Human recombined human tgf β

Magnetic beads isolated peripheral T lymphocytes, tumour-infiltrating T lymphocytes, and various genes modified T cells were expanded with Dynabeads Human T-Activator CD3/CD28 (ThermoFisher, CA). For in vitro generation of Treg cell, naive T cells were isolated by human naive T-cell isolation kit II (Miltenyi, Koln, Germany), and further stimulated with human recombined human TGF-β (1 ng ml⁻¹) (Peprotech, NJ) for 7 days. For detection of intracellular cytokines, T cells were stimulated at 37 °C for 5 h with Leukocyte Activation Cocktail (BD Pharmingen). Dead cells were excluded based on staining with Live/Dead fixable dye. Thereafter, cells were, fixed, permeabilized with IntraPrep reagent (BD Pharmingen), and then stained with flurochrome-conjugated labelled antibodies including CD4 (20 μl per test555346), CD8a (20 μl per test, 561949), CD25 (20 μl per test, 555432), Foxp3 (20 μl per test, 560082) and IFN-γ (20 μl per test-559327) (BD Pharmingen). Data were acquired on BD FACSVerse flow cytometer (BD Pharmingen).