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Detection, phenotyping, and quantification of antigen-specific T cells using a peptide-MHC dodecamer

Significance The recognition of foreign peptide-MHCs by T cells is a central event in adaptive immunity that triggers antigen-specific immune responses against infections and cancer. To study antigen-specific T cells, we devised a peptide-MHC dodecamer that can sensitively detect and specifically stain these T cells, especially low-affinity and rare ones. This dodecamer technology is superior to most current peptide-MHC multimers, compatible with existing reagents, inexpensive to make, and easy to use. It has been successfully applied to studies of human and murine antigen-specific αβ and γδ T cells by flow cytometry and mass cytometry. Thus, this dodecamer constitutes an important tool for the investigation of antigen-specific T cells in basic and clinical research. Here we report a peptide-MHC (pMHC) dodecamer as a “next generation” technology that is a significantly more sensitive and versatile alternative to pMHC tetramers for the detection, isolation, and phenotypic analysis of antigen-specific T cells. In particular, dodecamers are able to detect two- to fivefold more antigen-specific T cells in both human and murine CD4+ and CD8+ αβ T-cell compartments compared with the equivalent tetramers. The low-affinity, tetramer-negative, dodecamer-positive T cells showed comparable effector cytokine responses as those of high-affinity, tetramer-positive T cells. Dodecamers are able to detect early stage CD4+CD8+ double-positive thymocytes on which T-cell receptors are 10- to 30-fold less dense than mature T cells. Dodecamers also show utility in the analysis of γδ T cells and in cytometry by time-of-flight applications. This construct has a simple structure with a central scaffold protein linked to four streptavidin molecules, each having three pMHC ligands or other molecules. The dodecamer is straightforward and inexpensive to produce and is compatible with current tetramer technology and commercially available streptavidin conjugates.