Fluorescence-activated cell sorting and cloning of bona fide CD8+ CTL with reversible MHC-peptide and antibody Fab' conjugates.

Details

Serval ID
serval:BIB_D2C4FBD22187
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Fluorescence-activated cell sorting and cloning of bona fide CD8+ CTL with reversible MHC-peptide and antibody Fab' conjugates.
Journal
Journal of immunology
Author(s)
Guillaume P., Baumgaertner P., Angelov G.S., Speiser D., Luescher I.F.
ISSN
0022-1767
Publication state
Published
Issued date
2006
Peer-reviewed
Oui
Volume
177
Number
6
Pages
3903-3912
Language
english
Notes
Publication types: Journal Article ; Research Support, Non-U.S. Gov't
Publication Status: ppublish
Abstract
The isolation of subsets of Ag-specific T cells for in vitro and in vivo studies by FACS is compromised by the fact that the soluble MHC-peptide complexes and Abs used for staining, especially when combined, induce unwanted T cell activation and eventually apoptosis. This is especially a problem for CD8+ CTL, which are susceptible to activation-dependent cell death. In this study, we show that reversible MHC-peptide complexes (tetramers) can be prepared by conjugating MHC-peptide monomers with desthiobiotin (DTB; also called dethiobiotin) and multimerization by reaction with fluorescent streptavidin. While in the cold these reagents are stable and allow good staining, they rapidly dissociate in monomers at elevated temperatures, especially in the presence of free biotin. FACS cloning of Melan-A (MART-1)-specific CTL from a melanoma-infiltrated lymph node with reversible HLA-A2 Melan-A26-35 multimers yielded over two times more clones than when using the conventional biotin-containing multimers. CTL clones obtained by means of reversible multimers killed Melan-A-positive tumor cells more efficiently as compared with clones obtained with the stable multimers. Among the CTL obtained with the reversible multimers, but much less among those obtained with the stable multimers, a high proportion of clones exhibited high functional and physical avidity and died upon incubation with soluble MHC-peptide complexes. Finally, we show that Fab' of an anti-CD8 Ab can be converted in reversible DTB streptavidin conjugates the same way. These DTB reagents efficiently and reversibly stained murine and human CTL without affecting their viability.
Keywords
Animals, Biotin/analogs &amp, derivatives, Cell Separation, Clone Cells, Flow Cytometry, H-2 Antigens, HLA-A Antigens, Histocompatibility Antigens Class I, Humans, Immunoglobulin Fab Fragments, Mice, Peptides, T-Lymphocytes, Cytotoxic/chemistry, T-Lymphocytes, Cytotoxic/cytology
Pubmed
Web of science
Create date
28/01/2008 11:19
Last modification date
20/08/2019 15:52
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