Regulation of T cell differentiation during enteric viral infection

描述（由申请方提供）：诺如病毒（NV）胃肠炎是全球发病率和死亡率的主要原因，但对NV免疫力知之甚少，也不存在有效的疫苗。 最近发现的鼠诺如病毒（MNV），一个新的基因组的自然肠道小鼠病原体能够在组织培养生长，帮助建立了一个关键的作用，T细胞在NV清除。尽管取得了这些进展，但仍存在一些重要的问题，包括：（1）诺如病毒感染期间T细胞分化的动力学是什么？ 以及（2）为什么某些诺如病毒株在免疫活性宿主中持续存在？ 为了解决这些问题，我们已经确定了新的免疫显性T细胞表位，并构建了MHC-肽四聚体。这些新工具使我们能够首次追踪MNV特异性T细胞亚群及其功能特性。基于初步结果，我们假设，除了对感染的初始清除至关重要之外，肠粘膜中的表位特异性T细胞亚群可以提供广泛和持久的诺如病毒免疫-相反，在某些情况下MNV特异性T细胞功能的丧失（耗竭）导致病原体持续存在。这一假设将通过以下相互关联的具体目标来实现。(1)MNV特异性T细胞分化的动力学是什么，T细胞在哪里启动？ 对粘膜感染的免疫需要独特的T细胞分化状态和引发机制。首先，使用四聚体试剂，我们将在粘膜与全身感染后直接追踪和计数表位特异性效应和记忆T细胞。第二，我们将使用过继转移来确定粘膜与外周途径致敏的MNV特异性T细胞亚群的保护能力。(2)树突状细胞（DC）疫苗接种能否诱导针对诺如病毒的强大T细胞免疫？目前的诺如病毒疫苗导致弱的T细胞应答，这可能是由于VLP未能募集粘膜T细胞引发所需的特异性DC亚群所致。为了解决这种可能性，将来自肠与外周部位的DC暴露于免疫显性MNV T细胞表位，并用于接种未处理小鼠。 首先，我们将定义来自DC免疫动物的MNV特异性T细胞在再激发之前和之后的性质。 第二，我们将使用治疗性DC疫苗来治疗持续性MNV感染。这些实验将通过定义保护性T细胞免疫的DC相关性来与目标1和3相结合。 (3)在持续感染期间出现的MNV特异性T细胞的分化状态和保护特性是什么？ 某些诺如病毒株建立长期感染。首先，我们将通过定义慢性与急性感染小鼠的MNV特异性CD 4和CD 8 T细胞的表型、功能和保护能力来测试在持续性MNV感染期间是否发生T细胞功能障碍。其次，我们将通过免疫学和遗传学方法选择性地破坏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制见解，并作为治疗策略的平台。

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.