Mechanisms of initiation of T-cell signaling by the TCR-CD3 complex

项目总结 T细胞受体(TCR)-CD3复合体在细胞介导的抗原识别中起重要作用 适应性免疫反应。免疫学中一个基本的悬而未决的问题是，配体如何结合 TCR介导将关键信息从外部环境中的抗原识别部位传递到 通过CD3信号复合体进入细胞内，导致信号的启动。一个完整的 对TCR-CD3信号复合体的分子组织和功能有广泛的了解 发展以VERT为重点的免疫治疗干预新策略的意义 免疫激活的最早阶段。这个项目的总体目标是了解抗原识别是如何 导致细胞外TCR-CD3复合体的分子变化，从而进一步传播分子变化 细胞内免疫受体酪氨酸激活基序(ITAM)导致功能结果。我们的 假说是力诱导或自发的重定向和/或构象变化 细胞外TCR-CD3复合体和细胞内ITAM对激动剂配体结合是必需的 蛋白酪氨酸激酶(Lck，ZAP-70)充分结合以启动近端信号事件。基于我们的 最近发表的工作产生了TCR-CD3细胞外组装的结构模型 复杂和我们的初步数据表明，通过LCK介导的CD8存在反馈机制 与延长TCR-pMHC捕获键的激动剂pMHC的结合，我们提出了以下三个目的来测试 我们的假设。在第一个目标中，我们将使用顺磁驰豫增强，生物膜力探针 (BFP)分析和光交联法确定TCR-CD3复合体的胞外排列 影响细胞内信号传递。在第二个目标中，我们将使用核磁共振化学位移微扰分析和 BFP将研究细胞内CD3与LCK/ZAP-70的相互作用以确定CD3的结构变化 胞质结构域影响蛋白酪氨酸激酶的相互作用。在第三个目标中，我们将使用BFP 结合基于并行FRET的近端信号组件成像，以确定如何改变 在TCR-CD3细胞外相互作用影响CD3-LCK/ZAP-70相互作用、TCR-pMHC结合和TCR-LCK/ZAP-70 PMHC-CD8相互作用。通过实现这些目标，我们期望确定信号的机械基础 T细胞识别过程中TCR-CD3复合体的转导过程。提供详细的 对力的参与和这些分子相互作用的细节的理解将允许更多的 完全了解T细胞抗原识别过程中信号转导过程的分子基础。 这项工作与公共卫生的相关性在于有可能提高应答率和总体 免疫疗法的结果。潜在的治疗策略可能包括使用抗体、修饰的T细胞 允许操纵免疫反应的细胞、嵌合抗原受体或小分子 癌症、感染、炎症和自身免疫，这仍然是人类健康的一个重大问题。

PROJECT SUMMARY The T cell receptor (TCR)-CD3 complex plays an important role in antigen recognition in cell mediated adaptive immune responses. A fundamental unanswered question in immunology is how ligand engagement of TCR mediates critical information transfer from the antigen recognition site in the external environment to the intracellular compartment via the CD3 signaling complex, resulting in signal initiation. A complete understanding the molecular organization and function of the TCR-CD3 signaling complex has wide significance for development of new strategies for immune therapeutic intervention focusing on the very earliest stages of immune activation. The overall goal of this project is to understand how antigen recognition leads to molecular changes in the extracellular TCR-CD3 complex that further propagates molecular changes in intracellular immunoreceptor tyrosine-based activation motifs (ITAM) to result in functional outcomes. Our hypothesis is that force-induced or spontaneous reorientations and/or conformational changes in the extracellular TCR-CD3 complex and intracellular ITAMs upon agonist ligand binding are necessary for sufficient binding of protein tyrosine kinases (Lck, ZAP-70) to initiate proximal signaling events. Based on our recently published work that generated a structural model of the extracellular assembly of the TCR-CD3 complex and our preliminary data demonstrating that there is feedback mechanism through Lck mediated CD8 binding to agonist pMHC that prolongs TCR-pMHC catch-bonds we propose the following three aims to test our hypothesis. In the first aim, we will use paramagnetic relaxation enhancement, biomembrane force probe (BFP) assay and photo-crosslinking to determine how extracellular arrangements in the TCR-CD3 complex influence intracellular signaling. In the second aim, we will use NMR chemical shift perturbation analysis and BFP to study the interaction of intracellular CD3 with Lck/ZAP-70 to determine how structural changes in CD3 cytoplasmic domains influence protein tyrosine kinases interaction. In the third aim, we will employ BFP combined with concurrent FRET based imaging of proximal signaling components to determine how changes in TCR-CD3 extracellular interaction influence CD3-Lck /ZAP-70 interactions, TCR-pMHC binding and TCR- pMHC-CD8 interactions. By undertaking these aims we expect to determine the mechanistic basis of the signal transduction process through the TCR-CD3 complex during T cell recognition. Providing a detailed understanding of the involvement of force and specifics of these molecular interactions will allow for a more complete understanding of the molecular basis of signal transduction process during T cell antigen recognition. The relevance of this work to public health is in the potential to improve the response rate and overall outcomes of immunotherapies. Potential treatment strategies could include the use of antibodies, modified T cells, chimeric antigen receptors or small molecules which would allow for manipulation of immune responses to cancer, infections, inflammation and autoimmunity, which remains a significant problem in human health.