Project Summary Sarcoidosis is a systemic granulomatous disorder of unknown etiology that affects the lung in greater than 90% of cases. The disease is characterized by the accumulation of activated CD4+ T cells in the lung and other sites of disease activity. Evidence suggests that these T cells are intimately involved in the pathogenesis of sarcoidosis. In the previous version of this proposal, we identified lung CD4+ T cells from HLA-DR3-expressing Löfgren’s syndrome (LS) subjects expressing related T cell receptors (TCRs) and determined that these TCRs recognized peptides derived from NAD-dependent protein deacetylase (NDPD) expressed in a common airborne mold species, Aspergillus nidulans. Using HLA-DR3-NDPD tetramers and IFN-γ ELISPOT, we validated those findings and showed that a significantly greater number of NDPD-responsive CD4+ T cells exists in the lungs of LS subjects; thus,we have identified a potential causative agent in the genesis of LS. This study of Swedish subjects with LS serves as a “proof of concept” for the current renewal whose focus is to identify T cell epitopes for CD4+ T cells derived from the lungs of sarcoidosis subjects expressing HLA-DRB1*11:01, the HLA allele strongly linked to sarcoidosis in Caucasians and African-Americans in the US. Thus, we hypothesize that expanded CD4+ T cells in the lungs of HLA-DRB1*11:01-expressing US sarcoidosis patients are accumulating in response to etiologic sarcoidosis antigen(s) and recognize those antigens in an HLA- DRB1*11:01-restricted fashion. This proposal harnesses the strengths of a multidisciplinary research team and focuses on a sarcoidosis cohort in the US. Using a single cell RT-PCR approach, Aim 1 will characterize αβTCR pairs expressed on CD4+ T cells derived from the lungs of US sarcoidosis patients and generate hybridomas expressing disease-relevant TCRs. The second specific aim will determine the peptides that stimulate the CD4+ T cell hybridomas expressing the TCRs of interest. The final aim will use functional assays and HLA-DR11-peptide tetramers to identify and enumerate antigen-specific CD4+ T cells in the lungs of sarcoidosis patients and determine if the frequency of these T cells can serve as a biomarker for diagnosis and/or prognosis. Thus, using a novel yet proven scientific approach, we will address critical knowledge gaps in the etiologic T cell antigens involved in the pathogenesis of sarcoidosis in US patients, further advancing our understanding of this enigmatic disease.