In specific, IFN-c is expressed by virus-specific Tregs in the CNS of mice infected with the rJ2.2 strain of mouse hepatitis virus (MHV) [8] ; we used information obtained from this experimental system as the basis for the approach described here. To determine whether cytokines had a role in IFN-c expression by Tregs, we initially focused on cytokines that are up-regulated in the CNS of MHV-infected mice and remain elevated as the infection resolves [15] . Among these cytokines, IL-12, IL-6 and IFN-c have roles in T cell polarization and Treg development [16] [17] [18] [19] [20] , so we first evaluated their participation in Treg-mediated IFN-c expression. Lymphocytes were prepared from the lymph nodes of naïve B6 mice, labeled with CFSE and treated with each cytokine and anti-CD3 mAb for 66 hours. Cells were then stimulated for 4 hours with PMA and ionomycin and evaluated for T cell proliferation and IFN-c expression.