MHC–peptide tetramers for the analysis of antigen-specific T cells

Figures & data

Figure 1. Tetramer construction and stain.

(A) Soluble versions of the heavy-chain MHC class I molecules are synthesized in Escherichia coli. (B) The heavy chain is refolded with β₂-microglobulin and the epitope of interest. This refolded MHC molecule is biotinylated with the enzyme BirA at the specific BirA-recognition sequence, at the carboxyl terminus of the MHC molecule. (C)A tetramer is constructed by the addition of four MHC–biotin complexes, binding to a single streptavidin molecule, using the biotin–avidin interaction. This streptavidin molecule is conjugated to a fluorochrome to allow analysis by fluorescence-activated cell sorting. (D) Tetramers are mixed with the cell population that is to be analyzed. The tetramer will bind to CD8⁺ T cells that are expressing a T-cell receptor that is capable of recognizing the specific peptide and, with the addition monoclonal antibodies, cell populations can then be identified.

APC: Allophycocyanin; PE: Phycoerythrin.

Figure 2. Photoliable peptide cleavage.

The MHC complex containing the photoliable peptide is irradiated with UV light, cleaving the peptide and producing two fragments that dissociate from the MHC-binding groove. The resulting empty MHC complex is unstable; incubating this MHC with a peptide of interest creates a stable form that can then be used for analysis.

Figure 3. Tetramer-sorted cells.

(A) Tetramer-positive mice CD8⁺ cells for murine cytomegalovirus epitope m45 7 days postinfection, pre-sort. (B) Fluorescence-activated cell sorting plot shows the purity of these tetramer-positive cells post-sort.

FITC: Fluorescein isothiocyanate.

Figure 4. Combinatorial tetramer staining: representation of a single sample stained with seven different tetramers.

Each tetramer was made by tetramerizing the MHC class I molecule with either one fluorochrome APC, PE and FITC or a mixture, allowing many epitope-specific cell populations to be analyzed from a limited number of fluorochromes.

APC: Allophycocyanin; FITC: Fluorescein isothiocyanate; PE: Phycoerythrin.