Determining Protective Features of Human Memory T-cells to Inform Mycobacterium tuberculosis Vaccine Development

项目摘要/摘要 由结核分枝杆菌(Mtb)感染引起的结核病是一种可导致1.4人死亡的疾病 每年有几百万人。目前还没有可靠的疫苗来预防结核病，但许多潜伏感染的人正在 尽管在地方性环境中大量接触结核分枝杆菌，但防止进展为活动性结核病。CD4+T细胞 对于宿主预防结核病至关重要，因为它们直接与结核杆菌感染的细胞相互作用，分泌细胞因子和 细胞溶解分子，并招募或增强其他免疫细胞。然而，候选人和其他人发现， 所有针对结核分枝杆菌抗原的T细胞都能够识别结核分枝杆菌感染的巨噬细胞，而巨噬细胞是结核分枝杆菌的利基细胞。一个 疫苗开发尚未满足的关键需求是定义T细胞的抗原特异性和功能， 识别受感染的巨噬细胞，防止进展为活动性结核病。在目标1中，候选人使用自体 体外共培养和T细胞抗原受体(TCR)测序以确定比例、抗原特异性 以及识别结核分枝杆菌感染巨噬细胞的记忆CD4+T细胞的功能。在目标2中，候选人 在此系统上扩展以比较记忆CD4T细胞对感染的巨噬细胞的反应 在生活在结核病流行环境中但对活动性疾病的易感性不同的两组人中 疾病。利用单细胞转录组学，候选人比较了Mtb-1的功能和TCR谱系。 从未患活动性肺结核(“稳定”潜伏期)的暴露人群中分离出特异性记忆的CD4+T细胞 结核分枝杆菌感染)与后来发展为活动性结核病的个人(“结核病前期”进展者)之间的关系。本项目的成果 将定义与预防活动性结核病有关的保护性记忆CD4+T细胞的关键特征，提供 疫苗开发和改善结核病风险分层的基准。 这个为期5年的K08项目提供人类免疫学方面的指导、培训、翻译研究和 史蒂芬·卡彭特博士的单细胞转录，他是T细胞免疫学家和传染病内科医生， 凯斯西储大学(CWRU)。CWRU的制度环境结合了现有的 结核病研究单位，BSL-3流式细胞仪，细胞分类，单细胞RNA测序，以及生物信息学专业知识 以及一流的科学互动和课程研究生课程。历史悠久的乌干达- CWRU结核病研究合作使结核病流行环境中的临床和转化工作成为可能。这个 候选人正在建立一个实验室，其长期目标是了解保护性的定义特征 记忆T细胞对结核分枝杆菌的应答。他招募了在转化性结核病研究方面拥有丰富专业知识的导师， T细胞生物学和单细胞转录学，包括CWRU的Henry Boom和Mark Cameron博士，以及Dr。 萨姆·贝哈尔(加州大学马萨诸塞州医学院)。K08项目的完成将使应聘者转变为 独立研究人员，将他的研究计划定位为使用强大的单细胞免疫图谱 追踪和研究抗原特异性人类记忆T细胞的方法。

Project Summary / Abstract Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis (Mtb), is a disease that kills 1.4 million people every year. There is no reliable vaccine to prevent TB, yet many latently-infected individuals are protected from progressing to active TB despite heavy Mtb exposure living in an endemic setting. CD4+ T cells are critical for host protection against TB as they interact directly with Mtb-infected cells, secrete cytokines and cytolytic molecules, and recruit or augment other immune cells. However, the candidate and others find that not all T cells specific for Mtb antigens are able to recognize Mtb-infected macrophages, the niche cell for Mtb. A critical unmet need for vaccine development is to define the antigen specificities and functions of T cells that can recognize infected macrophages and prevent progression to active TB. In Aim 1, the candidate uses autologous ex vivo co-culture and T cell antigen receptor (TCR) sequencing to determine the proportion, antigen specificities and functions of memory CD4+ T cells that recognize Mtb-infected macrophages. In Aim 2, the candidate expands on this system to compare the repertoires of memory CD4 T cells that respond to infected macrophages among two groups of individuals who live in a setting endemic for TB yet differ in their susceptibility to active disease. Using single-cell transcriptomics, the candidate compares the functions and TCR repertoires of Mtb- specific memory CD4+ T cells isolated from exposed individuals who do not develop active TB (“stable” latent Mtb infection) vs. individuals who will later progress to active TB (“pre-TB” progressors). Results from this project will define key features of protective memory CD4+ T cells that are linked to the prevention of active TB, providing benchmarks for vaccine development and improvement of TB risk stratification. This 5-year K08 program provides mentoring, training in human immunology, translational research and single-cell transcriptomics for Dr. Stephen Carpenter, a T cell immunologist and infectious disease physician at Case Western Reserve University (CWRU). The institutional environment at CWRU combines an established TB research unit, BSL-3 flow cytometry, cell sorting, single-cell RNA sequencing, and expertise in bioinformatics together with a premier graduate program for scientific interaction and courses. The longstanding Uganda- CWRU Research Collaboration for TB enables clinical and translational work in a TB endemic setting. The candidate is establishing a lab with the long-term goals of understanding the defining features of protective memory T cell responses to Mtb. He has recruited mentors with substantial expertise in translational TB research, T cell biology and single-cell transcriptomics, including Drs. Henry Boom and Mark Cameron at CWRU, and Dr. Sam Behar (UMass Medical School). Completion of this K08 project will transition the candidate into an independent investigator, positioning his research program to use powerful single-cell immune profiling approaches to track and study antigen-specific human memory T cells.