Immune responses must be balanced in order to provide protective immunity, while preventing damage to surrounding tissues. In organs where the loss of cells can be irreversible, such as the central nervous system (CNS), the need to regulate an immune response is crucial. T regulatory cells (Tregs) are a subset of immune cells with potent immunosuppressive capacity and immunotherapeutic potential. During chronic Toxoplasma gondii infection, the effector T cell response in the CNS must be balanced to prevent immunopathology. The factors that regulate Tregs responses in vivo during T. gondii infection of the CNS, and under neuroinflammatory conditions in general, remain unknown. Our preliminary studies show that Tregs within the CNS during T. gondii infection are Th1 polarized and express CXCR3 and ICOS, which are driven by the Th1 transcription factor, T-bet. The absence of CXCR3 or the blockade of ICOS ligand (ICOSL) leads to the development of immunopathology in the CNS during infection. Our intravital imaging studies reveal that Tregs form long-lasting contacts with CD11c+ cells in the CNS of infected mice. Previous studies have shown that Tregs can exert suppressive function directly on CD11c-expressing dendritic cells (DCs) in vitro12-15; however, the molecules that facilitate the interactions between these cell types in vivo have not been identified. Therefore, we hypothesize that Th1 polarization of Tregs is required for Treg function in the CNS by driving CXCR3 and ICOS expression, which allows Tregs to interact with DCs and exert suppressive function. This hypothesis will be addressed in the following specific aims: Aim 1. Is Treg function in the CNS dependent on Th1 polarization? Studies in Aim 1 will use FoxP3GFPcreERT2 x Tbx21fl/fl mice, which allow for tamoxifen-induced depletion of T-bet from Tregs. Using this system, the behavior and function of T-bet-deficient Tregs will be compared to T-bet-sufficient Tregs. Aim 2. How does the T-bet-dependent chemokine receptor, CXCR3, contribute to Treg migration and function in the CNS? In Aim 2, the cellular sources of CXCR3 chemokines within the brain will be identified using reporter mice; the influence of CXCR3-signals on Treg migration will be tested using CXCR3, CXCL9, and CXCL10 blocking antibodies; and transfer of CXCR3-sufficient Tregs to infected Cxcr3-/- mice will determine whether CXCR3-expressing Tregs can ameliorate immunopathology. Aim 3. How does the T-bet-driven co- stimulatory molecule, ICOS, impact the control of inflammation in the CNS? Studies in Aim 3 will utilize ICOSL blocking antibodies to determine whether ICOS mediates Treg contact with DCs, influences DC function, prevents immunopathology, and promotes Treg viability and suppressive function. The studies outlined in this proposal will address how Th1 polarization influences Treg migration and suppression in the CNS. The findings from this proposal may be broadly relevant to Treg function during neuroinflammation; and, thus will impact the development of therapies to manage inflammation in the CNS.