Role of DM in Generation of Immunodominant Epitope(s)

描述（由申请人提供）：本提案的目的是评估DM（一种非经典的MHC II类异源二聚体）在从给定蛋白质中选择免疫显性表位中的作用。我们最近的数据有力地表明，DM选择性地解离复合物的肽-MHC，具有松软的构象，并留下紧凑的肽/MHC复合物不受影响。我们提出，这种与MHC II类的特定构象的优选相互作用，可能会影响从可以结合到MHC II类的所有其他表位的库中选择表位。本文设计了一种新的检测方法来研究DM在给定的II类MHC决定簇选择中的作用。使用纯化的空HLA-DR 1、可溶性HLA-DM和纯化的流感血凝素（HA）作为模型抗原，我们将产生在DM存在下保持与DR 1结合的抗原表位。我们将开始于纯化的流感血凝素，其在表达DR 1的个体中具有已知的HA 306 - 318免疫显性表位。使用在抗原加工中具有已知活性的酶，如组织蛋白酶，HA蛋白将在Aim I中存在DR 1和DM的情况下进行酶消化和DR 1结合。在上述条件下从DR 1洗脱的HPLC分级肽的质谱分析将揭示HA 306 -318是否被选为主要肽。目的二将采用与目的一相似的方法研究HLA-DO在糖尿病发病中的作用及免疫优势表位的选择。目的3将在体外检查纯化的DO与DM的相互作用，目的IV将在体内测试鉴定的表位的生物活性。一旦确定了该方法的可行性，它应该适用于鉴定任何复杂蛋白质的抗原表位从病原生物。抗原表位可用于II类四聚体测定，用于体内特异性活化T细胞的染色和疫苗的设计，以及作为免疫功效的免疫相关物。该研究对发现与生物防御相关的病原体免疫优势表位具有重要意义。

DESCRIPTION (provided by applicant): The goals of this proposal are to evaluate the role of DM, a non-classical MHC class II heterodimer, in the selection of immunodominant epitopes from a given protein. Our recent data strongly suggested that DM selectively dissociates complexes of peptide-MHC that have floppy conformation and leaves compact peptide/MHC complexes unaffected. We propose that this preferred interaction with a specific conformation of MHC class II, might influence epitope selection from the pool of all other epitopes that can bind to MHC class II. A new assay is designed here to investigate the role of DM in determinant selection by a given MHC class II. Using purified empty HLA-DR1, soluble HLA-DM, and purified Influenza Hemagglutinin (HA) as a model antigen, we will generate antigenic epitopes that remain bound to DR1 in the presence of DM. We would begin with purified influenza hemagglutinin that has a known immunodominant epitope of HA306- 318 in DR1 expressing individuals. Using enzymes with known activity in antigen processing, such as cathepsins, HA protein will be subjected to enzymatic digestion and DR1 binding in the presence of DR1 and DM in Aim I. Mass spectrometric analyzes of the HPLC fractionated peptides eluted from DR1 under the above condition will reveal whether HA306-318 is selected as a predominant peptide. Aim 2 will investigate effects of HLA-DO on regulation of DM and selection of immunodominant epitopes using similar approach to Aim I. Aim 3 will examine interactions of purified DO with DM in vitro and Aim IV will test biological activity of identified epitopes in vivo. Once the feasibility of the method is established, it should be applicable for identification of antigenic epitopes of any complex proteins from pathogenic organisms. The antigenic epitopes might be used in class II tetramer assays for staining of specific activated T cells in vivo and in design of vaccines and as correlates of immunity for immunization efficacy. This research has great impacts on discovering immunodominant epitopes of infectious agents relevant to biodefense.