To evaluate the function of Tregs in the presence of IL-12, responder T cells were enriched from naïve B6/Thy1.1 mice using a Pan T cell Isolation Kit II, and Tregs were isolated from B6 mice using a CD4 + CD25 + Regulatory T cell Isolation Kit (Miltenyi Biotec). For suppression assays, Tregs were mixed with CFSElabeled (2.5 mM) responder T cells at the indicated ratios (Tregs plus responders = 5610 4 cells/well). Cells were cultured in the presence of 2610 5 irradiated splenocytes and anti-CD3 mAb with or without IL-12 in 96-well round bottom plates. After 66 hr, Thy1.1 + Tconvs and Thy1.1 + CD8 T cells were analyzed for CFSE dilution by flow cytometry. The Division Index (DI) was obtained using FlowJo software (Tree Star, Inc., Ashland, OR). A normalized DI was calculated as follows: % normalized DI = 100% 6(DI of responders plus Tregs/DI of responders only).