Plate coated anti cd3 antibody

Cryopreserved human peripheral blood mononuclear cells (PBMCs) were purchased from ImmunoSpot (Cleveland, OH). Human CD3⁺T cells were separated from PBMCs using an EasySep Human T cell Isolation Kit (STEMCELL Technologies). For experiments where T cell proliferation was measured, purified CD8⁺T cells were labeled with CellTrace Violet (CTV; Invitrogen) prior to cell culture. The proliferation of CTV-labeled CD8⁺T cells was evaluated by quantification of CTV dilution. The CD8⁺T cells were stimulated with 293FT cells expressing MUC21 in the presence of plate-coated anti-CD3 antibody (Clone: OKT3, BioLegend) or p815-OKT3-tet-MUC21 as artificial antigen presenting cell. IFN-γ secretion in culture medium was analyzed by ELISA (BioLegend).

To investigate the differentiation of iTreg, CD4⁺ naïve T cells were purified using a commercially available isolation kit according to the manufacturer’s instructions (Miltenyi Biotec, Germany) and stimulated with plate-coated anti-CD3 antibody (1 mg/ml, Biolegend, USA) and soluble anti-CD28 antibody (1 mg/ml, Biolegend, USA) in X-VIVO15 medium (Lonza, Switzerland). Three days after plating, TGF-b (1 ng/ml, Biolegend, USA) and interleukin 2 (IL-2, 50 U/ml, Biolegend, USA) were added in the culture, and the cells were continued to cultivate for 2 days. According to different experimental purposes, reagents were added together with the polarization cytokines, including methyl-b-cyclodextrin-cholesterol (cholesterol, 10–20 mg/ml, Sigma, USA), dimethyloxalylglycine (DMOG, 100 mM, MCE, USA), PX-478 (10 mM, MCE, USA), MT (10 mM, Sigma, USA) and IL-1b (100 ng/ml, Biolegend, USA).