Regulation of T cell Differentiation

关键细胞内因子，特别是Stat4、T-bet和GATA-3的相互关联变化促进了Th1的发育。在这里，我们发现来自T-bet-/-小鼠的CD4+细胞通过高内源性GATA-3水平向Th2分化倾斜，但如果GATA-3水平在任何早期阶段通过抗il -4阻断IL-4R信号传导来调节，则表现出几乎正常的Th1分化。此外，在这些条件下，T-bet-/-小鼠的Th1细胞通过组蛋白乙酰化和Dnase I超敏检测显示IFNG启动子可及性。在相关研究中，我们发现gada -3对T-bet-/-细胞中Th1分化的负面影响与其抑制Stat4水平的能力有关，因为如果表达Stat4的逆转录病毒阻止了这一点，则可以再次观察到正常的Th1分化。最后，我们发现逆转录病毒T-bet在发育和建立的Th2细胞中的表达导致GATA-3水平的下调。总的来说，这些发现导致了一个T细胞分化模型，该模型认为，除非GATA-3水平或功能受到T-bet的调节，否则幼稚T细胞倾向于通过诱导GATA-3并随后下调Stat4/IL-12Rb2链向Th2分化。因此，在该模型中，T-bet在Th1细胞发育中的主要功能是抑制GATA-3，而不是正向调节IFNG基因。

Th1 development is facilitated by inter-related changes in key intracellular factors, particularly Stat4, T-bet and GATA-3. Here we showed that CD4+ cells from T-bet-/- mice are skewed toward Th2 differentiation by high endogenous GATA-3 levels, but exhibit virtually normal Th1 differentiation provided GATA-3 levels are regulated at any early stage by blockade of IL-4R signaling by anti-IL-4. In addition, under these conditions, Th1 cells from T-bet-/- mice manifest IFNG promotor accessibility as detected by histone acetylation and Dnase I hypersensitivity. In related studies, we showed that the negative effects of GATA-3 on Th1 differentiation in T-bet-/- cells are related to its ability to suppress Stat4 levels, since if this is prevented by a Stat4-expressing retrovirus, normal Th1 differentiation is again observed. Finally, we showed that retroviral T-bet expression in developing and established Th2 cells leads to down-regulation of GATA-3 levels. Collectively, these findings lead to a model of T cell differentiation which holds that naive T cells tend toward Th2 differentiation through induction of GATA-3 and subsequent down-regulation of Stat4/IL-12Rb2 chain unless GATA-3 levels or function are regulated by T-bet. Thus, in this model, the principal function of T-bet in developing Th1 cells is to repress GATA-3 rather than to positively regulate the IFNG gene. Project 2: In previous studies we showed that CD25+(CD4+) natural regulatory cells bear surface TGF-beta in the form of TGF-beta linked to latency-associated protein (LAP)(an inactive form of TGF-beta). This finding, plus the fact that the regulatory function of CD25+ cells could be inhibited in vitro and in vivo with anti-TGF-beta led us to postulate that TGF-beta was an important effector molecule in regulatory T cell suppressor function. In the present study we gather new data supporting the essential role of TGF-beta in CD25+ regulatory cell suppressor activity. First, using confocal microscopy, we showed that cells containing intra-cellular foxp3, a molecule specifically associated with suppressor T cell function are co-extensive with cells bearing TGF-beta. Second,using a novel assay system in which mink lung epithelial cells transfected with a SMAD-responsive promoter driving a luciferase reporter gene is used to detect active TGF-beta on the surface of cells and in solution, we showed that CD25+ T cells express TGF-beta in a functionally active form: upon cell-cell contact with the mink lung indicator cells, CD25+ cells induced activation of the SMAD-responsive promoter and induction of a luciferase signal. Third, we showed that the suppressor function of CD25+ T cells in standard in vitro suppressor assays that depend on the proliferation of CD25- cells as a read-out, is blocked in a dose-dependent fashion by addition of a specific inhibitor of the TGF-betaR1 component of the TGF-beta receptor. This inhibition was seen in an assay in which the T cells were stimulated in the absence of APC's by anti-CD3-coated bead-driven as well as in anti-CD3-APC-driven assay systems. Fourth and finally, we showed that cross-linking of CTLA-4 on the surface of CD25+ T cells with beads coating with anti-CTLA4 or B7-1, led to capping of the TGF-beta at the point of cell-bead contact. These data thus explain previous data that CTLA-4 also plays a role in CD25+ suppressor function.