Enhancing Glioma-Specific Immunity

描述（由申请人提供）：尽管几十年的疫苗研究，中枢神经系统肿瘤仍然是一个重要的健康问题。因此，了解中枢神经系统癌症的细胞免疫反应对于开发治疗这些疾病的免疫治疗策略至关重要。多形性胶质母细胞瘤（GBM）是最致命的癌症之一，尽管接受治疗，其平均生存期为12-15个月。在绝大多数病例中，完全手术切除肿瘤是不可行的。因此，开发能够在体内产生肿瘤抗原特异性T细胞反应的新型疫苗至关重要。我们的中心假设是，有效的胶质瘤特异性CD8 T细胞介导的免疫是通过适当的肿瘤抗原处理来实现的。为了解决我们的中心假设，我们将使用建立的GL261模型系统，使用我们实验室产生的新型重组小核糖核酸病毒和条件敲除小鼠来分析体内抗胶质瘤杀伤T细胞反应。我们还将评估注射小核糖核酸病毒疫苗在体内促进保护性免疫的途径，以及肿瘤抗原呈递和加工的机制。使用表达模型T细胞表位的工程GL261细胞系（称为“Quad Cassette”），我们在初步研究中确定，肿瘤特异性杀伤T细胞反应可以在体内对这种胶质瘤产生。此外，我们提出的小核糖核酸病毒疫苗接种方法可以使用该模型阻止或根除中枢神经系统中已建立的肿瘤的进展。因此，我们为这个胶质瘤模型系统生成的新试剂和技术将使我们能够通过执行以下特定目标来解决我们的中心假设：特定目标#1 -使用新型小核糖核酸病毒疫苗优化CD8 T细胞对内源性肿瘤抗原的反应。特异性目标#2 -确定通过有条件地沉默肿瘤抗原加工和在各种抗原呈递细胞（APCs）中呈递而产生的保护性CD8 T细胞介导的针对胶质瘤的免疫的机制。该项目具有创新性，因为它提供了一种多学科的方法，使用一种新的小核糖核酸病毒疫苗接种方法在体内产生中枢神经系统衍生的肿瘤特异性抗原。在此过程中，还将确定专业抗原提呈细胞对非法CD8 T细胞免疫脑癌的能力。我们已经建立了在活体动物中使用生物发光成像和MRI来监测GL261胶质瘤生长动力学的方法。我们的期望是基于小核糖核酸病毒的疫苗将被证明是有效的产生抗肿瘤CD8 T细胞反应。然而，本提案还将评估所有apc在中枢神经系统对肿瘤免疫中的重要性。这将对利用该系统优化增强抗肿瘤杀伤T细胞反应的新型免疫治疗策略产生重大影响。此外，所提出的研究具有转译性，因为可修饰的人小核糖核酸病毒可以作为治疗迄今无法治愈的癌症的新临床方法。为了实现这些目标，我们将采用：(a)流式细胞术，(b)行为研究，(c)高分辨率共聚焦显微镜和免疫组织化学，(d)病毒学，(e)蛋白质生物化学，(f)生物发光成像，(g)小型哺乳动物MRI。

DESCRIPTION (provided by applicant): CNS tumors remain a significant health concern despite decades of vaccine research. For this reason, understanding the cellular immune response that is mounted towards CNS cancers is of paramount importance in the development of immunotherapeutic strategies to treat these conditions. Glioblastoma multiforme (GBM) is among the most lethal of cancers, which despite treatment, presents with an average survival of 12-15 months. Complete surgical resection of the tumor is not feasible in the vast majority of cases. Therefore, it is critical to develop novel vaccines to generate tumor antigen specific T cel responses in vivo. Our central hypothesis is that is effective glioma-specific CD8 T cell-mediated immunity is achieved through appropriate tumor antigen processing To address our central hypothesis, we will use the established GL261 model systems to analyze anti-glioma killer T cell responses in vivo using novel recombinant picornaviruses and conditional knockout mice generated by our laboratory. We will also assess the route of injection of picornavirus vaccine in promoting protective immunity in vivo, as well as the mechanism of tumor antigen presentation and processing. Using an engineered GL261 cell line (termed "Quad Cassette") that expresses model T cell epitopes, we have determined in our preliminary studies that tumor-specific killer T cell response can be generated towards this glioma in vivo. Furthermore, we present that the proposed picornavirus vaccination approach can halt or eradicate progression of established tumors in the CNS using this model. Therefore, the novel reagents and techniques we have generated for this model system of glioma will enable us to address our central hypothesis through conducting the following specific aims: Specific Aim #1 - Optimize CD8 T cell responses to endogenous tumor antigens using novel picornavirus vaccination. Specific Aim #2 - Define the mechanism by which protective CD8 T cell mediated immunity against gliomas is generated through conditional silencing of tumor antigen processing and presenting in various antigen presenting cells (APCs). This project is innovative because it provides a multidisciplinary approach to generate CNS derived tumor- specific antigens in vivo using a novel picornavirus vaccination approach. In the process, the capacity of professional antigen presenting cells to illicit CD8 T cell immunity against brain cancer will also be determined. We have established the methodology to monitor growth kinetics of the GL261 glioma in vivo using bioluminescence imaging and MRI in live animals. Our expectation is that picornavirus based vaccines will prove to be effective in generating anti-tumor CD8 T cell responses. However, the importance of all APCs in CNS immunity to tumors will also be assessed in this proposal. This would have significant impact in that novel immunotherapeutic strategies to enhance anti-tumor killer T cell responses could be optimized using this system. Furthermore, the proposed studies are translational in that modifiable human picornaviruses could be employed as novel clinical approach to an as yet incurable cancer. To accomplish these aims, we will employ: (a) flow cytometry, (b) behavioral studies, (c) high resolution confocal microscopy and immunohistochemistry, (d) virology, (e) protein biochemistry, (f) bioluminescence imaging, and (g) small mammal MRI.