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

描述（由申请人提供）：这个创新的提案侧重于肽- 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，可溶性pMHC即使在非常高浓度的溶液中也无法触发，抗原提呈细胞（APCs）上的pMHC触发TCR并高效激活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可以被锚定在表面的极少数（1至10）单体激动剂pmhc触发，而不依赖于内源性pmhc或真正apc表面的其他分子。除了表面锚定外，触发也严重依赖于活跃的t细胞粘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——受体变形模型。我们假设TCR信号是由动态t细胞骨架产生的拉力引起的TCR/CD3复合物的构象变化引发的，并通过pMHC-TCR相互作用传递，并具有足够的阻力在力下破裂。该模型不仅提供了TCR信号启动的直接机制，而且解释了TCR触发的非凡敏感性和特异性。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具有强大TCR触发能力的表面的物理和机械特性。我们假设pMHC锚定的相对固定、坚硬和适度粘附的表面将使细胞骨架力通过pMHC-TCR结合最有效地传递给TCR。在Specific Aim 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。外部机械力将施加在TCR上，以测试其对触发的影响。用原子力显微镜（AFM）来测定TCR的可扩展性（受力下的构象变化）。最后，将使用原子力显微镜测量pMHC- tcr结合的破裂力（力下结合强度的参数），以验证我们的假设，即破裂力决定pMHC的效力。在我们看来，通过纳入被忽略的TCR和pMHC之间二维相互作用的动力学方面，该模型代表了TCR触发理论发展的新一步，从关注亲和到三维动力学，再到二维动力学，再到我们的动态二维动力学。在pMHC-TCR结合中引入外力，为免疫受体生物学的研究提供了一个新的维度。项目简介：TCR触发作为t细胞抗原识别的第一步，是适应性免疫应答中的关键事件。本文提出了一种新的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，并采用生物物理方法和单分子研究设计对该模型进行了验证。了解TCR触发机制对开发增强或抑制免疫反应以治疗人类疾病的新方法具有深远的意义。

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.