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，一种非经典的MHC II类异源二聚体，在从给定蛋白质中选择免疫优势表位的作用。我们最近的数据有力地表明，DM选择性地解离具有松弛构象的多肽-MHC复合体，而不影响致密的多肽/MHC复合体。我们认为，这种与MHC II类特定构象的优先相互作用，可能会影响从所有其他可以与MHC II类结合的表位池中选择表位。本文设计了一种新的方法来研究DM在给定MHC II类决定簇选择中的作用。以纯化的空HLA-DR1、可溶性HLA-DM和纯化的流感血凝素(HA)为模式抗原，我们将产生在DM存在的情况下仍与DR1结合的抗原表位。我们首先从纯化的流感血凝素开始，它在表达DR1的个体中具有已知的免疫优势表位HA306-318。在AIM I中，使用具有已知活性的酶，如组织蛋白酶，在DR1和DM的存在下，HA蛋白将被酶消化和DR1结合，在上述条件下洗脱的DR1的高效液相色谱分级多肽的质谱分析将揭示HA306-318是否被选为优势肽。目的2将采用类似于Aim I的方法研究HLADO对DM的调节作用和免疫优势表位的选择。Aim 3将在体外检测纯化的DO与DM的相互作用，Aim IV将在体内检测已鉴定的表位的生物学活性。一旦该方法的可行性确定，它应该适用于从病原生物中鉴定任何复杂蛋白的抗原表位。这些抗原表位可用于体内特异性活化T细胞的二类四聚体染色和疫苗设计，并可作为免疫效果的相关免疫指标。这项研究对发现与生物防御相关的感染性病原体的免疫优势表位具有重大影响。

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.