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 shown that oxidation of LDL (OxLDL) renders it immunogenic and cloned spontaneously arising IgM autoantibodies (Abs) from spleens of apo +mice (EO Abs) that were selected for binding to OxLDL. All were subsequently shown to bind oxidized phospholipids (OxPL) 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 blocked macrophage uptake of OxLDL, as well as phagocytosis of apoptotic cells. These data indicate that OxPL 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% 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 had 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 Abs 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. We will determine the impact of similar experiments in mice genetically unable to make the T15 clone and in mice that overexpress B-1 cell Abs. 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 oxidation-specific """"""""neo-self"""""""" ligands to which anti-OxLDL/T15 B cells respond.
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