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，疫苗载体和佐剂的发展进步极大地提高了公共卫生质量。白细胞介素12 （IL-12）是一种细胞因子，已被证明具有强大的佐剂活性，因此这种细胞因子可以改善针对多种细胞内病原体和肿瘤的治疗设计。关于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。本研究项目的目的是确定白细胞介素12 （IL-12）和白细胞介素10 （IL-10）在树突状细胞感染模型病原体单核增生李斯特菌时产生的机制，李斯特菌调节幼稚CD8+ T细胞（CTL）的激活和保护性记忆细胞群的发展。我们的实验室已经证明，在培养过程中，IL-10的中和增强了初始CTL的激活，而IL-12的中和则具有相反的效果。因此，我们假设细胞因子IL-12和IL-10调节初始CD8+ T细胞和李斯特菌感染的树突状细胞在启动过程中的相互作用，这种调节决定了获得保护性免疫能力的效应物的数量。为了验证这一假设，我们提出以下具体目标：具体目标1：确定细胞因子IL-12和IL-10在进入树突状细胞时产生的细胞因子如何调节体外干扰素γ测量的初始T细胞的效应功能以及这些细胞在体内的保护能力。特异性目的2：确定IL-12或IL-10是否调节体外初始CD8+ T细胞与李斯特菌感染的树突状细胞之间相互作用的频率和持续时间。为了解决这些问题，幼稚的ctl将暴露于李斯特菌感染的野生型，IL-12敲除或IL-10敲除dc。去除这些细胞因子的影响将通过监测干扰素γ产生，IL-12受体信号和极化，以及对单核细胞增生李斯特菌过继转移后的体内保护试验来分析。此外，当IL-12或IL-10被中和时，ctl和DCs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共轭测定来监测。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CD8+ T细胞的功能结果。通过这项工作产生的发现可以转化为改进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。

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.