Molecular Basis for gamma/delta T Lineage Specification

说明（由申请人提供）：双方对？？在胸腺发育过程中，它们的效应命运发生谱系和特异性；然而，我们对控制这些命运决定的发育线索的理解仍然不完整。越来越多的证据表明两者都是？？谱系承诺和效应器命运受T细胞受体（TCR）信号强度差异的影响。TCR信号强度的差异通过诱导E蛋白DNA结合拮抗剂Id3影响命运。虽然E蛋白明显发挥着核心作用，但它们对发育结果的影响几乎肯定是由其他转录因子和控制其表达和功能的细胞外信号调节的。因此，这些命运决定太过复杂，无法通过专注于一个基因或途径来理解，因此需要Murre开创的基于网络的综合方法。这个程序的总体目标是确定如何？？产生不同强度的TCR信号，并了解E蛋白活性和协同dna结合蛋白的最终改变在影响谱系和？？效应的命运。全面了解这样一个多方面的发育过程超出了任何一个实验室的能力范围，因为它需要大量的实验方法来操纵决定命运的线索，并评估它们对命运的影响，无论是在功能上还是在分子上，在体外还是体内。组成该计划的调查人员拥有必要的独特而又互补的技能来做到这一点。Wiest实验室（项目1）已经建立了体内和体外模型，其中可以通过改变TCR信号强度来操纵发育命运。Zuniga-Pflucker博士（项目3）建立了优雅的体外和体内系统，其中Notch和细胞因子输入可以被操纵以评估对效应器命运的影响。Drs。庄（项目2）和Murre（项目4）是E蛋白及其Id家族拮抗剂的遗传和分子分析专家。最后，Murre博士（基因组学核心）将利用他新颖的生物信息学方法来协助所有项目在分子定义关键里程碑？？通过组装围绕E蛋白靶点组装的全球调控网络来发展。总的来说，这些努力承诺通过全面定义控制过程来推动该领域向前发展。从细胞外信号到细胞核内靶标网络的谱系承诺和效应器命运。

DESCRIPTION (provided by applicant): Both the commitment of progenitors to the ?? lineage and specification of their effector fates occurs during development in the thymus; however, our understanding of the developmental cues controlling these fate decisions remains incomplete. Accumulating evidence suggests that both ?? lineage commitment and effector fate are influenced by differences in T cell receptor (TCR) signal strength. The differences in TCR signal strength influence fate by inducing Id3, an antagonist of E protein DNA binding. While E proteins clearly play a central role, their influence on developmental outcomes is almost certainly modulated by additional transcription factors and the extracellular signals that control their expression and function. Accordingly, these fate decisions are too complex to be understood by focusing on one gene or pathway and so require the comprehensive, network-based approach pioneered by Murre. The overall goal of this program is to determine how ?? TCR signals of varying intensities are generated and understand the role that the resultant alterations in E protein activity and cooperating DNA-binding proteins play in influencing lineage and ?? effector fate. Gaining a comprehensive understanding of such a multifaceted developmental process is beyond the scope of any individual laboratory, as it requires facility with numerous experimental approaches to manipulate fate-determining cues and assess their effect on fate, both functionally and molecularly, in vitro and in vivo. The investigators comprising this program possess the necessary distinct, yet complementary, skills to do so. The Wiest lab (Project 1) has generated in vivo and in vitro models in which developmental fates can be manipulated by altering TCR signal intensity. Dr. Zuniga-Pflucker (Project 3) has established elegant in vitro and in vivo systems in which Notch and cytokine input can be manipulated to assess the impact on effector fate. Drs. Zhuang (Project 2) and Murre (Project 4) are experts in the genetic and molecular analysis of E proteins and their Id family antagonists. Finally, Dr. Murre (Genomics Core) will utilize his novel bioinformatic approach to assist all projects in molecularly defining critical milestones in ?? development by assembling global regulatory networks assembled around E protein targets. Collectively, these efforts promise to move the field forward by comprehensively defining the processes controlling ?? lineage commitment and effector fate from extracellular signals to the network of targets in the nucleus. RELEVANCE: ?? T cells are increasingly understood to play critical roles in immune responses to pathogens and tumors that are unique and thus not overlapping with those of ???lineage T cells. Therefore, a greater understanding of how the development of ?? T cells if controlled at the molecular level may enable the manipulation of their production or function for therapeutic benefit. Project 1: Influence of ligand on specification of gamma/delta fate and function Project Leader (PL): Wiest, D DESCRIPTION (provided by applicant): The goal of this proposal is to understand the molecular processes controlling ?? lineage commitment and specification of effector fate. Both events occur during development in the thymus; however, our understanding of the developmental cues controlling these fate decisions remains incomplete. Accumulating evidence suggests that both ?? lineage commitment and effector fate are influenced by differences in T cell receptor (TCR) signal strength. The differences in TCR signal strength influence fate by inducing Id3, an antagonist of E protein DNA binding. While E proteins clearly play a central role, their influence on developmental outcomes is almost certainly modulated by additional transcription factors and the extracellular signals that control their expression and function. Accordingly, these fate decisions are too complex to be understood by focusing on one gene or pathway and so require the comprehensive, network-based approach pioneered by Murre. We will employ this approach to elucidate the E protein targets that are crucial for these fate decisions, as well as, the DNA-binding proteins and signaling cascades with which they cooperate. In doing so, we will exploit the ?? TCR transgenic (Tg) model, KN6, whose known selecting ligand, the non-classical MHC-I molecule T10d, can be manipulated to alter TCR signaling. In Aiml, we will employ KN6 Tg mice, and endogenous T10/22 reactive ?? progenitors, to determine how specific ablation of the T10/22 ligand affects ?? lineage commitment, repertoire selection, and effector function. Aim2 will exploit the network approach described above to determine how Id3 is able to promote development of V?2+ and V?3+ ?? T cells, but restrains the development of V?1.1+ innate ?? T cells. Aim3 will exploit our novel marker of ?? lineage commitment, CD73, to determine whether ?? lineage commitment and specification of effector fate are separable or occur simultaneously. The molecular processes defining lineage commitment will be elucidated through the comprehensive network being assembled in Project 4, and by genetic tests of the resulting molecular model. These efforts require the capabilities of all program members and promise to reveal critical new insights into how ?? T cell development is controlled. RELEVANCE: ?? T cells regulate inflammation, preserve epithelial barriers, and are particularly adept at killing cutaneous tumors. Accordingly, understanding the molecular processes controlling their development and function may enable their manipulation for therapeutic benefit. Moreover, our investigation of molecular effectors controlling T lineage commitment is also of fundamental importance for other developmental processes, since control of cell growth and differentiation is a recurring theme in development and transformation.