Inhibitory activity of Treg cells from untreated and IL-2/anti-IL-2 mAb complexes treated NOD mice at the activation phase. As described in the materials and methods, NOD mice were left untreated or treated with IL-2/anti-IL-2 mAb complexes. To investigate the role of Treg cells during the activation phase, freshly isolated Treg cells and 8.3 CD8+ T cells were cultured together in the presence of the anti-CD3/anti-CD28 Ab conjugated Dynabeads and human IL-2 for 3–4 days. On the day of assay (the last day of activation of CD8 T cells), CD8+ T cells were separated from Treg cells by negative selection using the mouse Dynabeads CD4 (L3T4) from Invitrogen. The NIT-1 cells were adjusted to 2000/well. The 8.3 CD8+ T cells were used at an E/T ratio of 5:1. The 8.3 CD8+ T cells and NIT-1 cells plus IGRP peptide were mixed together and incubated overnight (12 to 16 hours). After overnight incubation, 8.3 T cells were excluded by washing three times with cell culture medium while adherent NIT-1 cells remained in the testing wells. Subsequently, ATP contents reflecting the NIT-1 cell remaining viable was measured by the addition of 200 μl of 50% Cell Titer Glo. The data shown are representative of three experiments. (A) Tregs from untreated NOD mice. (B) Similar to A, but Tregs were isolated from IL-2/anti-IL-2 mAb complexes treated NOD mice. Mean and SD of triplicate samples were shown; and the data shown are representative of three independent experiments.