Next-generation detection of antigen-responsive T cells using DNA barcode-labeled peptidemajor histocompatibility complex I multimers

Identification of antigenic peptides recognized by T cells is important for understanding and treatingimmune related diseases. Current cytometry-based approaches are limited to simultaneous screening of T cell reactivity towards 10-100 distinct peptide specificities, which poorly match the large diversity of T cell recognition in humans. Consequently it has been impossible to comprehensively analyze T cell responsiveness in cancer, infectious and autoimmune diseases. We present and validate a novel technology that enables parallel detection of numerous different peptide-MHC responsive T cells in asingle sample using >1000 different peptide-MHC multimers labeled with individual DNA barcodes. After isolation of MHC multimer binding T cells their recognition are revealed by amplification and sequencing of the MHC multimer-associated DNA barcodes. The relative frequency of the sequenced DNA barcodes originating from a given peptide-MHC motif relates to the size of the antigenresponsive T cell population. We have demonstrated the use of large panels of >1000 DNA barcoded MHC multimers for detection of rare T cell populations of virus and cancer-restricted origin in various tissues and compared with combinatorial encoding of fluorescent-labeled MHC multimers. Finally, we have demonstrated that this technology can be applied for multiplex T cell detection both in limited biological samples, such as uncultured tumor material, and for simultaneous assessment of target recognition and functional capability of T cells. This technology enables true high-throughput detection of antigen-responsive T cells and will advance our understanding of immune recognition from model antigens to genomewide immune assessments on a personalized basis.