New methods for monitoring the immune system, in individual cells and in vivo

描述(由申请人提供)：了解单细胞中天然免疫信号网络的动态是免疫学的基本目标。利用结合了活细胞成像、高通量图像分析、微流控细胞培养和计算网络建模的最新技术，Covert Lab通过测量NFkB对刺激和时间依赖刺激组合的单细胞反应，研究了细胞如何解码复杂的环境信息(自然，2010；科学信号2009)。虽然这些和类似的方法在表征细胞群体中的表型异质性方面非常有用(例如，在研究P53)，但可以从中得出的结论受到可测量输出数量相对较少以及这样一个事实的限制，即到目前为止，几乎所有这种研究都是在培养细胞中进行的。我们建议极大地扩大免疫系统活细胞动态成像的范围，开发新技术来显著增加可测量输出的数量，并使体内测量成为可能。我们的具体目标是：(1)创建一个构建和细胞库，能够同时在单个细胞中监测各种因素，包括多个平行的信号通路和内源性表达水平。(2)为了了解网络动力学如何控制基因表达，我们建议通过将最近发展的RNA FISH技术与我们的活细胞成像技术相结合，开发将转录因子的动态与单个细胞中内源基因表达的动态相关联的方法。这将是第一次将动态转录因子的活性与单个细胞中的基因表达进行直接比较。(3)Covert实验室将与加州大学圣迭戈分校的Tannishtha Reya合作，将我们在单细胞中成像和量化蛋白质定位的方法与她监测体内细胞运动的开创性工具相结合。通过结合这些方法，我们将第一次观察转录因子在单个细胞中通过完整动物骨髓的动态。为了实现这些目标，我们希望对环境信息如何在信令网络动态中进行编码有一个更加详细和系统级别的理解，并为科学界创造出一些同类技术的先河。 与公共健康相关：在单个细胞和体内项目中监测免疫系统的新方法近年来，科学家们已经意识到我们身体中的每个单独细胞是多么重要，即使是同类的两个相邻细胞的表现也可能截然不同。有一些方法可以实时观察单个细胞，特别是关键蛋白质的激活，但这项技术有很多局限性。我们建议开发一些新技术，使追踪和免疫反应变得更容易，因为它发生在单个细胞中--即使在活着的动物体内细胞移动时也是如此。

DESCRIPTION (provided by applicant): To understand the dynamics of the innate immune signaling network in single cells is a fundamental goal of immunology. Using an approach that combines the latest technologies for live-cell imaging, high-throughput image analysis, microfluidic cell culture and computational network modeling, the Covert Lab studies how cells decode complex environmental information by measuring the single-cell responses of NFkB to combinations of stimuli and time-dependent stimuli (Nature, 2010; Science Signaling 2009). Although these and similar approaches have been extremely useful in characterizing phenotypic heterogeneity within a population of cells (also in studying p53, for example), the conclusions that can be drawn from them are limited by the relatively low number of measureable outputs as well as the fact that until now, virtually all of this kind of research has been performed in cultued cells. We propose to dramatically expand the scope of live-cell dynamic imaging of the immune system, developing new technologies to dramatically increase the number of measureable outputs, and enable in vivo measurements. Our Specific Aims are: (1) to create a library of constructs and cells that will enable monitoring of a variety of factors, encompassing multiple parallel signaling pathways and at endogenous expression levels, simultaneously in individual cells. (2) To understand how network dynamics control gene expression, we propose to develop methods to correlate the dynamics of transcription factors with the dynamics of endogenous gene expression in single cells, by integrating recently developed techniques for RNA FISH with our live cell imaging technology. This will be the first time that dynamic transcription factor activity has ever been directly compared with gene expression in individual cells. (3) The Covert Lab will partner with Tannishtha Reya at UCSD to integrate our methods for imaging and quantifying protein localization in single cells with her pioneering tools for monitoring cellular movement in vivo. By combining these approaches, we will be the first to observe the dynamics of transcription factors in individual cells as they move through the bone marrow of intact animals. In achieving these goals, we expect to achieve a significantly more detailed and system-level understanding of how environmental information is encoded in signaling network dynamics, and to have produced some first-of-its-kind technology for the scientific community. PUBLIC HEALTH RELEVANCE: New methods for monitoring the immune system, in individual cells and in vivo Project Narrative In recent years, scientists have realized how important each individual cell in our bodies are, and how even two neighboring cells of the same kind can behave quite differently. There are some ways to observe individual cells, and specifically the activation of key proteins, in real- time, but there are many limitations of the technology. We are proposing to develop some new technology that will make it much easier to track and the immune response as it occurs in single cells -- even as the cells move around in a living animal.