ROLE OF GRANZYMES IN CYTOTOXIC LYMPHOCYTE FUNCTIONS

颗粒酶在细胞毒性淋巴细胞功能中的作用

• 批准号: 8168718
• 负责人: TIMOTHY J. LEY
• 金额: $ 0.97万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-10 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168718
• 关键词: Autoimmune Diseases; Autologous; CD8B1 gene; Cell Death; Cell physiology; Cells; Cessation of life; Cleaved cell; Computer Retrieval of Information on Scientific Projects Database; Cytoplasmic Granules; Disease; Funding; Goals; Grant; Granzyme; Health; Human; Immune; Immune Targeting; In Vitro; Individual; Institution; Lymphocyte; Lymphocyte Function; Mass Spectrum Analysis; Mediating; Molecular; Mus; Oncogenic Viruses; Orphan; Pathway interactions; Process; Proteomics; Reagent; Regulation; Regulatory T-Lymphocyte; Research; Research Personnel; Resources; Role; Source; Surface; Synapses; United States National Institutes of Health; Viral; cytotoxic; gel electrophoresis; granzyme A; in vivo; killings; novel; perforin; trafficking; tumor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The long term goal of this project is to understand the molecular and cellular mechanisms by which cytotoxic lymphocytes (CL) kill their target cells in health (eg. viral and tumor clearance) and disease (eg. Graft vs. Host Disease, and autoimmune states). CL can kill by secreting the contents of their cytotoxic granules (including perforin and granzymes) onto the surface of target cells via the "secretory synapse." Perforin is responsible for delivering and/or trafficking the granzymes, which induce target cell death by cleaving a variety of substrates. Granzymes A and B induce cell death via non-overlapping pathways, but several "orphan" granzymes are expressed in both mice and humans, and may be relevant for specific CL functions. We have made (or are currently making) pure 129/SvJ mice deficient for granzymes A, B, C (and all combinations thereof), and perforin. With this unique set of reagents, we will further examine granzyme activities and substrates via the following specific aims: Specific Aim 1: We will define the roles of individual granzymes for perforin-mediated cytotoxity in vivo. Perforin is required for the clearance of many tumors and viruses, and for CDS8+ mediated GvHD, but the roles of individual granzymes in these processes remains extremely controversial. We will use the precisely strain-matched mice described above to better define the roles of these granzymes for CD8+ and NK mediated killing in vitro and in vivo. Specific Aim 2: We will define the role of the perforin/qranzyme pathway for CD4+ mediated immune regulation in mice. We have shown that human regulatory T cells contain perforin and granzymes, and that they can kill autologous immune targets in a perforin-dependent fashion. We will use precisely strainmatched 129/SvJ mice deficient for perforin or granzymes to assess the importance of these molecules for T regulatory cell function in vitro and in vivo. Specific Aim 3: We will use proteomic approaches to define the substrates of qranzymes. We will use 2-D differential in-gel electrophoresis (2D-DIGE) and mass spectrometry to identify the substrates and cleavage products of granzymes A, B, and C. Novel substrates will be evaluated for their importance in mediating granzyme-induced death in vitro and in vivo.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 该项目的长期目标是了解细胞毒性淋巴细胞（CL）在健康情况下杀死其靶细胞的分子和细胞机制（例如，病毒和肿瘤清除）和疾病（例如，移植物抗宿主病和自身免疫状态）。CL可通过分泌突触将其细胞毒性颗粒（包括穿孔素和颗粒酶）分泌到靶细胞表面而杀伤细胞。穿孔蛋白负责递送和/或运输颗粒酶，颗粒酶通过切割各种底物诱导靶细胞死亡。颗粒酶A和B通过非重叠途径诱导细胞死亡，但几种“孤儿”颗粒酶在小鼠和人中表达，并且可能与特定的CL功能相关。我们已经制造了（或目前正在制造）缺乏颗粒酶A、B、C（及其所有组合）和穿孔素的纯129/SvJ小鼠。有了这套独特的试剂，我们将通过以下具体目标进一步研究颗粒酶的活性和底物：具体目标1：我们将定义个体颗粒酶在体内穿孔素介导的细胞毒性中的作用。穿孔蛋白是清除许多肿瘤和病毒以及CDS 8+介导的GvHD所必需的，但单个颗粒酶在这些过程中的作用仍然存在极大的争议。我们将使用上述精确品系匹配的小鼠来更好地定义这些颗粒酶在体外和体内对CD 8+和NK介导的杀伤的作用。具体目标2：我们将确定穿孔素/qranzyme途径在小鼠中CD 4+介导的免疫调节中的作用。我们已经表明，人类调节性T细胞含有穿孔素和颗粒酶，并且它们可以以穿孔素依赖的方式杀死自体免疫靶标。我们将使用精确匹配的穿孔素或颗粒酶缺陷的129/SvJ小鼠来评估这些分子对体外和体内T调节细胞功能的重要性。具体目标3：我们将利用蛋白质组学的方法来确定qranzyme的底物。我们将使用2-D差异凝胶电泳（2D-DIGE）和质谱来鉴定颗粒酶A、B和C的底物和裂解产物。新的底物将在体外和体内介导颗粒酶诱导的死亡的重要性进行评估。