Dissecting the Role of IL-12 in Naive CD8+ T cell Activation by Dendritic Cells

描述（由申请人提供）：近几十年来，疫苗载体和佐剂开发的进步极大地改善了公共卫生质量。白细胞介素12（IL-12）是一种细胞因子，已被证明具有有效的佐剂活性，因此这种细胞因子可以改善针对各种细胞内病原体以及肿瘤设计的治疗。IL-12及其拮抗剂白细胞介素10如何决定细胞毒性淋巴细胞活化（CTL）的完整理解仍有待确定。本研究的目的是确定模型病原体单核细胞增生李斯特菌（Listeria monocytogenes）感染树突状细胞后产生的白细胞介素12（IL-12）和白细胞介素10（IL-10）调节幼稚CD 8 + T细胞（CTL）活化和记忆细胞保护性群体的发育的机制。我们的实验室已经证明，中和培养物中的IL-10在引发期间增强幼稚CTL活化，而中和IL-12具有相反的效果。因此，我们假设细胞因子IL-12和IL-10调节初始CD 8 + T细胞和李斯特菌感染的树突状细胞之间的相互作用，并且这种调节决定了获得赋予保护性免疫能力的效应物的数量。为了检验这一假设，我们提出了以下具体目的：具体目的1：确定在树突状细胞中的细胞质进入后产生的细胞因子IL-12和IL-10如何调节初始T细胞的效应子功能，如通过干扰素γ在体外测量的，以及这些细胞在体内的保护能力。具体目标二：确定IL-12或IL-10是否调节初始CD 8 + T细胞与李斯特菌感染的树突状细胞之间的体外相互作用的频率和持续时间。为了解决这些问题，将在体外将幼稚CTL暴露于李斯特菌感染的野生型、IL-12敲除或IL-10敲除DC。将通过监测干扰素γ产生、IL-12受体信号传导和极化以及在幼稚CTL过继转移后针对单核细胞增生李斯特菌的体内保护测定来分析去除这些细胞因子的影响。此外，当IL-12或IL-10被中和时，CTL和DC之间相互作用的持续时间和频率将通过延时视频显微镜和基于流式细胞术的缀合物测定来监测。通过完成我的论文项目，我将阐明IL-12和IL-10如何在分子水平上决定CD 8 + T细胞的功能结果。通过这项工作产生的发现可以转化为目前使用的针对各种细胞内病原体和肿瘤的疫苗和治疗方法的改进。

DESCRIPTION (provided by applicant): Advancements in the development of vaccine vectors and adjuvants has vastly improved the quality of public health in recent decades. Interleukin 12 (IL-12) is a cytokine that has been shown to possess potent adjuvant activity, thus this cytokine could improve treatments designed against a variety of intracellular pathogens as well as tumors. A complete understanding of how IL-12, and its antagonist interleukin 10, dictates cytotoxic lymphocyte activation (CTLs) remains to be determined. The goal of this research project is to determine the mechanisms by which interleukin 12 (IL-12) and interleukin 10 (IL-10) produced upon dendritic cell infection with the model pathogen, Listeria monocytogenes regulate naive CD8+ T cell (CTL) activation and the development of a protective population of memory cells. Our lab has demonstrated that the neutralization of IL-10 in culture augments naive CTL activation during priming, while the neutralization of IL-12 has the opposite effect. Thus, we hypothesize that the cytokines IL-12 and IL-10 regulate the interactions between naive CD8+ T cells and Listeria-infected dendritic cells during priming and this regulation dictates the number of effectors that acquire the ability to confer protective immunity. In order to test this hypothesis we propose the following Specific Aims: Specific Aim 1: To determine how the cytokines IL-12 and IL-10 generated upon listerial cytoplasmic entry in dendritic cells regulate effector function of naive T cells as measured by interferon gamma in vitro and the protective capacity of these cells in vivo. Specific Aim 2: To determine if IL-12 or IL-10 regulate the frequency and duration of interactions between naive CD8+ T cells and Listeria- infected dendritic cells in vitro. In order to address these questions naive CTLs will be exposed to Listeria infected wild-type, IL-12 knockout, or IL-10 knockout DCs in vitro. The affects of the removal of these cytokines will be analyzed by monitoring interferon gamma production, IL-12 receptor signaling and polarization, and in vivo protection assays against Listeria monocytogenes after the adoptive transfer of naive CTLs. In addition, the duration and frequency of interactions between CTLs and DCs when IL-12 or IL-10 is neutralized will be monitored via time lapse video microscopy and flow cytometric based conjugate assays. By completion of my thesis project, I will have elucidated how IL-12 and IL-10 dictate the functional outcome of CD8+ T cells on the molecular level. The findings generated through this work could be translated into the improvement of currently used vaccines and therapeutics against a variety of intracellular pathogens and tumors.