title: MHC-Peptide Tetramers to Visualize Antigen-Specific T Cells.
abstract: Mature T lymphocytes of the CD8 or CD4 classes bear αβ T cell receptors (TCR) that are specific for a molecular complex consisting of a major histocompatibility complex class I or II (MHC class I or II) molecule bound to a unique self or foreign peptide. Until recently, methods for monitoring antigen-specific T cell immune responses were restricted primarily to functional assays based on limiting dilution analysis, because the lack of specific molecular reagents to identify clonal T cells obviated approaches to identify and enumerate specific T cells. Development of efficient methods to express and refold MHC class I molecules with synthetic peptides coincided with identification of specific protein sequences that provide the substrate for enzymatic biotinylation. This combination has led to the development of a straightforward method for generating synthetic TCR ligands, making them tetravalent to provide increased avidity, and labeling them through a streptavidin moiety with useful fluorescent tags such as allophycocyanin or R-phycoerythrin. This unit describes the preparation of MHC class I/peptide tetramers in detail, including bacterial expression and refolding of the MHC class I light chain, β2-microglobulin (β2m), as well as the formation of a complex consisting of the MHC class I heavy chain of interest, β2m, and a chosen peptide. © 2016 by John Wiley & Sons, Inc.
authors: Emory University School of Medicine, Atlanta, Georgia.
authors: John D
authors: JD
authors: Altman
authors: Stanford University School of Medicine and The Howard Hughes Medical Institute, Palo Alto, California.
authors: Mark M
authors: MM
authors: Davis
mesh_terms: D019921:Biotinylation; D003062:Codon; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D000949:Histocompatibility Antigens Class II / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D006801:Humans; D008285:Major Histocompatibility Complex / Q000276:immunology*; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D011485:Protein Binding / Q000276:immunology; D015202:Protein Engineering; D055503:Protein Multimerization; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013194:Staining and Labeling; D019809:Streptavidin; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism*
chemical_list: D003062:Codon; D018984:Epitopes, T-Lymphocyte; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; C408902:MHC binding peptide; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D019809:Streptavidin
keywords: MHC tetramers; T cells; flow cytometry