Characterization of degranulation regulators in human mast cells

项目总结/摘要 该项目的长期目标是了解人类肥大细胞（MC）功能的调节， 阐明MCs在人类健康和疾病中的作用。MCs是IgE相关免疫反应中的主要效应细胞。 响应（例如，哮喘、过敏、寄生虫免疫）。这些细胞也可能有助于某些自身免疫性疾病 疾病和启动对病原体和其他试剂的反应。一旦激活，MC经历 脱粒，导致许多介质（包括储存的产物，例如，组胺、肝素 和蛋白酶），以及脂质介质的产生，例如，LTC 4和PGD 2以及许多细胞因子， 生长因子根据环境，MC可以具有效应子和/或免疫调节作用。最 研究MC发育和功能的研究采用了各种小鼠模型。但我们 对调节人类MC激活的机制知之甚少。同样，相对 关于人类MC中信号通路的扰动如何有助于MC， 相关病理学这反过来又阻碍了用于治疗和/或预防癌症的治疗剂的设计。 预防人类受试者的过敏和其它肥大细胞相关疾病。我们最近开发了一个 采用功能基因组学的技术平台，加上高分辨率单细胞共聚焦成像， 其可以快速鉴定人肥大细胞中的脱粒调节剂。我们现在建议使用这个 平台，以确定培养的供体细胞中IgE/FceRI依赖性信号转导途径的关键调节因子， 衍生的原代人肥大细胞。具体而言，我们将尝试在原代人肥大细胞中验证， 选择先前在啮齿动物肥大细胞中鉴定的主要脱粒调节剂。我们还将使用针对性 排列人CRISPR-Cas9文库以鉴定新的（即，先前未鉴定的）IgE/FceRI的调节剂- 在原代人肥大细胞中依赖信号通路。此外，我们将研究功能 通过使用基于CRISPR-Cas9的方法在肥大细胞增多症患者中检测到的KIT D816 V突变的意义 基因编辑技术诱导/纠正人HMC-1细胞系和原代人HMC-1细胞系中的相同突变 肥大细胞为了实现我们的目标，我们提出了两个目标。在目标1中，我们将鉴定新的人肥大细胞 脱粒调节剂和系统地定义IgE/FceRI依赖性信号传导途径， 人肥大细胞在目标2中，我们将使用CRISPR-Cas9“引物编辑”来进行HMC的突变研究。 1人肥大细胞系和原代人肥大细胞。这项工作将有助于创建一个详细的模型 并将开始分析KIT D816 V突变是如何 影响人类肥大细胞生物学。

PROJECT SUMMARY/ABSTRACT This project’s long-term goals are to understand the regulation of human mast cell (MC) function and to elucidate the roles of MCs in human health and disease. MCs are major effector cells in IgE-associated responses (e.g., asthma, allergy, parasite immunity). These cells also may contribute to certain autoimmune disorders and in the initiation of responses to pathogens and other agents. Upon activation, MCs undergo degranulation, leading to the secretion of many mediators (including stored products, e.g., histamine, heparin and proteases), as well as the production of lipid mediators, e.g., LTC4 and PGD2, and many cytokines and growth factors. Depending on the setting, MCs can have effector and/or immunoregulatory roles. Most research investigating MC development and function has employed various mouse models. However, we know considerably less about the mechanisms that regulate the activation of human MCs. Similarly, relatively little is known about how the perturbation of signaling pathways in human MCs can contribute to MC- associated pathology. This, in turn, has hampered the design of therapeutic agents for the treatment and/or prevention of allergies and other mast cell-associated diseases in human subjects. We recently developed a technology platform employing functional genomics, coupled with high-resolution single-cell confocal imaging, which can rapidly identify regulators of degranulation in human mast cells. We now propose to use this platform to identify key regulators of IgE/FceRI-dependent signal transduction pathways in cultured, donor- derived, primary human mast cells. Specifically, we will attempt to validate, in primary human mast cells, selected major degranulation regulators previously identified in rodent mast cells. We also will use targeted arrayed human CRISPR-Cas9 libraries to identify novel (i.e., previously unidentified) regulators of IgE/FceRI- dependent signaling pathways in primary human mast cells. In addition, we will investigate the functional significance of the KIT D816V mutation detected in mastocytosis patients by using the CRISPR-Cas9-based gene editing technologies to induce/correct the same mutation in human HMC-1 cell lines and primary human mast cells. To achieve our goals, we propose two aims. In Aim 1, we will identify novel human mast cell degranulation regulators and systematically define IgE/FceRI-dependent signaling pathways in primary human mast cells. In Aim 2, we will use CRISPR-Cas9 “prime editing” to perform mutational studies of HMC- 1 human mast cell lines and primary human mast cells. Together, this work will help create a detailed model of mast cell degranulation in human mast cells and will begin to analyze how the KIT D816V mutation may influence human mast cell biology.