Development of 3D Alveolar Tissue Models, CRISPR-editing and Microbiota-immune Response Assay Platforms for Deciphering Human Lung Immunity

项目总结技术开发项目 技术开发项目(Tech Dev)的目标是开发以下方法、工具和分析 满足杰克逊实验室人类免疫学合作中心的需求 提高科学界解决有关人类肺部免疫的问题的能力 免疫-微生物区系相互作用和免疫细胞的基本机制。与肺免疫有关的主要问题 功能仍未得到回答-例如免疫细胞和肺上皮细胞之间的细胞间相互作用 影响对外来物质的反应，或呼吸道或肺间隔内微生物群的存在 影响病毒感染和其他肺部疾病的发病机制。研究的重大技术障碍 人类免疫-肺动力学是人类肺的绝对复杂性--它不断地过滤空气中的空气 通过肺上皮与居民之间的动态相互作用产生的颗粒物、感染性生物和空气 免疫细胞，如巨噬细胞或树突状细胞。这种复杂性不容易在动物身上建模。 系统或使用死亡的人类肺组织。为了克服这些挑战，技术开发人员将专注于三个方面 创新的人体组织平台：1)肺和上呼吸道的三维(3D)生物打印模型 研究暴露于病毒或新陈代谢期间肺免疫功能相互作用组的环境 试剂；2)基于CRISPR/Cas9的工具，用于对原始人类免疫细胞亚群进行基因工程，造血学 干细胞和/或肺上皮祖细胞用于探测细胞功能；以及3)一个功能强大的体外平台，用于 筛选驻留在肺部的微生物区系并确定它们对人类肺部免疫反应的影响。每一个 这些平台解决了应用程序中未得到满足的特定需求，并将使我们能够分别研究 人类肺免疫在动态和生理相关的微环境中，以询问特定的 预测对病毒感染有反应的细胞类型和分子途径，并评估细菌的影响 从人体呼吸道分离的代谢物对抗病毒反应的影响。通过这些努力，JAX CCHI将 具备解决以前难以解决的与肺免疫动力学相关的问题的能力，朝着更 从机理上理解肺免疫功能。我们的具体目标是： 目的1.利用三维生物打印技术建立人肺组织免疫相互作用的体外模型。 目标2：优化基于CRISPR的基因工具，用于工程原代人体免疫和肺上皮细胞 细胞。 目的3：建立功能免疫分析平台，确定人体免疫调节功能 肺和呼吸道微生物区系。

PROJECT SUMMARY TECHNOLOGY DEVELOPMENT PROJECT The goal of the Technology Development project (Tech Dev) is to develop approaches, tools and assays that address the needs of The Jackson Laboratory Cooperative Center on Human Immunology (JAX CCHI) and that advance the capabilities of the scientific community to tackle questions regarding human lung immunity, human immune-microbiota interactions and basic mechanisms of immune cells. Major questions related to lung immune function remain unanswered—such as the cell-to-cell interactions between immune and lung epithelial cells that shape responses to foreign agents, or how the presence of microbiota in the airways or within lung compartments influences the pathogenesis of viral infections and other lung diseases. A significant technical barrier to studying human immune-lung dynamics is the sheer complexity of the human lung—which constantly filters airborne particles, infectious organisms and air through dynamic interactions between the lung epithelium and resident immune cells such as macrophages or dendritic cells. This complexity cannot be easily modeled in animal systems or using deceased human lung tissue. To surmount these challenges, Tech Dev will focus on three innovative human tissue platforms: 1) three-dimensional (3D) bioprinted models of the lung and upper respiratory environment for investigating the functional lung-immune interactome during exposure to viral or metabolic agents; 2) CRISPR/Cas9-based tools to genetically engineer primary human immune cell subsets, hematopoietic stem cells and/or lung epithelial progenitors to probe cell function; and 3) a functional in vitro platform for screening lung-resident microbiota and determining their impact on human lung immune responses. Each of these platforms addresses a specific unmet need in the application and will enable us, respectively, to study the human lung immunity within a dynamic and physiologically relevant microenvironment, to interrogate specific cell types and molecular pathways predicted to respond to viral infections, and to assess the impact of bacterial metabolites isolated from human airways on antiviral responses. Through these efforts, the JAX CCHI will be equipped to address previously inaccessible questions related to lung-immune dynamics, towards a more mechanistic understanding of lung immune function. Our Specific Aims are: Aim 1. Develop in vitro models of human lung tissue-immune interactions using 3D bioprinting. Aim 2: Optimize CRISPR-based genetic tools for use in engineering primary human immune and lung epithelial cells. Aim 3: Develop a functional immune assay platform to determine the immunomodulatory landscape of human lung and airway microbiota.