Retrogenic humanized mice for the study of T1D

描述（由申请人提供）：1型糖尿病（T1D）在人类中直接研究尤其具有挑战性。当患者被诊断为T1D时，免疫系统对β细胞的破坏几乎已经完成，这使得研究疾病发展的机制变得困难。为了解决这个问题，我们建议开发一种新的人源化T1D小鼠模型，允许研究人类胰岛自身反应性T细胞的体内生成和功能。这种新模式是基于最近的技术突破。1)人胰岛自身反应性t细胞克隆的产生。2)人类造血干细胞（HSC）高效转导慢病毒技术的发展。3)新型NOD-scid IL2rgnull （NSG）小鼠的可用性，这些小鼠在植入人类HSC后支持产生功能性的人类免疫系统。4)人类胎儿肝脏/胸腺/胰腺模型的开发，在该模型中，人类免疫系统在自体功能人类胰腺的存在下产生。结合这些新的尖端技术，我们将产生人类TCR转基因（“逆转录”）小鼠，植入富含人类TCR转基因（Tg）自身反应性T细胞的功能性人类免疫系统。为了实现这一目标，我们开发了“下一代”转基因NSG小鼠，包括HLA Tg NSG小鼠。我们已经在实验室中建立了人HSC的慢病毒转导，并从人IGRP265-273特异性的hla - a2限制性CD8 T细胞克隆中克隆了tcr；(b)来自人类GAD555-567特异性hla - dr4限制性CD4 T细胞克隆的TCR和(c)阳性对照hiv特异性TCR。我们还开发了一种自发性糖尿病nrg -秋田小鼠品系，该品系可以植入人类胎儿肝脏、胸腺和胰腺，以产生人类免疫系统，该免疫系统在自体人类胎儿胰腺移植物的存在下发育成熟并调节葡萄糖稳态。我们将利用这些新技术产生“逆转录”的NSG-HLA Tg小鼠，并将其植入人类免疫系统，表达针对IGRP265-273或GAD555-567的Tg TCRs。我们将测试人类TCR Tg自身反应性CD4和CD8 T细胞单独或共同诱导人源化小鼠糖尿病的总体假设。Specific Aim 1将组装并验证创建具有人类T1D反应性免疫系统的逆转录小鼠所需的组件。具体目标2将确定人类TCR逆转录模型是否发展为胰岛素炎或T1D，并将分析环境扰动调节这一过程的能力。这些研究将导致人源化T1D小鼠模型的发展，其中所有人类免疫成分都存在于疾病的发生和进展中。这项研究的结果将为研究T1D的发展提供一个重要的临床前模型系统，而不会使患者处于危险之中。这些新的动物模型的产生、验证和使用将允许研究调节人类自身反应性T细胞发育和功能的机制，并为治疗方法如何调节这一过程提供见解。

DESCRIPTION (provided by applicant): Type 1 diabetes (T1D) is particularly challenging to study directly in humans. By the time a patient is diagnosed with T1D, the destruction of beta cells by the immune system is nearly complete, rendering it difficult to investigate the mechanisms by which the disease develops. To address this, we propose to develop a novel humanized mouse model of T1D that permits study of the in vivo generation and function of human islet autoreactive T cells. This new model is based on recent technological breakthroughs. 1) Generation of human islet autoreactive T-cell clones. 2) Development of lentiviral technology enabling efficient transduction of human hematopoietic stem cells (HSC). 3) Availability of novel NOD-scid IL2rgnull (NSG) mice that support the generation of a functional human immune system following engraftment with human HSC. 4) Development of a human fetal liver/thymus/pancreas model in which a human immune system is generated in the presence of an autologous, functioning human pancreas. Combining these new cutting edge technologies, we will generate human TCR-transgenic ("retrogenic") mice engrafted with a functional human immune system enriched in human TCR transgenic (Tg) autoreactive T cells. To accomplish this, we have developed "next generation" genetically modified NSG mice, including HLA Tg NSG mice. We have established lentiviral transduction of human HSC in our laboratory, and have cloned into the virus (a) TCRs from HLA-A2-restricted CD8 T cell clones specific for human IGRP265-273; (b) TCRs from HLA-DR4-restricted CD4 T cell clones specific for human GAD555-567 and (c) a positive control HIV-specific TCR. We have also developed a spontaneously diabetic NRG-Akita mouse strain that can be engrafted with human fetal liver, thymus, and pancreas to generate a human immune system that develops in the presence of the autologous human fetal pancreas graft that matures and regulates glucose homeostasis. We will use these new technologies to generate "retrogenic" NSG-HLA Tg mice engrafted with a human immune system expressing Tg TCRs against IGRP265-273 or GAD555-567. We will test the overall hypothesis that human TCR Tg autoreactive CD4 and CD8 T cells either alone or together will induce diabetes in humanized mice. Specific Aim 1 will assemble and validate the components needed to create retrogenic mice with human T1D reactive immune systems. Specific Aim 2 will determine whether the human TCR retrogenic models develop insulitis or T1D and will analyze the ability of environmental perturbations to modulate this process. These studies will lead to the development of a humanized mouse model of T1D in which all of the human immune components are present to participate in disease initiation and progression. The results of this study will provide an important pre-clinical model system for investigation of T1D development without putting patients at risk. The generation, validation, and use of these new animal models will permit investigation of mechanisms that regulate human autoreactive T cell development and function and provide insights into how therapeutics may modulate this process.