Pe conjugated anti human cd69

OKT3 mAb (anti-human CD3; Thermo Fisher Scientific, cat. # MA1-10,175) at a concentration of 1 μg/mL was used to pretreat 96-well plates. Human T cells were negatively selected and purified from PBMCs (AllCells) using an EasySep™ Human T-Cell Isolation Kit (Stemcell, cat. # 17,951). T cells were added to the wells at 1.5 × 10⁵ cells/well and cultured for 24 h and 6 days in the presence of 10 μg/mL anti-PVRIG or control antibody. The control treatment was not precoated with the OKT3 mAb. After 24 h and 6 days, the cells were collected and stained for cell surface markers before flow cytometry analysis. The following antibodies were used in the flow cytometry analyses: AF488-conjugated anti-human CD4 (BioLegend, cat # 300,519), PerCP/Cyanine5.5-conjugated anti-human CD8a (BioLegend, cat. # 300,924), PE-conjugated anti-human CD69 (BioLegend, cat. # 310,906), BV421-conjugated anti-human CD25 (BioLegend, cat. # 356,114), and PE/Cyanine7-conjugated anti-human CD3 (BioLegend, cat. # 300,316).