The role of IDO and Th17 in an experimental mouse model of glioma

描述(由申请人提供)： 诊断为多形性胶质母细胞瘤(GBM)的患者的中位生存期为一年。尽管手术、化疗和放射治疗不断改善，但自1950年以来，GBM的长期存活率并没有改善。据推测，为GBM提供更有效治疗的方法之一是通过免疫调节。为了研究这一假设，我们先前确定在人的GBM中发现了免疫抑制调节性T细胞(Tregs)，这是在实验性小鼠脑胶质瘤模型中概括的。利用实验性的小鼠胶质瘤模型，我们还确定Treg的耗竭增加了长期存活率。基于这些发现，这项研究计划旨在通过研究免疫调节酶吲哚胺2，3双加氧酶(IDO)来扩大观察范围，该酶目前正被用于乳腺癌、肺癌、胰腺癌以及黑色素瘤的第一阶段临床试验。IDO已被证明调节Treg向产生IL-17A的促炎CD4+T辅助细胞(Th17s)的转化。然而，之前没有研究确定IDO和Th17在GBM中的作用。因此，本研究旨在确定：1)IDO的作用；2)Th17s的作用；3)IDO在原位和转基因小鼠脑胶质瘤模型中调节Treg/Th17水平的机制。目的1将利用野生型(WT)、IDO-/-和CD4-/-小鼠、IDO抑制剂、shRNA和CD4+T-基因敲除IDO以及树突状细胞过继转移，对胶质瘤的体内转基因和原位小鼠模型进行分析。将分析IDO mRNA水平、IDO酶活性、IDO蛋白定位、Treg/Th17水平和胶质瘤侵袭性的变化，并与小鼠生存相关。目的2将使用WT-、IL-17A-/-、ROR3T-/-和CD4-/--小鼠，结合CD4+T细胞过继转移，对选择性IL-17A缺乏的原位胶质瘤小鼠模型进行关键测试。将分析Treg/Th17频率、Treg/Th17相关细胞表面和细胞因子表达、IL-17A蛋白表达、IDO酶活性和胶质瘤侵袭性的变化，并与小鼠生存相关。目的3研究IDO在体内调节Treg/Th17转化的机制，使用IL-17报告基因、CD4-/-、CD4+pSTAT5-/-和CD4+pSTAT3-/--原位小鼠模型，用CD4+T细胞过继转移抑制胶质瘤1IDO。分析CD4+T细胞Treg/Th17频率、pSTAT3和pSTAT5水平的变化，过继转移的Tregs和Th17s的转化率，Treg/Th17绝对值，以及CD4+CD25+、CD4+CD25+IL-17+、CD4+IL-17+-T细胞抑制能力。人脑胶质瘤是一种致命性胶质瘤，可导致Tregs和Th17s的浸润，以及IDO的选择性表达。这项建议旨在通过临床前的恶性胶质瘤实验小鼠模型来了解IDO在维持Tregs和Th17s之间的平衡方面所起的作用。 公共卫生相关性： 多形性胶质母细胞瘤(GBM)是一种起源于胶质细胞的高度恶性的中枢神经系统原发肿瘤，中位生存期为15个月。我们和其他人已经证明，CD4+T淋巴细胞渗入人的GBM，以及重现GBM的小鼠模型，导致研究表明，免疫调节是未来GBM治疗的潜在靶点。为了进一步进行这些研究，本提案旨在通过脑内注射和自发发育的小鼠GBM模型，研究吲哚胺2，3双加氧酶(IDO)的作用、辅助性T细胞(Th17s)的作用以及IDO调节Treg/Th17水平的分子机制。

DESCRIPTION (provided by applicant): The median survival for patients diagnosed with glioblastoma multiforme (GBM) is one year. Although surgery, chemotherapy and radioactive therapy have continuously improved, long-term survival from GBM has not improved since 1950. One of the methods that have been hypothesized to provide more effective treatment for GBM is through immunomodulation. To investigate this hypothesis, we previously determined that immunosuppressive regulatory T cells (Tregs) are found in human GBM, which is recapitulated in experimental mouse models of glioma. Using an experimental mouse model of glioma, we also determined that Treg depletion increases long-term survival. Based on those discoveries, this research proposal aims to extend those observations by investigating the immunomodulatory enzyme, indoleamine 2, 3 dioxygenase (IDO), which is currently being targeted in Phase 1 clinical trials for breast, lung, and pancreatic cancers, as well as melanoma. IDO has been shown to regulate the conversion of Treg into IL-17A-producing pro- inflammatory CD4+ T helper 17 cells (Th17s). However, no previous investigation has determined the roles of IDO and Th17s in GBM. Therefore, this study aims to identify: 1) the role of IDO, 2) the role of Th17s and 3) the mechanism by which IDO regulates Treg/Th17 levels in orthotopic and transgenic mouse models of glioma. Aim 1 will provide an analysis of in vivo transgenic and orthotopic mouse models of glioma in combination with select IDO deficiency using wild-type (WT), IDO-/- and CD4-/- mice, IDO inhibitors, IDO knockdown with shRNA and CD4+ T- and dendritic cell-adoptive transfers. The changes in IDO mRNA levels, IDO enzyme activity, IDO protein localization, Treg/Th17 levels and glioma invasiveness will be analyzed and correlated to mouse survival. Aim 2 will critically test in vivo orthotopic mouse models of glioma with selective IL-17A deficiency using WT-, IL-17A-/--, ROR3t-/-- and CD4-/--mice, in conjunction with CD4+ T cell adoptive transfers. The changes in Treg/Th17 frequency, Treg/Th17-related cell surface and cytokine expression, IL- 17A protein expression, IDO enzyme activity and glioma invasiveness will be analyzed and correlated to mouse survival. Aim 3 will investigate the mechanism by which IDO regulates Treg/Th17 conversion in vivo using IL-17 reporter-, CD4-/--, CD4+pSTAT5-/--, and CD4+pSTAT3-/--orthotopic mouse models of glioma 1IDO inhibition with CD4+ T cell adoptive transfers. The changes in Treg/Th17 frequency, pSTAT3 and pSTAT5 levels in CD4+ T cells, conversion of adoptively transferred Tregs and Th17s, absolute Treg/Th17 numbers, and CD4+CD25+-, CD4+CD25+IL-17+-, CD4+IL-17+-T cell suppressor capacity will be analyzed. Human GBM is a fatal type of glioma, which results in the infiltration of Tregs and Th17s, as well as the selective expression of IDO. This proposal aims to understand the roles that IDO plays in maintaining the balance between Tregs and Th17s using pre-clinical experimental mouse models of malignant glioma. PUBLIC HEALTH RELEVANCE: Glioblastoma multiforme (GBM) is a highly malignant primary tumor of the central nervous system of glial origin with a median survival time of 15 months. We and others have shown that CD4+ T lymphocytes infiltrate human GBM, as well as mouse models that recapitulate GBM, leading to studies demonstrating that, immunomodulation is a potential target for future treatment of GBM. To further these studies, this proposal aims to investigate the role of indoleamine 2, 3 dioxygenase (IDO), the role of t helper 17 cells (Th17s) and the molecular mechanism by which IDO regulates Treg/Th17 levels using intracranially-injected and spontaneously-developing mouse models of GBM.