Role of DM in Generation of Immunodominant Epitope(s)

描述（由申请人提供）：本提案的目标是评估DM（一种非经典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。我们最近的数据强烈表明，DM选择性地解离具有松散构象的肽-MHC复合物，而不影响紧凑的肽/MHC复合物。我们提出，这种与MHC II类特定构象的首选相互作用可能会影响所有其他可以与MHC II类结合的表位池中的表位选择。这里设计了一种新的测定方法来研究DM在给定MHC II类的决定因素选择中的作用。使用纯化的空HLA-DR1、可溶性HLA-DM和纯化的流感血凝素（HA）作为模型抗原，我们将生成在DM存在时仍与DR1结合的抗原表位。我们将从纯化的流感血凝素开始，该抗原表位在DR1表达个体中已知具有HA306- 318的免疫优势表位。使用在抗原加工中已知活性的酶，如组织蛋白酶，HA蛋白将在Aim i中DR1和DM存在的情况下进行酶切和DR1结合。在上述条件下，对从DR1中洗脱的HPLC分离肽进行质谱分析将揭示HA306-318是否被选择为优势肽。Aim 2将使用与Aim 1类似的方法研究HLA-DO对DM调控和免疫优势表位选择的影响。Aim 3将在体外研究纯化DO与DM的相互作用，Aim IV将在体内测试鉴定的表位的生物活性。一旦建立了该方法的可行性，它应该适用于鉴定来自病原生物的任何复杂蛋白的抗原表位。抗原表位可用于II类四聚体试验，用于体内特异性活化T细胞染色和疫苗设计，并作为免疫效果的免疫相关因素。本研究对发现与生物防御相关的感染因子的免疫显性表位具有重要意义。

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.