A simplified procedure for the preparation of MHC/peptide tetramers: chemical biotinylation of an unpaired cysteine engineered at the C-terminus of MHC-I.
Recently, a powerful approach for the detection of MHC/peptide-specific T cells has been made possible by the engineering of soluble-tetrameric MHC/peptide complexes, consisting of singly biotinylated MHC/peptide molecules bound to fluorescent-labeled streptavidin. These tetrameric molecules are thought to compensate for the low affinity and relative fast dissociation rate of the TCR/MHC-peptide interaction by increasing the avidity of this interaction, thus allowing the stable binding of MHC/peptide tetramers to TCR expressing cells. Here we describe a new more simplified procedure for obtaining MHC/peptide tetramers using the well-characterized H-2K(b)/VSV system. This procedure consists of the incorporation of an unpaired cysteine residue at the C-terminus of the H-2K(b) molecule, allowing site-specific biotinylation by a -SH-specific biotinylating reagent. The H-2K(b)/VSV tetramers bound only to hybridomas expressing H-2K(b)/VSV-specific TCRs. When coated on a plate, these tetramers were able to induce IL-2 release by those hybridomas. Furthermore, H-2K(b)/VSV tetramers bound to CTL populations obtained from mice immunized with VSV-peptide. The specificity of the binding was further refined by studying cross-recognition of VSV by CTL populations obtained from mice immunized with single amino acid substituted VSV peptide variants. H-2K(b)/VSV tetramers bound only to those CTL populations that cross-reacted with the wild-type VSV peptide. Our method provides a simple, efficient and inexpensive procedure for making MHC/peptide tetramers, a highly specific and very useful reagent with a number of important applications in basic and clinical T cell research.
Kalergis
A M
AM
Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Goyarts
E C
EC
Palmieri
E
E
Honda
S
S
Zhang
W
W
Nathenson
S G
SG
D019921:Biotinylation; D003429:Cross Reactions; D003545:Cysteine / Q000235:genetics* / Q000276:immunology; D005818:Genetic Engineering; D006183:H-2 Antigens / Q000235:genetics* / Q000276:immunology; D007376:Interleukin-2 / Q000096:biosynthesis; D008213:Lymphocyte Activation / Q000276:immunology; D019251:Nucleocapsid / Q000235:genetics* / Q000276:immunology; D019590:Nucleocapsid Proteins; D010455:Peptides / Q000235:genetics / Q000276:immunology; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D001613:beta 2-Microglobulin / Q000235:genetics / Q000276:immunology
D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; D007376:Interleukin-2; D019590:Nucleocapsid Proteins; D010455:Peptides; D016693:Receptors, Antigen, T-Cell, alpha-beta; D001613:beta 2-Microglobulin; D003545:Cysteine