Isolation and Culture of Human PBMCs and CD4+ T Cells

Human peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation over Ficoll-Hypaque solution (Haoyang Biological Technology Co., Tianjin, China) and stored at -80°C for quantitative real-time polymerase chain reaction (qRT–PCR). Human CD4⁺ T cells were isolated from PBMCs using human CD4 microbeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) as previously described (33 (link)). Human PBMCs and CD4⁺ T cells were cultured with RPMI-1640 medium (Gibco, California, USA) containing 10% foetal bovine serum (Gibco) for transfection. HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% foetal bovine serum (Gibco) at 37 °C in 5% CO₂.