Next-generation detection of antigen-responsive T cells using DNA barcode-labeled peptide-major 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 screeningof T cell reactivity towards 10-100 distinct peptide specificities, which poorly match the large diversityof T cell recognition in humans. Consequently it has been impossible to comprehensively analyze Tcell responsiveness in cancer, infectious and autoimmune diseases. We present and validate a noveltechnology 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 andsequencing of the MHC multimer-associated DNA barcodes. The relative frequency of the sequencedDNA barcodes originating from a given peptide-MHC motif relates to the size of the antigenresponsiveT cell population. We have demonstrated the use of large panels of >1000 DNA barcodedMHC multimers for detection of rareT cell populations of virus and cancer-restricted origin in various tissues and compared withcombinatorial encoding of fluorescent-labeled MHC multimers. Finally, we have demonstrated that thistechnology can be applied for multiplex T cell detection both in limited biological samples, such asuncultured tumor material, and for simultaneous assessment of target recognition and functionalcapability of T cells. This technology enables true high-throughput detection of antigen-responsiveT cells and will advance our understanding of immune recognition from model antigens to genomewideimmune assessments on a personalized basis.