B-LYMPHOCYTE MEDIATED IMMUNE MECHANISMS IN ATHEROGENESIS

现在有很多证据表明免疫系统与动脉粥样硬化的发生有关，本基金的目的是探索b淋巴细胞相关免疫机制的作用。我们之前已经证明LDL （OxLDL）的氧化使其具有免疫原性，并从apoE-/-小鼠的脾脏（EO Abs）中克隆出自发产生的IgM自身抗体（Abs），这些抗体被选择用于结合OxLDL。所有这些随后被证明结合氧化磷脂作为游离脂质或作为加合物与蛋白质。我们证明EO抗体（如EO6）与氧化应激下的凋亡细胞结合。EO6阻断巨噬细胞对OxLDL的摄取，并抑制凋亡细胞的吞噬。这些数据表明，氧化磷脂作为配体介导巨噬细胞对氧化修饰细胞和脂蛋白的识别。为了深入了解这些抗体的结构，我们克隆并测序了它们的可变区基因，发现VH/VL基因使用与T15抗磷胆碱（PC）抗体100%同源，这些抗体已经被广泛研究了30多年。T15抗体与肺炎链球菌结合，肺炎链球菌的细胞壁多糖上含有PC，并赋予抵抗这种病原体的“先天”保护。然而，即使在无菌小鼠中，T15克隆也会扩展，这表明它是基于先天的“管家作用”而被选择的，尽管在我们的研究之前，它所结合的新抗原还没有被定义。apoE-/-小鼠中T15 b细胞大量扩增的事实表明，它们对动脉粥样硬化负荷产生的氧化特异性表位有特异性免疫反应。在本提案中，我们将定义小鼠模型中动脉粥样硬化发展过程中血浆、病变和b细胞区室中T15 Ig和T15 b细胞的自然历史。我们将通过T15/EO6抗体本身或B-1细胞在小鼠中的被动转移实验来确定它们对动脉粥样硬化的影响。此外，我们将确定人类是否在肺炎球菌感染或肺炎球菌疫苗接种后产生抗OxLDL抗体，并在小鼠实验中确定肺炎球菌暴露或免疫接种是否会影响动脉粥样硬化过程。最后，我们将尝试克隆和表征类似的人类自身抗体。这些数据将提供关于T15 B细胞介导的动脉粥样硬化机制的新信息，并首次表征抗oxldl /T15 B细胞应答的“新自我”配体。

There is now much evidence to implicate the immune system in atherogenesis and the purpose of this grant is to explore the role of B-lymphocyte associated immune mechanisms. We have previously demonstrated that oxidation of LDL (OxLDL) renders it immunogenic and cloned spontaneously arising IgM autoantibodies (Abs) from the spleens of apoE-/- mice (EO Abs) that were selected for binding OxLDL. All were subsequently shown to bind oxidized phospholipids present as free lipids or as adducts with proteins. We demonstrated that the EO Abs (such as EO6) bound to apoptotic cells, which are known to be under oxidative stress. EO6 block macrophage uptake of OxLDL, as well as phagocytosis of apoptotic cells. These data indicate that oxidized phospholipids serve as ligands that mediate macrophage recognition of oxidatively modified cells and lipoproteins. To gain insight into the structure of these Abs, we cloned and sequenced their variable region genes and discovered a 100 percent homology of both VH/VL gene usage with T15 anti-phosphorylcholine (PC) Abs that have been extensively studied for over 30 years. T15 Abs bind to S. pneumonia, which contains PC on its cell wall polysaccharide, and confers "innate" protection against this pathogen. However, the T15 clone expands even in germ-free mice suggesting that it is selected based on an innate "housekeeping role," although the neo-antigens to which it binds have not been defined, prior to our studies. The fact that T15 B-cells are greatly expanded in the apoE-/- mice suggests a specific immune response to oxidation-specific epitopes generated by their atherosclerotic burden. In this proposal, we will define the natural history of T15 Ig and T15 B-cells in plasma, lesions and B-cell compartments during the course of development of atherogenesis in murine models. We will determine their impact on atherogenesis through passive transfer experiments in mice, with T15/EO6 Abs themselves or B-1 cells. In addition, we will determine if humans develop anti OxLDL Abs following pneumococcal infection, or after pneumococcal vaccination, and determine experimentally in mice whether pneumococcal exposure or immunization impacts the atherogenic process. Finally, we will attempt to clone and characterize similar autoAbs in man. These data should provide novel information on T15 B-cell mediated mechanisms in atherogenesis and for the first time characterize "neo-self" ligands to which anti-OxLDL/T15 B cells respond.