Mechanisms of antibody-mediated lung Injury after blood transfusion

项目摘要/摘要 输血对于许多失血或血液产生缺陷的患者来说是挽救生命的，但有 限制其有效性和/或导致医学后遗症的并发症。一个这样的后遗症是 发生急性肺损伤，或输血相关的急性肺损伤(TRALI)。TRALI长期以来一直是 输血相关死亡的头号原因和住院发病率的主要贡献者。这个 TRALI的发病机制尚不完全清楚，但已有共识认为TRALI是 以献血者血液制品中存在的人类白细胞抗原/MHC抗体为主的抗体介导性疾病 在易感受者身上引发肺损伤。事实上，我们已经开发了一种TRALI的小鼠模型，其基础是 向表达同源MHCI抗原的小鼠输注MHC-I类抗体。在此应用程序中，我们将 以此模型为平台，研究三个具体目标。在目标1中，我们将确定 通过测试被设计为缺陷或缺乏TRALI的小鼠来启动TRALI所需的同源抗原表达 在特定的细胞位置有足够的抗原表达。将使用新颖的鼠标工具选择性地删除 I类表达(B2mfl/fl)或选择性表达异型I类蛋白(Con-KD) 免疫细胞。使用这些免费的小鼠工具，我们将明确地解决抗原的关键部位 启动TRALI所需的表达式。在目标2中，我们将把注意力转向生物化学特性 与TRALI诱导相关的抗体。抗第I类(34-1-1)小鼠单抗 2S)被描述为产生TRALI，而许多其他针对同一抗原的抗体不能 引起TRALI；目前尚不清楚为什么这种抗体具有独特的致病性。我们将检验假设 抗体亲和力、免疫球蛋白G亚型、糖基化和交叉反应均影响TRALI的诱导。这些 研究将利用尖端的抗体工程以及结合和亲和分析来确定性质 在TRALI中传递致病性的抗体。在目标3中，我们将重点关注由此产生的下游事件 来自抗体的结合和免疫细胞的识别。Fcγ受体是近端的免疫受体 触发抗体介导的免疫反应。使用新的鼠标模型，允许有条件的 删除激活的FcγRs(Con-Fcγ-KO)，我们将确定负责抗体识别的关键细胞 在TRALI。我们将进一步确定抑制性FcγR(FcγRII)是否影响TRALI的严重性。最后，我们会 人源化34-1-2S抗体并将其注入经改造表达人Fcγ受体的小鼠体内 小鼠FcγR.34-1-2S将被表达为不同的人免疫球蛋白亚型(IgG1-IgG4)中的每一个 免疫球蛋白亚型影响人类TRALI的假说。这些实验的结果将明确地描绘出 对TRALI的诱导至关重要的细胞、抗体决定因素和受体。这一知识将有助于我们 了解输血治疗的这一严重并发症，并提出预防的翻译策略 或者治疗TRALI。

PROJECT SUMMARY/ABSTRACT Blood transfusions are life-saving for many patients with blood loss or blood production defects, but there are complications that limit their effectiveness and/or lead to medical sequelae. One such sequela is the development of acute lung injury, or transfusion-related acute lung injury (TRALI). TRALI has long been the number one cause of transfusion-related death and a major contributor to in-hospital morbidity. The pathogenesis of TRALI is incompletely understood, but there is consensus that TRALI is a classic example of antibody-mediated disease with a prominent role of HLA/MHC antibodies present in the donor blood product inciting lung injury in susceptible recipients. Indeed, we have developed a mouse model of TRALI based on transfusion of MHC Class I antibody into mice expressing the cognate MHCI antigen. In this application, we will use this model as a platform to investigate three specific aims. In Aim 1, we will determine the critical site of cognate antigen expression required to initiate TRALI by testing mice that are engineered to be deficient or sufficient in antigen expression in specific cellular locations. Novel mouse tools will be used to selectively delete Class I expression (B2mfl/fl) or selectively express allo-Class I protein (Con-Kd) on the endothelium or on specific immune cells. Using these complimentary mouse tools, we will definitively address the critical site of antigen expression required to initiate TRALI. In Aim 2, we will turn our attention to the biochemical characteristics of antibodies associated with the induction of TRALI. A single mouse monoclonal antibody against Class I (34-1- 2S) has been described to produce TRALI, whereas many other antibodies against the same antigen do not cause TRALI; it is unclear why this antibody is unique in its pathogenicity. We will test the hypotheses that antibody affinity, IgG subtype, glycosylation, and cross-reactivity each influence the induction of TRALI. These studies will utilize cutting-edge antibody engineering and binding and affinity assays to determine the properties of antibodies that impart pathogenicity in TRALI. In Aim 3, we will focus on the downstream events resulting from antibody engagement and recognition by immune cells. FcγRs are immune receptors that are the proximal trigger of antibody-mediated immune responses. Using a new mouse model that allows for the conditional deletion of activating FcγRs (Con-Fcγ-KO), we will determine the critical cells responsible for antibody recognition in TRALI. We will further determine if an inhibitory FcγR (FcγRII) influences TRALI severity. Finally, we will humanize the 34-1-2S antibody and infuse it into mice engineered to express human FcγRs in the absence of murine FcγRs. 34-1-2S will be expressed as each of the different human IgG subtypes (IgG1-IgG4) testing the hypothesis that IgG subtype affects TRALI in humans. The results of these experiments will definitively map the cells, antibody determinants, and receptors that are critical to TRALI induction. This knowledge will aid in our understanding of this serious complication of transfusion therapy and inform translational strategies to prevent or treat TRALI.