Intestinal surveillance by intraepithelial lymphocytes

肠上皮内淋巴细胞（IEL）形成粘膜免疫系统的关键分支之一， 潜在地提供针对病原体的第一道免疫防御线，这是由于它们位于关键部位， 肠腔和身体核心之间的界面。尽管近年来， 控制不同IEL群体发展的机制已经阐明， 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：缺乏允许特异性控制IEL发育和功能的遗传模型，IEL较差 离体存活和由于它们位于上皮隔室内而难以接近。这项建议 使用新工具和方法的组合来剖析功能， 这些细胞在体内。首先，为了解决体内IEL动力学，我们将使用多光子活体内以及组织 与光片显微术相关的透明化。使用这些技术，我们已经能够跟踪IEL 在幼稚动物和肠道感染期间，多个绒毛中同时存在动力学。我们观察到 TCR γ细胞+IEL是高度移动的细胞，其不断地观察肠上皮，快速响应肠上皮细胞的增殖。 通过改变它们的运动性和在肠上皮细胞之间穿梭的模式来感染病原性细菌 细胞（IEC），以及它们在绒毛中的位置。这些变化与基因表达变化有关 以及能源利用途径的变化。我们假设，一个协调的IEC-IEL响应鲁米那 干扰导致IEL行为和代谢的改变，最终导致对细菌的最佳抗性。 病原体入侵本提案中的研究主要集中在主要IEL群体TCR γ +细胞上， 定义他们响应肠道微生物利用gnotobiotic小鼠以及模型的胃肠道 感染.我们将描述IEL细胞动力学和代谢变化的相对贡献， 对肠道病原体的保护作用。我们还将研究IEC如何感知微生物 影响IEL对感染的反应。最终，利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 亚群和潜在的分子机制。通过结合基因报告基因、多种细胞特异性和 利用各种微生物刺激模型， 我们希望确定IEL的具体监测和保护特性。该提案提供 这是一种全新的方法来解剖不断扫描肠道表面的功能性T细胞。因此，在本发明中， 从这项研究中获得的知识将扩大我们对肠道上皮细胞获得性免疫的理解， 屏障，提供关于宿主-微生物相互作用的有价值的信息。定义关键IEL功能 在肠道防御过程中，是理解粘膜防御的初始过程的重要一步。 免疫力，并可能导致更好的治疗干预胃肠道感染的策略。

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.