Regulation of T cell differentiation during enteric viral infection

描述(申请人提供)：诺如病毒(NV)胃肠炎是导致全球发病率和死亡率的主要因素，但对NV免疫知之甚少，也没有有效的疫苗存在。最近发现的小鼠诺如病毒(MNV)是一种能够在组织培养中生长的新的自然小鼠肠道病原体基因组，它帮助确立了T细胞在NV清除中的关键作用。尽管取得了这些进展，但仍然存在一些重要的问题，包括：(1)诺沃克病毒感染过程中T细胞分化的动力学是什么？ 以及，(2)为什么某些诺沃克病毒株会持续存在于具有免疫能力的宿主体内？为了解决这些问题，我们已经确定了新的免疫优势T细胞表位，并构建了MHC-肽四聚体。这些新工具使我们第一次能够追踪MNV特异性T细胞亚群及其功能特性。根据初步结果，我们推测，除了对感染的初始清除至关重要，肠粘膜中的表位特异性T细胞亚群还可以提供广泛和持久的诺沃克病毒免疫。相反，在某些情况下，MNV特异性T细胞功能的丧失(衰竭)会导致病原体的持久性。这一假设将被以下相互关联的具体目标所追求。(1)MNV特异性T细胞分化的动力学是什么，T细胞是在哪里启动的？对粘膜感染的免疫需要独特的T细胞分化状态和启动机制。首先，使用四聚体试剂，我们将直接跟踪和计数黏膜感染与全身感染后的表位特异性效应T细胞和记忆T细胞。其次，我们将使用过继转移来确定黏膜途径与外周途径启动的MNV特异性T细胞亚群的保护能力。(2)树突状细胞(DC)疫苗能否诱导强大的T细胞对诺如病毒的免疫？目前的诺如病毒疫苗导致较弱的T细胞反应，这可能是由于VLP未能招募粘膜T细胞启动所需的特定DC亚群所致。为了解决这种可能性，来自肠道和外周的DC将暴露于免疫优势的MNV T细胞表位，并用于接种初生鼠。首先，我们将定义来自DC免疫动物的MNV特异性T细胞在再攻击前后的特性。第二，我们将使用治疗性DC疫苗接种来治疗持续的MNV感染。这些实验将通过定义保护性T细胞免疫的DC相关来与AIMS 1和3对接。(3)在持续感染期间，MNV特异性T细胞的分化状态和保护特性是什么？某些诺沃克病毒株会建立长期感染。首先，我们将通过确定MNV特异性CD4和CD8T细胞的表型、功能和保护能力来测试持续MNV感染期间是否发生T细胞功能障碍。其次，我们将通过使用免疫学和遗传学方法选择性地干扰关键的抑制途径来定义MNV特异性T细胞耗竭的可逆性。这些创新的体内研究将为T细胞在MNV感染中的作用提供机械性见解，并作为治疗策略的平台。 公共卫生相关性：诺如病毒是全球疾病的主要贡献者。在初步实验中，利用小鼠诺如病毒感染性肠炎模型，我们定义了高度保守的免疫优势T细胞表位，并构建了MHC-肽四聚体试剂。我们建议使用这些新的分子工具来(1)确定T细胞免疫在诺如病毒感染中的作用；(2)探索新的疫苗接种途径；(3)确定导致诺如病毒持续感染的免疫机制。

DESCRIPTION (provided by applicant): Norovirus (NV) gastroenteritis is a major contributor to global morbidity and mortality, yet little is known about NV immunity and no effective vaccine exists. The recent discovery of murine noroviruses (MNV) a new genogroup of natural enteric mouse pathogens capable of growth in tissue culture has helped establish a key role for T cells in NV clearance. Despite these advances, a number of important questions remain, including: (1) what are the dynamics of T cell differentiation during norovirus infection~ and, (2) why do certain strains of norovirus persist in immunocompetent hosts? To address these questions we have identified novel immunodominant T cell epitopes and have constructed MHC-peptide tetramers. These new tools have allowed us, for the first time, to track MNV-specific T cell subsets and their functional properties. Based on preliminary results, we hypothesize that, in addition to being essential for initial clearance of infection, epitope- speciic T cell subsets in the intestinal mucosa can provide broad and long-lasting norovirus immunity~ conversely, loss of MNV-specific T cell function (exhaustion) in some settings leads to pathogen persistence. This hypothesis will be pursued by the following interrelated Specific Aims. (1) What are the dynamics of MNV-specific T cell differentiation, and where are T cells primed? Immunity to mucosal infection requires unique T cell differentiation states and priming mechanisms. First, using tetramer reagents, we will directly track and enumerate epitope-specific effector and memory T cells following mucosal vs. systemic infection. Second, we will use adoptive transfer to define the protective capacity of MNV-specific T cell subsets primed by mucosal vs. peripheral route. (2) Can dendritic cell (DC) vaccination induce robust T cell immunity to norovirus? Current norovirus vaccines result in weak T cell responses and this may be caused by failure of VLPs to recruit specific DC subsets necessary for mucosal T cell priming. To address this possibility, DCs from intestinal vs. peripheral sites will be exposed to immunodominant MNV T cell epitopes, and used to vaccinate na¿ve mice. First, we will define the properties of MNV-specific T cells from DC- immunized animals before and after rechallenge. Second, we will use therapeutic DC vaccination to treat persistent MNV infection. These experiments will interface with Aims 1 and 3 by defining DC correlates of protective T cell immunity. (3) What are the differentiation states and protective properties of MNV- specific T cells that arise during persistent infection? Certain norovirus strains establish long-term infection. First, we will test whether T cell dysfunction occurs during persistent MNV infection by defining the phenotype, function, and protective capacity of MNV-specific CD4 and CD8 T cells from chronically vs. acutely infected mice. Second, we will define the reversibly of MNV-specific T cell exhaustion by using immunologic and genetic methods to selectively disrupt key inhibitory pathways. These innovative in vivo studies will provide mechanistic insights into the role of T cells in MNV infection, and serve as a platform for therapeutic strategies. PUBLIC HEALTH RELEVANCE: Noroviruses are major contributors to global disease. In preliminary experiments using the murine norovirus model of infectious enteritis we have defined highly conserved immunodominant T cell epitopes and have constructed MHC-peptide tetramer reagents. We propose to use these new molecular tools to (1) define the role of T cell immunity during norovirus infection~ (2) explore novel vaccination approaches~ and (3) define immune mechanisms leading to persistent norovirus infection.