Interplay Between Pathogenic and Regulatory T Cells in EAE

描述（由申请人提供）：摘要T辅助（h）17细胞是新近描述的CD 4 + T细胞的亚群，其与Th 1细胞沿着参与自身免疫性疾病如多发性硬化（MS）及其动物模型实验性自身免疫性脑脊髓炎（EAE）的发展。另一方面，调节性T细胞保护自身免疫的发展。Th 17细胞和与其分化相关的因子促进自身免疫的机制尚未完全阐明。我们先前已经确定，Th 17细胞的分化需要TGF-2加上IL-6和随后的IL-21的联合作用。另一方面，IL-23 p19对于维持Th 17应答和EAE的发展至关重要，因为IL- 23 p19缺陷的小鼠对EAE具有抗性。我们发现IL-23受体（IL-23 R）除了在Th 17细胞上表达外，还在先天免疫系统的细胞如巨噬细胞和小胶质细胞上表达。然而，IL-23对非T细胞的作用迄今为止尚不清楚。我们已经产生了一种新的IL-23 R绿色荧光蛋白（GFP）敲入（KI）报告小鼠，其中所有表达IL-23 R的细胞将同时表达GFP，并变得对IL-23无应答。IL-23 R GFP KI小鼠将帮助我们确定IL- 23对Th 17细胞和巨噬细胞/小胶质细胞在EAE发生和进展中的相对贡献。我们已经进一步确定，在EAE发作时，Th 17细胞迅速迁移到中枢神经系统（CNS），并且它们的数量与疾病的严重程度相关。然而，Th 17细胞的数量迅速下降，而Th 1细胞的数量保持，这表明Th 17细胞可能具有主导作用的早期在初始阶段，而Th 1细胞更多地参与在慢性期的EAE。我们已经产生了IL-17 F RFP/DTR小鼠，其中Th 17细胞可以通过注射白喉毒素在任何给定时间被删除，我们将能够确定Th 17在疾病的不同阶段的作用。最后，在我们的努力，以进一步确定的机制，Th 17细胞具有增强的致病活性，我们已经分离出一种表面分子选择性表达的Th 17细胞命名podoplanin。Podoplanin的表达以前从未在T细胞上报道过，但Podoplanin以前曾参与肿瘤细胞的迁移。我们的初步数据，在用髓磷脂抗原免疫的小鼠体内注射抗podoplanin抗体，显示与对照动物相比，治疗动物CNS中存在的Th 17细胞数量增加，表明podoplanin可能在Th 17细胞的迁移中起作用。 公共卫生相关性：基于我们的初步数据并使用我们生成的新试剂，我们设计了实验来解决几个对我们理解自身免疫性疾病很重要的突出问题：1）确定EAE期间效应Th 17和Th 1细胞以及T-reg细胞之间的相互作用，3）确定IL-23如何通过其对Th 17和先天免疫系统细胞的作用而加剧EAE的发展。这些研究将补充正在进行的关于Th 17细胞和调节性T细胞的研究，并有助于我们全面了解自身免疫及其调节的发展机制。

DESCRIPTION (provided by applicant): Abstract T helper (h) 17 cells are a newly described subset of CD4+ T cells which has been implicated, along with Th1 cells in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Regulatory T cells, on the other hand, protect from the development of autoimmunity. The mechanisms by which Th17 cells and factors associated with their differentiation promote autoimmunity have not been fully elucidated. We have previously determined that the differentiation of Th17 cells required the combined action of TGF-2 plus IL-6 and later IL-21. On the other hand IL-23 p19 is critical for sustaining Th17 responses and for the development of EAE since mice deficient for IL- 23 p19 are resistant to EAE. We found that IL-23 receptor (IL-23R), besides being expressed on Th17 cells, is also expressed on cells of the innate immune system such as macrophages and microglia. However, the role of IL-23 on non-T cells is to date unknown. We have generated a novel IL-23R green fluorescent protein (GFP) knockin (KI) reporter mouse in which all cells expressing the IL-23R will concomitantly express the GFP and become non responsive to IL-23. The IL-23R GFP KI mice will help us determine the relative contribution of IL- 23 on Th17 cells and macrophages/microglia in the development and progression of EAE. We have further determined that Th17 cells migrate quickly to the central nervous system (CNS) at the onset of EAE and their numbers correlate with the disease severity. However, the number of Th17 cells rapidly decline while the number of Th1 cells is maintained suggesting that Th17 cells may have a dominant role early on at the initiation phase and that Th1 cells are more involved during the chronic phase of EAE. We have generated an IL-17F RFP/DTR mouse in which Th17 cells can be deleted at any given time through the injection of diphtheria toxin and we will be able to determine the role of Th17 at different stages of the disease. Finally, in our efforts to further determine the mechanisms by which Th17 cells have enhanced pathogenic activity, we have isolated a surface molecule selectively expressed on Th17 cells named podoplanin. The expression of podoplanin has never been reported on T cells before but podoplanin has previously been implicated in the migration of tumor cells. Our preliminary data, injecting anti-podoplanin antibody in vivo in mice immunized with myelin antigen show an increase number of Th17 cells present in the CNS of the treated animals compared to control animals suggesting that podoplanin may play a role in the migration of Th17 cells. PUBLIC HEALTH RELEVANCE: Based on our preliminary data and using novel reagents that we have generated, we have designed experiments to address several outstanding questions that are important for our understanding of autoimmune diseases: 1) Determine the interplay between effector Th17 and Th1 cells, and T-reg cells during EAE, 2) Determine the role of podoplanin in the development of Th17 cells 3) Determine how IL-23 exacerbate the development of EAE through its action on Th17 and cells of the innate immune system. Project Narrative These studies will complement ongoing studies on Th17 cells and regulatory T cells and contribute to our overall understanding of the mechanisms involved in the development of autoimmunity and their regulation.