Synaptic Control of Cytotoxic T cell Function

 描述（由申请方提供）：细胞毒性T淋巴细胞（CTL）通过破坏感染或转化的靶细胞在细胞免疫应答中发挥核心作用。它们强大的抗肿瘤活性使它们成为几种有希望的抗癌免疫策略的核心。CTL通过与它们的靶细胞形成紧密的、径向对称的接触来工作，称为免疫突触（IS）。然后，它们分泌溶细胞分子进入突触空间以诱导靶细胞死亡。一般认为IS的细胞骨架框架增强了这种反应。皮质丝状肌动蛋白（F-肌动蛋白）在IS的外围富集，从中心耗尽，形成特征环。同时，中心体，作为一个细胞内的小泡货物的焦点，重新定位到一个位置的IS中心下方。已经提出，这些事件通过使含有溶细胞因子的颗粒靠近突触膜而将分泌物集中到靶细胞。然而，这一模式尚未经过严格测试。此外，在IS的细胞骨架动力学的其他可能的作用，如机械信号的转移到靶细胞，仍然没有探索。在过去的五年里，我们已经定义了塑造突触细胞骨架的两种关键机制，即引导中心体的二酰基甘油依赖性途径和控制F-肌动蛋白环形成的磷酸肌醇3-激酶（PI 3 K）依赖性途径。我们现在将研究这些机制如何促进CTL功能。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-肌动蛋白动力学一起提供的CTL效应反应的强度和特异性。将追求以下特定目的：1）确定中心体极化在引导CTL分泌应答中的作用; 2）确定F-肌动蛋白清除的机制和功能;和3）确定PI 3 K依赖性力施加如何增强细胞毒性。对于第一个目标，将使用基于支架蛋白SAS 4的条件性缺失的新的遗传策略来从CTL去除中心体。对于第二个目标，将使用功能获得和丧失实验来评估凝溶胶蛋白家族的肌动蛋白重塑因子如何影响细胞骨架极化和CTL效应子应答。对于第三个目标，生物物理方法将用于量化IS处的机械转导，并检查力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。它还提出了关于IS结构如何促进化学和机械信息向目标转移的创新概念 cell.拟议的研究是重要的，因为它们将导致突触结构如何指定CTL功能的全面理解，这将有助于在免疫环境中利用和控制CTL活性的努力。因此，我们的建议与NIH的使命相关，因为它将有助于提高可以改善人类健康的知识。

 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.