Profiling intraepithelial lymphocyte populations in health and CrohnâÂÂs disease

项目摘要。 屏障表面的免疫监视对于提供针对肠毒素的防御是必不可少的。 病原体和维持粘膜稳态。破坏了促炎性细胞因子 和调节性免疫反应可导致粘膜耐受性丧失和慢性 炎症性疾病，如克罗恩病（CD）。CD中的大多数炎症反应 已经归因于位于固有层中的先天性和适应性免疫细胞;然而， 关于上皮内淋巴细胞（IEL）的贡献，我们知道的相对较少 CD发病机制。IEL，包括上皮内T细胞（IET）和上皮内ILC（IE-ILC）， 有助于维持上皮屏障的完整性，并作为第一道防线， 管腔微生物由于难以从患者处获得足够数量的IEL， 为了进行深入的功能分析，我们建议利用单细胞RNA 测序技术和RNA成像来描述表型和功能特征 健康和急性CD中的回肠IEL亚群。使用转录组学作为指导， 为单个IEL子集的签名，我们将开发单分子荧光原位杂交 在一些实施方案中，使用多路荧光原位杂交（sm-FISH）探针以使患者活检中的这些细胞群可视化。合作 与IBD遗传学联盟合作，我们将从纵向内窥镜活检中获得组织切片 已接受回肠切除术的CD患者队列，并确定IEL 表型、定位和效应子功能与疾病发病机制相关。这些实验将 不仅提供了人类回肠IEL隔室的第一个细胞图谱，而且还可能导致 开发用于治疗CD的新型预后生物标志物或治疗干预。

PROJECT SUMMARY. Immunological surveillance at barrier surfaces is essential to provide defense against enteric pathogens and maintain mucosal homeostasis. Disruption of the balance between pro-inflammatory and regulatory immune response can lead to a loss of mucosal tolerance and development of chronic inflammatory disease, such as Crohn's disease (CD). The majority of inflammatory responses in CD have been attributed to both innate and adaptive immune cells located in the lamina propria; however, relatively little is known regarding the contribution of the intraepithelial lymphocyte (IEL) compartment to CD pathogenesis. IELs, which include intraepithelial T cells (IET) and intraepithelial ILCs (IE-ILC), contribute to the maintenance of epithelial barrier integrity and function as first line of defense against luminal microorganisms. Due to the difficulty in obtaining a sufficient number of IELs from patient biopsies for in-depth functional analyses, we propose to leverage advances in single-cell RNA sequencing technology and RNA imaging to profile the phenotypic and functional characteristics of ileal IEL subpopulations in health and acute CD. Using transcriptomics as a guide to identify molecular signatures for individual IEL subsets, we will develop single-molecule fluorescence in situ hybridization (sm-FISH) probe multiplexes to visualize these cell populations in patient biopsies. In collaboration with the IBD Genetics Consortium, we will obtain tissue sections from longitudinal endoscopic biopsies of a CD patient cohort that has undergone ileal resection and determine the extent to which IEL phenotype, localization and effector function correlate to disease pathogenesis. These experiments will not only provide the first cell atlas of the human ileal IEL compartment, but may also lead to the development of novel prognostic biomarkers or therapeutic interventions for the treatment of CD.