Cd3 cd28 t cell activation dynabeads

White blood cells from healthy donors were obtained from the Blood Center of Shanghai (Shanghai, China). Human PBMCs were isolated from whole blood by Ficoll-Paque gradient separation (GE Healthcare, Boston, MA, United States). CD3⁺ T cells were enriched by negative immunomagnetic bead selection according to the manufacturer’s protocol (Miltenyi Biotec, Germany). Isolated CD3⁺ T cells were stimulated with CD3/CD28 T-cell activation Dynabeads (Gibco) for 48 h. For infection, 1 × 10⁶ stimulated CD3⁺ cells were transduced with concentrated pseudovirus supernatant (MOI = 10) plus 7 μg/ml polybrene (Yeason) for 12 h, and then, the second round of infection was carried out with the same procedure. T cells were expanded in complete T cell medium containing 90% vivo15 (Lonza, Basel, Switzerland) supplemented with 10% FBS and 100 U/ml penicillin/streptomycin. Cells were also fed with 5 ng/ml IL-2 (R&D, St. Paul, MN, United States), 2 ng/ml IL-7 (R&D) and 2 ng/ml IL-15 (R&D) every 48 h. 4-7 days after LV transduction, CAR⁺ cells were enriched by staining with FITC-conjugated goat anti-human F(ab’)₂ antibody (Jackson ImmunoResearch Laboratories, West Grove, United States) and sorted by a FACS Fusion I (BD Biosciences, Grand Island, NY, United States). Genetically modified T cells were used for functional assays after sorting.