Regulation of T cell Differentiation

T 细胞分化的调节

• 批准号: 7196663
• 负责人: WARREN STROBER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7196663
• 关键词: biological signal transduction; cell cell interaction; cell differentiation; cell line; confocal scanning microscopy; crosslink; cytokine receptors; flow cytometry; genetically modified animals; helper T lymphocyte; immunogenetics; immunomagnetic separation; immunoregulation; interferon gamma; interleukin 12; interleukin 4; laboratory mouse; protein protein interaction; suppressor T lymphocyte; transcription factor; transforming growth factors

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.

Th1的发展受到关键细胞内因子，特别是STAT4、T-bet和GATA-3相互关联的变化的促进。在这里，我们显示了T-bet-/-小鼠的CD4+细胞由于内源性GATA-3水平的升高而偏向Th2分化，但如果GATA-3水平在任何早期阶段都被抗IL-4阻断IL-4R信号调节，则显示出几乎正常的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超敏反应检测到IFNG启动子的可及性。在相关研究中，我们发现GATA-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，因为如果这一作用被表达STAT4的逆转录病毒阻止，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在发育中和已建立的Th2细胞中的表达导致GATA-3水平下调。总而言之，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的水平或功能受到T-bet的调节，否则初始T细胞通过诱导GATA-3并随后下调STAT4/IL-12Rb2链而趋向于Th2分化。因此，在这个模型中，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。 项目2： 在以前的研究中，我们发现CD25+(CD4+)自然调节细胞以与潜伏期相关蛋白(LAP)(一种非活性形式的转化生长因子-β)相连的转化生长因子-β的形式表达表面转化生长因子-β。这一发现，再加上CD25+细胞的调节功能在体外和体内都可以被抗转化生长因子-β抗体抑制的事实，使我们假设转化生长因子-β是调节T细胞抑制功能的重要效应分子。在目前的研究中，我们收集了新的数据，支持转化生长因子-β在CD25+调节细胞抑制活性中的重要作用。首先，使用共聚焦显微镜，我们发现含有细胞内Foxp3的细胞与携带转化生长因子-β的细胞共同扩张。Foxp3是一种与抑制T细胞功能相关的分子。其次，利用一种新的检测系统，将SMAD反应启动子导入水貂肺上皮细胞，驱动荧光素酶报告基因，以检测细胞表面和溶液中活性的转化生长因子-β，我们发现CD25+T细胞以一种功能活跃的形式表达转化生长因子-β：当细胞与水貂肺指示细胞接触时，CD25+细胞诱导SMAD反应启动子的激活和荧光素酶信号的诱导。第三，我们发现在标准的体外抑制试验中，依赖于CD25-细胞的增殖作为读出的CD25+T细胞的抑制功能，通过添加转化生长因子-β受体的转化生长因子-β1组分的特异性抑制剂，以剂量依赖的方式被阻断。这种抑制作用见于在没有APC的情况下用抗CD3包被的微珠驱动的T细胞刺激的实验以及在抗CD3-APC驱动的实验系统中看到的。第四，也是最后一点，我们发现CD25+T细胞表面的CTLA-4与包被抗CTLA-4或B7-1的微球交联，导致了细胞-微珠接触点的转化生长因子-β的封顶。这些数据解释了先前的数据，即CTLA-4也在CD25+抑制功能中发挥作用。