Fluorescence-activated Cell Sorting and Cloning of Bona Fide Cd8+ Ctl with Reversible Mhc-peptide and Antibody Fab' Conjugates.
From: Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland.
Journal of immunology (Baltimore, Md. : 1950)
- Publish Date: Sep 2006
- ISSN: 0022-1767
- Volume: 177
- Issue: 6
- Pages: 3903-12
- Medium: Print
- Language: English
- Citation (JAMA): Guillaume Philippe, Baumgaertner Petra, Angelov Georgi S, et al. Fluorescence-activated Cell Sorting and Cloning of Bona Fide Cd8+ Ctl with Reversible Mhc-peptide and Antibody Fab' Conjugates.. J. Immunol. Sep 2006;177:3903-12
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.
Mesh Headings (Keywords): Animals, Biotin, 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
Check for Full Text / PubMed Unique Identifier (PMID): 16951353
This abstract is part of PubMed, a service of the U.S. National Library of Medicine. PubMed includes more than 17 million citations from MEDLINE and other life science journals for biomedical articles. See Copyright and Disclaimers.
Linked medical terms appearing on this page are added by Healia to help readers find more information and are not part of the original PubMed document.
The data herein was last updated on July 8th, 2008 and may not reflect the most current and accurate data available from NLM.