Interplay Between Pathogenic and Regulatory T Cells in EAE

T辅助(h) 17细胞是CD4+ T细胞的一个新发现的亚群，与Th1细胞一起参与自身免疫性疾病的发展，如多发性硬化症（MS）及其动物模型实验性自身免疫性脑脊髓炎（EAE）。另一方面，调节性T细胞可以防止自身免疫的发展。Th17细胞及其分化相关因子促进自身免疫的机制尚未完全阐明。我们之前已经确定Th17细胞的分化需要TGF-2 + IL-6和后来的IL-21的联合作用。另一方面，IL- 23p19对于维持Th17反应和EAE的发展至关重要，因为缺乏IL- 23p19的小鼠对EAE具有抗性。我们发现IL-23受体（IL-23R）除了在Th17细胞上表达外，也在巨噬细胞和小胶质细胞等先天免疫系统细胞上表达。然而，IL-23对非t细胞的作用至今尚不清楚。我们培育了一种新的IL-23R绿色荧光蛋白（GFP）敲入（KI）报告小鼠，在这种小鼠中，所有表达IL-23R的细胞都会同时表达GFP，而对IL-23无反应。IL- 23r GFP KI小鼠将帮助我们确定IL-23在EAE发生和进展中对Th17细胞和巨噬细胞/小胶质细胞的相对贡献。我们进一步确定，在EAE发病时，Th17细胞迅速迁移到中枢神经系统（CNS），其数量与疾病严重程度相关。然而，Th17细胞的数量迅速下降，而Th1细胞的数量保持不变，这表明Th17细胞可能在EAE的早期起始期起主导作用，而Th1细胞在EAE的慢性期更多地参与。我们已经生成了IL-17F RFP/DTR小鼠，其中Th17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Th17在疾病不同阶段的作用。最后，为了进一步确定Th17细胞增强致病活性的机制，我们分离出一种在Th17细胞上选择性表达的表面分子podoplanin。以前从未报道过podoplanin在T细胞上的表达，但podoplanin先前与肿瘤细胞的迁移有关。我们的初步数据显示，在髓鞘抗原免疫小鼠体内注射抗podoplanin抗体，与对照组相比，治疗动物中枢神经系统中Th17细胞的数量增加，这表明podoplanin可能在Th17细胞的迁移中起作用。

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.