Synaptic Control of Cytotoxic T cell Function

 描述(由申请人提供)：细胞毒性T淋巴细胞(CTL)通过破坏感染或转化的目标细胞，在细胞免疫反应中发挥核心作用。它们强大的抗肿瘤活性使其成为几种有希望的抗癌免疫治疗策略的核心。CTL的作用方式是与其靶细胞形成紧密的、径向对称的接触，称为免疫突触(IS)。然后，它们将细胞溶解分子分泌到突触间隙，诱导靶细胞死亡。一般认为，IS的细胞骨架加强了这种反应。皮质细丝肌动蛋白(F-肌动蛋白)在IS的外围丰富，从中心消失，形成一个特征性的环。同时，中心体作为细胞内囊泡货物的焦点，重新定位到IS中心正下方的位置。有人提出，这些事件通过使含有细胞溶解因子的颗粒靠近突触膜而将分泌集中到靶细胞。然而，这种模式并没有经过严格的测试。此外，细胞骨架动力学在IS中的其他可能作用，如将机械信号传递到靶细胞，仍未被探索。在过去的五年中，我们定义了两种塑造突触细胞骨架的关键机制，一种是指导中心体的二酰甘油依赖途径，另一种是控制F-肌动蛋白环形成的磷脂酰肌醇3-激酶(PI3K)依赖途径。我们现在将研究这些机制是如何促进CTL功能的。我们的总体假设是，中心体极化、中央F-肌动蛋白清除和外周F-肌动蛋白动力学共同为CTL效应器反应提供了强度和特异性。我们将追求以下具体目标：1)确定中心体极化在引导CTL分泌反应中的作用；2)确定F-肌动蛋白清除的机制和功能；3)确定PI3K依赖的力量如何增强细胞毒作用。对于第一个目的，将使用一种基于有条件删除支架蛋白SAS4的新的遗传策略来从CTL中移除中心体。对于第二个目标，我们将使用获得和丧失功能的实验来评估明胶蛋白家族的肌动蛋白重塑因素如何影响细胞骨架极化和CTL效应反应。对于第三个目标，将使用生物物理方法来量化IS的机械转导，并检查力量施加如何增强靶细胞杀伤。这项工作在技术上是创新的，因为它结合了最先进的成像方式、遗传工具和生物物理方法，以新的方式探索淋巴细胞功能。它还提出了创新的概念，即IS的结构如何促进将化学信息和机械信息转移到目标 手机。这项拟议的研究很重要，因为它们将导致对突触结构如何指定CTL功能的全面理解，这将有助于在免疫治疗背景下利用和控制CTL活性。因此，我们的建议与美国国立卫生研究院的任务相关，因为它将有助于促进知识的进步，从而改善人类健康。

 DESCRIPTION (provided by applicant): Cytotoxic T lymphocytes (CTLs) play a central role in cellular immune responses by destroying infected or transformed target cells. Their potent anti-tumor activity has made them the centerpiece of several promising immunotherapeutic strategies to fight cancer. CTLs operate by forming a close, radially symmetric contact with their target cell known as an immunological synapse (IS). Then, they secrete cytolytic molecules into the synaptic space to induce target cell death. It is generally thought that the cytoskeletal framework of the IS potentiates this response. Cortical filamentous actin (F-actin) is enriched in the periphery of the IS and depleted from the center, forming a characteristic ring. Concomitantly, the centrosome, which serves as a focal point for intracellular vesicular cargo, reorients to a position just beneath the center of the IS. It has been proposed that these events focus secretion toward the target cell by bringing granules containing cytolytic factors close to the synaptic membrane. This model has not been rigorously tested, however. In addition, other possible roles for cytoskeletal dynamics at the IS, such as the transfer of mechanical signals to the target cell, remain unexplored. Over the past five years, we have defined two key mechanisms that shape the synaptic cytoskeleton, a diacylglycerol dependent pathway that guides the centrosome and a phosphoinositide 3-kinase (PI3K) dependent pathway that controls F-actin ring formation. We will now examine how these mechanisms contribute to CTL function. Our overall hypothesis is that centrosome polarization, central F-actin clearance, and peripheral F-actin dynamics together provide strength and specificity to CTL effector responses. The following Specific Aims will be pursued: 1) Determine the role of centrosome polarization in guiding CTL secretory responses; 2) Determine the mechanism and function of F-actin clearance; and 3) Determine how PI3K dependent force exertion potentiates cytotoxicity. For the first Aim, a novel genetic strategy based on conditional deletion of the scaffolding protein SAS4 will be used to remove the centrosome from CTLs. For the second Aim, gain- and loss-of-function experiments will be used to evaluate how actin- remodeling factors of the gelsolin family influence cytoskeletal polarization and CTL effector responses. For the third Aim, biophysical approaches will be used to quantify mechanotransduction at the IS and examine how force exertion potentiates target cell killing. This work is technically innovative because it incorporates state-of- the-art imaging modalities, genetic tools, and biophysical methods to explore lymphocyte function in new ways. It also advances innovative concepts about how the structure of the IS facilitates the transfer chemical and also mechanical information to the target cell. The proposed studies are important because they will lead to a comprehensive understanding of how synaptic architecture specifies CTL function, which will aid efforts to harness and control CTL activity in immunotherapeutic contexts. Hence, our proposal is relevant to the NIH mission in that it will contribute to the advancement of knowledge that could improve human health.