MHC-peptide tetramers to visualize antigen-specific T cells.
Mature T lymphocytes of the CD8 or CD4 classes bear alphabeta 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 the T cell immune response to a viral or tumor antigen 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 fluorescein or 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, beta2-microglobulin (beta2m), as well as the formation of a complex consisting of the MHC class I heavy chain of interest, beta2m, and a chosen peptide.
Altman
John D
JD
Emory University School of Medicine, Atlanta, Georgia.
Davis
Mark M
MM
Stanford University School of Medicine and The Howard Hughes Medical Institute, Palo Alto, California.
D000818:Animals; D000941:Antigens / Q000276:immunology*; D018984:Epitopes, T-Lymphocyte / Q000032:analysis* / Q000276:immunology*; D006801:Humans; D008285:Major Histocompatibility Complex / Q000276:immunology*; D010455:Peptides / Q000032:analysis / Q000276:immunology*; D013194:Staining and Labeling / Q000379:methods*
D000941:Antigens; D018984:Epitopes, T-Lymphocyte; D010455:Peptides