Invariant NKT cell responses to viral infections and tumors

描述(由申请人提供)：不变自然杀伤T细胞(INKT)细胞是一种独特的淋巴细胞亚群，表达不变T细胞抗原受体(TCR)阿尔法链，在小鼠和人类之间高度保守。这种独特的TCR及其抗原特异性的保守性表明，在小鼠模型上进行的实验可能与人类研究有特别的相关性。这些细胞参与了对几种细菌的反应，这些细菌的糖脂抗原可以参与其典型的TCR。此外，最近的研究表明，iNKT细胞在对包括病毒在内的各种微生物的先天反应中发挥了作用，这些微生物不编码其不变的TCR抗原。我们的初步研究表明，与感染了小鼠巨细胞病毒(MCMV)的树突状细胞(DC)亚型培养后，iNKT细胞在体外被激活并产生细胞因子。此外，我们已经开始确定体内iNKT细胞在感染MCMV后36小时内启动先天免疫反应过程中的作用。MCMV是一种特性和研究都很好的病毒，是研究病毒感染后iNKT细胞如何激活的理想模型病原体。该方案的第一个目的是确定TLR9、IL-12和CD1d辐射的抗原提呈在MCMV感染激活iNKT细胞中的体内作用。我们大量的体外初步数据表明，在Flt3配体(FltSL)来源的DC培养中，具有激活MCMV感染的iNKT细胞的DC，而在GM-CSF来源的DC中不存在。体外激活需要TLR9的表达和IL-12的合成，但在大多数实验中，不需要CD1d的表达。这些数据表明，识别CD1d提呈的自身抗原并不是激活培养48小时的iNKT细胞所必需的。我们将在体内进行实验，以确定MCMV感染后体外产生的数据的相关性。第二个目的是确定在巨细胞病毒感染后激活iNKT细胞的相关DC类型(S)。由于FltSL来源的DC培养是DC亚型的异质性混合，我们将纯化DC亚群并确定那些负责iNKT细胞激活的人。这将使我们能够确定iNKT细胞在体外和体内激活的机制。我们的初步数据表明，FltSL DC培养中存在的浆细胞样树突状细胞可能有助于iNKT细胞的激活。因此，我们将使用PDCA-1特异性抗体在体内耗尽这些细胞，并评估这是否会改变体内MCMV感染时iNKT细胞的激活。此外，我们还将检测连续感染MCMV的体外纯化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.