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.
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