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

摘要 这一提议将确定颗粒酶A(GzmA)介导的抑制 结核分枝杆菌(Mtb)的生长，以更好地了解宿主的免疫反应。四分之一的人 世界人口都感染了结核分枝杆菌。目前，卡介苗菌株是唯一可用于保护的疫苗 抗结核病(TB)。γ9δ2T细胞在肺中发现频率较高，这是结核分枝杆菌的主要部位 感染。我们的实验室先前已经证明，TB特异性γ9δ2T细胞在受到 分枝杆菌抗原。此外，我们的实验室已经证明，当结核特异性γδT释放GZMA时 细胞，抑制病原体在细胞内的复制。GzmA是一种丝氨酸蛋白酶，存在于细胞毒素中 NK细胞、CD8+CTL和γδT细胞的颗粒。它具有切割Arg或Lys后的碱性残基的类胰蛋白酶活性， 最初被认为是以穿孔素依赖的方式在靶细胞中诱导细胞凋亡 与颗粒酶B相似，然而，最近的文献表明，GzmA在生理浓度下 (NM)不会引起细胞凋亡。相反，它诱导单核/巨噬细胞产生促炎因子。 细胞因子。我们的实验室已经证明，GzmA以非穿孔素的方式诱导感染的巨噬细胞 分泌促炎细胞因子，如肿瘤坏死因子-α和白介素1β，并抑制细胞内生长 分枝杆菌。进行了转录分析，但未能确定GzmA- 中介抑制。我们假设，鉴于GzmA的蛋白质分解性质，一个合理的方法来 要研究这种机制，就必须进行蛋白质组学分析。GZmA进入途径与酶 将在目标1中研究催化，以更好地了解抑制的必要步骤。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 这些通路是在蛋白质组学初步实验中发现的，并将通过敲除来调节 在细胞系和/或原代细胞中的基因或抑制关键因素。最后，更多的全球蛋白质组筛查将 来检测AIM 3中其他差异表达的蛋白质。总的来说，这个项目将有助于 为结核病保护设计新的免疫治疗和/或化疗解决方案，并将有助于世卫组织的 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 令人兴奋的学习，这将使我发展所需的技能，使我的职业目标成为一个成功的 独立的医生兼科学家。

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.