Molecular aspects of antigen presentation in multiple sclerosis

小胶质细胞是中枢神经系统的抗原呈递细胞，可能参与多发性硬化症（MS）的发生。本申请将探索从HLA-DR2转基因动物中获得的细胞生物学和生物化学，并使用适当限制的人类T细胞刺激作为读数来表征这些细胞的抗原呈递能力。详细检查两种抗原：髓鞘碱性蛋白（MBP）和髓鞘/少突胶质细胞糖蛋白（MOG）。通过体外转录/翻译获得的MBP和MOG制剂将作为天然放射性标记抗原的来源，其处理过程将使用生化技术进行监测。这些实验不仅将在DR2转基因小鼠中进行，还将在溶酶体蛋白酶组织蛋白酶B、D、L或s突变纯合的DR2转基因小鼠中进行。这些酶可能是MBP和MOG蛋白水解转化为可与DR2结合的肽的候选者。通过这种方式，可以检测重要自身抗原的加工途径。鉴定MBP和MOG转化为引发疾病的有害的dr2肽复合物所必需的蛋白酶是鉴定抑制剂的第一步，这些抑制剂可以阻断这些酶，从而在治疗ms中具有治疗价值。我们将描述Ii类分子和抗原（MOG）在人类小胶质细胞中的运输。这种材料的有限可用性必然意味着更有限的实验范围，但在DR2转基因小鼠小胶质细胞上进行的研究，其功能特性的表征以及对MBP和MOG加工所需的相关蛋白酶的评估将允许有充分论证的实验选择来验证为小鼠模型阐述的概念。所提出的实验不仅可以揭示小胶质细胞特有的细胞生物学特性和启动自身免疫T细胞反应的必要条件，而且还可以为治疗多发性硬化症指明新的策略。

Microglial cells are the antigen presenting cells of the central nervous system and are likely involved in the initiation of multiple sclerosis (MS). The present application will explore their cell-biological and biochemical be obtained from HLA-DR2 transgenic animals and used to characterize the antigen presentation capacity of such cells, using stimulation of appropriately restricted human T cells as a readout. Two antigens will be examined in detail: myelin basic protein (MBP) and myelin/oligodendrocytes glycoprotein (MOG). Preparations of MBP and MOG obtained by in vitro transcription/translation will serve as a source of native, radiolabeled antigen, the processing of which will be monitored using biochemical techniques. These experiments will be performed not only in DR2 transgenic mice, but also in DR2-transgenics bred to homozygosity for mutations in the lysosomal proteases Cathepsin B, D, L or S. These enzymes are likely candidates for the proteolytic conversion of MBP and MOG into peptides that can bind to DR2. In this manner, the processing pathway of important autoantigens can be examined. The identification of the protease(s) essential for conversion of MBP and MOG into the offending DR2-peptide complex that initiates disease is a first step towards identification of inhibitors that could block these enzymes and thus be of therapeutic value in the treatment of ms. We shall characterize the trafficking of Class Ii molecules and antigen (MOG) in human microglia. The limited availability of this material necessarily implies a more restricted range of experiments, but the studies performed on DR2 transgenic murine microglia, the characterization of their functional properties and the assessment of the relevant proteases required for processing of MBP and MOG will allow a well-argued choice of experiments to validate the concepts elaborated for the murine model. The proposed experiments should not only uncover cell biological properties unique to microglia and essential for initiation of autoimmune T cell responses, but also point the way to new strategies for treatment of MS.