Fig. A.28 Cytokine-expressing cells can be tracked in vivo using cytokine gene knock-in reporter mice. To identify cells expressing a specific cytokine in intact animals, the locus encoding the cytokine is modified by homologous recombination (see Fig. A.44 and Section A-35). An internal ribosome entry site (IRES) and the gene for a fluorescent protein such as eGFP are inserted 3ʹ of the last exon of the cytokine gene, downstream of the cytokine protein stop codon and upstream of the mRNA transcription termination and polyadenylation signal (the poly-A site). The IRES element allows the ribosome to initiate translation of a second protein-coding sequence at an internal site on the mRNA. When the modified locus is transcribed and spliced to form the mature mRNA, both the intact cytokine protein and the fluorescent reporter protein (e.g., eGFP) are produced from the same transcript. This allows the identification and characterization of cytokine-expressing cells, such as by flow cytometry, based on the detection of eGFP.
Fig. A.29 Fluorescent proteins are available in a rainbow of colors. Derivatives of GFP and a red-fluorescent coral protein can generate eight different fluorescent colors. A beach scene is drawn with bacterial strains expressing each fluorescent protein. Courtesy of Roger Tsien.
A-24 Identification of T-cell receptor specificity using peptide:MHC tetramers.
10,000 1000 100 10 1 1 10 100 HLA-A2 + CMV-speciÿc tetramer staining CD8 staining 1000 10,000 Peptide:MHC tetramers are bound by T cells expressing receptors of the appropriate speciÿcity streptavidin MHC class I The peptide:MHC tetramer is made from recombinant MHC molecules with speciÿc peptides, bound to streptavidin via biotin
For many years, the ability to identify antigen-specific T cells directly through their receptor specificity eluded immunologists. Foreign antigen could not be used directly to identify T cells because, unlike B cells, T cells do not recognize antigen alone but rather the complexes of peptide fragments of antigen bound to self MHC molecules. Moreover, the affinity of interaction between the T-cell receptor and the peptide:MHC complex is in practice so low that attempts to label T cells with their specific peptide:MHC complexes routinely failed. The breakthrough in labeling antigen-specific T cells came with the idea of making multimers of the peptide:MHC complex, so as to increase the avidity of the interaction.
Peptides can be biotinylated using the bacterial enzyme BirA, which recognizes a specific amino acid sequence. Recombinant MHC molecules containing this target sequence are used to make peptide:MHC complexes, which are then biotinylated. Avidin, or its bacterial counterpart streptavidin, contains four sites that bind biotin with extremely high affinity. Mixing the biotinylated peptide:MHC complex with avidin or streptavidin results in the formation of a peptide:MHC tetramer—four specific peptide:MHC complexes bound to a single molecule of streptavidin (Fig. A.30). Routinely, the streptavidin moiety is labeled with a fluorochrome to allow detection of those T cells capable of binding the peptide:MHC tetramer.