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.
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.
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