Peptide:MHC tetramers have been used to identify populations of antigen-specific T cells in, for example, patients with acute Epstein–Barr virus infections (infectious mononucleosis), showing that up to 80% of the peripheral T cells in infected individuals can be specific for a single peptide:MHC complex. They have also been used to follow responses over timescales of years in individuals with HIV or, in the example we show, cytomegalovirus infections. These reagents have also been important in identifying the cells responding, for example, to nonclassical MHC class I molecules such as HLA-E or HLA-G, in both cases showing that these nonclassical molecules are recognized by subsets of NK receptors.
Fig. A.30 Peptide:MHC complexes coupled to streptavidin to form tetramers are able to stain antigen-specific T cells. Peptide:MHC tetramers are formed from recombinant refolded peptide:MHC complexes containing a single defined peptide epitope. MHC molecules that contain biotin can be chemically synthesized, but usually the recombinant MHC heavy chain is linked to a bacterial biotinylation sequence, a target for the Escherichia coli enzyme BirA, which is used to add a single biotin group to the MHC molecule. Streptavidin is a tetramer, each subunit having a single binding site for biotin; hence the streptavidin:peptide:MHC complex creates a tetramer of peptide:MHC complexes (top panel). Although the affinity between the T-cell receptor and its peptide:MHC ligand is too low for a single complex to bind stably to a T cell, the tetramer, by being able to make a more avid interaction with multiple peptide:MHC complexes binding simultaneously, is able to bind to T cells whose receptors are specific for the particular peptide:MHC complex (middle panel). Routinely, the streptavidin molecules are coupled to a fluorochrome, so that the binding to T cells can be monitored by flow cytometry. In the example shown in the bottom panel, T cells have been stained simultaneously with antibodies specific for CD3 and CD8, and with a tetramer of HLA-A2 molecules containing a cytomegalovirus peptide. Only the CD3+ cells are shown, with the staining of CD8 displayed on the vertical axis and the tetramer staining displayed along the horizontal axis. The CD8– cells (mostly CD4+), on the bottom left of the figure, show no specific tetramer staining, while the bulk of the CD8+ cells, on the top left, likewise show no tetramer staining. However, a discrete population of tetramer positive CD8+ cells, at the top right of the panel, comprising some 5% of the total CD8+ cells, can clearly be seen. Data courtesy of G. Aubert.
A-25 Biosensor assays for measuring the rates of association and dissociation of antigen receptors for their ligands.