Soluble anti cd3 and anti cd28 antibodies

Transwell co-culture experiments were performed in 24-well plates with 0.4 μm pore sizes in inner wells (Greiner, Frickenhausen, Germany) to physically separate CD8⁺ T cells and SKOV3/A2780 cells. Approximately 2 × 10⁵ SKOV3/A2780 cells per well were grown in the outer wells using 1.5 mL 1640-medium containing 10% human AB serum. Next, 6 × 10⁵ isolated human CD8⁺ T cells were added into the inner wells in 500 μL of the same medium. For TGF-β1 neutralization experiments, 10 μg/mL anti-TGF-β1 neutralizing antibody (R&D Systems, Minneapolis, MN, USA) was added to the SKOV3/A2780 co-culture system to block TGF-β1 function. After 5 days incubation, CD8⁺ T cells were harvested and washed with PBS. Approximately 1 × 10⁶ cells were collected for flow cytometry analysis, and 2 × 10⁶ cells were collected for western blot analysis. The remaining cells were dispensed into 96-well plates at 4×10⁴ cells per well and stimulated with 1 mg/mL soluble anti-CD3 and anti-CD28 antibodies (eBioscience, San Diego, CA, USA). After 6 and 24 h stimulation, CD8⁺ T cells were collected for evaluation of cytokine mRNA expression levels by RT-PCR.