Abstract
Fluorescently labeled multimeric complexes of peptide-MHC, the molecular entities recognized by the T cell receptor, have become essential reagents for detection of antigen-specific CD8+ T cells by flow cytometry. Here we present a method for high-throughput parallel detection of antigen-specific T cells by combinatorial encoding of MHC multimers. Peptide-MHC complexes are produced by UV-mediated MHC peptide exchange and multimerized in the form of streptavidin-fluorochrome conjugates. Eight different fluorochromes are used for the generation of MHC multimers and, by a two-dimensional combinatorial matrix, these eight fluorochromes are combined to generate 28 unique two-color codes. By the use of combinatorial encoding, a large number of different T cell populations can be detected in a single sample. The method can be used for T cell epitope mapping, and also for the monitoring of CD8+ immune responses during cancer and infectious disease or after immunotherapy. One panel of 28 combinatorially encoded MHC multimers can be prepared in 4 h. Staining and detection takes a further 3 h.
Original language | English |
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Journal | Nature Protocols (Print) |
Volume | 7 |
Issue number | 5 |
Pages (from-to) | 891-902 |
Number of pages | 12 |
ISSN | 1754-2189 |
DOIs | |
Publication status | Published - 2012 |
Externally published | Yes |
Keywords
- Biochemistry, Genetics and Molecular Biology (all)
- antigen
- epitope
- fluorescent dye
- H2 antigen
- HLA antigen
- quantum dot
- animal
- article
- CD8+ T lymphocyte
- chemistry
- epitope mapping
- fluorescence imaging
- human
- immunology
- metabolism
- methodology
- mouse
- Animals
- Antigens
- CD8-Positive T-Lymphocytes
- Epitope Mapping
- Epitopes, T-Lymphocyte
- Fluorescent Dyes
- H-2 Antigens
- HLA Antigens
- Humans
- Mice
- Optical Imaging
- Quantum Dots
- BIOCHEMICAL
- CLASS-I LIGANDS
- P/MHC MICROARRAYS
- EPITOPES
- TECHNOLOGY
- DISCOVERY
- CYTOMETRY
- TETRAMERS
- EXCHANGE