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

剖析 IL-12 在树突状细胞激活幼稚 CD8 T 细胞中的作用

• 批准号: 7322072
• 负责人: Curtis James Henry
• 金额: $ 4.1万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7322072
• 关键词: Address; Adjuvant; Adoptive Transfer; Affect; Antigen-Presenting Cells; Antigens; Biological Assay; CD80 gene; CD8B1 gene; Cell Communication; Cells; Dendritic Cells; Development; Effector Cell; Excision; Exhibits; Frequencies; Goals; Immunity; In Vitro; Infection; Inflammatory Response; Interferon Type II; Interleukin-10; Interleukin-12; Knock-out; Listeria; Listeria monocytogenes; Lymphocyte Activation; Measures; Mediating; Memory; Modeling; Molecular; Monitor; Numbers; Population; Process; Production; Public Health; Receptor Signaling; Regulation; Research Project Grants; Role; T-Cell Activation; T-Lymphocyte; Testing; Time; Translating; Video Microscopy; Work; base; cytokine; cytotoxic; day; functional outcomes; improved; in vivo; interleukin-12 receptor; pathogen; receptor; research study; therapeutic vaccine; therapy design; tumor; vaccine development; vector vaccine

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.

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，已被证明具有有效的佐剂活性，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激活 (CTL) 的完整理解仍有待确定。该研究项目的目标是确定树突状细胞感染模型病原体单核细胞增生李斯特氏菌后产生白细胞介素 12 (IL-12) 和白细胞介素 10 (IL-10) 调节幼稚 CD8+ T 细胞 (CTL) 激活和记忆细胞保护性群体的发展。我们的实验室已证明，培养物中 IL-10 的中和可增强启动过程中初始 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 在启动过程中调节初始 CD8+ 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器的数量。为了检验这一假设，我们提出以下具体目标： 具体目标 1：确定李斯特菌进入树突状细胞胞质时产生的细胞因子 IL-12 和 IL-10 如何调节初始 T 细胞的效应功能（通过干扰素 γ 测量）体外和这些细胞的体内保护能力。具体目标 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相互作用的频率和持续时间。为了解决这些问题，幼稚 CTL 将在体外暴露于李斯特菌感染的野生型、IL-12 敲除型或 IL-10 敲除型 DC。将通过监测干扰素 γ 的产生、IL-12 受体信号传导和极化，以及在过继转移初始 CTL 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。完成我的论文项目后，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目前使用的针对多种细胞内病原体和肿瘤的疫苗和疗法的改进。