Tgf 1

Blood (120 ml) was collected by venepuncture from two times four donors for RNA-IC and OOPS. Peripheral blood mononuclear cells (PBMCs) were isolated by standard Ficoll gradient (Pancoll R ) centrifugation and CD4 + T cells were isolated from 1x10 8 cells using CD4 + Microbeads (Miltenyi) to arrive at 2-3x10 7 cells. These were resuspended at 2x10 6 /ml in T cell medium ((AIM-V (Invitrogen), 10% heat-inactivated human serum, 2mM Lglutamine, 10 mM HEPES and 1,25 µg/ml Fungizone), supplemented with 500 ng/ml PHA (Murex) and 100 IU IL-2/ml (Novartis). Cells were dispensed in 24-well plates at 2 ml per well and on day 3 old medium was replaced by fresh T cell medium and cell were harvested and counted at day 4,5. For generating iTreg, naïve CD4 + T cells were isolated from PBMCs using Microbeads (Naive CD4+ T Cell Isolation Kit II, Miltenyi) and resuspended at 1x10 6 cells/ml in T cell medium supplemented with 500 ng/ml PHA (Murex), 500 IU IL-2/ml (Novartis), 500 nM Rapamycin (Selleckchem), and 5 ng/ml TGF-ß1 (Miltenyi). Cells were cultured in 24-well plates at 2 ml per well together with 1x10 6 irradiated (40 Gy) PBMCs derived from three donors. On day 3, old medium was replaced by fresh T cell medium including supplements and the cells harvested and counted at day 5.