ANTIGEN PRESENTATION IN HUMAN AUTOIMMUNE DISEASES

不同免疫学研究领域的融合为研究多发性硬化症（一种中枢神经系统髓鞘炎症性疾病）患者的T细胞识别抗原提供了一个更综合的方法。这些包括阐明TCR识别抗原/MHC的晶体结构；鉴定潜在的生化技术，提供对II类限制性呈现的细胞生物学的见解，特别强调在这一过程中涉及的蛋白酶。在第一个项目中，我们将利用新开发的MHC四聚体技术来确定HLA- DR2 MS患者CSF和血液中抗原特异性T细胞的频率、激活状态和细胞因子谱，这几乎肯定被限制稀释技术低估了。髓磷脂肽的APC在MS病变中的表现将使用由髓磷脂特异性T细胞克隆的cdna生成的TCR四聚体进行研究，并用于对MS斑块组织进行染色。在第二个项目中，组合肽文库将用于检测寡克隆CSF T细胞的抗原特异性。这些研究也将在具有明确的中枢神经系统感染的受试者中进行，并将确定与MS患者的中枢神经系统T细胞相比，MS患者的中枢神经系统T细胞表达的TCRs在识别组合肽库方面是否退化，而与识别病毒或细菌抗原的中枢神经系统T细胞相比，TCRs在识别组合肽库方面是否退化。将确定寡克隆T细胞和MBP/DR2四聚体结合T细胞是否反映了原始脑脊液群体。项目2将探索中枢神经系统小胶质细胞的细胞生物学和生化特性。研究髓磷脂自身抗原MBP和MOG的加工途径以及II类分子和MOG在人小胶质细胞中的转运。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。这些实验将检验溶酶体蛋白酶组织蛋白酶B、D、L或S的作用，它们可能是抗原加工中MBO和MOG蛋白水解转化的候选者。项目1中携带抗原的APCs的MOG和MBP原位可视化将用于检测这些抗原的加工过程。项目1中携带抗原的APCs的原位可视化将用于项目3中分离的中枢神经系统小胶质细胞的检测。利用敏感技术分析抗原特异性CD4+ T细胞将对理解MS的发病机制和通过任何形式的治疗进行免疫监测具有重要意义。

A confluence of different areas of immunological research offers an opportunity for a more synthetic approach to investigate T cell recognition of antigens in patients with MS, an inflammatory disease of the CNS myelin. These include the elucidation of the crystal structure for TCR recognition of antigen/MHC; identification of potential biochemical techniques that provide insights into the cell biology of class II restricted presentation with particular emphasis on the proteases involved in this process. In the first project, we shall utilize the newly developed MHC tetramer technology to determine the frequence, activation state and cytokine profile of antigen specific T cells in the CSF & blood of HLA- DR2 MS patients, which has almost certainly been underestimated by limiting dilution techniques. The presentation of myelin peptides by APC's in MS lesions will be investigated using TCR tetramers generated with cDNAs from myelin specific T cell clones and used to stain MS plaque tissue. In the second project, combinatorial peptide libraries will be used to examine the antigen specificity of oligoclonal CSF T cells. These investigations will also be performed on subjects with defined CNS infections and will determine whether TCRs expressed on CNS T cells in patients with MS are degenerate in their recognition of combinatorial peptide libraries as compared to CNS T cells in patients with MS are degenerate in their recognition of combinatorial peptide libraries as compared to CNS T cells recognizing viral or bacterial antigens. It will be determined whether oligoclonal T cells and MBP/DR2 tetramer binding T cells are reflective of the original CSF population Project 2 will explore the cell-biological and biochemical properties of CNS microglial cells. The processing pathway of the myelin autoantigens MBP and MOG and the trafficking of Class II molecules and MOG in human microglia will be examined. 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 examine the role of lysosomal proteases Cathepsin B, D, L or S, likely candidates for the proteolytic conversion of MBO and MOG in antigen processing. MOG and MBP visualization in situ of antigen-bearing APCs in project 1 will be used to examine the processing of these antigens. The visualization in situ of antigen-bearing APCs in project 1 will be used to examine CNS microglial cells isolated in project 3. Analysis of antigen-specific CD4+ T cells with sensitive techniques will have important implications for understanding the pathogenesis of MS and for immune monitoring by any form of therapy.