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

描述(由申请人提供)：CD4+CD25+FoxP3+T细胞被诱导以及它们抑制自身免疫诱导的分子和细胞机制尚未阐明。这个T细胞亚群在调节多发性硬化症(MS)动物模型EAE中的作用才刚刚开始研究。为了在体内从单细胞水平研究FoxP3+T细胞，我们通过同源重组的方法将绿色荧光蛋白(GFP)特异性地导入内源性FoxpS基因座，获得了FoxPS基因敲除小鼠(FoxPSKI)。在FoxpSKI小鼠中，利用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，发现在EAE过程中，MOG特异性T-regs在中枢神经系统(CNS)积聚，并产生IL-10和意外的干扰素-g。为了研究T-regs在EAE中的作用，我们将利用DTR/GFP(白喉毒素受体与绿色荧光蛋白融合)报告基因导入内源性FoxPS基因座的小鼠，允许通过给予白喉毒素在体内有条件地耗尽FoxPS表达细胞。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，方法是在EAE的不同阶段将它们删除。我们还发现，在体外，在转化生长因子-b和IL-6的共同作用下，CD4+T细胞可以分化为致病的Th17细胞，或者在转化生长因子-b单独存在的情况下，T-reg可以分化为致病Th17细胞。这些结果表明，在活体内这两个亚群的功能存在相互作用，并且它们可能相互调节。然而，炎症环境和致病Th17细胞是否可以调节T-reg细胞的生成和抑制活性尚不清楚。为了解决Th17和T-reg之间相互作用的这些重要问题，我们培育了一种新型的报告小鼠，在这种小鼠中，表达IL-17的细胞IL-17可以被红色荧光蛋白(RFP)跟随，这些细胞可以通过注射白喉毒素有条件地被耗尽。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：MOG特异性Tregs的不同亚群的起源和功能是什么？AIM2：IL-6在EAE的Treg功能中起什么作用？Aim3：T-regs和Th17在EAE中有什么相互作用？

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?