Coculture of ECs and CD3+ T cells
CD3+ cells were sorted from BMMNCs with a CD3 MicroBead kit (Miltenyi Biotec, NRW, Germany), and were cocultured with 7-day primary cultured ECs in a direct contact way at a 1:10 number ratio. The previous medium for ECs was changed to 1 ml RPMI medium (Gibco) containing 10% FBS per well for coculture.
Immunophenotype analysis of the cocultured T cell subsets
After 3 days of coculture, CD3+ T cells were treated with cell stimulation cocktail (500×, eBioscience, CA, USA) as the manufacturer’s instructions to induce CD3+ T cells activation and cytokine secretion. Then, we evaluated the IL-4, IFN-γ, IL-17 and Foxp3 levels by flow cytometry using the hereinafter monoclonal antibody panel: CD3-APC-H7, CD8-V500, CD25-PE-Cy7, IL-4-PE, IFN-γ-Percpcy5.5, IL-17-FITC, and Foxp3-APC (BD Biosciences)[5, 28-30]. The immunophenotyping of T cell subsets including Th1, Th2, Th17, Tc1, Tc2 and regulatory T cells (Tregs) were in keeping with our previous studies[5, 28-31].