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 该提案将挖掘负责粒酶A（GZMA）介导的侵犯的机制 结核分枝杆菌（MTB）生长，以更好地了解宿主免疫反应。 世界人口被MTB感染。 针对结核病（TB）。 感染。 分枝杆菌抗原。 细胞，抑制病原体的细胞内反映。 NK细胞的颗粒，CD8+ CTL和γδT细胞具有胰蛋白酶活性在ARG或LYS之后裂解基本居民 最初是以深度蛋白的方式成为目标细胞中凋亡的诱导剂 类似于GranzymeB。然而，最近的文献表明，GZMA处于 （NM）不会引起凋亡。 细胞因子。 分泌促炎性细胞因子，例如TNF-ALPHA AL-1β，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- 介导的遗传性。 研究这种机制将进行蛋白质组学分析。 将在AIM 1中研究催化，以更好地了解AIM 2中的必要步骤。 内质网（ER）应力反应和嘌呤能通道激活将弯曲：这两个 在预后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lin和/或原代细胞中的关键因素。 进行AIM 3中检测到其他差异表达的蛋白质。总的来说，该项目将在 新颖的免疫治疗和/或化学治疗剂的设计用于结核病保护，将有助于谁 到2030年，目前将结核病死亡人数降低90％的目标。 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，以成为成功 独立的医师科学家。

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