To evaluate the function of IFN-c producing Tregs, lymphocytes were prepared from Foxp3 gfp /Thy1.1 mice and stimulated with anti-CD3 mAb in the presence of IL-12 or medium as control. After 66 hr, cells were harvested, washed and enriched for CD4 + T cells using a CD4 T Cell Isolation Kit II (Miltenyi Biotech). GFP + Tregs were then sorted. Approximately .90% and ,3% of Tregs expressed IFN-c in the IL-12 and medium groups, respectively. Responder Tconvs were isolated from naïve Foxp3 gfp (Thy1.2) mice. For suppression assays, Tregs were co-cultured with CFSE-labeled (2.5 mM) responder Tconvs at the indicated ratios (Tregs plus responders = 5610 4 cells/well) in 96-well round bottom plates in the absence of IL-12. Wells also contained 2610 5 T-cell depleted splenocytes (irradiated at 2500 rad) and anti-CD3 mAb. After 66 hr, Thy1.1 2 Thy1.2 + Tconv cells were analyzed for CFSE dilution by flow cytometry.