Inhibitory activity of Treg cells from NOD and B6 mice at the effector phase. (A) IL-2/anti-IL-2mAb complexes enhanced the immmunosuppressive activities of endogenous regulatory T-cells at the effector phase. (B) Impact at the effector phase of varied numbers of Treg cells isolated from B6 mice treated with IL-2/anti-IL-2mAb complexes. (C) Impact at the effector phase of varied numbers of Treg cells isolated from NOD treated with IL-2/IL-2mAb complex. 8.3 CD8 T cells were isolated from TCR transgenic 8.3 mice using the CD8+ T cell isolation kit from Miltenyi. They were then in vitro activated for 3 days with CD3/CD28 Dynabeads and human IL-2. On the day of the assay, Treg cells from mice untreated or treated with IL-2/anti-IL-2mAb complexes for 3 days (i.p) were isolated using the CD4+CD25+ Treg isolation kit from Miltenyi. The 8.3 CD8 T cells activated in the culture were separated from Dynabeads by magnet. NIT-1 cells were plated at 2000/well (50 μl), equal to 4x104 cells/ml. 8.3 CD8+ T cells were used at an E/T ratio of 5:1. Various concentrations of Treg were mixed with the 8.3 CD8+ T cells and then added to NIT-1 cells. After overnight incubation, Treg cells and 8.3 CD8+ T cells were removed by washing three times with cell culture medium while adherent NIT-1 cells remained in the testing wells. Subsequently, the viability of remaining NIT-1 cells was measured by the addition of 200 μl of 50% Cell Titer Glo. Mean and SD of triplicate samples were shown; and the data shown are representative of three independent experiments.