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(申请人提供)：吸入铍可被肺泡巨噬细胞吞噬，并可诱导慢性铍病(CBD)患者肺内抗原介导的铍(Be)特异性CD4+T细胞的激活。这种过继免疫反应导致T细胞克隆性增殖，产生Th-1型细胞因子，并导致肉芽肿炎症。当T细胞抗原受体(TCR)的特定亚类与表面具有正确的主要组织相容性(MHC)II类分子/BE抗原复合体的抗原提呈细胞接触时，这些T细胞介导的事件就会发生。以往的研究已经确定和表征了TCR和HLAII类在CBD中的遗传学和功能相关性，并已证明铍调节T细胞的增殖、细胞因子基因的表达和蛋白质的产生，特别是当Be特异的TCRs与人类白细胞抗原DPB1与第69位氨基酸的谷氨酸连接时。然而，位于人类白细胞抗原-DPB1和TCR之间的铍抗原的组成和结构尚不清楚。这项R21应用的中心目标是证明使用新的生化和物理化学工具来确定铍抗原与人类白细胞抗原-DPB1和人类白细胞抗原相关多肽的精确化学性质的可行性。为此，将进行一系列实验，以确定使用表达相关或不相关的HLA-DPB1分子的成熟的BE特异性抗原提呈细胞系分离BE抗原并对其进行化学表征是可行的。实验将证明，使用一种新的和独特的分子络合物10Be-铁蛋白来分离和纯化那些与铍结合的人类白细胞抗原-DPB1和多肽是可能的。与劳伦斯·利弗莫尔国家实验室的研究人员合作，实验将首次使用加速器质谱学(AMS)来识别抗原呈递细胞中的铍相关蛋白，特别是人类白细胞抗原II类结合位点和多肽绑定10Be，检测水平低至1×10-18M。免疫亲和纯化的人类白细胞抗原-DPB1-10Be-抗原分子将从抗原呈递细胞中提取、洗脱、分离和鉴定。AMS将用于确定CBD来源细胞中的HLA11类结合多肽复合体是否由10Be组成，该10Be仅与HLADPB1结合，仅与相关抗原肽结合，还是两者都结合。结果将通过使用来自CBD患者的CBD反应者T细胞株来测试假定的BE抗原来证实，以测量铍特定的T细胞增殖。这项拟议的转化性研究将把物理化学家、生物化学家、免疫学家和内科科学家聚集在一起，专注于定义CBD中BE抗原的确切化学性质。这些结果将对我们理解金属抗原的行为；金属抗原如何与人类白细胞抗原II类限制元件相互作用；金属抗原如何触发导致肉芽肿性炎症的过继免疫反应；以及促进我们对环境暴露和基因相互作用导致肉芽肿性疾病的基本机制的理解具有重要意义。