Intestinal surveillance by intraepithelial lymphocytes

肠道上皮内淋巴细胞(IEL)是黏膜免疫系统的重要分支之一， 由于病原体在危急时刻的位置，潜在地提供了针对病原体的第一道免疫防御 肠腔和身体核心之间的界面。尽管近几年来一些 控制不同IEL群体发育的机制已被阐明，其所起的作用 胃肠道感染期间的IEL仍然难以捉摸。有几个主要障碍阻碍了解决这些问题的努力 缺陷：缺乏能够对IEL发育和功能进行特定控制的遗传模型，IEL质量差 由于它们位于上皮腔内，在体外存活和接触困难。这项建议 使用新工具和方法的组合来解决这些主要障碍，以剖析功能 这些细胞在体内的数量。首先，为了解决体内IEL动力学问题，我们将使用生命内和组织内的多光子 与光片显微镜相关的清除。使用这些技术，我们已经能够跟踪IEL 幼稚动物和肠道感染时多个绒毛的动态变化。我们观察到 TcrIEL是一种高度移动的细胞，它不断地观察肠道上皮，快速响应 通过改变肠道上皮细胞的运动性和洗牌模式引起的病原菌感染 细胞(IECs)以及它们在绒毛内的位置。这些变化与基因表达的变化有关。 以及能源利用途径的变化。我们假设对鲁米那的协同IEC-IEL反应 扰动导致IEL行为和新陈代谢的改变，最终导致对 病原体入侵。主要集中在主要的IEL群体，TCR细胞，在这项建议中的研究将 利用灵知生菌小鼠和胃肠道模型定义它们对肠道微生物的反应 感染。我们将表征IEL细胞动力学和代谢变化在 对肠道病原体的保护作用的反应。我们还将研究IECS如何感知微生物 影响IEL对感染的反应。最终，使用基因靶向策略和分子生物学 技术，我们将研究转录因子T-bet在IEL活性调节中的作用 子集和潜在的分子机制。通过结合基因报告、多种细胞特异性和 利用各种微生物刺激模式的时间控制的基因打靶和体内成像策略， 我们希望确定IEL的具体监测和保护特征。这项提议提供了 全新的方法来解剖不断扫描肠道表面的功能T细胞。因此， 从这项研究中获得的知识将扩大我们对肠道上皮获得性免疫的理解 障碍，有助于提供关于宿主-微生物相互作用的宝贵信息。定义关键IEL函数 在肠道防御过程中，是了解粘膜参与的初始过程的重要一步 免疫，并可能导致胃肠道感染的治疗干预更好的策略。

Intestinal intraepithelial lymphocytes (IELs) form one of the key branches of the mucosal immune system, potentially providing a first line of immune defense against pathogens due to their location at the critical interface between the intestinal lumen and the core of the body. Although in recent years some of the mechanisms controlling the development of different IEL populations have been elucidated, the role played by IELs during gastrointestinal infection remains elusive. A few main obstacles hamper efforts to address these deficits: lack of genetic models that would allow specific control of IEL development and function, poor IEL survival ex vivo and difficult access due to their location within the epithelial compartment. This proposal addresses these main impediments using a combination of novel tools and approaches to dissect the function of these cells in vivo. First, to address IEL dynamics in vivo we will use multi-photon intra-vital as well as tissue clearing associated with light sheet microscopy. Using these techniques we have been able to track IEL dynamics in multiple villi simultaneously in naïve animals and during enteric infections. We observed that TCR+ IELs are highly mobile cells that constantly survey the intestinal epithelium, rapidly responding to pathogenic bacterial infection by changing their motility and pattern of shuffling between intestinal epithelial cells (IECs), as well as their location within the villi. These changes were linked to gene expression changes and changes in energy utilization pathways. We hypothesize that a coordinated IEC-IEL response to luminal perturbations results in modification of IEL behavior and metabolism, ultimately leading to optimal resistance to pathogen invasion. Mostly focusing on the main IEL population, TCR+ cells, the studies in this proposal will define they respond to intestinal microbes utilizing gnotobiotic mice as well as models of gastrointestinal infections. We will characterize the relative contribution of IEL cell dynamics and metabolic changes in response to enteric pathogens to their protective function. We will also study how sensing of microbes by IECs influences IEL responses against infections. Ultimately, using gene-targeting strategies and molecular biology techniques, we will investigate the role of the transcription factor T-bet in the modulation of activity of IEL subsets and the underlying molecular mechanisms. By combining gene-reporter, multiple cell-specific and temporally controlled gene targeting and in vivo imaging strategies with various model of microbial stimulation, we expect to identify specific surveillance and protective characteristics for IELs. This proposal offers completely novel approaches to dissect the function T cells that constantly scan the intestinal surface. Thus, the knowledge gained from this study will expand our understanding of adaptive immunity at the gut epithelial barrier, contributing valuable information regarding host-microbial interactions. Defining key IEL functions during intestinal defense is an important step for understanding the initial processes involved in mucosal immunity and may lead to better strategies for therapeutic intervention in gastrointestinal infections.