Trafficking and the role of myelin specific Regulatory T cells in EAE

描述（由申请人提供）：CD4+CD25+FoxP3+ T细胞诱导和抑制自身免疫诱导的分子和细胞机制尚未阐明。这一T细胞亚群在多发性硬化症（MS）动物模型EAE中的调节作用才刚刚开始被研究。为了在体内单细胞水平上研究FoxP3+ T细胞，我们专门将绿色荧光蛋白（GFP）通过同源重组引入内源性FoxpS基因组位点，并产生FoxpS敲入小鼠（FoxPSKI）。在FoxpSKI小鼠中，使用MOG lAb特异性四聚体，我们在体内跟踪了MOG特异性调节细胞（T-regs）的产生，发现MOG特异性T-regs在EAE过程中在中枢神经系统（CNS）积累，并产生IL-10和意想不到的IFN-g。为了研究T-regs在EAE中的功能，我们将利用将DTR/GFP（白喉毒素受体与绿色荧光蛋白融合）报告基因引入内源性FoxPS位点的小鼠，通过给药白喉毒素在体内有条件地消耗表达FoxPS的细胞。利用这些小鼠，我们将能够研究MOG特异性T-regs的产生、运输和体内功能，通过在EAE的不同阶段删除它们。我们还发现CD4+ T细胞可以在体外分化为致病性Th17细胞，在TGF-b加IL-6存在或T-reg存在单独TGF-b。这些结果表明，这两个亚群在体内的功能存在互惠性，并且它们可能相互调节。然而，炎症环境和致病性Th17细胞是否能调节T-reg细胞的产生及其抑制活性尚不清楚。为了解决这些与Th17和T-reg之间相互作用相关的重要问题，我们产生了一种新的报告小鼠，在这种小鼠中，IL-17表达细胞IL-17可以被红色荧光蛋白（RFP）跟踪，这些细胞可以通过白喉毒素有条件地耗尽。基于我们的新试剂和初步数据，我们设计了实验来直接回答以下问题：目的1:MOG特异性treg不同亚群的起源和功能是什么？目的2:IL-6在EAE中对Treg功能的作用是什么？目的3:T-regs和Th17inEAE之间的相互作用是什么？

DESCRIPTION (provided by applicant): The molecular and cellular mechanisms by which CD4+CD25+FoxP3+ T cells are induced and by which they inhibit induction of autoimmunity have not been elucidated. The role of this subset of T cells in the regulation of EAE, an animal model of Multiple Sclerosis (MS), is just beginning to be investigated. To study FoxP3+ T cells at the single cell level in vivo, we specifically introduced the green fluorescent protein (GFP) into the endogenous FoxpS genomic locus by homologous recombination and generated a FoxPS knock-in mouse (FoxPSKI). In the FoxpSKI mice and with the use of MOG lAb specific tetramer, we followed in vivo the generation of MOG specific regulatory cells (T-regs) and found that MOG specific T-regs accumulate in the central nervous system (CNS) during the course of EAE, and produced IL-10 and unexpectedly IFN-g. To study the function of T-regs in EAE, we will take advantage of mice in which a DTR/GFP (Diphteria Toxin Receptor fused with the Green Fluorescent Protein) reporter has been introduced into the endogenous FoxPS locus, allowing in vivo conditional depletion of FoxPS expressing cells through administration of diphteria toxin. Using these mice we will be able to study the generation, trafficking and in vivo function of MOG specific T-regs, by deleting them at different stages of EAE. We also discovered that CD4+ T cells can differentiate in vitro into either pathogenic Th17 cells in the presence of TGF-b plus IL-6 or T-reg in the presence of TGF-b alone. These results suggest the existence of reciprocity in the function of these two subsets in vivo, and that they may regulate each other. However, whether the inflammatory milieu and pathogenic Th17 cells can modulate the generation and the suppressive activity of T-reg cells has not been elucidated. To address these important questions related to the interplay between Th17 and T-reg, we have generated a novel reporter mouse in which IL-17 expressing cells IL-17 can be followed by the Red Fluorescent Protein (RFP) and these cells can be conditionally depleted through administration of diphteria toxin. Based on our novel reagents and preliminary data, we have designed experiments to directly answer the following questions: Aim1: What is the origin and function of different subsets of MOG specific Tregs? Aim2: What is the role of IL-6 on Treg function in EAE? Aim3: What is the interplay between T-regs and Th17inEAE?