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1年。虽然手术、化疗和放射治疗不断改善，但自1950年以来，GBM的长期生存率没有改善。已经假设为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，我们以前确定，免疫抑制调节性T细胞（TCFs）中发现的人GBM，这是在胶质瘤的实验小鼠模型重演。使用神经胶质瘤的实验小鼠模型，我们还确定Treg耗竭增加长期存活。基于这些发现，这项研究提案旨在通过研究免疫调节酶吲哚胺2，3双加氧酶（IDO）来扩展这些观察结果，该酶目前正在针对乳腺癌、肺癌和胰腺癌以及黑色素瘤进行1期临床试验。已显示IDO调节Treg转化为产生IL-17 A的促炎性CD 4 + T辅助17细胞（Th 17）。然而，以前的研究还没有确定IDO和Th 17在GBM中的作用。因此，本研究旨在确定：1）IDO的作用，2）Th 17的作用和3）IDO调节胶质瘤原位和转基因小鼠模型中Treg/Th 17水平的机制。目的1将提供使用野生型（WT）、IDO-/-和CD 4-/-小鼠、IDO抑制剂、用shRNA和CD 4 + T-和树突状细胞过继转移的IDO敲减的胶质瘤的体内转基因和原位小鼠模型与选择IDO缺陷的组合的分析。将分析IDO mRNA水平、IDO酶活性、IDO蛋白定位、Treg/Th 17水平和胶质瘤侵袭性的变化，并将其与小鼠存活相关联。目的2将使用WT-、IL-17 A-/--、ROR 3 t-/--和CD 4-/--小鼠结合CD 4 + T细胞过继转移，严格测试具有选择性IL-17 A缺陷的胶质瘤的体内原位小鼠模型。将分析Treg/Th 17频率、Treg/Th 17相关细胞表面和细胞因子表达、IL-17 A蛋白表达、IDO酶活性和胶质瘤侵袭性的变化，并将其与小鼠存活相关联。目的3将使用IL-17报告基因-、CD 4-/--、CD 4 + pSTAT 5-/--和CD 4 + pSTAT 3-/--胶质瘤1 IDO抑制与CD 4 + T细胞过继转移的原位小鼠模型来研究IDO在体内调节Treg/Th 17转化的机制。将分析Treg/Th 17频率、CD 4 + T细胞中的pSTAT 3和pSTAT 5水平、过继转移的Th 17和Th 17的转化、Treg/Th 17绝对数量以及CD 4 + CD 25 +-、CD 4 + CD 25 +IL-17+-、CD 4 +IL-17+-T细胞抑制能力的变化。人GBM是一种致命的胶质瘤类型，其导致T细胞亚群和Th 17细胞的浸润以及IDO的选择性表达。本提案旨在使用恶性胶质瘤的临床前实验小鼠模型来了解IDO在维持Tcl 4和Th 17之间的平衡中所起的作用。 公共卫生相关性： 多形性胶质母细胞瘤（GBM）是一种高度恶性的原发性肿瘤的中枢神经系统的胶质源性与中位生存时间为15个月。我们和其他人已经表明，CD 4 + T淋巴细胞浸润人GBM，以及重演GBM的小鼠模型，导致研究表明，免疫调节是未来治疗GBM的潜在靶点。为了进一步研究这些问题，本研究的目的是利用颅内注射和自发发育的GBM小鼠模型研究吲哚胺2，3双加氧酶（IDO）的作用、辅助性T细胞17（Th 17）的作用以及IDO调节Treg/Th 17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.