Ctla 4

In vitro induced T_reg (iTreg) cells were differentiated from naive CD4⁺ T cells and activated by precoated 5 µg/mL CD3 Monoclonal Antibody (OKT3) (eBioscience,16-0037-85) overnight at 4 °C. The coating plate first washed once with phosphate-buffered saline to remove unbound soluble CD3 antibodies, followed by adding resuspended naïve CD4⁺ T cells at 3 × 105 cells per mL in culture medium (1,640 + 10% fetal bovine serum [FBS]) including 2 µg/mL CD28 Monoclonal Antibody (CD28.2) (eBioscience, 16-0289-85), 10 ng/mL IL-2 (PeproTech, 200-02-B), and 20 ng/mL TGF-β (R&D, 240-B-002). Two hundred microliters of cells suspension was added per well in a 96-well plate (6 × 10⁴ cells per well), and cultured at 37 °C for 4 d. Naive CD4⁺ T cells were isolated from human PBMCs using the MACS human naive CD4 T-cell isolation kit II (Miltenyi Biotec, 130-094-131). Generated iTreg cells were confirmed using a staining antibody mixture against human CD4 (Biolegend, 317408), CD25 (Biolegend, 356108), CTLA-4 (Biolegend, 349908), and FoxP3 (Biolegend, 320208). Pan-T cells were isolated from human PBMCs using a Pan T Cell Isolation Kit (Meltenyi, 130-096-535). The obtained pan-T cells were confirmed using staining mixture against human CD3 (Biolegend, 300406) and CTLA-4 (Biolegend, 369611). The iTreg and pan-T cells were labeled with Calcein AM (Invitrogen, C34851) and used in the ADCC assay.