Mechanisms of antibody-mediated lung Injury after blood transfusion

项目概要/摘要 对于许多失血或造血缺陷的患者来说，输血可以挽救生命，但也存在一些问题。 限制其有效性和/或导致医疗后遗症的并发症。其中一个后遗症就是 发生急性肺损伤或输血相关急性肺损伤 (TRALI)。 TRALI 长期以来一直是 是输血相关死亡的第一大原因，也是院内发病率的主要原因。的 TRALI 的发病机制尚不完全清楚，但一致认为 TRALI 是一个典型的例子 抗体介导的疾病，其主要作用是供体血液制品中存在的 HLA/MHC 抗体 引起易感接受者的肺损伤。事实上，我们已经开发了一个基于 TRALI 的小鼠模型 将 MHC I 类抗体输注至表达同源 MHCI 抗原的小鼠中。在这个应用程序中，我们将 使用该模型作为平台来研究三个具体目标。在目标 1 中，我们将确定关键位置 通过测试被设计为有缺陷或有缺陷的小鼠来启动 TRALI 所需的同源抗原表达 足以在特定细胞位置表达抗原。新颖的鼠标工具将用于选择性删除 I 类表达 (B2mfl/fl) 或在内皮或特定细胞上选择性表达同种异体 I 类蛋白 (Con-Kd) 免疫细胞。使用这些免费的小鼠工具，我们将明确解决抗原的关键位点 启动 TRALI 所需的表达。在目标 2 中，我们将把注意力转向 与 TRALI 诱导相关的抗体。针对 I 类 (34-1- 2S）已被描述为产生 TRALI，而针对同一抗原的许多其他抗体则不会 导致TRALI；目前还不清楚为什么这种抗体具有独特的致病性。我们将测试以下假设 抗体亲和力、IgG 亚型、糖基化和交叉反应性均会影响 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力测定来确定特性 赋予 TRALI 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 来自免疫细胞的抗体结合和识别。 FcγR 是最接近的免疫受体 触发抗体介导的免疫反应。使用允许有条件的新鼠标模型 删除激活的 FcγR (Con-Fcγ-KO)，我们将确定负责抗体识别的关键细胞 在特拉利。我们将进一步确定抑制性 FcγR (FcγRII) 是否影响 TRALI 严重程度。最后，我们将 将 34-1-2S 抗体人源化，并将其注入经过改造的小鼠体内，以在缺乏 FcγR 的情况下表达人 FcγR 鼠 FcγR。 34-1-2S 将表示为每个不同的人类 IgG 亚型 (IgG1-IgG4)，用于测试 IgG 亚型影响人类 TRALI 的假设。这些实验的结果将明确地绘制出 对 TRALI 诱导至关重要的细胞、抗体决定簇和受体。这些知识将有助于我们 了解输血治疗的这种严重并发症，并为预防的转化策略提供信息 或治疗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.