Signal Initiation from the T-cell Antigen Receptor by Mechanical Force

描述(申请人提供)：这项创新的建议集中在由多肽-MHC配体(PMHC)启动T细胞受体(TCR)信号的基本问题。TCR触发的一个独特特征是，尽管可溶性的pMHC即使在很高的浓度下也不能在溶液中触发，但抗原提呈细胞(APC)上的pMHC可以触发TCR并激活高效力的T细胞。使用具有特定成分的人工抗原呈递系统，我们确定TCR可以由极少数(1到10)锚定在表面的单体激动剂pMHC触发，而不依赖于内源性pMHC或真实APC表面的其他分子。除了表面锚定，触发也关键依赖于活跃的T细胞黏附和完整的细胞骨架功能。考虑到这三种成分在TCR触发中的充分性，T细胞-APC相互作用的动态性，以及pMHC-TCR相互作用的恒定机械应力，我们提出了一种新的TCR触发模型--受体形变模型。我们假设TCR信号是由动态T细胞骨架的拉力引起TCR/CD3复合体的构象变化而启动的，并通过具有足够抵抗力的pMHC-TCR相互作用传递。这个模型不仅为TCR信号的启动提供了一个简单的机制，而且解释了TCR触发的非凡的敏感性和特异性。我们将从两个角度检验我们的假设。在具体目标1中，我们将定义赋予pMHC强大TCR触发能力的表面的物理和机械性质。我们假设，对于pMHC锚定，相对固定、僵硬和中等粘附性的表面将能够最有效地通过pMHC-TCR结合将细胞骨架力传递到TCR。在具体目标2中，我们将使用单分子研究设计直接测试机械力在TCR触发中的作用。将在TCR上施加外部机械力，以测试它们对触发的影响。TCR的延伸性(在力作用下的构象变化)将使用原子力显微镜(AFM)来确定。最后，将使用AFM测量pMHC-TCR结合的破裂力(受力下结合强度的参数)，以验证我们的假设，即这决定了pMHC的效力。在我们看来，通过纳入TCR和pMHC之间2D相互作用的被省略的动力学方面，该模型代表了TCR触发理论从关注亲和力到3D动力学、2D动力学再到我们的动态2D动力学的新的一步。PMHC-TCR结合外力的引入为免疫受体生物学的研究提供了新的维度。项目简介：作为T细胞识别抗原的第一步，TCR的触发在获得性免疫反应中是一个关键事件。在这里，我们提出了一种新的TCR触发模型-受体变形模型，该模型解释了TCR触发的非凡敏感性和特异性，并使用生物物理方法和单分子研究设计对该模型进行了验证。对TCR触发机制的了解对于开发新的方法来增强或抑制免疫反应以治疗人类疾病具有深远的意义。

DESCRIPTION (provided by applicant): This innovative proposal focuses on the fundamental question of T-cell receptor (TCR) signal initiation by peptide-MHC ligands (pMHCs). A unique feature of TCR triggering is that while soluble pMHC is incapable of triggering in solution even at very high concentrations, pMHCs on antigen presenting cells (APCs) trigger TCR and activate T-cells with high potency. Using artificial antigen presentation systems with defined components, we determined that TCR can be triggered by very few (1 to 10) monomeric agonist pMHCs anchored on a surface, independent of endogenous pMHCs or other molecules on the surface of real APCs. In addition to surface-anchoring, the triggering also critically depends upon active T-cell adhesion and intact cytoskeletal function. Considering the sufficiency of these three components in TCR triggering, the dynamic nature of T cell-APC interaction, and the constant mechanical stresses on pMHC-TCR interaction, we propose a new model for TCR triggering, the receptor deformation model. We hypothesize that TCR signaling is initiated by conformational change of the TCR/CD3 complex induced by a pulling force originating from the dynamic T-cell cytoskeleton, and transmitted through pMHC-TCR interactions with sufficient resistance to rupture under force. This model not only offers a straightforward mechanism for TCR signal initiation, but also explains the extraordinary sensitivity and specificity of TCR triggering. We will test our hypothesis from two perspectives. In Specific Aim 1, we will define the physical and mechanical properties of a surface that confers pMHC with potent TCR triggering capacity. We hypothesize that a relatively immobile, stiff, and moderately adhesive surface for pMHC anchoring will enable transmission of cytoskeletal force to TCR through pMHC-TCR binding most efficiently. In Specific Aim 2, we will directly test the role of mechanical force in TCR triggering using single molecule research design. External mechanical forces will be exerted on the TCR to test their effect on triggering. The extensibility (conformational change under force) of TCR will be determined using atomic force microscopy (AFM). Finally, the rupture force (a parameter of binding strength under force) of pMHC-TCR binding will be measured using AFM, to test our hypothesis that this determines pMHC potency. In our view, by incorporating the omitted dynamic aspect of the 2D interaction between TCR and pMHC, this model represents a new step in the evolution of TCR triggering theory, from the focus on affinity to 3D kinetics, to 2D kinetics, to our dynamic 2D kinetics. The introduction of an external force to pMHC-TCR binding provides a new dimension to research on immune receptor biology. PROJECT NARRATIVE: As the very first step of antigen recognition by T-cells, TCR triggering is a critical event in the adaptive immune response. Here, we propose a new model for TCR triggering, the receptor deformation model, which explains the extraordinary sensitivity and specificity of TCR triggering, and test this model using biophysical methods and single molecule research design. An understanding of the mechanism of TCR triggering has profound implications in developing new approaches for enhancing or subduing immune responses to treat human disease.