Antigen Presentation in Human Autoimmune Diseases

描述(申请人提供)：该计划项目将继续关注多发性硬化症患者的抗原呈递和T细胞识别。在过去三年中，在建立和验证新技术方面作出了相当大的努力，这些新技术现已到位，以便继续实施私营部门保护计划。这些新的方法将被用来可视化中枢神经系统中的抗原呈递，并从分子上确定参与疾病过程的T细胞和B细胞群体。项目1已经开发了一种新的方法来产生MAC II类梗，该方法将用于量化和表征MS患者的抗原特异性CODT细胞和人源化的疾病小鼠模型。项目2已经从多发性硬化症患者的脑脊液中分离出扩增的T细胞群体，并将表征T细胞和B细胞群体的抗原受体。组合多肽文库和MHC II类载体将用于确定从脑脊液中建立的T细胞克隆的抗原特异性。通过激光捕获显微切割技术从脑脊液中分离出B细胞，并用激光捕获显微切割技术从MS斑块组织中分离出B细胞，从而根据分离的重链和轻链序列表达重组抗体。这种重组抗体提供了丰富的这些蛋白质的来源，用于确定它们的靶标特异性，将通过争论的方法建立，并评估致病性。项目3将使用一种新创建的小鼠模型来研究抗原提呈细胞进入中枢神经系统的情况，在该模型中，GFP部分通过敲入技术连接到MHC II类Beta链上。这些表达GFP的抗原提呈细胞与浸润性T细胞的相互作用将通过双光子显微镜在活体动物的视神经中进行研究。每个研究人员都为这一重要的临床问题贡献了独特的专业知识和创新的方法。由此产生的计划是高度协同的，并将允许将蛋白质组学和生物化学中的强大新工具应用于人类炎症性疾病的病理生理学。

DESCRIPTION (provided by applicant): This program project will continue to focus on antigen presentation and T cell recognition in patients with multiple sclerosis. During the past three years considerable efforts were directed at establishing and validating new technology, now in place for the continuation of the PPG. These novel approaches will be used to visualize antigen presentation in the central nervous system and to molecularly define the T cell and B cell populations involved in the disease process. Project 1 has developed a novel approach for the generation of MAC class II terriers, which will be used to quantify and characterize antigen-specific COD T cells in MS patients and a humanized mouse model of the disease. Project 2 has isolated expanded populations of T cells from the cerebrospinal fluid of patients with MS and will characterize the antigen receptors of T cell and B cell populations. Combinatorial peptide libraries and MHC class II terriers will be used to define the antigen specificity of T cell clones established from the cerebrospinal fluid. B cells will be isolated from cerebrospinal fluid by FACES and MS plaque tissue by laser capture micro dissection, so that recombinant antibodies can be expressed based on the isolated heavy and light chain sequences. Such recombinant antibodies provide an abundant source of these proteins for definition of their target specificity, to be established by a polemics approach, and assessment of pathogenicity. Project 3 will examine the entry of antigen presenting cells into the CNS using a newly created mouse model in which a GFP moiety is attached to the MHC class II beta chain through knock-in technology. The interaction of these GFP expressing antigen presenting cells with infiltrating T cells will be investigated in the optic nerve of live animals by 2-photon microscopy. Each of the investigators contributes a unique area of expertise and innovative approaches to this important clinical problem. The resulting program is highly synergistic and will allow the application of powerful new tools in proteomics and biochemistry to the pathophysiology of human inflammatory disease.