Direct binding of peptides to MHC class I molecules on living cells. Analysis at the single cell level.

Détails

ID Serval
serval:BIB_06CD7A92FE75
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
Direct binding of peptides to MHC class I molecules on living cells. Analysis at the single cell level.
Périodique
Journal of immunology
Auteur(s)
López J.A., Luescher I.F., Cerottini J.C.
ISSN
0022-1767
Statut éditorial
Publié
Date de publication
1992
Peer-reviewed
Oui
Volume
149
Numéro
12
Pages
3827-3835
Langue
anglais
Notes
Publication types: Journal Article
Publication Status: ppublish
Résumé
To directly assess the binding of exogenous peptides to cell surface-associated MHC class I molecules at the single cell level, we examined the possibility of combining the use of biotinylated peptide derivatives with an immunofluorescence detection system based on flow cytometry. Various biotinylated derivatives of the adenovirus 5 early region 1A peptide 234-243, an antigenic peptide recognized by CTL in the context of H-2Db, were first screened in functional assays for their ability to bind efficiently to Db molecules on living cells. Suitable peptide derivatives were then tested for their ability to generate positive fluorescence signals upon addition of phycoerythrin-labeled streptavidin to peptide derivative-bearing cells. Strong fluorescent staining of Db-expressing cells was achieved after incubation with a peptide derivative containing a biotin group at the C-terminus. Competition experiments using the unmodified parental peptide as well as unrelated peptides known to bind to Kd, Kb, or Db, respectively, established that binding of the biotinylated peptide to living cells was Db-specific. By using Con A blasts derived from different H-2 congenic mouse strains, it could be shown that the biotinylated peptide bound only to Db among > 20 class I alleles tested. Moreover, binding of the biotinylated peptide to cells expressing the Dbm13 and Dbm14 mutant molecules was drastically reduced compared to Db. Binding of the biotinylated peptide to freshly isolated Db+ cells was readily detectable, allowing direct assessment of the relative amount of peptide bound to distinct lymphocyte subpopulations by three-color flow cytometry. While minor differences between peripheral T and B cells could be documented, thymocytes were found to differ widely in their peptide binding activity. In all cases, these differences correlated positively with the differential expression of Db at the cell surface. Finally, kinetic studies at different temperatures strongly suggested that the biotinylated peptide first associated with Db molecules available constitutively at the cell surface and then with newly arrived Db molecules.
Mots-clé
Adenovirus E1A Proteins/metabolism, Amino Acid Sequence, Animals, Antibody Specificity, Antigen-Antibody Reactions/physiology, Binding, Competitive, Dose-Response Relationship, Immunologic, Flow Cytometry, Fluorescent Antibody Technique, H-2 Antigens/metabolism, Histocompatibility Antigens Class I/metabolism, Mice, Molecular Sequence Data, Protein Binding, Time Factors
Pubmed
Web of science
Création de la notice
28/01/2008 11:20
Dernière modification de la notice
20/08/2019 12:29
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