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

描述(申请人提供)：TCR-CD3复合体、CD4和CD28是重要的检查点分子，允许T细胞检测抗原和抗原提呈细胞(APC)表面的激活诱导分子。然后，他们将这种抗原和APC特异的信息传递给T细胞的细胞内信号装置。最终，这些信息指导T细胞命运的决定，驱动发育、激活、分化和效应器功能的执行。在过去的25年里，这些分子对免疫监测和人类健康的基本重要性导致了关于它们的结构和功能的几项研究。因此，现在人们对它们的单独结构、它们与各自配体的相互作用以及这些相互作用所产生的信号级联都有了很大的了解。但是，我们还没有确定它们作为驱动T细胞命运决定的分子机制的组件如何匹配和共同工作，这阻碍了我们理解这个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瞄准这一目标的能力。 具有试剂的分子机械，旨在增强对疫苗、肿瘤或病原体的反应，或减弱对移植或自身抗原的反应。我们的目标是解构、理解并最终操纵这台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CD3亚单位的细胞内信号域；2)确定这些信号亚单位如何与修饰它们的酶紧密结合；以及3)确定稳定这一高阶机制的结构的表面如何影响体内CD4+T细胞的命运决定。我们开发了一个新的实验平台，将经典的生化和分子生物学技术与现代多顺反子逆转录病毒系统、基于激酶的二聚化分析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，采取高度受控的还原方法来实现目标1和目标2。此外，我们正在建立新的小鼠模型系统来实现目标3。具体地说，我们将研究我们已经确定的介导TCR-CD3复合体稳定性和TCR二聚化的表面，以确定这些相互作用是如何进行的。它们是将pMHC特异性信息从细胞外部传递到细胞内部的高阶大分子机制的核心，影响T细胞的命运 体内的决定。总之，这些实验将对决定CD4+T细胞命运的分子机制产生重要的见解，并确定翻译免疫调节试剂开发的潜在靶点。

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.