Live Imaging of Immunity to M. tuberculosis

项目摘要 由结核分枝杆菌引起的结核病每年导致的死亡人数比 有没有其他传染病，包括艾滋病毒。消除结核病的障碍之一是缺乏 足够有效的疫苗。尽管有强有力的证据表明CD4T细胞在结核病免疫中起着重要作用， 自然产生的T细胞反应并不能可靠地消除结核病中的病原体，我们也不完全了解 限制CD4T细胞对结核分枝杆菌应答有效性的机制。在早期的工作中，我们 发现为了最佳的免疫控制，CD4T细胞必须直接识别和制造分子间 在肺部感染部位与结核分枝杆菌感染细胞接触。根据这一要求， 亲密接触，我们和其他人发现(在小鼠、非人类灵长类动物和人类中)CD4T细胞是 位于肉芽肿的边缘，而不是位于感染细胞所在的核心。因此，我们 假设限制对结核病免疫的一个主要机制是CD4T细胞无法接触和接触 通过感染细胞，克服这一缺陷将提高T细胞对结核病的免疫效力。几个 可能的机制可以解释体内结核分枝杆菌感染细胞和CD4T细胞的空间分离， 然而，这些不能用固定的静态图像或从肉芽肿中取出的细胞来分析 环境。因此，在这个项目中，我们建议结合一个独特的多光子显微镜系统来 活体成像，包含在图卢兹IPBS的生物安全3级设施中，使用独特的报告小鼠和 在加州大学旧金山分校培养和鉴定结核分枝杆菌菌株，以表征抗原特异性的相互作用 活体小鼠肺中带有结核分枝杆菌感染和旁观者细胞的CD4T细胞。这些结合在一起 创新的实验系统使我们能够测试特定的假设，这些假设可以解释空间和 肺中CD4T细胞和结核分枝杆菌感染细胞的功能分离。从以下方面获得的知识 这些研究将用于：1)为实验设计提供信息，以确定限制 体内CD4T细胞与结核分枝杆菌感染细胞的相互作用；2)为比较研究提供基础 不同的候选结核病疫苗，以确定其中哪种疫苗促进T细胞的发展，获得最佳的 肉芽肿核心内结核分枝杆菌感染细胞。在其他实验中，我们将扩展最新的发现 提示先天淋巴样细胞(ILC)在结核病保护性免疫中的重要作用。具体地说，我们将遵循 研究发现，2型ILC(ILC2)在体内经历了戏剧性的表型转化为ILC1样细胞。 对结核分枝杆菌感染的反应，通过实时成像ILC2和ILC1样细胞与结核杆菌的相互作用。 在感染的初始阶段和慢性阶段，结核感染细胞和CD4T细胞。我们的研究 有很高的潜力提供有价值的见解和结核病免疫的新范例，目的是向 开发有效的疫苗和宿主导向疗法。

Project Summary Tuberculosis (TB), caused by the bacterium, Mycobacterium tuberculosis, kills more humans every year than does any other infectious disease, including HIV. Among the obstacles to eliminating TB is the lack of a sufficiently-efficacious vaccine. Despite strong evidence for essential roles of CD4 T cells in TB immunity, naturally-occurring T cell responses do not reliably eliminate the pathogen in TB, and we do not fully understand the mechanisms that limit the effectiveness of CD4 T cell responses to M. tuberculosis. In earlier work, we discovered that for optimal immune control, CD4 T cells must directly recognize and make intermolecular contacts with M. tuberculosis-infected cells at the site of infection in the lungs. In light of this requirement for intimate contact, we and others have found (in mice, nonhuman primates, and humans) that CD4 T cells are located at the periphery of granulomas, rather than in the core where infected cells reside. We therefore hypothesize that a major mechanism limiting immunity to TB is the failure of CD4 T cells to contact and engage with infected cells, and that overcoming this failure will increase the efficacy of T cell immunity to TB. Several potential mechanisms can explain the spatial separation of M. tuberculosis-infected cells and CD4 T cells in vivo, yet these are not amenable to analysis using fixed, static images or cells removed from the granuloma environment. Therefore, in this project, we propose to combine a unique multiphoton microscopy system for intravital imaging, contained in a Biosafety Level 3 facility at the IPBS in Toulouse, with unique reporter mice and M. tuberculosis strains developed and characterized at UCSF, to characterize interactions of antigen-specific CD4 T cells with M. tuberculosis-infected and bystander cells in the lungs of live mice. The combination of these innovative experimental systems allows us to test specific hypotheses that can account for the spatial and functional separation of CD4 T cells and M. tuberculosis-infected cells in the lungs. The knowledge gained from these studies will be used: 1) to inform design of experiments to define the molecular mechanisms that restrict CD4 T cell interactions with M. tuberculosis-infected cells in vivo; 2) to provide the basis for studies comparing distinct candidate TB vaccines, to determine which of them promote development of T cells that optimally access M. tuberculosis-infected cells in granuloma cores. In additional experiments, we will extend recent findings indicating important roles for innate lymphoid cells (ILC) in protective immunity to TB. Specifically, we will follow up on findings that type 2 ILC (ILC2) undergo dramatic phenotypic transformations to ILC1-like cells in vivo in response to M. tuberculosis infection, by live imaging of the interactions of ILC2 and ILC1-like cells with M. tuberculosis-infected cells and with CD4 T cells, during the initial and the chronic stages of infection. Our studies have high potential to provide valuable insights and new paradigms of immunity to TB, with the goal of informing development of efficacious vaccines and host-directed therapies.