CD8 T cell derived Granzyme K activates complement that drives synovial fibroblast inflammation

CD8 T 细胞衍生的颗粒酶 K 激活补体，驱动滑膜成纤维细胞炎症

• 批准号: 10733690
• 负责人: Michael B. Brenner
• 金额: $ 30.7万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-22 至 2028-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10733690
• 关键词: Address; Affect; Affinity; Anaphylatoxins; Apoptosis; Arthritis; Automobile Driving; Binding; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; Cartilage; Caspase; Cell Degranulation; Cell membrane; Cell surface; Cells; Charge; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Collagen Arthritis; Complement; Complement 3 Convertase; Complement 3a; Complement 3b; Complement 4b; Complement 5a; Complement Activation; Complement Factor D; Complement Membrane Attack Complex; Complement Receptor; Complement component C1s; Cytotoxic T-Lymphocytes; Deposition; Disease; Electrostatics; Elements; FRAP1 gene; Fibroblasts; Generations; Glycosaminoglycans; Granzyme; Human; Inflammation; Inflammatory; Inflammatory Arthritis; Joints; Lectin; Leukocytes; Lymphocyte; MASP2 gene; Mediating; Membrane; Metabolic; Metabolic Activation; Modeling; Mus; Organoids; Pathology; Pathway interactions; Play; Process; Production; Protein Secretion; Proteins; Publishing; Recombinants; Research; Rheumatoid Arthritis; Role; Serine Protease; Serum; Source; Surface; Synovial Membrane; Synovial joint; Synovitis; T-Lymphocyte; T-Lymphocyte Subsets; TNF gene; Testing; Tissues; Work; bone; cell type; complement C2a; complement C5b; complement pathway; disability; drug development; immunopathology; in vivo; insight; joint inflammation; joint injury; mast cell; mouse model; new therapeutic target; response

The role of T cells in RA is established, but nearly all research has focused on CD4 T cells. We have shown that CD8 T cells are similarly expanded and activated in inflamed RA synovium yet do not belong to the typical granzyme (Gzm) B+ cytotoxic T lymphocyte (CTL) subset. Instead, they express high levels of GzmK whose function is not well defined. Remarkably, we find that GzmK drives a new pathway of complement activation. We show that GzmK can cleave C4 and C2 into C4b and C2a to generate an active C3 convertase (C4b2a) that cleaves C3 to C3a and C3b. We show that fibroblasts express the highest levels of complement components C2, C3 and C4 in the synovium and can secrete these proteins in response to CD8 T cell-derived TNF and IFNg. We show that GzmK can elicit formation of an active C3 covertase by cleaving complement components secreted by fibroblasts. Further, we demonstrate that GzmK generated C3a and C3b are bioactive and can drive mast cell degranulation and opsonize cells, respectively. We hypothesize that GzmK-mediated complement activation by GzmK in vivo occurs on cell surfaces as we show GzmK binds strongly to plasma membranes. Recently published research by others shows that complement activation drives inflammatory priming of murine synovial fibroblasts, mediated by mTOR activation with downstream metabolic changes and production of pro-inflammatory molecules. We propose to determine if human CD8 T cell-derived GzmK can induce inflammatory priming of human synovial fibroblasts by mediating local cellular complement activation and whether GzmK can drive complement activation and inflammatory arthritis in murine models. In Aim 1, we study GzmK binding to cell surfaces and determine its ability to assemble a C5 convertase, resulting in the generation of the anaphylatoxin C5a and a C5b-9 membrane attack complex. In Aim 2, we define the ability of GzmK to induce inflammatory priming of synovial fibroblasts via complement activation, by determining the effects of 1) recombinant and live CD8 T cell-derived GzmK on the inflammatory and metabolic activation of synovial fibroblasts. Then, using CRISPR GzmK-deleted T cells or CRISPR complement factor or complement receptor deleted fibroblasts, we confirm if these effects are mediated directly by GzmK and complement. In Aim 3, we use GzmK globally deficient mice and targeted CD8 T cell GzmK-deficient mice to demonstrate the role of GzmK in vivo in driving inflammation, complement activation, joint damage and fibroblast activation in the CIA mouse model of inflammatory arthritis. Together, these studies define a new pathway of complement activation mediated by lymphocyte- derived GzmK, and its role in inflammatory fibroblast activation and joint pathology. These studies will unravel key elements of how the pathway works and determine its role in arthritis in vivo. These findings provide new insights into the immunopathology of RA and point to GzmK and CD8 T cells as potential new therapeutic targets.

建立了T细胞在RA中的作用，但几乎所有研究都集中在CD4 T细胞上。我们有 表明CD8 T细胞在发炎的RA滑膜中类似地扩展和激活，但不属于 典型的颗粒（GZM）B+细胞毒性T淋巴细胞（CTL）子集。相反，它们表达了高水平的GZMK 其功能的定义不当。值得注意的是，我们发现GZMK推动了一种新的补充途径 激活。我们表明GZMK可以将C4和C2裂解到C4B和C2A中以生成活动C3转化酶 （C4B2A）将C3切割至C3A和C3B。我们表明成纤维细胞表达最高水平的补体 滑膜中的成分C2，C3和C4，可以响应CD8 T细胞衍生的这些蛋白质分泌这些蛋白 TNF和IFNG。我们表明，GZMK可以通过切割补体来形成活跃的C3覆盖酶 成纤维细胞分泌的组件。此外，我们证明了GZMK生成的C3A和C3B是生物活性的 并可以分别驱动肥大细胞脱粒和调理细胞。我们假设GZMK介导 GZMK在体内的补体激活在细胞表面上发生，因为我们显示GZMK与等离子体强烈结合 膜。其他人最近发表的研究表明，补体激活会驱动炎症 鼠滑膜成纤维细胞的启动，由MTOR激活介导，下游代谢变化和 促炎分子的产生。我们建议确定人类CD8 T细胞衍生的GZMK是否可以 通过介导局部细胞补体激活来诱导人类滑膜成纤维细胞的炎症启动 以及GZMK是否可以驱动鼠模型中的补体激活和炎症性关节炎。 在AIM 1中，我们研究了与细胞表面结合的GZMK，并确定其组装C5的能力 转化酶，导致过敏毒素C5a和C5B-9膜攻击复合物的产生。在 AIM 2，我们定义了GZMK通过补体诱导滑膜成纤维细胞炎症启动的能力 激活，通过确定1）重组和活CD8 T细胞衍生的GZMK的影响 滑膜成纤维细胞的代谢激活。然后，使用CRISPR GZMK污染的T细胞或CRISPR 补体因子或补体受体删除的成纤维细胞，我们确认这些作用是否介导 直接由GZMK和补充。在AIM 3中，我们使用GZMK全球缺陷的小鼠和靶向CD8 T细胞 缺乏GZMK的小鼠，以证明GZMK在体内的作用在驱动炎症，补体激活中的作用， CIA小鼠炎症性关节炎模型中的关节损伤和成纤维细胞激活。 总之，这些研究定义了由淋巴细胞介导的补体激活的新途径 衍生的GZMK及其在炎症成纤维细胞激活和关节病理中的作用。这些研究将解散 途径如何工作并确定其在体内关节炎中的作用的关键要素。这些发现提供了新的 对RA的免疫病理学的见解，并将GZMK和CD8 T细胞指向潜在的新治疗 目标。