Cd28 antibody

1 ml of heparinized blood was incubated with 1 µl CD28 antibody (BD Biosciences, Clone CD28.2 RUO) and 50 µl tetanus/diphtheria vaccine (2 I.U. tetanus-toxoid and 0.2 I.U. diphtheria-toxoid; Sanofi Pasteur) 37 °C and 5% CO₂. After 2 h of incubation Brefeldin-A (Sigma Aldrich) was added to a final concentration of 2 µg/ml. After a total of 6 h of incubation, the blood was lysed using RBC Lysis Buffer (BioLegend) for 10 min and pelleted at 280 g for 10 min at room temperature. The samples were washed twice with PBS/BSA and prepared for analysis by flow cytometry.

T cells were cultured in RPMI 1640 with 10% fetal bovine serum, 100 IU/ml penicillin/streptomycin, and 100 IUml IL-2 (Peprotech). T-cell cultures were expanded and maintained by stimulation by coculture with a mixture of irradiated human PBMCs (4x10⁶/mL), purified anti-CD3 antibody (60ng/mL) and purified CD28 antibody (60ng/mL) (both from BD Biosciences).

For TCR-induced degranulation assay, 6 x 10⁵ CIK cells in 100 μl PBS were stimulated with 1 μg/ml CD28 antibody (BD Biosciences), 1 μg/ml CMV-pp65 antigen polypeptides (Miltenyi) and stained with 5μμl PE-CD107a antibody (Miltenyi). For NKG2D-induced degranulation assay, 6x10⁵ CD8+ CIK cells were mixed with 6x10⁶ or 6x10⁵ K562 cells in 200 μl, followed by addition of 5 μl PE-CD107a antibody. After incubation at 37°C for 4 hr, the cells were added 3 ml PBS and pelleted by centrifugation at 400 g for 5 min. The cell pellet was resuspended in 100 μl MACS buffer, added with 20 μl FITC-CD8 antibody (BD Biosciences), and incubated at 4°C in the dark for 30 min. Then, the cells were washed once with 3 ml PBS, resuspended in 500 μl PBS, and subjected to flow cytometry analysis of CD8+ CD107a+ cells. The same procedures were performed for OCT4 and Sox2 polypeptide antigens. The sequence of OCT4 peptide was DVVRVWFCNRRQKGK, while that of Sox2 was DYKYRPRRKTKTLMKKDKYTLPG. The antigenic polypeptides used were synthesized by Shanghai Qiangyao Co. Ltd (Shanghai, China), purified by HPLC to purity of 98%, and stored at -80°C with stock concentration of 5 mg/ml.

Human PBMCs, obtained from healthy volunteer donors (GCRBC, Houston, TX), were stimulated on plate-bound CD3 (1 ng/mL) and CD28 antibodies (1 ng/mL) (BD Biosciences, Mountain View, CA), in media containing 45% Click’s media (Irvine Scientific, Santa Ana, CA), 45% RPMI 1640, 10% fetal bovine serum (Hyclone), 1% L-glutamine (Invitrogen, Carlsbad, CA), IL-7 (10 ng/mL) (PeproTech, Rocky Hill, NJ), and IL-15 (5 ng/mL) (PeproTech, Rocky Hill, NJ). Stimulated PBMCs were transduced with the retroviral vector encoding the CARs. Transduced cells were fed with IL-7 (10 ng/mL) and IL-15 (5 ng/mL) two-three times per week for 12–14 days of culture before subsequent analysis.

To determine the interactions of lymphocyte–neutrophil, lymphocytes (1.0 × 10⁵ per well) were plated in 96-well plates precoated with CD3 (2 μg/ml, BD Bioscience, USA) antibodies and supplemented with CD28 antibodies (5 μg/ml, BD Biosciences, USA) to reverse the hyporesponsiveness of lymphocytes. Neutrophils (2.0 × 10⁵ per well) from wild-type (WT) and PD-L1-deficient (PD-L1^−/−) mice were added to cultures together. Transwell experiments, in which cell–cell contact between neutrophils and lymphocytes, were prevented. Neutrophils were added to the upper chambers of 24-well transwell trays, and lymphocytes were added to the lower chambers. Antibodies (BD Bioscience, USA) were added according to the manufacturer’s protocol. Changes in lymphocyte function after in vitro culture were assessed by FCM.