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.

T 细胞在 RA 中的作用已经确定，但几乎所有研究都集中在 CD4 T 细胞上。我们有 结果表明，CD8 T 细胞在发炎的 RA 滑膜中同样扩增和激活，但不属于 典型的颗粒酶 (Gzm) B+ 细胞毒性 T 淋巴细胞 (CTL) 亚群。相反，它们表达高水平的 GzmK 其功能没有明确定义。值得注意的是，我们发现 GzmK 驱动了一条新的补体途径 激活。我们证明 GzmK 可以将 C4 和 C2 裂解为 C4b 和 C2a 以生成活性 C3 转化酶 (C4b2a) 将 C3 裂解为 C3a 和 C3b。我们发现成纤维细胞表达最高水平的补体 滑膜中的成分 C2、C3 和 C4 可以分泌这些蛋白质以响应 CD8 T 细胞衍生的 TNF 和 IFNg。我们证明 GzmK 可以通过裂解补体引发活性 C3 转化酶的形成 成纤维细胞分泌的成分。此外，我们证明 GzmK 生成的 C3a 和 C3b 具有生物活性 并可分别驱动肥大细胞脱粒和调理细胞。我们假设 GzmK 介导 GzmK 体内的补体激活发生在细胞表面，因为我们表明 GzmK 与血浆强烈结合 膜。其他人最近发表的研究表明，补体激活会导致炎症 小鼠滑膜成纤维细胞的启动，由 mTOR 激活和下游代谢变化介导 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诱导人滑膜成纤维细胞炎症启动 以及 GzmK 是否可以在小鼠模型中驱动补体激活和炎症性关节炎。 在目标 1 中，我们研究 GzmK 与细胞表面的结合并确定其组装 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 目标 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 通过确定 1) 重组和活 CD8 T 细胞衍生的 GzmK 对炎症的影响来激活 和滑膜成纤维细胞的代谢激活。然后，使用 CRISPR GzmK 删除的 T 细胞或 CRISPR 补体因子或补体受体缺失的成纤维细胞，我们确认这些效应是否是介导的 直接由 GzmK 补码。在目标 3 中，我们使用 GzmK 全局缺陷小鼠和靶向 CD8 T 细胞 GzmK 缺陷小鼠证明 GzmK 在体内驱动炎症、补体激活、 炎症性关节炎 CIA 小鼠模型中的关节损伤和成纤维细胞活化。 这些研究共同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活化和关节病理学中的作用。这些研究将揭开 该途径如何发挥作用并决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 免疫病理学的深入了解并指出 GzmK 和 CD8 T 细胞作为潜在的新疗法 目标。