Role of autophagy-related protein Vps34 in antigen presentation and self-tolerance

项目摘要 将自身和外源抗原呈递给T淋巴细胞涉及多种细胞过程，包括 蛋白水解、内吞作用、吞噬作用、囊泡运输和自噬。我们最近的研究调查了 自噬的作用，自我降解机制，以及相关的细胞过程中的MHC I类和类 II限制性抗原呈递给T淋巴细胞，长期目标是操纵这些途径， 预防或治疗人类疾病。我们关注的是自噬中的一个关键角色，III类磷脂酰肌醇- 3激酶（PI 3 K）空泡蛋白分选34（Vps 34）。我们已发表和未发表的初步研究 提供了Vps 34在MHC I类和II类限制性抗原呈递中的关键作用的证据， 树突状细胞（DC），其在诱导针对自身抗原的耐受性和启动免疫应答中起关键作用。 针对外来抗原的适应性免疫反应。我们已经证明，经典的MHC I类和I类 II抗原呈递途径在Vps 34缺陷型DC中增强，并且这些细胞具有特异性的 凋亡细胞相关抗原向MHC I类限制性CD 8 + T细胞交叉呈递的缺陷。 此外，使用胸腺上皮细胞（TEC）中携带选择性Vps 34缺陷的小鼠，我们 显示Vps 34介导的细胞过程在胸腺内T淋巴细胞发育中的关键作用。指导 在这个科学前提下，我们提出了一个总体假设，即Vps 34介导的自噬相关的 这些过程在向T淋巴细胞呈递抗原和促进自身耐受中起关键作用。我们 我们将在以下具体目标中检验这一假设：目标1将研究Vps 34介导的 在非常规MHC I类和II类限制性抗原呈递中的功能，并将探讨 涉及自噬依赖性和非自噬依赖性的分子机制。目标2将询问Vps 34的作用 胸腺内发育和T细胞库的选择。目的3将探讨Vps 34在以下方面的作用： 维持外周耐受和控制T淋巴细胞对自身抗原的应答。在这些研究中， 我们将采用多种实验方法，包括体外和体内抗原呈递分析， 和各种基因工程动物。由于全球Vps 34基因缺陷与生命不相容，我们将 使用在DC或TEC中选择性缺乏Vps 34表达的小鼠。我们将补充这些研究 使用Vps 34功能的药理学调节剂与鼠和人细胞的培养物接触。我们还将采取 表达neo自身抗原和/或抗原的各种报道动物和转基因小鼠的优点是 特异性T细胞受体。总的来说，这些拟议的研究将为我们提供一个全面的机制， 鉴于自噬相关蛋白Vps 34在抗原呈递、T细胞发育和免疫应答中的作用， 选择、外周耐受和T细胞免疫应答。这些拟议的研究将有助于 用于开发针对微生物病原体和肿瘤的改进疫苗和免疫疗法，以及 用于设计诱导对自身或外来抗原耐受的方法。

PROJECT SUMMARY The presentation of self and foreign antigens to T lymphocytes involves a variety of cellular processes, including proteolysis, endocytosis, phagocytosis, vesicle trafficking and autophagy. Our recent studies have investigated the role of autophagy, a self-degradation mechanism, and related cellular processes in MHC class I- and class II-restricted antigen presentation to T lymphocytes, with the long-term goal of manipulating these pathways to prevent or treat human disease. We have focused on a key player in autophagy, the class III phosphatidylinositol- 3 kinase (PI3K) vacuolar protein sorting 34 (Vps34). Our published and unpublished preliminary studies have provided evidence for a key role of Vps34 in both MHC class I- and class II-restricted antigen presentation by dendritic cells (DCs), which are critical players in the induction of tolerance against self-antigens and for initiating adaptive immune responses against foreign antigens. We have shown that the classical MHC class I and class II antigen presentation pathways are enhanced in Vps34-deficient DCs, and that these cells harbor a specific defect in the cross-presentation of apoptotic cell-associated antigens to MHC class I-restricted CD8+ T cells. Moreover, using mice carrying a selective deficiency of Vps34 in their thymic epithelial cells (TECs), we have shown a critical role of Vps34-mediated cellular processes in intrathymic T lymphocyte development. Guided by this scientific premise we propose the overall hypothesis that Vps34-mediated, autophagy-related processes play critical roles in presenting antigens to T lymphocytes and promoting self-tolerance. We will test this hypothesis in the following specific aims: Aim 1 will investigate the contribution of Vps34-mediated functions in unconventional MHC class I- and class II-restricted antigen presentation, and will explore the autophagy-dependent and -independent molecular mechanisms involved. Aim 2 will interrogate the role of Vps34 in the intrathymic development and selection of the T cell repertoire. Aim 3 will explore the role of Vps34 in maintaining peripheral tolerance and controlling T lymphocyte responses against autoantigens. In these studies, we will employ a variety of experimental approaches, including in vitro and in vivo antigen presentation assays, and various genetically engineered animals. As global Vps34 gene-deficiency is incompatible with life, we will employ mice selectively deficient in Vps34 expression in either DCs or TECs. We will complement these studies with cultures of murine and human cells using pharmacological modulators of Vps34 function. We will also take advantage of a variety of reporter animals and transgenic mice expressing neo self-antigens and/or antigen- specific T cell receptors. Collectively, these proposed studies will provide us with a comprehensive mechanistic view of the contribution of the autophagy-related protein Vps34 in antigen presentation, T cell development and selection, peripheral tolerance and T cell immune responsiveness. These proposed studies will be instrumental for the development of improved vaccines and immunotherapies against microbial pathogens and tumors, and for devising methods to induce tolerance against self or foreign antigens.