Invariant NKT cell responses to viral infections and tumors

描述（由申请人提供）：不变自然杀伤T（iNKT）细胞是一种独特的淋巴细胞亚群，表达小鼠和人类之间高度保守的不变T细胞抗原受体（TCR）α链。这种独特的TCR及其抗原特异性的保守性表明，在小鼠模型中进行的实验可能与人类研究特别相关。这些细胞参与了对几种细菌的反应，这些细菌具有可以参与其经典TCR的糖脂抗原。此外，最近的研究表明，iNKT细胞在对各种微生物（包括病毒）的先天性应答中发挥作用，这些微生物不编码其不变TCR的抗原。我们的初步研究表明，iNKT细胞在体外被激活，产生细胞因子后，与树突状细胞（DC）亚型，已感染小鼠巨细胞病毒（MCMV）的培养。此外，我们已经开始确定在感染后36小时内先天免疫应答启动期间，iNKT细胞在体内对MCMV感染的应答中的作用。MCMV是一种充分表征和研究的病毒，是研究病毒感染后iNKT细胞如何活化的理想模型病原体。该建议的第一个目的是确定TLR 9、IL-12和CD 1d介导的抗原呈递在MCMV感染激活iNKT细胞中的体内作用。我们广泛的初步体外数据表明，在MCMV感染后，能够活化iNKT细胞的DC出现在Flt 3配体（FltSL）衍生的DC培养物中，但不在GM-CSF衍生的DC中。体外活化需要TLR 9表达和IL-12的合成，但在大多数实验中，不需要CD 1d表达。这些数据表明，CD 1d呈递的自身抗原的识别对于激活培养48小时的iNKT细胞不是绝对必要的。我们将进行体内实验，以确定MCMV感染后体外产生的数据的相关性。第二个目标是鉴定MCMV感染后激活iNKT细胞的相关DC类型。由于FltSL衍生的DC培养物是DC亚型的异质混合物，我们将纯化DC亚群并鉴定负责iNKT细胞活化的那些。这将使我们能够确定负责iNKT细胞在体外和体内活化的机制。我们的初步数据表明，FltSL DC培养物中存在的浆细胞样DC可能有助于iNKT细胞活化。因此，我们将使用PDCA-1特异性抗体在体内耗尽这些细胞，并评估这是否改变了体内MCMV感染后iNKT细胞的活化。此外，我们将测试用MCMV顺序感染的离体纯化的DC亚群在体外激活iNKT细胞的能力。

DESCRIPTION (provided by applicant): Invariant Natural Killer T (iNKT) cells are a unique subpopulation of lymphocytes that expresses an invariant T cell antigen receptor (TCR) alpha chain that is highly conserved between mice and humans. This unique TCR, and the conservation of its antigenic specificity, suggests that experiments performed in mouse models may have a particular relevance for human studies. These cells have been implicated in the responses to several bacteria that have glycolipid antigens that can engage their canonical TCR. Additionally, recent studies have demonstrated that iNKT cells have a role in the innate response to diverse microbes, including viruses, that do not encode antigens for their invariant TCR. Our preliminary studies have demonstrated that iNKT cells are activated in vitro to produce cytokines after culture with dendritic cell (DC) subtypes that have been infected with mouse cytomegalovirus (MCMV). Additionally, we have begun to identify the role of iNKT cells in vivo in response to infection with MCMV during the initiation of the innate immune response within 36 hours after infection. MCMV is a well characterized and studied virus and is ideal model pathogen for investigating how iNKT cells are activated following virus infection. The first aim in this proposal is to identify the in vivo roles of TLR9, IL-12, and CD1d-rnediated antigen presentation for the activation of iNKT cells by MCMV infection. Our extensive preliminary in vitro data indicates that DC capable of iNKT cells activation upon MCMV infections arise in Flt3 ligand (FltSL) derived DC cultures, but not in DC derived in GM-CSF. Activation in vitro requires TLR9 expression and the synthesis of IL-12, but in most experiments, did not require CD1d expression. These data suggest that recognition of self-antigens presented by CD1d is not absolutely necessary to activate iNKT cells in culture for 48 hrs. We will carry out experiments in vivo to identify the relevance of the data generated in vitro following MCMV infection. The second aim will identify the relevant DC type(s) that activate iNKT cells after MCMV infection. Since the FltSL derived DC culture is a heterogeneous mix of DC subtypes, we will purify DC subsets and identify those responsible for iNKT cell activation. This will enable us to define the mechanisms responsible for iNKT cell activation in vitro and in vivo. Our preliminary data suggests that plasmacytoid DC present in the FltSL DC cultures may contribute iNKT cell activation. Therefore, we will deplete these cells in vivo using a PDCA-1- specific antibody and assess whether this alters activation of iNKT cells upon MCMV infection in vivo. Additionally, we will test the ability of ex vivo purified DC subsets sequentially infected with MCMV to activate iNKT cells in vitro.