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

颗粒酶A介导的γ9δ2 T细胞结核病保护作用的机制研究

• 批准号: 10669084
• 负责人: Valerio Rasi
• 金额: $ 5.27万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-22 至 2024-07-21
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10669084
• 关键词: Adult; Aerosols; Antigens; Apoptosis; Apoptosis Promoter; BCG Vaccine; Biological Assay; Biological Models; Biological Process; CD8B1 gene; Catalysis; Cause of Death; Cell Line; Cell physiology; Cell secretion; Cells; Cessation of life; Chemicals; Communicable Diseases; Control Groups; Cytoplasmic Granules; Cytoprotection; Disease; Dose; Environment; Enzymes; Epidemic; Extracellular Protein; Frequencies; Gene Expression; Genes; Genus Mycobacterium; Goals; Granzyme; Growth; HIV/AIDS; Immune response; Immunocompromised Host; Immunotherapeutic agent; In Vitro; Individual; Infection prevention; Inflammatory; Inflammatory Response; Interleukin-1 beta; Investigation; Knock-out; Literature; Lung; Macrophage; Mediating; Monitor; Multi-Drug Resistance; Mycobacterium tuberculosis; Mycobacterium tuberculosis antigens; Natural Killer Cells; Nature; Pathology; Pathway interactions; Patients; Pharmaceutical Preparations; Physicians; Physiological; Point Mutation; Population; Post-Translational Protein Processing; Predisposition; Protein Analysis; Proteins; Proteome; Proteomics; Recombinants; Research Proposals; Resistance; Role; Route; Scientist; Serine Protease; Site; Spottings; Surface; T-Cell Receptor; T-Lymphocyte; TNF gene; Testing; Therapeutic; Training; Tryptase; Tuberculosis; Vaccinated; Vaccination; Variant; Visualization; biological adaptation to stress; career; cytokine; cytotoxic; design; differential expression; endoplasmic reticulum stress; experimental study; extracellular; human model; immunocytochemistry; infection rate; inhibitor; knockout gene; medication compliance; monocyte; mycobacterial; nonhuman primate; novel; novel vaccines; overexpression; pathogen; perforin; protective effect; protein expression; receptor; skills; transmission process; vaccine access; vaccine trial; γδ T cells

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.

抽象的 该建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）生长，以更好地了解宿主免疫反应。四分之一 世界人口感染了MTB。目前，BCG菌株是唯一可保护的疫苗 针对结核病（TB）。发现γ9δ2T细胞是在肺中较高的频率下发现的，肺是MTB的主要部位 感染。我们的实验室以前已经表明，TB特异性γ9δ2T细胞在用 分枝杆菌抗原。此外，我们的实验室证明了GZMA，当由TB特异性γδT释放时 细胞，抑制病原体的细胞内复制。 GZMA是在细胞毒性中发现的连续蛋白 NK细胞，CD8+ CTL和γδT细胞的颗粒。它具有胰蛋白酶活性在arg或lys之后切割碱性残基， 最初被认为是靶细胞中凋亡的一种诱导的诱导的方式 类似于GranzymeB。但是，最近的文献表明，GZMA处于生理浓度 （NM）不会引起凋亡。相反，它诱导单核细胞/巨噬细胞产生促炎性 细胞因子。我们的实验室表明，以培养素独立的方式GZMA诱导了感染的巨噬细胞 分泌促炎性细胞因子，例如TNF-α和IL-1β，并抑制细胞内生长 分枝杆菌。进行了转录组分析，该分析未能确定GZMA-的机制 介导的抑制作用。我们假设鉴于GZMA的蛋白水解性质，这是一种合理的方法 研究这种机制将是进行蛋白质组学分析。 GZMA进入和酶 AIM 1的催化将进行催化，以更好地了解抑制的必要步骤。在AIM 2中 内质网（ER）应力反应和嘌呤能通道激活将受到询问：这两个 在初步的蛋白质组学实验中发现了途径，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素。最后，其他全局蛋白质组学屏幕将 进行AIM 3中检测其他不同表达的蛋白质。总的来说，该项目将有助于 新型免疫治疗和/或化学治疗解决方案的设计，用于结核病保护，将有助于WHO的 目前的目标是减少到2030年的结核病死亡人数为90％。拟议的综合培训计划包括 激动人心的研究将使我能够为自己的职业目标发展所需的技能才能成为成功 独立的物理科学家。

项目成果

Improved Purification of Human Granzyme A/B and Granulysin Using a Mammalian Expression System.

• DOI: 10.3389/fimmu.2022.830290
• 发表时间: 2022
• 期刊: Frontiers in immunology
• 影响因子: 7.3
• 作者: Rasi V;Hameed OA;Matthey P;Bera S;Grandgenett DP;Salentinig S;Walch M;Hoft DF
• 通讯作者: Hoft DF