title: Detection, phenotyping, and quantification of antigen-specific T cells using a peptide-MHC dodecamer.
abstract: 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.
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305; mmdavis@stanford.edu huangjun@uchicago.edu.
authors.forename: Jun
authors.initials: J
authors.lastname: Huang
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305;
authors.forename: Xun
authors.initials: X
authors.lastname: Zeng
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305;
authors.forename: Natalia
authors.initials: N
authors.lastname: Sigal
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305;
authors.forename: Peder J
authors.initials: PJ
authors.lastname: Lund
authors.affiliation: Department of Medicine, Division of Rheumatology, University of Pennsylvania, Philadelphia, PA 19104;
authors.forename: Laura F
authors.initials: LF
authors.lastname: Su
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305;
authors.forename: Huang
authors.initials: H
authors.lastname: Huang
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305;
authors.forename: Yueh-hsiu
authors.initials: YH
authors.lastname: Chien
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305; Stanford Institute for Immunity, Transplantation, and Infection, Stanford University, Stanford, CA 94305; Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305 mmdavis@stanford.edu huangjun@uchicago.edu.
authors.forename: Mark M
authors.initials: MM
authors.lastname: Davis
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000469:parasitology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000502:physiology; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D008285:Major Histocompatibility Complex; D008822:Mice, Transgenic; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism
chemical_list: D010455:Peptides; D011948:Receptors, Antigen, T-Cell
keywords: antigen-specific T cell; avidity; dodecamer; peptide-MHC