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

细胞毒性淋巴细胞杀伤肿瘤细胞机制的定量分析方法

• 批准号: 1562905
• 负责人: Morgan Huse
• 金额: $ 53.5万
• 依托单位: Sloan Kettering Institute For Cancer Research
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1562905&HistoricalAwards=false
• 关键词: Quantitative; approaches; mechanistic; analysis; tumor

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.

细胞毒性 T 细胞通过选择性破坏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几种有前途的抗癌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 细胞如何运作不仅具有生物学意义，而且具有潜在的临床意义。 T 细胞首先通过与其靶标形成紧密的细胞-细胞界面（称为免疫突触）来进行杀伤。 然后，它们将有毒蛋白质的混合物分泌到突触中，从而损害目标的质膜和细胞内内容物。最近的生物物理学研究表明，T 细胞在突触上施加大量机械力，这可能会改变靶细胞的形状和物理特性。这项研究中的实验将调查以下假设：这些力量通过增强分泌到突触的有毒蛋白质的活性来促进杀伤力。机械力和化学信号以这种方式配合的想法尚未得到重视，但它可能代表了理解细胞间通讯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带薪暑期实习计划，为她们提供进入研究生涯的入门途径。这些研究将重点关注突触力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，突触形成增加了靶细胞膜张力，从而增强了穿孔素孔的形成。为了研究这一假设，生物物理方法将与免疫学测定相结合。聚丙烯酰胺水凝胶基质将用于探索靶细胞张力与穿孔素孔形成之间的关系。光陷阱方法将用于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用于检查力施加和穿孔素分泌的时空协调。这些研究目标的成功完成可以将机械增强（即物理和化学信号之间的协同作用）确立为细胞间通讯的重要途径，这将广泛影响细胞间相互作用和机械生物学的当前概念和未来研究。