Live Imaging of Immunity to M. tuberculosis

项目摘要 结核病（TB），由结核分枝杆菌引起，每年杀死的人比 是否患有任何其他传染病，包括艾滋病毒。消除结核病的障碍之一是缺乏一个 非常有效的疫苗。尽管有强有力的证据表明CD 4 T细胞在结核免疫中起着重要作用， 自然发生的T细胞反应并不能可靠地消除结核病中的病原体， 限制CD 4 T细胞对M.结核在早期的工作中，我们 发现，为了获得最佳的免疫控制，CD 4 T细胞必须直接识别并制造分子间 与M的联系肺内感染部位的结核感染细胞。根据这一要求， 亲密接触，我们和其他人已经发现（在小鼠，非人灵长类动物和人类），CD 4 T细胞是 位于肉芽肿的外围，而不是感染细胞所在的核心。因此我们 假设限制对结核病免疫的主要机制是CD 4 T细胞无法接触和参与 与受感染的细胞，克服这一失败将增加T细胞免疫结核病的功效。几 可能的机制可以解释M的空间分离。结核感染的细胞和体内的CD 4 T细胞， 但是这些不适于使用固定的、静态的图像或从肉芽肿中取出的细胞进行分析 环境因此，在这个项目中，我们建议将联合收割机与一个独特的多光子显微镜系统相结合， 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， M.结核菌株在UCSF开发和表征，以表征抗原特异性 CD 4 T细胞与M.肺结核感染和旁观者细胞在活小鼠的肺。结合这些 创新的实验系统使我们能够测试特定的假设，可以解释空间和 CD 4 T细胞和M.肺里的结核感染细胞知识来自于 这些研究将用于：1）为实验设计提供信息，以确定限制 CD 4 T细胞与M.结核感染细胞在体内; 2）为研究比较 不同的候选结核病疫苗，以确定它们中的哪一种促进T细胞的发育， M.肉芽肿核心中的结核感染细胞。在另外的实验中，我们将扩展最近的发现， 表明先天性淋巴样细胞（ILC）在对TB的保护性免疫中的重要作用。具体来说，我们将遵循 2型ILC（ILC 2）在体内经历了向ILC 1样细胞的显著表型转化， 回应M。结核感染，通过ILC 2和ILC 1样细胞与M. 结核感染的细胞和CD 4 T细胞，在感染的初始和慢性阶段。我们的研究 具有提供有价值的见解和新的结核病免疫范例的巨大潜力，其目标是提供信息， 开发有效的疫苗和针对宿主的疗法。

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.