Defining the Beryllium Antigen Complex in Berylliosis

描述（由申请人提供）：吸入的铍可被肺泡巨噬细胞内吞，并可诱导慢性铍病（CBD）患者肺中铍（be）特异性CD4+ T细胞的抗原介导活化。这种过继性免疫反应导致T细胞克隆扩增、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 -抗原复合物的抗原呈递细胞结合时，这些T细胞介导的事件就会发生。先前的研究已经确定并表征了CBD中TCR和HLA II类的遗传学和功能相关性，并证明铍调节T细胞增殖、细胞因子基因表达和蛋白质产生，特别是当铍特异性TCR与氨基酸位置69的谷氨酸连接HLA- dpb1。然而，位于HLA-DPB1和TCR之间的凹槽中的铍抗原的组成和结构尚不清楚。本R21申请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HLA-DPB1和hla相关肽的精确化学性质的可行性。为了做到这一点，将进行一系列实验，以确定使用已建立的表达相关或不相关HLA-DPB1分子的be特异性抗原呈递细胞系分离be抗原并进行化学表征是可行的。实验将证明，利用一种新的、独特的分子复合物10be -铁蛋白，可以分离和纯化那些与铍结合的HLA-DPB1和肽。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（AMS）来鉴定铍相关蛋白，特别是HLA ii类结合位点，以及抗原提呈细胞中结合10Be的肽，在Be检测水平低至1 x 10-18 m时，免疫亲和纯化的HLA- dpb1 -10Be抗原分子将从抗原提呈细胞中提取，洗脱，分离和鉴定。AMS将用于确定cbd来源细胞中HLA类ll结合肽复合物是否由仅与HLA- dpb1结合，仅与相关抗原肽结合，或两者结合的10Be组成。结果将通过使用来自CBD患者的CBD应答T细胞系测试假定的be抗原来证实，以测量铍特异性T细胞的增殖。这项拟议的转化研究将汇集物理化学家、生物化学家、免疫学家和内科科学家，专注于确定CBD中be抗原的精确化学性质。这些结果将对我们理解金属抗原的行为产生影响；金属抗原如何与HLA II类限制性元件相互作用；金属抗原如何引发过继性免疫反应，导致肉芽肿性炎症；并推进我们对环境暴露和基因相互作用导致肉芽肿病的基本机制的理解。

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.