Peripheral blood mononuclear cell (PBMC) cultures and stimulation
A total of 5mL whole venous blood was collected in ethylene diaminetetraacetic acid (EDTA) anticoagulant vials from the normal controls. PBMCs were isolated and plated in 24-well plates at 1 × 106 per well density on the day of sample collection, and they were cultured using RPMI1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and 100 U/mL penicillin-streptomycin (Gibco, Carlsbad, CA, USA). Cells were stimulated with the anti-CD3 antibody (0.5μg/mL) and anti-CD28 (0.5 μg/mL) (Miltenyi Biotec, Palo Alto, CA, USA), and then cultured at 37°C in a moist atmosphere containing 5% CO2 for 72 hours.