Anti cd28 mab

PBMCs were activated in plates coated with 1 µg/mL anti-CD3 mAb (clone: OKT3, Miltenyi Biotec) and cultured in the presence of 3 µg/mL anti-CD28 mAb (clone: 15E8, Miltenyi Biotec), 25 U/mL human IL-7 (Miltenyi Biotec) and 50 U/mL human IL-15 (Miltenyi Biotec) for 3 days. Afterward, 8 × 10⁴ activated human PBMCs were transduced with 0.5 µL VSV-LV (MOI 4–5) via spinfection (at 850 × g, 90 min and 32 °C) and further cultivated for 3 days.

Human peripheral blood mononuclear cell (PBMC) samples were obtained from healthy donors isolated by using MACSprep™ PBMC Isolation Kit (Miltenyi Biotec, Germany), according to the manufacturer’s instructions. Written informed consents were obtained from all volunteer. In brief, The PBMCs were activated for 48 h in 24-well tissue culture–treated plates (2 × 10⁶/well) with anti-CD3 mAb (Miltenyi, Biotec, 1 μg/ml) and anti-CD28 mAb (Miltenyi, Biotec, 1 μg/ml) in a complete medium containing 90% RPMI 1640 and supplemented with 10% FBS (Gibco), 200 U/ml IL-2, 25 mM HEPPES, 55 μM 2-M, 100 U/ml penicillin, and 100 μg/ml streptomycin. After 2 days, the 2 × 10⁵ activated T cells were infected with 250 μL of CAR-encoding lentiviral supernatant in a tissue culture–treated 24-well plate. After lentiviral infection for 24 h, the lentiviral supernatant was replaced with a fresh complete medium, and cell culture were maintained at 37°C with 5% CO₂. On the 7th day after transfection, the T cells were collected for subsequent experiments.

Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors (n = 4) after informed consent, isolated by using MACSprep™ PBMC Isolation Kit (Miltenyi Biotec, Germany), according the manufacturer’s instructions. The PBMCs were activated for 48 h in tissue culture-treated 24-well plates (2 × 10⁶/well) with anti-CD3 mAb (Miltenyi, Biotec, plate-bound, 1 ug/ml) and anti-CD28 mAb (Miltenyi, Biotec, soluble, 1 ug/ml) in complete medium (90% RPMI-1640 supplemented with 10% FBS (Gibco), 2 mM L-glutamine, 25 mM HEPES, 55 μM 2-M,100 U/ml penicillin, 100 μg/ml streptomycin and different cytokine combinations), and various cytokine conditions as shown in Figure 1B. After 2 days, 2 × 10⁵ activated T cells were transfected with 500 μL the CAR-containing lentiviral supernatant in 24-well tissue culture-treated plates, and the supernatant was replaced with the fresh corresponding medium at 24 h after transfection. Culture medium change with fresh addition of cytokines was performed every other day. The T cells were transferred to 12-well tissue culture plates or 6-well tissue culture plates depending on the total cell number, and cell cultures were maintained at 37°C with 5% CO₂. On the 12th day after transfection, the T cells were collected for subsequent experiments.