Defining the Beryllium Antigen Complex in Berylliosis

定义铍中毒中的铍抗原复合物

• 批准号: 7210181
• 负责人: LEE S NEWMAN
• 金额: $ 26.95万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7210181
• 关键词: Alveolar Macrophages; American; Amino Acids; Antigen-Presenting Cells; Antigens; Behavior; Berylliosis; Beryllium; Binding; Binding Sites; Biochemical; Breathing; Bronchoalveolar Lavage; CD4 Positive T Lymphocytes; Cell Line; Cells; Chemicals; Chronic berylliosis; Class; Cleaved cell; Clinical; Clonal Expansion; Collaborations; Complex; Computer Simulation; Cytokine Gene; Data; Detection; Development; Digestion; Disease; Elements; Endocytosis; Environmental Exposure; Event; Ferritin; Foundations; Gene Expression; Generations; Genes; Genetic; Genotype; Glutamic Acid; Goals; Granulomatous; HLA-DPB1 gene; HLA-DR Antigens; Haptens; High Pressure Liquid Chromatography; Histocompatibility; Human; Immune response; Immunologist; Inflammation; Inflammatory; Interleukin-2; Investigation; Ions; Isotopes; Laboratories; Lung; Major Histocompatibility Complex, Class II, DP Beta 1; Maps; Mass Spectrum Analysis; Measures; Mediating; Metals; Methods; Molecular; Monoclonal Antibodies; Nature; Pathogenesis; Pathway interactions; Patients; Peptide Fragments; Peptides; Physicians; Population; Positioning Attribute; Prevention; Production; Proteins; Rare Diseases; Research; Research Personnel; Research Project Grants; Rest; Risk; Role; Sarcoidosis; Scientist; Series; Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure; Study Section; Surface; T memory cell; T-Cell Activation; T-Cell Proliferation; T-Cell Receptor; T-Lymphocyte; Testing; Th1 Cells; Time; Transcriptional Activation; Translational Research; Up-Regulation; Ursidae Family; Work; Workplace; adduct; antigen binding; carboxylate; cytokine; innovation; novel; programs; protein aminoacid sequence; research study; response; tandem mass spectrometry; tool; translational study

DESCRIPTION (provided by applicant): Inhaled beryllium can be endocytosed by alveolar macrophages and can induce antigen-mediated activation of beryllium (Be)-specific CD4+ T cells in the lungs of patients with chronic beryllium disease (CBD). This adoptive immune response results in T cell clonal expansion, production of Th-1 type cytokines, and granulomatous inflammation. These T cell-mediated events occur when specific subclasses of T cell antigen receptor (TCR) engage antigen-presenting cells that bear the correct major histocompatibility (MHC) class II molecule/Be-antigen complexes on their surface. Previous studies have identified and characterized the genetics and functional relevance of the TCR and HLA class II in CBD, and have demonstrated that beryllium regulates T cell proliferation, cytokine gene expression and protein production, especially when beryllium-specific TCRs ligate HLA-DPB1 with a glutamic acid in amino acid position 69. However the composition and structure of the beryllium antigen that lies in the groove between HLA-DPB1 and the TCR remains unknown. The central goal of this R21 application is to demonstrate the feasibility of using novel biochemical and physical chemical tools to determine the precise chemical nature of beryllium antigen, in relation to HLA-DPB1 and HLA-associated peptides. To do this, a series of experiments will be performed to establish that it is feasible to isolate and chemically characterize Be-antigen using well-established Be-specific antigen presenting cell lines that express either relevant or irrelevant HLA-DPB1 molecules. The experiments will demonstrate that it is possible to isolate and purify those HLA-DPB1 and peptides that are bound to beryllium using a new and unique molecular complex, 10Be-ferritin. In collaboration with investigators at Lawrence Livermore National Laboratory, experiments will employ accelerator mass spectroscopy (AMS), for the first time, to identify beryllium-associated proteins, specifically HLA class II-binding sites, and peptides bind 10Be in the antigen presenting cells, at levels of Be detection as low as 1 x 10-18 M. Immunoaffinity-purified HLA-DPB1-10Be-antigen molecules will be extracted from antigen presenting cells, eluted, separated, and identified. AMS will be used to determine if the HLA class ll-bound peptide complex in CBD-derived cells consists of 10Be that is bound only to HLA-DPB1, only to associated antigenic peptides, or to both. Results will be confirmed by testing putative Be-antigen using CBD responder T cell lines that have been derived from patients with CBD, to measure beryllium-specific T cell proliferation. This proposed translational research study will bring together physical chemists, biochemists, immunologists and physician scientists focused on defining the precise chemical nature of Be-antigen in CBD. The results will have implications for our understanding of the behavior of metal antigens; how metal antigens may interact with HLA class II restriction elements; how metal antigens trigger an adoptive immune response that results in granulomatous inflammation; and advance our understanding of the fundamental mechanisms by which environmental exposures and genes interact, causing granulomatous disease.

描述（由申请人提供）：吸入的铍可通过肺泡巨噬细胞内吞噬，并且可以诱导慢性铍病（CBD）患者肺中特异性CD4+ T细胞的抗原介导的激活。这种过继的免疫反应导致T细胞克隆膨胀，Th-1型细胞因子的产生和肉芽肿性炎症。当T细胞抗原受体（TCR）的特定亚类接合具有正确的主要组织相容性（MHC）II类分子/BE抗原复合物在其表面上时，就会发生这些T细胞介导的事件。 Previous studies have identified and characterized the genetics and functional relevance of the TCR and HLA class II in CBD, and have demonstrated that beryllium regulates T cell proliferation, cytokine gene expression and protein production, especially when beryllium-specific TCRs ligate HLA-DPB1 with a glutamic acid in amino acid position 69. However the composition and structure of the beryllium antigen that lies in the HLA-DPB1和TCR之间的凹槽仍然未知。此R21应用的核心目标是证明使用新型的生化和物理化学工具来确定与HLA-DPB1和HLA相关肽有关的确切化学性质的可行性。为此，将进行一系列实验，以确定使用良好的Be-Tem be-tem抗原呈现出表达相关或无关的HLA-DPB1分子的细胞系，可以隔离和化学表征Be-Antigen。实验将证明，可以使用一种新的和独特的分子复合物10BE-Ferritin隔离和纯化那些与铍结合的HLA-DPB1和肽。在与Lawrence Livermore国家实验室的研究人员合作的情况下，实验将首次采用加速器质谱（AMS），以识别与铍相关的蛋白质，特定于HLA II类结合位点，特异性HLA HLA蛋白质，并在抗原呈现的细胞中与PEPTIDES进行特定的peptides，在抗原呈现的细胞中与未接收的水平相结合。 HLA-DPB1-10BE-抗原分子将从抗原呈递细胞中提取，洗脱，分离和鉴定。 AM将用于确定CBD衍生细胞中HLA类LL结合的肽复合物是由仅与HLA-DPB1结合的10BE，仅与相关的抗原肽或两者兼而有之。将通过使用CBD响应者T细胞系测试推定的BE抗原来确认结果，该细胞已从CBD患者中得出，以测量铍特异性T细胞增殖。这项拟议的翻译研究将汇集物理化学家，生物化学家，免疫学家和医师科学家，致力于定义CBD中BE-antigen的确切化学性质。结果将对我们对金属抗原行为的理解产生影响。金属抗原如何与HLA II类限制元件相互作用；金属抗原如何触发产生颗粒炎症的继发性免疫反应；并促进我们对环境暴露和基因相互作用的基本机制的理解，从而导致肉芽肿性疾病。