Novel Tools for Immune Monitoring in Autoimmune Diseases

在目前的资助期间，Wucherpfennig博士的实验室开发了新的方法来 自身免疫性疾病中自身抗体和CD4T细胞的定量检测这项工作是按照以下方式进行的 研究项目不可或缺的组成部分，这些技术现在已经成熟到了这样一个程度，即 由Wucherpfennig博士监督的核心可以最有效地产生试剂。协同工作 导致了自身抗原四聚体的产生，使 人类脱髓鞘疾病自身抗体的灵敏检测。髓鞘四聚体 少突胶质细胞糖蛋白(MOG)被证明可以敏感地检测MOG自身抗体。 急性脱髓鞘脑脊髓炎(ADEM)和儿科MS患者的第一个主要目标 核心将是产生这样的抗原四聚体，用于量化针对其他自身抗原的自身抗体 多发性硬化症和1型糖尿病。该核心将产生两种放射性标记的自身抗体版本 用于表征和分离自身抗原特异性B细胞的定量和荧光形式 在MS和1型糖尿病患者中，以及在相关的动物模型中。 在目前的资助期间，伍氏实验室还开发了一种新的方法，用于 创建MHC II类四聚体。MHC II类分子通过共价结合片段表达 在生物合成和纯化过程中保护结合部位的多肽。所述低亲和力片段多肽为 在接头裂解后迅速释放，因此这些MHC II类/CLIP前体可用于 从单一的蛋白质制剂中产生各种不同的MHC II类四聚体。这 在MS和1型动物模型中实现自身抗原特异性CD4T细胞定量的方法 糖尿病。特别是，它允许同时显示Foxp3-GFP中的调节性和效应性T细胞 在实验性自身免疫性脑脊髓炎(EAE)过程中敲入小鼠。第二个目标是 因此，这一核心将是为该PPG的所有项目生成MHC II类四聚体，并进一步 通过开发可用于检测低价态的更高价态的多聚体来开发这项技术 亲和力CD4T细胞群。

During the current funding period, Dr. Wucherpfennig's lab has developed novel approaches for the quantification of autoantibodies and CD4 T cells in autoimmune diseases. This work was conducted as integral component of research projects, and the technologies have now matured to a point where these reagents can be generated most efficiently by a core supervised by Dr. Wucherpfennig. Collaborative work between the Wucherpfennig and Hafler labs resulted in the creation of autoantigen tetramers that enable sensitive detection of autoantibodies in human demyelinating diseases. Tetramers of myelin oligodendrocyte glycoprotein (MOG) were shown to permit sensitive detection of MOG autoantibodies in patients with acute demyelinating encephalomyelitis (ADEM) and pediatric MS. The first major aim for this core will be to generate such antigen tetramers for quantification of autoantibodies against other selfantigens in MS and type 1 diabetes. The core will generate both radiolabeled versions for autoantibody quantification as well as fluorescent versions for characterization and isolation of autoantigen-specific B cells in patients with MS and type 1 diabetes, and in relevant animal models. During the present funding period, the Wucherpfennig lab has also developed a novel approach for the creation of MHC class II tetramers. MHC class II molecules are expressed with a covalently bound CLIP peptide that protects the binding site during biosynthesis and purification. The low affinity CLIP peptide is rapidly released following linker cleavage, and these MHC class II/CLIP precursors can therefore be used for the generation of a variety of different MHC class II tetramers from a single protein preparation. This approach enabled quantification of autoantigen-specific CD4 T cells in animal models of MS and type 1 diabetes. In particular, it allowed simultaneous visualization of regulatory and effector T cells in Foxp3-GFP knock-in mice during the course of experimental autoimmune encephalomyelitis (EAE). The second aim for this core will therefore be to generate MHC class II tetramers for all projects of this PPG and to further develop this technology by generation of multimers with higher valency that can be used to detect lower affinity CD4 T cell populations.