Probing the mechanistic basis for T cell fate decisions (R01)

描述（由申请人提供）：TCR-CD 3复合物、CD 4和CD 28是重要的检查点分子，允许T细胞在抗原呈递细胞（APC）表面上检测抗原和活化诱导分子。然后，它们将这种抗原和APC特异性信息转移到T细胞的细胞内信号装置。最终，这些信息指导T细胞的命运决定，这些决定驱动发育、活化、分化和效应功能的执行。这些分子对免疫监视和人类健康的根本重要性导致了过去25年来对其结构和功能的几项研究。因此，现在对它们的个体结构、它们与各自配体的相互作用以及由这些相互作用产生的信号级联有了很多了解。但是，我们还没有确定它们如何作为驱动T细胞命运决定的分子机制的组成部分相互配合和共同工作，这阻碍了我们理解这种分子机制作为一个整体是如何执行其功能的。此外，这种知识的缺乏阻碍了我们从战略上确定这一目标的能力。 分子机械，其具有设计用于增强对疫苗、肿瘤或病原体的反应或减弱对移植物或自身抗原的反应的试剂。我们的目标是解构，理解，并最终操纵这种复杂的大分子机器的形式和功能。为此，我们将：1）确定抗原特异性信息如何通过TCR跨膜传递到CD 3亚基的胞内信号传导结构域; 2）确定这些信号传导亚基如何与修饰它们的酶紧密接近;和3）确定稳定这种高阶机制的结构的表面如何影响体内CD 4 + T细胞命运决定。我们开发了一种新的实验平台，将经典的生物化学和分子生物学技术与现代多顺反子逆转录病毒系统、基于激酶的二聚化测定和活细胞荧光视频成像（包括使用全内反射荧光显微镜（TIRFM）和福斯特共振能量转移（FRET））相结合，以采取高度受控的还原论方法来解决目标1和2。此外，我们正在建立新的小鼠模型系统，以实现目标3。具体来说，我们将研究我们已经确定为介导TCR-CD 3复合物稳定性和TCR二聚化的表面，以确定这些相互作用如何影响T细胞命运，这些相互作用是将pMHC特异性信息从细胞外部传递到细胞内部的高级大分子机制的核心 决定在体内。总之，这些实验将产生重要的见解的分子机制，CD 4 + T细胞的命运决定的基础，并确定翻译免疫调节试剂的发展的潜在目标。

DESCRIPTION (provided by applicant): The TCR-CD3 complex, CD4, and CD28 are vital checkpoint molecules that allow T cells to survey for antigens and activation induced molecules on the surfaces of antigen presenting cells (APCs). They then transfer this antigen- and APC-specific information to the T cell's intracellular signaling apparatus. Ultimately, this informatio directs the T cell fate decisions that drive development, activation, differentiation, and the execution of effector functions. The fundamental importance of these molecules to immune surveillance and human health has resulted in several studies regarding their structure and function over the past 25+ years. As a result, much is now known about their individual structures, their interactions with their respective ligands in isolation, and the signaling cascads that result from these interactions. But, we have yet to determine how they fit and work together as components of the molecular machinery that drives T cell fate decisions and this has prevented us from understanding how this molecular machinery, as a whole, executes its functions. Further, this lack of knowledge has handicapped our ability to strategically target this molecular machinery with reagents designed to either enhance responses to vaccines, tumor, or pathogens, or to attenuate responses to transplants or auto-antigens. Our goal is to deconstruct, understand, and ultimately manipulate the form and function of this complex macromolecular machine. To this end, we will: 1) determine how antigen-specific information is relayed across the membrane by the TCR to the intracellular signaling domains of the CD3 subunits; 2) determine how these signaling subunits are positioned in close proximity with the enzymes that modify them; and 3) determine how the surfaces that stabilize the architecture of this higher order machinery influence CD4+ T cell fate decisions in vivo. We have developed a novel experimental platform that combines classic biochemical and molecular biology techniques with modern polycistronic retroviral systems, kinase- based dimerization assays, and live cell fluorescent video imaging (including the use of total internal reflection fluorescence microscopy (TIRFM) and Forster Resonance Energy Transfer (FRET)), to take a highly controlled reductionist approach to addressing Aim 1 and 2. In addition, we are building novel mouse model systems to accomplish Aim 3. Specifically we will study the surfaces that we have already identified as mediating TCR- CD3 complex stability and TCR dimerization to determine how these interactions, which are at the core of the higher-order macromolecular machinery that transfers pMHC-specific information from the outside to the inside of a cell, influence T cell fate decisions in vivo. Altogether, these experiments will yield important insights into the molecular mechanisms that underlie CD4+ T cell fate decisions and identify potential targets for the development of translational immune-modulating reagents.