Novel humanized mouse model of mucosal immunity

项目总结 尽管人源化小鼠已经成功地用于艾滋病毒感染的体内研究，但癌症和 免疫疗法、人类幼稚免疫反应和适应性免疫反应仍然是次优的，交叉反应有限。 鼠和人的细胞因子之间被认为是一个关键的促成因素。因此，人类的粘膜 免疫系统没有完全发育，缺少几个组成部分，包括人类的存在 肺上皮细胞在其自然的肺环境中。为了解决这些研究差距，我们提出了一个综合的 基因和细胞编辑方法开发用于人类研究的下一代人源化小鼠 粘膜免疫。我们的方法有两个目标：在目标1中，我们将构造和认证hNSGF- SGM3-IL6-TSLP-TSLPR小鼠用于人类免疫细胞发育。我们将产生小鼠表达一种 人TSLP受体的完整及其与人造血祖细胞的植入检测 通过测量骨髓、脾、肠道、呼吸道、肺和血液中免疫细胞的细胞组成。 为了检测人类的免疫功能，我们将使用一种粘膜配方的流感疫苗(Flumist)作为 免疫触发和抗原源，分析血液中的细胞因子反应 和脾；人类细胞迁移到粘膜部位、脾和骨髓；以及疫苗抗原的诱导- 脾中的特异性T细胞和B细胞。在目标2中，我们将构建人源化的呼吸道上皮，并在 HNSGF-SGM3-IL6-TSLP-TSLPR小鼠。我们将研究人类呼吸道上皮细胞的发育。 人支气管上皮祖细胞在NSGF-SGM3-IL6-TSLP-TSLPR小鼠体内的移植(人源化或人源化) 不是来自同一捐赠者的HPC)通过组织免疫荧光染色的人类特异性靶标，包括 为了建立人类呼吸道上皮细胞的功能，我们将用活的小鼠 流感病毒和包括IL-33在内的物种特异性警报的诱导；I型干扰素签名 人体上皮细胞作为体内功能以及组织组成和吸引力的衡量标准 人类免疫细胞对人类呼吸道上皮细胞的作用。我们的假设是，一个完整的TSLP信号通路 而人类上皮细胞将通过促进串扰提高人源化小鼠的人类黏膜免疫力 在基质细胞和免疫细胞之间。该模型随后可用于I.粘膜疫苗的开发，以及 二、对其他呼吸道病毒的研究，包括SARS-CoV-2，它需要人类特异性受体来 确定感染。因此，一旦获得认证，我们的模型将为未来的机械研究奠定基础 在遗传可变供体背景下的活体粘膜免疫以及使粘膜研究成为可能 佐剂、过敏原、疫苗和生物制品。

PROJECT SUMMARY Although humanized mice have been successfully used for in vivo studies of HIV infection, cancer and immunotherapies, human naïve and adaptive immune responses remain suboptimal, with limited cross-reactivity between murine and human cytokines considered as a key contributing factor. As a result, the human mucosal immune system is not fully developed, and several components are missing, including the presence of human lung epithelial cells in their natural lung environment. To address these research gaps, we propose a combined genetic and cellular editing approach to develop the next generation of humanized mice for studies of human mucosal immunity. Our approach is structured in two aims: in Aim 1, we will construct and credential hNSGF- SGM3-IL6-TSLP-TSLPR mice for human immune cell development. We will generate mice expressing a complete human TSLP receptor and examine engraftment with human hematopoietic progenitor cells (HPCs) by measuring cellular composition of immune cells in the bone marrow, spleen, gut, airways, lungs, and blood. To examine human immune function, we will use a mucosal formulation of influenza vaccine (Flumist) as an immune trigger and antigen source as we have done in the past and analyze cytokine responses in the blood and spleen; human cell migration to mucosal sites, spleen, and bone marrow; and induction of vaccine antigen- specific T and B cells in the spleen. In Aim 2, we will construct and credential humanized airway epithelium in hNSGF-SGM3-IL6-TSLP-TSLPR mice. We will examine the development of human airway epithelium upon transplant of human bronchial epithelial progenitor cells in NSGF-SGM3-IL6-TSLP-TSLPR mice (humanized or not with HPCs from the same donor) by tissue immunofluorescence staining of human-specific targets including HLA class I. To establish the functionality of human airway epithelial cells, we will challenge mice with live influenza virus and measure the induction of species-specific alarmins including IL-33; type I interferon signature in human epithelial cells as a measure of their functionality in vivo, as well as tissue composition and attraction of human immune cells to human airway epithelium. Our hypothesis is that a complete TSLP signaling pathway and human epithelial cells will improve human mucosal immunity in humanized mice by facilitating the crosstalk between stromal cells and immune cells. This model can then be used for i. mucosal vaccine development, and ii. studies of other respiratory viruses including SARS-CoV-2, which requires human-specific receptors to establish infection. Thus, once credentialed, our model will lay the groundwork for future mechanistic studies of mucosal immunity in vivo in the context of genetically variable donors as well as enable studies of mucosal adjuvants, allergens, vaccines, and biologicals.