Sarcoidosis is a systemic granulomatous disorder of unknown etiology which affects the lung in greater than 90% of cases. The disease is characterized by an accumulation of activated CD4+T cells in the lung and other sites of disease activity, and evidence strongly suggests that these T cells are intimately involved in the pathogenesis of sarcoidosis. T cell receptor (TCR) repertoire analysis of BAL CD4+ T cells has shown increased expression of particular TCR V region subsets, in particular Valpha2.3. Sequencing of these expanded TCR V regions has shown oligoclonal T cell populations suggesting their accumulation in response to conventional antigen stimulation. Individuals with expanded bronchoalveolar lavage (BAL) Valpha2.3 subsets usually express the HLA DR allele, DRB1*0301. These expansions are only present in the lung and disappear with disease remission, reinforcing their importance in the disease process. One difficulty in working with BAL T cell clones is that these cells can be expanded in culture for only a short period of time before dying. In this proposal, we will enroll active sarcoidosis patients expressing both DRB1*0301 and TCR Valpha2.3 BAL expansions and use a novel approach to define the T cell ligands in sarcoidosis. This approach will utilize a baculovirus-generated HLA-DRB1*0301 peptide library in which SF9 insect cells will each express a different DR3/peptide complex. Approximately ten soluble Valpha2.3 TCRs will be generated also in a baculovirus system. These soluble TCRs can then be screened against the DR3 peptide library using flow cytometry. This approach allows for a rapid screening of the library. Then, positively staining SF9 insect cells can be sorted on the cytofluorograph and greatly enriched. The recognized peptide in the positively staining SF9 cell can be sequenced. This novel approach will generate """"""""mimotopes"""""""" which can be screened against known peptide/protein sequences and will provide new insight into the etiologic sarcoidosis antigen.
