Human t activator cd3 cd28 beads

Human peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll density centrifugation from healthy anonymous blood donors. CD4+ T cells were negatively selected using magnetic beads as per manufacturer’s instructions (Human CD4+ T Cell Isolation Kit, Stemcell Technologies, Vancouver, BC, Canada; Cat.# 17952). CD4+ T cells were cultured in RPMI 1640 supplemented with 10% FBS, 100 I.U. Penicillin, 100 μg/mL Streptomycin, 2 mM L-glutamine, and 30 Units/mL of rIL-2 (Peprotech, Rocky Hill, NJ, USA; Cat.# 200-02). Cells were stimulated with Human T-Activator CD3/CD28 Beads (Thermo Fisher Scientific, Waltham, MA, USA; Cat.#11132D) following manufacturer’s instructions and expanded in the presence of 30 Units/mL of rIL-2. Cells were infected by spinoculation at 1200× g for 1.5 h in multi-well plates. Experiments with primary T-cells were repeated with cells from eight different donors. CD14+ monocytes were isolated using magnetic beads as per manufacturer’s instructions (EasySep Human Monocyte Isolation Kit, Stemcell Technologies, Vancouver, BC, Canada; Cat.# 19359). Primary macrophages were further differentiated from the purified monocytes with recombinant human GM-CSF (10 ng/mL).

For each shRNA construct, lentiviral supernatants from three 100-mm dishes of HEK293 cells (10 ml each) were pooled at 48 h post-transfection, filtered through a 0.45-μm syringe filter and subjected to ultracentrifugation at 23,000 × g (SW28 rotor) for 90 min at 4°C. Viral pellets were gently resuspended in 300 μl serum-free DMEM medium and either used immediately for transductions or snap-frozen in liquid nitrogen and stored at −80°C for future use. Briefly, human peripheral CD4⁺ T cells were isolated from blood by negative selection using the Dynabeads Untouched Human CD4 T-cell isolation kit (Life Technologies Corp.). 2.5 × 10⁷ CD4⁺ T cells were stimulated per well of a 6-well plate by plate-bound CD3 mAb (UCHT-1, 10 μg/ml), soluble CD28 mAb (CD28.2, 1 μg/ml) and recombinant human IL-2 (50 U/ml; AbDSerotec) for 5 h prior to transduction. Cells were then harvested, suspended in 800 μl RPMI, 10% FBS plus 200 μl concentrated lentiviral supernatant, 5 μg/ml Polybrene, 1 μg/ml CD28 mAb and 50 U/ml IL-2 and reapplied to a CD3 mAb coated 6-well for overnight incubation. Cells were harvested next day and incubated with Human T-activator CD3/CD28 beads (Life Technologies) at a bead:cell ratio of 1:5 in the presence of 50 U/ml IL-2.

Human peripheral blood mononuclear cells (PBMC) were purchased from SAILY Biotechnology (Shanghai) Co., Ltd. (Shanghai, China) To obtain pre-activated T cells (ATC), 5 × 10⁶ PBMC were resuspended in 10 mL complete RPMI-1640 medium containing 30 U/mL human IL-2 (R&D Systems, Minneapolis, MN, USA) and 1 × 10⁷ human T-activator CD3/CD28 beads (Gibco, Grand Island, NY, USA) at 37 °C with 5% CO₂ and 95% humidity for 5–7 days.