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].