 (OKT3; eBioscience) or anti-human CD28 (CD28.2; BD Pharmingen) monoclonal antibodies.

Monocyte purity, proper Mo-DC differentiation and the basal state of activation of both cells were assessed by flow cytometry using the following anti-human monoclonal antibodies: CD14 (M5E2, Biolegend, San Diego, CA, USA), CD1b (SN13, Biolegend), CD1d (51.1, Biolegend), CD11c (3.9, eBioscience), CD80 (2D10, Biolegend), HLA-DR (LN3, eBioscience).
iNKT and T cell determinations were performed in total PBMCs or in CD14⁻ fractions, using CD1d-PBS57 tetramers (NIH Tetramer Core Facility, Emory University, Atlanta, GA, USA) and the following anti-human monoclonal antibodies: CD3 (OKT3, eBioscience), CD4 (OKT4, Biolegend), and CD8 (RPA-T8, eBioscience). The purity of T cell clones VM-D5 and JS63 was assessed by using CD1d-PBS57 tetramers (NIH Tetramer Core Facility, Emory University, Atlanta, GA, USA) together with anti-human CD3 (OKT3, eBioscience) monoclonal antibody. The purity of T cell clones s33d, GG33A, and DS1C9b was assessed by using anti-human TCR Vβ13.1 (IMMV222), Vβ18 (BA62.6), and Vβ7.1 (ZOE), monoclonal antibodies from Immunotec (Immunotec Research Inc, Canada). Cells were acquired in a FACS Canto II (BD Biosciences, San Diego, CA, USA) using the BD FACSDiva™ software (BD Biosciences). Data analysis was performed with FlowJo® v10 (FlowJo LLC, Ashland, OR, USA).

Isolated PBMCs were activated using anti-human CD3 (OKT3) and anti-human CD28 antibody (CD28.2) (eBioscience, USA) at the final concentration of 0.5-1x10⁶ cell/well in a 24-well culture plate. Recombinant IL-2 was added at a final concentration of 200 IU/ml and incubated for 72 hours. Activated T cells were transduced overnight at the final concentration of 0.5-1x10⁶ cell/well in a retronectin coated 24-well culture plate (20 ug/ml of retronectin in DPBS for 2 hours, room temperature) using either second (In house) or third generation LVV (VIVE Biotech, Spain). Transduced T cells were then expanded for seven days in the presence of 200 IU/ml of recombinant IL-2. Cells were then harvested and stored in the gas phase of the liquid nitrogen until used.