ROLE OF GRANZYMES IN CYTOTOXIC LYMPHOCYTE FUNCTIONS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这个项目的长期目标是了解细胞毒性淋巴细胞(CL)在健康中杀死其目标细胞的分子和细胞机制(例如。病毒和肿瘤清除)和疾病(例如移植物与宿主疾病和自身免疫状态)。CL可以通过“分泌性突触”将其细胞毒性颗粒(包括穿孔素和颗粒酶)的内容分泌到靶细胞表面而杀死细胞。穿孔素负责运送和/或运输颗粒酶，颗粒酶通过裂解各种底物来诱导靶细胞死亡。颗粒酶A和B通过不重叠的途径诱导细胞死亡，但在小鼠和人类中都表达了几种“孤儿”颗粒酶，可能与特定的CL功能有关。我们已经(或目前正在制作)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，我们将通过以下特定目标进一步检测颗粒酶活性和底物：特定目标1：我们将确定单个颗粒酶在体内穿孔素介导的细胞毒中的作用。穿孔素是许多肿瘤和病毒清除所必需的，也是CDS8+介导的GvHD所必需的，但单个颗粒酶在这些过程中的作用仍存在极大争议。我们将使用上述精确匹配的小鼠来更好地确定这些颗粒酶在体外和体内对CD8+和NK介导的杀伤的作用。具体目标2：我们将确定穿孔素/qranzyme途径在小鼠体内CD4+介导的免疫调节中的作用。我们已经证明，人类调节性T细胞含有穿孔素和颗粒酶，它们可以以穿孔素依赖的方式杀死自体免疫靶点。我们将使用缺乏穿孔素或颗粒酶的精确匹配的129/SVJ小鼠来评估这些分子在体外和体内对T调节细胞功能的重要性。具体目标3：我们将使用蛋白质组学方法来确定qranzyme的底物。我们将使用二维差示凝胶内电泳(2D-DGE)和质谱仪来鉴定颗粒酶A、B和C的底物和裂解产物。新的底物将在体内外评估它们在介导颗粒酶诱导的死亡中的重要性。

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.