Human T cell priming assay (hTCPA) for the identification of contact allergens based on naive T cells and DC – IFN-γ and TNF-α readout

By Anne Richter - Sonja S. Schmucker - Philipp R. Esser - Verena Traska - Verena Weber - Lisa Dietz - Hermann-Josef Thierse - Davide Pennino - Andrea Cavani - Stefan F. Martin

Many small protein reactive organic and inorganic chemicals can cause allergic contact dermatitis, a T cell mediated inflammatory skin disease. In vitro alternatives to animal testing are needed for the identification of chemicals that pose such risks to human health. We here publish the standard operation procedure for a human T cell priming assay developed primarily for the identification of contact allergens within the integrated EU project Sens-it-iv. This multiparametric flow cytometry based assay identifies chemical specific T cells based on their frequency and antigen-specific production of the cytokines IFN-γ and TNF-α at the single cell level. Using sorted naïve T cells and monocyte-derived dendritic cells pulsed with the test chemical or with chemical-protein conjugates, the successful priming of an antigen-specific T cell response is controlled after antigen-specific restimulation by cytokine readout. As the most specific response of the immune system to contact allergens the analysis of the chemical-specific T cell response may be a useful in vitro assay for hazard identification in immunotoxicology. This assay may be a valuable, highly specific element of an integrated testing strategy for the identification of chemicals and drugs that cause T cell mediated respiratory or gastrointestinal tract hypersensitivities or adverse drug reactions.