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. ? ? ?
