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 c ells has been made possible by the engineering of soluble-tetrameric MHC/pe ptide complexes, consisting of singly biotinylated MHC/peptide molecules bo und to fluorescent-labeled streptavidin. These tetrameric molecules are tho ught to compensate for the low affinity and relative fast dissociation rate of the TCR/MHC-peptide interaction by increasing the avidity of this inter action, thus allowing the stable binding of MHC/peptide tetramers to TCR ex pressing cells. Here we describe a new more simplified procedure for obtain ing MHC/peptide tetramers using the well-characterized H-2K(b)/VSV system. This procedure consists of the incorporation of an unpaired cysteine residu e at the C-terminus of the H-2K(b) molecule, allowing site-specific biotiny lation by a -SH-specific biotinylating reagent. The H-2K(b)/VSV tetramers b ound only to hybridomas expressing H-2K(b)/VSV-specific TCRs. When coated o n a plate, these tetramers were able to induce IL-2 release by those hybrid omas. Furthermore, H-2K(b)/VSV tetramers bound to CTL populations obtained from mice immunized with VSV-peptide. The specificity of the binding was fu rther refined by studying cross-recognition of VSV by CTL populations obtai ned from mice immunized with single amino acid substituted VSV peptide vari ants. 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, effic ient and inexpensive procedure for making MHC/peptide tetramers, a highly s pecific and very useful reagent with a number of important applications in basic and clinical T cell research. (C) 2000 Elsevier Science B.V. All righ ts reserved.