Identification of antigen-specific T-cells and protein biomarkers for diagnosis of sarcoidosis

Abstract

Sarcoidosis is a multisystem granulomatous disease in humans with unknown aetiology and no current definitive diagnostic method. Intradermal inoculation of Kveim reagent (sarcoidosis tissue) was historically used for diagnosis, by causing a granuloma at injection site within 4-6 weeks, with high sensitivity and specificity. The immune mechanisms of this are poorly understood and the antigenic targets of granuloma-associated CD4+ T-cells are unknown. This T-cell infiltrate is oligoclonal, implicating a small number of antigenic targets. Previous work has shown that these are likely proteins. Aim 1 of this project involved identification of candidate protein antigens within Kveim reagent using proteomics, an established technology for the in depth quantitative differential analysis of complex protein mixtures. These sarcoidosis-specific proteins were analysed using gel electrophoresis with subsequent mass spectrometry and protein database interrogation. More than 80 proteins were identified as appearing specific to Kveim reagent or of significantly different abundance in sarcoidosis tissue compared to controls. These previously unreported proteins represent novel information that may help in the further understanding of the cellular pathways involved in disease. Additionally, multiplex cytokine analysis was performed on supernatant from sarcoidosis peripheral blood mononuclear cells incubated with both Kveim reagent and Kveim protein fractions to identify a secreted Th1 signal specific to disease. Information from the proteomics approach combined with this cytokine signal may be beneficial in creating an ex vivo immune based assay for diagnosis. Aim 2 of this project involved the direct analysis of serum and bronchoalveolar lavage (BAL) fluid by multiplex cytokine analysis to investigate signals which could serve as diagnostic biomarkers for the disease. BAL patterns in sarcoidosis were found indistinguishable from tuberculosis but significantly different to healthy volunteers. Significant differences were also found between sarcoidosis and tuberculosis serum, allowing for the creation of a model to reliably distinguish between the two diseases.