MODULATION OF CLASS II ANTIGEN PROCESSING BY HLA DO

主要组织相容性复合体（MHC） II类分子是异二聚体糖蛋白，它结合来自外源蛋白的肽并将其呈递到CD4 T细胞。与残余不变链片段相关的MHC II类分子[II类相关不变链肽（CLIP）]通过II类分子HLA-DM的催化作用装载抗原肽，HLA-DM也具有II类伴侣和肽编辑器的功能。HLA-DO （DO）是另一种在B细胞、树突状细胞和胸腺上皮中表达的II类分子，在运输到MHC II类室（MIIC）期间和之后都与DM有物理关联。MIIC中的DM-DO关联表明这两种分子的功能是相连的。DO被证明可以阻断DM的功能，因为纯化的DM-DO复合物在体外不能催化CLIP交换肽。在II+ DO-类细胞系中，DO的表达引起II- clip复合物在细胞表面的积累，表明DO在体内阻断了DM功能。DM功能的抑制通过降低细胞表面II类肽复合物的水平导致II类加工途径的下调。所观察到的do介导的DM功能抑制的生化、功能和免疫学后果尚不清楚。此外，目前尚不清楚DO是否具有其他功能。本申请中提出的研究旨在定义DO表达的生物学后果，并确定在II类加工途径中是否存在DO的替代功能。这将通过三种不同的生化方法来完成。首先，在HLA-DO存在或不存在的情况下，将生成表达II类抗原加工途径已知组分的真实抗原提呈细胞系，并使用生化方法进行分析。其次，在没有糖尿病的情况下，HLA-DO分子可以从内质膜转运，但保留了与糖尿病相关的能力，我们生成并检查了这些分子，以分析DO的结构和功能。第三，将确定B细胞受体的内化或B细胞的激活是否导致Do从糖尿病中释放，从而激活DM。提出的工作应回答关于Do在II类加工途径中的作用的重要问题，并与肿瘤监测和自身免疫等领域相关。

Major histocompatibility complex (MHC) class II molecules are heterodimeric glycoproteins that bind peptides derived from exogenous proteins and present them to CD4 T cells. MHC class II molecules associated with residual invariant chain fragments [class II-associated invariant chain peptides (CLIP)] are loaded with antigenic peptides by the catalytic action of the class II- like molecule, HLA-DM that also functions as a class II chaperone and peptide editor. HLA-DO (DO), yet another class II-like molecule that is expressed in B cells, dendritic cells and thymic epithelium, physically associates with DM both during and after transport to the MHC class II compartments (MIIC). The DM-DO association in the MIIC suggested that the functions of both molecules are linked. DO was shown to block DM function as purified DM-DO complexes could not catalyze CLIP exchange for peptide in vitro. Expression of DO in class II+ DO- cell lines caused the accumulation of class II-CLIP complexes at the cell surface, suggesting that DO blocked DM function in vivo. The inhibition of DM function results in down modulation of the class II processing pathway by decreasing the level of class II-peptide complexes on the cell surface. The biochemical, functional and immunological consequences of the observed DO-mediated inhibition of DM function remain unknown. Additionally, it is not known if DO has additional functions. The studies proposed in this application are designed to define the biological consequences of DO expression and to determine if there are alternative functions for DO in the class II processing pathway. This will be accomplished by three different biochemical approaches. First, bonafide antigen presenting cell lines that express the known components of the class II antigen processing pathway in the presence and absence of HLA-DO will be generated and analyzed using a biochemical approach. Second, HLA-DO molecules that can transport from the ER in the absence of DM but that retain their ability to associate with DM we be generated and examined to analyze DO structure and function. Third, it will be determined if internalization of the B cell receptor or if activation of B cells results in the release of Do from DM, thereby activating DM. The proposed work should answer important questions about the role of DO the class II processing pathway and has relevance in such areas as tumor surveillance and autoimmunity.