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

T细胞在类风湿关节炎中的作用已经确定，但几乎所有的研究都集中在CD4T细胞上。我们有 结果表明，CD8T细胞在炎症的RA滑膜中类似地被扩张和激活，但不属于 典型颗粒酶(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 其功能没有明确界定。值得注意的是，我们发现GzmK驱动了一条新的补体途径 激活。我们发现GzmK可以将C4和C2裂解成C4b和C2a，从而产生活性的C3转换酶 (C4b2a)将C3裂解成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C2、C3和C4组分，并能分泌这些蛋白来响应CD8T细胞来源的 肿瘤坏死因子和肿瘤坏死因子。我们发现GzmK可以通过裂解补体来诱导活性C3转移酶的形成 成纤维细胞分泌的成分。进一步，我们证明了GzmK产生的C3a和C3b具有生物活性 并可分别驱动肥大细胞脱颗粒和调理细胞。我们假设GzmK介导的 体内GzmK激活补体发生在细胞表面，因为我们发现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 MTOR激活及下游代谢变化介导的小鼠滑膜成纤维细胞的启动 产生促炎分子。我们建议确定人类CD8 T细胞来源的GzmK是否可以 通过介导局部细胞补体活化诱导人滑膜成纤维细胞炎性启动 以及GzmK是否能在小鼠模型中驱动补体激活和炎症性关节炎。 在目标1中，我们研究了GzmK与细胞表面的结合，并确定了它组装C5的能力 转化酶，导致产生过敏性毒素C5a和C5b-9膜攻击复合体。在……里面 目的2，我们确定了GzmK通过补体诱导滑膜成纤维细胞炎症启动的能力 通过测定1)重组和活体CD8T细胞来源的GzmK对炎症反应的影响来激活 以及滑膜成纤维细胞的代谢活化。然后，使用CRISPR GzmK缺失的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被介导 直接通过GzmK和补码。在目标3中，我们使用GzmK全局缺陷小鼠和靶向CD8 T细胞 GzmK缺陷小鼠在体内证明GzmK在推动炎症，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激活。 总之，这些研究定义了一种由淋巴细胞介导的补体激活的新途径-- 衍生的GzmK，及其在炎性成纤维细胞激活和关节病理中的作用。这些研究将会瓦解 关键要素的途径如何运作，并决定其在体内关节炎中的作用。这些发现提供了新的 对RA免疫病理的洞察并指出GzmK和CD8 T细胞是潜在的新治疗方法 目标。

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.