Vegf a

For primary CD4⁺ T-cell isolation, PBMCs were acquired utilizing Ficoll Paque Plus (GE Healthcare, USA) gradient cell separation according to the manufacturer’s instructions. Next, the CD4⁺ T Cell Isolation Kit, human (Miltenyi Biotec, Germany) was utilized to isolate CD4⁺ T cells. Then, the cells were resuspended at 1 × 10⁶ cells/mL and cultured in advanced RPMI1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% 2-mercaptoethanol (2-ME; Gibco) and 1% penicillin–streptomycin (Gibco) in 48-well plates precoated with 2 μg/mL anti-human CD3 antibody (BioLegend, USA). For VEGF-R-related assays, after incubation with 5 μg/mL anti-VEGF-R1 antibody (R&D Systems, USA) and/or 10 μg/mL anti-VEGF-R2 antibody (Abcam, USA) for 1 h, CD4⁺ T cells were treated with blank (CON) or 15 ng/mL VEGF-A (R&D Systems, USA) for 2 days respectively. GolgiPlug Protein Transport Inhibitor (BD Bioscience, USA) was administered for the last 6 h to detect the cytotoxic molecules. Regarding AKT/mTOR inhibition assays, CD4⁺ T cells were treated with blank (CON), 15 ng/mL VEGF-A, VEGF-A + 10 μM MK-2206 2HCl (AKT inhibitor, MedChemExpress, USA), VEGF-A + 100 nM rapamycin (mTOR inhibitor, Selleck, China) or VEGF-A + 10 μM MK-2206 2HCl + 10 μM MHY1485 (mTOR activator, MedChemExpress, USA) for 2 days in the presence of GolgiPlug Protein Transport Inhibitor for the last 6 h.

Cells were cultured in medium contained exogenous VEGFA (100 ng/mL, R&D, USA) and bevacizumab (inhibitor of VEGFA, 125 μg/mL, Selleck, USA), respectively.