T helper type 1 (Th cells are of critical importance in regulating immune responses in health and disease states. Altering the cytokine-producing potential of polarized Th1 cells has been proposed as a potential therapeutic intervention for many autoimmune, allergic and infectious diseases. However, the phenotype of polarized Th cells is generally difficult to alter. We conducted a series of experiments to explore the molecular basis for this stable Th1 cell phenotype. Using an in vitro Th cell differentiation system, we found the following: I) naive CD4+ T cells from IFNgammaR-/- mice can differentiate into IFNgamma producers, but failed to develop further into committed Th1 cells (They retained their IL-4-producing potential); 2) failure of IFNgammaR-/- Th1 cells to commit cannot be corrected by prolonged exposure to IL- 12; and 3) IL-4-induced STAT6 phosphorylation and GATA3 expression was significantly enhanced in IFNgammaR-/- Th1 cells. Based on those observations, we hypothesize that IFNgamma mediates Th1 cell commitment by suppressing the Jak/STAT6 signaling pathway in a STAT1-dependent manner.
Three specific aims are designed to test our hypothesis.
Aim 1. Determine whether IFNgamma mediates Th1 cell commitment. IFNgamma producing CD4 T cells will be isolated from wild type and IFNgammaR-/- Th1 cell cultures by IFNgamma affinity matrix technique and single cell cloning. The sorted cells will be further tested whether they have committed into a stable Thl phenotype. To examine whether the IFNgamma effects are direct, we will restore IFNgammaR for IFNgammaR-/- Th1 cells and determine whether restoration of IFNgammaR stabilizes IFNgammaR-/- Th1 cells.
Aim 2. Investigate whether Jak-STAT6 signaling pathway is the target for IFNgamma mediated inhibition of the IL-4-producing potential in committed Th1 cells. We will compare IL-4 receptor signaling components in wild-type and IFNgammaR-/- Th1 cells, Immunoprecipitation and western blot analysis will be used to examine whether IL-4-induced phosphorylation of IL-4R alpha chain, Jak 1, Jak3 and STAT6 is greater in the IFNgammaR-/- Th1 cells. To examine whether IFNgamma can suppress STAT6 phosphorylation directly, we will add IFNgamma back to IFNgamma-/- Th1 cells to examine whether IFNgamma can suppress IL-4-induced STAT6 phosphorylation.
Aim 3. Examine the signaling mechanism(s) by which IFNgamma suppresses Jak-STAT6 signaling pathway in committed Th1 cells. STATI-/- Th1 cells will be analyzed to determine whether they exhibit the same phenotype as the IFNgammaR-/- Th1 cells. In addition, constitutively active STAT 1 will be introduced into the IFNgammaR-/- Th1 cells to test whether it can suppress Jak-STAT6 signaling. We will determine the role of suppressors of cytokine signaling (SOCS-l, 3) and protein tyrosine phosphatases and novel genes in suppressing Jak-STAT6 signaling. Information obtained from these studies will redefine the Th1 cell developmental pathway and will elucidate the mechanism(s) by which IFNgamma suppresses IL-4-producing potential in committed Th1 cells, These information will also provide molecular targets for therapeutic interventions for many Th1 cell-mediated diseases.
