Molecular Basis of Th1 Development

这个项目代表了定义Th1发育的分子基础的努力。在基础水平上对Th1发展进行分析的动机原理源于CD4 T细胞，特别是Th1细胞在包括1型糖尿病在内的几种自身免疫性疾病中发挥的核心作用。这里描述的项目是根据上一个筹资周期取得的进展所产生的广泛初步数据。在对IL-12信号通路的研究中，我们认识到Th1的发育首先是Stat4依赖阶段，然后是Stat4独立阶段。后一阶段代表更完全分化的Th1细胞，其中TCR单独激活后可以产生高水平的IFNgamma，而不通过IL-12信号通路。重要的是，该结果直接表明，在Th1发育的这一阶段，Stat4激活是诱导IFNgamma产生的一个极其微弱的信号，并且它对TCR激活产生的IFNgamma贡献很小。因此，我们得出了这样的假设，即Stat4的作用是引起Th1特异性转录因子的表达，这些转录因子在响应TCR衍生信号时直接促进IFNgamma的产生。因此，我们得出的假设是，Stat4的作用是引起Th1特异性转录因子的表达，然后直接促进IFNgamma的产生，以响应TCR衍生的信号。我们已经开始使用几种克隆方法来鉴定这些转录因子。我们的初步数据表明，我们已经确定了一种这样的转录因子，ERM，我们证明它是由IL-12单独通过Stat4激活诱导的。我们还开发了一种重要的方法，将有助于我们分析Th1的发展和这些转录因子的作用。也就是说，我们现在已经开发出一种技术，可以通过逆转录病毒以非常高的效率感染原代T细胞，并表达感兴趣的转录因子。我们证明，在stat4缺陷T细胞中表达ERM可以显著增加其IFNgamma的产生。我们建议使用现有的方法以非常直接的方式跟踪这些发现。我们将进行一项分析，以全面表征ERM在Th1发育中的作用，并将继续寻找其他stat4诱导因子。此外，我们将分析Stat4激活基因特异性的基础，并定义T激活与其他STATs（特别是Stat1）之间的差异。最后，我们将重点放在小鼠和人类系统在Stat4激活方面的特定差异上，我们认为这是一个需要解决的重要问题，因为小鼠系统被大量用作人类疾病的模型。

This Project represents an effort to define the molecular basis of Th1 development. The motivating rationale for pursuing an analysis of Th1 development at a fundamental level stems from the central role that CD4 T cells, particularly Th1 cells, play in several types of autoimmune diseases, including the type 1 diabetes. The project described here is based on extensive preliminary data that have resulted fro, progress made in the previous funding cycle. While pursuing studies of the IL-12 signaling pathway, we recognized that Th1 development proceeds to first a Stat4-dependent, and then a Stat4- independent phase. This latter phase represents more fully differentiated Th1 cells in which high IFNgamma production can occur after TCR activation alone, without contribution through the IL-12 signaling pathway. Importantly, this result directly showed that in this phase of Th1 development, Stat4 activation is an extremely weak signal to induce IFNgamma production, and that it contributes very little to the IFNgamma produced by a TCR activation. Therefore, we arrived at the hypothesis that the role of Stat4 was to cause the expression of Th1- specific transcription factors which them directly promote the production of IFNgamma in response to TCR derived signals. Therefore, we arrived at the hypothesis that the role of Stat4 was to cause the expression of Th1- specific transcription factors when then directly promote the production of IFNgamma in response to TCR derived signals. We have set about to identify such transcription factors using several cloning approaches. Our preliminary data show that we have identified one such transcription factor, ERM, which we demonstrate is induced by IL-12, through Stat4 activation alone. We have also developed an important methodology that will facilitate our analysis of Th1 development and the role of these transcription factors. That is, we have now developed the technique to infect primary T cells by retrovirus at very high efficiency and express the transcription factors of interest. We demonstrate that expression of ERM in Stat4-deficient T cells can significantly augment their IFNgamma production. We propose to follow these findings in very direct ways using established methods. We will carry out an analysis to completely characterize the role of ERM in Th1 development, and will continue to search for additional Stat4-induced factors. Furthermore, we will analyze the basis for the specificity of gene activation by Stat4, and define the differences between T activation compared to other STATs, notably Stat1. Finally, we focus on a specific difference between the mouse and human systems in the activation of Stat4, which we argue is an important issue to resolve, since murine systems are used heavily as models for human diseases.