Cd3 cd28 tetrameric antibody complex

Production of AFP-specific TCR-T cells is similar to that described previously [33 (link)]. In brief, peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by density gradient centrifugation with Ficoll-hypaque. T cells were purified with T cell isolation kit (STEMCELL Technologies, Cambridge, MA) and stimulated with CD3/CD28 tetrameric antibody complex (STEMCELL Technologies, Cambridge, MA) for 24 h. Activated T cells were transduced with lentiviral particles containing the TCR gene. After 48 h, the expression of TCR was measured by flow cytometry and CD3/CD28 antibody complex was rinsed away. For TCR-T cells culture, RPMI-1640 medium containing 10% FBS and 50 IU/mL of interleukin-2 (IL-2) (Quanqi, Shandong, China) was refreshed every other day.

Human T cells were isolated from the buffy coat of healthy donors by negative selection and activated by the CD3/CD28 tetrameric antibody complex (Stemcell Technologies, Vancouver, Canada) for three days before transducing with lentivirus. The CD3/CD28 antibody complex was rinsed away four days after lv transduction. 10 ng/ml of human recombinant interleukin-2 (rIL-2, PeproTech, Rocky Hill, NJ, USA) was used in the culture system through the process. T cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% heated fetal bovine serum (Gibco), 100 U/mL penicillin (Gibco) and 0.1 mg/mL streptomycin (Gibco), 10 mM HEPAS (Gibco), 1× MEM NEAA (Gibco) and 55 μM 2-Mercaptoethanol (Gibco). When the cell density exceeds 2.5 × 10⁶ cells/ml or the medium turns yellow, split cultures back to a density of 0.5–1 × 10⁶ cells/ml in a culture medium containing 10 ng/ml rIL-2.