Multiplex and functional detection of antigen-specific human T cells by ITRA--indirect T cell recognition assay

The identification and functional characterization of pathogen-specific T cells plays a critical role in immunological research and diagnostics. In addition to the present standard technologies such as intracellular cytokine staining (ICS), enzyme-linked immunospot (ELISPOT) and peptide-major-histocompatibility-complex (MHC) multimer staining, we aimed to develop a multiplex detection assay, which provides fast in vitro functional data for both human CD4 and CD8 T cells with different antigen specificities in one sample. In this study, we have exploited the expression of CD83 on B cells to develop the cell array-based indirect T cell recognition assay (ITRA). In detail, B cells are pulsed with different pathogen peptide pools and fluorescently barcoded. Thereafter the B cells are pooled and co-cultured with autologous T cells. Subsequently each B cell population is analyzed via flow cytometry for CD83 expression, which indicates antigen-specific interaction with CD4 T cells. Moreover, we revealed donor dependent variations of cytotoxic activity of pathogen-specific CD4 T cells and CD8 T cells, evidenced by specific lysis of peptide-pulsed B cells. Taken together, ITRA is a novel antigen presenting cell (APC) array based method to analyze the presence and function of various antigen-specific T cells in one sample. It has the potential to be used in the future for epitope/antigen screening in research and for analysis of anti-tumor, anti-pathogen or autoimmune T cell responses in patient samples.