Characterization of degranulation regulators in human mast cells

项目概要/摘要 该项目的长期目标是了解人类肥大细胞（MC）功能的调节并 阐明 MC 在人类健康和疾病中的作用。 MCs是IgE相关的主要效应细胞 反应（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 疾病以及对病原体和其他物质的反应的启动。激活后，MC 会经历 脱颗粒，导致许多介质的分泌（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 LTC4 和 PGD2，以及许多细胞因子和 生长因子。根据具体情况，MC 可以发挥效应器和/或免疫调节作用。大多数 调查 MC 发育和功能的研究采用了各种小鼠模型。然而，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导致 MC- 知之甚少 相关病理学。这反过来又阻碍了用于治疗和/或治疗的治疗剂的设计。 预防人类受试者的过敏和其他肥大细胞相关疾病。我们最近开发了一个 采用功能基因组学的技术平台，结合高分辨率单细胞共聚焦成像， 它可以快速识别人类肥大细胞脱颗粒的调节因子。我们现在建议使用这个 该平台可识别培养的供体中 IgE/FceRI 依赖性信号转导途径的关键调节因子 衍生的原代人类肥大细胞。具体来说，我们将尝试在原代人类肥大细胞中验证， 选择先前在啮齿动物肥大细胞中发现的主要脱粒调节因子。我们还将使用有针对性的 排列人类 CRISPR-Cas9 文库来识别 IgE/FceRI 的新型（即以前未识别的）调节因子 原代人类肥大细胞中的依赖性信号通路。此外，我们还将研究功能 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 D816V 突变的意义 基因编辑技术诱导/纠正人类 HMC-1 细胞系和原代人类中的相同突变 肥大细胞。为了实现我们的目标，我们提出两个目标。在目标 1 中，我们将鉴定新型人类肥大细胞 脱颗粒调节剂并系统地定义原发性中 IgE/FceRI 依赖性信号通路 人类肥大细胞。在目标2中，我们将使用CRISPR-Cas9“prime edit”来进行HMC-的突变研究 1 人类肥大细胞系和原代人类肥大细胞。这项工作将共同帮助创建一个详细的模型 人类肥大细胞中肥大细胞脱颗粒的研究，并将开始分析 KIT D816V 突变如何可能 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.