Mechanistic investigations of Granzyme A-mediated γ9δ2 T cell TB protective effects

摘要 这一提议将确定负责颗粒酶A（GzmA）介导的抑制的机制。 结核分枝杆菌（Mtb）的生长，以更好地了解宿主的免疫反应。的四分之一 世界人口感染结核病。目前，BCG菌株是唯一可用的疫苗， 结核病（TB）。γ 9 δ 2 T细胞在肺中以较高的频率发现，肺是Mtb的主要部位。 感染我们的实验室先前已经表明，TB特异性γ 9 δ 2 T细胞在用以下刺激后分泌GzmA： 分枝杆菌抗原此外，我们的实验室已经证明，当TB特异性γ δ T释放GzmA时， 细胞，抑制病原体的细胞内复制。GzmA是一种丝氨酸蛋白酶，发现细胞毒性 NK细胞颗粒、CD8 + CTL和γ δ T细胞。它具有类胰蛋白酶活性，切割Arg或Lys后的碱性残基， 并且最初被认为是以穿孔素依赖的方式在靶细胞中诱导凋亡 与粒酶B类似。然而，最近的文献表明，GzmA在生理浓度 (nM)不会引起细胞凋亡。相反，它诱导单核细胞/巨噬细胞产生促炎性细胞因子。 细胞因子我们的实验室已经表明，GzmA以不依赖穿孔素的方式诱导受感染的巨噬细胞 分泌促炎细胞因子，如TNF-α和IL-1 β，并抑制细胞内生长， 分枝杆菌进行了转录组分析，未能确定GzmA的机制， 介导的抑制。我们假设，鉴于GzmA的蛋白水解性质， 研究这种机制将是进行蛋白质组学分析。GzmA进入途径和酶 在目标1中将研究催化，以更好地理解抑制的必要步骤。在目标2中， 内质网（ER）应激反应和嘌呤能通道激活将被询问：这两个 在初步的蛋白质组学实验中发现了这些途径，并将通过敲除 基因或抑制细胞系和/或原代细胞中的关键因子。最后，额外的全球蛋白质组学筛选将 检测Aim 3中其他差异表达蛋白。总的来说，这个项目将有助于 设计用于结核病保护的新型免疫和/或化疗解决方案，并将有助于世界卫生组织 目前的目标是到2030年将结核病死亡人数减少90%。拟议的全面培训计划包括以下内容 令人兴奋的研究，这将使我发展所需的技能，为我的职业目标，成为一个成功的 独立的物理学家和科学家

ABSTRACT This proposal will determine the mechanism responsible for Granzyme A (GzmA)-mediated inhibition of Mycobacterium tuberculosis (Mtb) growth, to better understand the host immune response. One fourth of the world’s population is infected with Mtb. Currently, BCG strains are the only vaccines available to protect against tuberculosis (TB). γ9δ2 T cells are found at higher frequencies in lungs, which is the primary site of Mtb infection. Our lab has previously shown that TB specific γ9δ2 T cells secrete GzmA upon stimulation with mycobacterial antigens. Furthermore, our lab has demonstrated that GzmA, when released by TB-specific γδ T cells, inhibits the intracellular replication of the pathogen. GzmA is a serine protease found within cytotoxic granules of NK cells, CD8+ CTL, and γδ T cells. It has tryptase activity cleaving basic residues after Arg or Lys, and was originally thought to be an inducer of apoptosis in the target cell in a perforin-dependent manner similar to Granzyme B. However, more recent literature has shown that GzmA at a physiologic concentration (nM) does not cause apoptosis. Instead, it induces monocytes/macrophages to produce pro-inflammatory cytokines. Our lab has shown that GzmA in a perforin-independent manner induces the infected macrophage to secrete pro-inflammatory cytokines, such as TNF-alpha and IL-1β, and inhibits the intracellular growth of mycobacteria. A transcriptomal analysis was performed, which failed to identify the mechanism of GzmA- mediated inhibition. We hypothesize that given the proteolytic nature of GzmA, a rational approach to investigate this mechanism would be to perform a proteomic analysis. GzmA route of entry and enzyme catalysis will be studied in Aim 1 to better understand the necessary steps for inhibition. In Aim 2, the Endoplasmic Reticulum (ER) stress response and purinergic channel activation will be interrogated: these two pathways were discovered during preliminary proteomic experiments, and will be modulated by knocking out genes or inhibiting key factors in cell lines and/or primary cells. Finally, additional global proteomic screens will be conducted to detect other differentially expressed proteins in Aim 3. Overall, this project will help in the design of novel immunotherapeutic and/or chemotherapeutic solutions for TB protection and will aid the WHO’s current goal to decrease TB deaths of 90% by 2030. A proposed comprehensive training plan includes these exciting studies that will allow me to develop needed skills for my career goal to become a successful independent physician-scientist.