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的质量。白介素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。对IL-12及其拮抗剂IL-10如何决定细胞毒性淋巴细胞激活(CTL)的完整了解仍有待确定。本研究的目的是确定单核细胞增多性李斯特氏菌感染树突状细胞产生白介素12(IL-12)和白介素10(IL-10)调节初始CD8 T细胞(CTL)激活和记忆细胞保护性群体发育的机制。我们的实验室已经证明，在启动过程中，中和培养物中的IL-10可以增强幼稚的CTL激活，而中和IL-12则具有相反的作用。因此，我们假设细胞因子IL-12和IL-10在启动过程中调节初始CD8T细胞和感染李斯特菌的树突状细胞之间的相互作用，这种调节决定了获得保护性免疫能力的效应器的数量。为了验证这一假说，我们提出了以下具体目标：具体目标1：确定李斯特菌胞浆进入树突状细胞产生的细胞因子IL-12和IL-10在体外如何调节干扰素-γ测定的初始T细胞的效应器功能以及这些细胞在体内的保护能力。特定目的2：确定IL-12或IL-10是否在体外调节初始CD8 T细胞与李斯特菌感染的树突状细胞之间的相互作用的频率和持续时间。为了解决这些问题，幼稚的CTL将在体外暴露于李斯特菌感染的野生型、IL-12基因敲除或IL-10基因敲除的DC。这些细胞因子的去除将通过监测干扰素产生、IL-12受体信号和极化，以及过继转移初始CTL后对单核细胞增多性李斯特菌的体内保护试验来分析。此外，当IL-12或IL-10被中和时，CTL和DC之间的相互作用的持续时间和频率将通过基于时间推移的视频显微镜和基于流式细胞术的结合分析来监测。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CD8T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.