Natural Killer Dendritic Cells in Listeria Infection

描述（由申请人提供）：虽然普遍认为干扰素-γ（IFN-γ）对单核细胞增生李斯特菌的清除至关重要，但IFN-γ的细胞来源一直存在争议。一些研究人员得出结论，NK细胞负责，而其他人则涉及T细胞。最近，我们已经确定了一种新的细胞类型，分泌大量的IFN-γ，并存在于正常小鼠的脾脏和其他器官。由于这些独特的细胞直接裂解靶细胞，在体外和体内激活初始T细胞，并表达自然杀伤细胞标志物NK1.1和树突状细胞标志物CD 11 c，我们将其标记为自然杀伤树突状细胞（NKDC）。其他一些研究人员已经在啮齿动物和人类中发现了NKDC样细胞。我们有初步的数据，脾脏NKDC产生IFN-γ在李斯特菌感染小鼠。因此，我们假设NKDC对针对李斯特菌的免疫应答至关重要。虽然NKDC的裂解和抗原呈递能力可能很重要，但我们选择专门关注其IFN-γ的生产，以用于这2年的应用。我们建议评估李斯特菌感染小鼠期间NKDC产生IFN-γ的生物学意义，并剖析其调节机制。在目标1中，我们将确定李斯特菌感染期间NKDC参与IFN-γ产生的程度。为了排除其他细胞在体内产生IFN-γ，但一旦它们从脾脏中的局部环境中去除就不能这样做的可能性，我们将利用最近描述的脾细胞体内细胞内细胞因子分析技术。目的2：建立李斯特菌感染过程中NKDC产生IFN-γ的机制。具体来说，我们将测试IL-12、IL-18和Toll样受体9，因为我们已经发现它们响应细菌CpG调节NKDC IFN-γ的产生。在目标3中，我们将确定NKDC产生的IFN-γ是否足以诱导针对李斯特菌的免疫应答。我们将从野生型小鼠中过继转移NKDC到IFN-γ缺陷小鼠中，然后评估它们对李斯特菌感染的反应。将NKDC与目标1中确定的产生IFN-γ的NK细胞和其他脾细胞亚群进行比较。我们提出的实验将开始定义NKDC在对李斯特菌免疫反应中的作用，李斯特菌是一种导致人类死亡的病原体，也是生物恐怖主义的潜在代理人，并提供对这种新型细胞类型如何参与其他类型免疫的更好理解。

DESCRIPTION (provided by applicant): While there is general consensus that interferon-gamma (IFN-gamma) is vital to the clearance of Listeria monocytogenes, the cellular source of IFN-gamma has been controversial. Some investigators have concluded that NK cells are responsible, while others have implicated T cells. Recently, we have identified a novel cell type that secretes large amounts of IFN-gamma and exists in the spleen and other organs of normal mice. Because these unique cells lyse targets directly, activate naive T cells in vitro and in vivo, and express both the natural killer cell marker NK1.1 and the dendritic cell marker CD11c, we have labeled them as natural killer dendritic cells (NKDC). A few other investigators have recognized NKDC-like cells in rodents and humans. We have preliminary data that splenic NKDC produce IFN-gamma during Listeria infection in mice. Therefore, we hypothesize that NKDC are critical to the immune response against Listeria. While the lytic and antigen-presenting abilities of NKDC may be important, we have chosen to focus exclusively on their IFN-gamma production for this 2-year application. We propose to assess the biologic significance of IFN-gamma production by NKDC during Listeria infection in mice and dissect the mechanisms by which it is regulated. In Aim 1, we will determine the extent of NKDC involvement in IFN-gamma production during Listeria infection. To exclude the possibility that other cells make IFN-gamma in vivo, but are unable to do so once they are removed from their local environment in the spleen, we will make use of a recently described technique of in vivo intracellular cytokine analysis of splenocytes. In Aim 2, we will establish the mechanism of IFN-gamma production by NKDC during Listeria infection. Specifically, we will test IL-12, IL-18, and Toll-like receptor 9 as we have found these to regulate NKDC IFN-gamma production in response to bacterial CpG. In Aim 3, we will determine whether NKDC production of IFN-gamma is sufficient to induce an immune response against Listeria. We will adoptively transfer NKDC from wild-type mice into IFN-gamma deficient mice and then assess their response to Listeria infection. NKDC will be compared to NK cells and other splenic cell subsets that make IFN-gamma as identified in Aim 1. Our proposed experiments will begin to define the role of NKDC in the immune response to Listeria, a pathogen that is a cause of human mortality and a potential agent for bioterrorism, and provide a greater understanding of how this novel cell type may participate in other types of immunity.