Molecular aspects of antigen presentation in multiple sclerosis

小胶质细胞是中枢神经系统的抗原提呈细胞，可能参与多发性硬化症(MS)的发病。本应用将探索它们的细胞生物学和生化，可从人类白细胞抗原DR2转基因动物中获得，并以适当限制的人类T细胞刺激为读数，用于表征此类细胞的抗原提呈能力。将详细检测两种抗原：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的制备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转基因小鼠身上进行，而且也将在DR2转基因小鼠中进行，这些转基因基因是为了组织蛋白酶B、D、L或S的溶酶体蛋白酶突变而培育的纯合子。这些酶可能是MBP和MOG蛋白水解性转化为能够与DR2结合的多肽的候选酶。通过这种方式，可以检查重要自身抗原的处理途径。鉴定出将MBP和MOG转化为致病的DR2-肽复合体所必需的蛋白水解酶(S)是鉴定可阻断这些酶的抑制剂的第一步，因此在治疗多发性硬化症方面具有治疗价值。我们将表征第二类分子和抗原(MOG)在人类小胶质细胞中的运输。这种材料的可获得性有限必然意味着实验范围更有限，但对DR2转基因小胶质细胞进行的研究、其功能特性的表征以及处理MBP和MOG所需的相关蛋白酶的评估将允许有充分理由的实验选择来验证为小鼠模型所阐述的概念。拟议中的实验不仅应该揭示小胶质细胞独有的、启动自身免疫T细胞反应所必需的细胞生物学特性，而且还将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.