Quantitative approaches for the mechanistic analysis of tumor cell killing by cytotoxic lymphocytes

细胞毒性T细胞通过选择性地摧毁病毒感染或癌变的靶细胞来发挥作用。近年来，这种靶向杀伤能力已成为抗击癌症的几种有希望的免疫治疗策略的核心组成部分。因此，更好地了解细胞毒性T细胞是如何运作的，不仅具有生物学意义，而且具有潜在的临床意义。T细胞首先与它们的靶标形成一个紧密的细胞-细胞界面，称为免疫突触，从而杀死它们。然后，它们分泌一种有毒蛋白质的混合物进入突触，破坏目标的质膜和细胞内内容物。最近的生物物理研究表明，T细胞在突触上施加了大量的机械力，这可能会改变靶细胞的形状和物理性质。这项研究中的实验将调查这样一种假设，即这些力量通过增强分泌到突触中的有毒蛋白质的活性来增强杀伤力。机械力和化学信号以这种方式合作的想法是相当不受欢迎的，可能代表着理解细胞间通信和免疫功能的一个重要的新概念。首席研究员还将开发一个针对高中和大学女生的机械生物学暑期带薪实习计划，为她们提供进入研究生涯的途径。研究将集中在突触力量和分泌的细胞溶解分子穿孔素之间的潜在协同作用，穿孔素在目标细胞膜上形成蛋白质孔。初步结果表明，突触的形成增加了靶细胞膜的张力，从而促进了穿孔素孔的形成。生物物理学方法将与免疫学分析相结合，以研究这一假说。将使用聚丙烯酰胺水凝胶底物来探索靶细胞张力与穿孔素孔形成之间的关系。光学陷阱方法将被用来量化突触形成对膜张力的影响。最后，聚二甲基硅氧烷微柱阵列将被用来检测作用力和穿孔素分泌的时空协调性。这些研究目标的成功完成可以确立机械电势(即物理和化学信号之间的协同作用)作为细胞间通信的重要途径，这将广泛影响细胞-细胞相互作用和机械生物学的当前概念和未来研究。

Cytotoxic T cells function by selectively destroying virally infected or cancerous target cells. In recent years, this targeted killing capacity has emerged as a core component of several promising immunotherapeutic strategies to fight cancer. A better understanding of how cytotoxic T cells operate is therefore not only of biological interest but also of potential clinical relevance. T cells kill by first forming a close cell-cell interface, called an immunological synapse, with their targets. They then secrete a mixture of toxic proteins into the synapse, which damage the target's plasma membrane and intracellular contents. Recent biophysical studies have indicated that T cells exert a substantial amount of mechanical force across the synapse, which could potentially alter the shape and physical properties of the target cell. The experiments in this research will investigate the hypothesis that these forces boost killing by enhancing the activity of the toxic proteins secreted into the synapse. The idea that mechanical force and chemical signals cooperate in this manner is quite unappreciated and could represent an important new concept in the understanding of intercellular communication and immune function. The principal investigator will also develop a paid summer internship program in mechanobiology targeting female students at both the high school and university levels to provide them an entryway into a research career.The studies will focus on potential synergy between synaptic forces and the secreted cytolytic molecule perforin, which forms proteinaceous pores on the target cell membrane. Preliminary results suggest that synapse formation increases target cell membrane tension, thereby potentiating perforin pore formation. Biophysical methods will be merged with immunological assays in order to investigate this hypothesis. Polyacrylamide hydrogel substrates will be used to explore the relationship between target cell tension and perforin pore formation. Optical trap methodology will be used to quantify the effects of synapse formation on membrane tension. Finally, polydimethylsiloxine micropillar arrays will be used to examine the spatiotemporal coordination of force exertion and perforin secretion. The successful completion of these research goals could establish mechanopotentiation (i.e. the synergy between physical and chemical signals) as an important avenue for intercellular communication, which would broadly influence current conceptions and future studies of cell-cell interactions and mechanobiology.