Utilizing Dendritic Cell Biology to Characterize the Innate Immune Response to Blood Coagulation Proteins

项目摘要 凝血糖蛋白因子VIII（FVIII）或因子IX（FIX）的遗传缺陷导致 出血性疾病血友病A和B。这些人需要静脉注射 治疗和预防出血事件的外源性因素。然而，30%的重度血友病A患者和 1-3%的血友病B患者会形成称为FVIII和FIX抑制剂的中和抗体 分别尽管对FVIII和FIX的免疫应答都依赖于CD 4 + T细胞应答， FIX抑制剂可诱导过敏性IgE介导的超敏反应和肾病综合征， 免疫复合物沉积，这在FVIII抑制剂患者中很少见。抗原呈递细胞 包括树突状细胞（DC）、B细胞和巨噬细胞在内的APC对于感测抗原和 将适应性免疫应答导向免疫或耐受。DC具有增强的捕获能力， 并加工抗原以在MHC复合物上呈递给幼稚T细胞。对以下方面的了解有限： 介导对FVIII和FIX的先天免疫的潜在机制和关键细胞组分。的 这项工作的主要目的是表征DC在对FVIII和FIX的先天免疫应答中的作用。 本研究的主要目的是：1）评价传统DC在CD 4 + T细胞依赖性免疫中的作用。 血友病A和B小鼠模型中的抗体形成，2）确定FVIII和FIX的机制 通过常规DC的识别和内化，和3）确定FVIII和FIX的性质， 介导DC活化和共刺激信号传导。在目标1中，T细胞应答和抗体形成将是 在用FVIII、FIX或卵清蛋白免疫的小鼠中靶向耗竭APC亚群后测量。的作用 通过C型凝集素受体DCIR 2的吞噬作用、巨胞饮作用和受体介导的内吞作用 和Dectin-2在正常条件下和炎症情况下的FVIII和FIX摄取中的作用， 确定Aim 2中DC识别和内化因子的机制。在目标3中， 将测量FVIII和FIX浓度的增加对体外DC活化的影响。此外，影响 抑制DC共刺激信号对T细胞活化、抗体应答和补体活化的影响， 通过静脉内、皮下或腹膜内注射FVIII免疫血友病A和B小鼠， 将对FIX进行评价。对这些机制的基本理解对于评估 血友病患者在早期因子暴露期间的免疫学变化和策略的发展 预防和消除抑制剂。概述的职业发展计划将涉及以下方面的培训 免疫学，分子生物学实验室技术和转化研究，以确保成功 向独立研究过渡。

Project Summary Inherited deficiencies of blood coagulation glycoproteins factor VIII (FVIII) or factor IX (FIX) result in the bleeding disorders hemophilia A and B, respectively. These individuals require intravenous infusions of exogenous factor to treat and prevent bleeding events. However 30% of patients with severe hemophilia A and 1-3% of patients with hemophilia B will form neutralizing antibodies called inhibitors against FVIII and FIX respectively. Although the immune response to FVIII and FIX are both dependent on CD4+ T cell responses, inhibitors to FIX can induce allergic IgE-mediated hypersensitivity responses and nephrotic syndrome due to immune complex deposition, which are rarely seen in patients with FVIII inhibitors. Antigen presenting cells (APCs), including dendritic cells (DCs), B cells, and macrophages, are essential for sensing antigens and directing adaptive immune responses towards immunity or tolerance. DCs have an enhanced ability to capture and process antigens for presentation on MHC complexes to naïve T cells. There is limited understanding of the underlying mechanisms and critical cellular components that mediate innate immunity to FVIII and FIX. The main objective of this work is to characterize the role of DCs in the innate immune response to FVIII and FIX. This proposal consists of 3 specific aims: 1) Evaluate the role of conventional DCs in CD4+ T cell dependent- antibody formation in murine models of hemophilia A and B, 2) Identify the mechanisms of FVIII and FIX recognition and internalization by conventional DCs, and 3) Determine the properties of FVIII and FIX that mediate DC activation and co-stimulatory signaling. In Aim 1, T cell responses and antibody formation will be measured after targeted depletion of APC subsets in mice immunized with FVIII, FIX, or ovalbumin. The role of phagocytosis, macropinocytosis, and receptor-mediated endocytosis through C-type lectin receptors DCIR2 and Dectin-2 in FVIII and FIX uptake under normal conditions and with inflammation will be evaluated to determine the mechanism of factor recognition and internalization by DCs in Aim 2. In Aim 3, the effect of increasing concentrations of FVIII and FIX on DC activation in vitro will be measured. Additionally, the effect of inhibiting DC co-stimulatory signals on T cell activation, antibody responses, and complement activation in hemophilia A and B mice immunized by intravenous, subcutaneous, or intraperitoneal injections of FVIII and FIX will be evaluated. A fundamental understanding of these mechanisms will be important for evaluating immunologic changes in hemophilia patients during early factor exposure and for the development of strategies that prevent and eradicate inhibitors. The career development plan outlined will address training in immunology, molecular biology laboratory techniques, and translational research to ensure successful transition to research independence.