title: Preparation of peptide-MHC and T-cell receptor dextramers by biotinylated dextran doping.
abstract: Peptide-major histocompatibility complex (pMHC) multimers enable the detection, characterization, and isolation of antigen-specific T-cell subsets at the single-cell level via flow cytometry and fluorescence microscopy. These labeling reagents exploit a multivalent scaffold to increase the avidity of individually weak T-cell receptor (TCR)-pMHC interactions. Dextramers are an improvement over the original streptavidin-based tetramer technology because they are more multivalent, improving sensitivity for rare, low-avidity T cells, including self/tumor-reactive clones. However, commercial pMHC dextramers are expensive, and in-house production is very involved for a typical biology research laboratory. Here, we present a simple, inexpensive protocol for preparing pMHC dextramers by doping in biotinylated dextran during conventional tetramer preparation. We use these pMHC dextramers to identify patient-derived, tumor-reactive T cells. We apply the same dextran doping technique to prepare TCR dextramers and use these novel reagents to yield new insight into MHC I-mediated antigen presentation.
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Bethune
authors.affiliation: Department of Surgery, University of California, Los Angeles, Los Angeles, CA.|Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA.
authors.forename: Begoña
authors.initials: B
authors.lastname: Comin-Anduix
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA.
authors.forename: Yu-Hsien
authors.initials: YH
authors.lastname: Hwang Fu
authors.affiliation: Department of Surgery, University of California, Los Angeles, Los Angeles, CA.|Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA.|Department of Medicine, Hematology, and Oncology, University of California, Los Angeles, Los Angeles, CA.
authors.forename: Antoni
authors.initials: A
authors.lastname: Ribas
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA.
authors.forename: David
authors.initials: D
authors.lastname: Baltimore
mesh_terms: D001709:Biotechnology / Q000379:methods*; D001710:Biotin; D003911:Dextrans / Q000737:chemistry / Q000378:metabolism*; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006649:Histocompatibility Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D020014:K562 Cells; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019809:Streptavidin; D014161:Transduction, Genetic
chemical_list: D003911:Dextrans; D005456:Fluorescent Dyes; D006649:Histocompatibility Antigens; D010455:Peptides; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D001710:Biotin; D019809:Streptavidin
keywords: T-cell receptor (TCR); biotinylated dextran; cancer immunotherapy; dextramer; immunology; ligand discovery; major histocompatibility complex (MHC); multimer; pMHC; peptide; peptide-specific MHC clustering; tetramer; tumor antigen discovery