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

项目摘要 遗传性凝血糖蛋白第VIII因子(FVIII)或第IX因子(FIX)缺陷导致 出血性疾病血友病分别为甲型和乙型。这些人需要静脉输注 外源性因素治疗和预防出血事件。然而，30%的严重血友病A和 1-3%的血友病B患者会形成中和抗体，称为FVIII和FIX抑制物 分别进行了分析。尽管对FVIII和FIX的免疫反应都依赖于CD4+T细胞反应， FIX抑制剂可诱导变态反应性IgE介导的过敏反应和肾病综合征 免疫复合体沉积，这在使用FVIII抑制剂的患者中很少见。抗原提呈细胞 (APC)，包括树突状细胞(DC)、B细胞和巨噬细胞，是感知抗原和 引导适应性免疫反应向免疫或耐受方向发展。分布式控制系统具有增强的捕获能力 并处理抗原，在MHC复合体上呈递给幼稚的T细胞。人们对……的了解有限 介导对FVIII和FIX先天免疫的潜在机制和关键细胞成分。这个 这项工作的主要目的是表征树突状细胞在FVIII和FIX的先天免疫反应中的作用。 这项建议包括3个具体目标：1)评估传统DC在CD4+T细胞依赖- 血友病A、B小鼠模型的抗体形成2)鉴定FVIII和FIX的作用机制 传统DC的识别和内化，以及3)确定FVIII的性质并修复 介导DC激活和共刺激信号。在目标1中，T细胞反应和抗体形成将是 在用FVIII、FIX或卵清蛋白免疫的小鼠的APC亚群靶向耗尽后测量。的作用 C型凝集素受体DCIR2的吞噬作用、巨噬细胞吞噬作用和受体介导的内吞作用 和Dectin-2在FVIII和FIX摄取在正常情况下和有炎症情况下将被评估为 在目标2中，确定DC识别和内化因子的机制。在目标3中， FVIII和FIX在体外对DC激活的浓度增加将被测量。此外， 抑制DC共刺激信号对T细胞激活、抗体反应和补体激活的影响 静脉、皮下或腹腔注射FVIII和FVIII免疫A和B血友病小鼠 将对修复进行评估。对这些机制的基本理解对于评估将是重要的 血友病患者在因子暴露早期的免疫学变化及其策略的发展 以防止和根除抑制物。概述的职业发展计划将涉及 免疫学、分子生物学实验室技术和转化研究，以确保成功 向研究独立过渡。

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.