A simplified procedure for the preparation of MHC/peptide tetramers: Chemical biotinylation of an unpaired cysteine engineered at the C-terminus of MHC-I

Alexis M. Kalergis, Earl C. Goyarts, Edith Palmieri, Shinichiro Honda, Weijia Zhang, Stanley G. Nathenson

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

Abstract

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- 2Kb/VSV system. This procedure consists of the incorporation of an unpaired cysteine residue at the C-terminus of the H-2Kb molecule, allowing site- specific biotinylation by a -SH-specific biotinylating reagent. The H- 2Kb/VSV tetramers bound only to hybridomas expressing H-2Kb/VSV-specific TCRs. When coated on a plate, these tetramers were able to induce IL-2 release by those hybridomas. Furthermore, H-2Kb/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-2Kb/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. (C) 2000 Elsevier Science B.V.

Original languageEnglish
Pages (from-to)61-70
Number of pages10
JournalJournal of Immunological Methods
Volume234
Issue number1-2
DOIs
StatePublished - 3 Feb 2000
Externally publishedYes

Keywords

  • Cysteine biotinylation
  • MHC-I tetramers
  • TCR activation
  • TCR cross-reactivity
  • TCR specificity

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