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MHC‐peptide multimers:tools of choice for detecting and sorting antigen‐specific T‐cells

Immunologists have long sought methods for tracking specific T cells in vivo in the course of an immune response against pathogens. The specificity of T-cell activation relies on the recognition by the T-cell receptor (TCR) of foreign peptides presented by MHC proteins. Peptides bound to class I MHC molecules stimulate the TCRs of CD8+ cells, whereas class II MHC-peptide complexes are recognized by TCRs of CD4+ cells. Until recently, the only available reagents with which to label T-cell clonotypes were idiotypic MoAbs, which are specific for a particular combination of TCR α and β chains. The production of such antibodies is sometimes difficult, requires prior knowledge of the TCRs that react to a given epitope, and cannot be used to assess the full extent of a normal heterogeneous immune response against a particular MHC-peptide complex. Methods for producing soluble MHC proteins loaded with relevant peptides have been developed,1,2 and researchers tried labeling specific T cells with these MHCpeptide complexes. However, because of the low affinity of the interaction between the TCR and the MHC-peptide complex (approx. 10–5 M, with a dissociation constant of <1 min3), initial attempts using soluble monomeric forms of MHC-peptide were unsuccessful. The problem was solved by constructing multimers of MHC-peptide to increase the overall avidity of the reagent. Two strategies were used: the approach of Dal Porto et al.4 was to make a chimeric MHCimmunoglobulin protein to produce divalent peptideMHC-immunoglobulin complexes, whereas Altman et al.5 produced tetravalent MHC-peptide complexes by crosslinking biotinylated MHC-peptide monomers with streptavidin. GENERATION OF MHC TETRAMERS The method described by Altman et al.,5 which is favored by most laboratories producing class I MHC or class II MHC-peptide multimers, relies on the use of an Escherichia coli enzyme, called BirA, which specifically recognizes a 15-mer peptide and biotinylates a lysine within this sequence.6 As depicted in Fig. 1, a BirA peptide-tagged MHC chain is produced in E. coli by ligating an oligonucleotide coding for the BirAspecific peptide to the 3 ́ end of the cDNA coding for the class I MHC heavy chain and by cloning the construct in the appropriate expression vector. The advantage of using enzymatic biotinylation is that it will occur only on the specific sequence at the tail of the MHC chain, whereas chemical biotinylation, which is commonly used for antibodies, would couple biotine to any lysine within the sequence and thus would potentially alter TCR recognition sites or result in a bad positioning of the monomer onto streptavidin. For class I MHC complexes, the MHC heavy chain and β2-microglobulin are produced separately as inclusion bodies in E. coli and dissolved in 8M urea, and both chains are then mixed with the peptide of interest in a large volume of refolding solution for 4 days to allow proper renaturation of the MHC-peptide-β2-microglobulin monomer. This is the critical step, and the amount of recovered, properly refolded monomer will vary with the isotype of the heavy chain (HLA-A, B, or C) and with the affinity of the peptide for the MHC molecule. After concentration, monomers are biotinylated with recombinant BirA enzyme and then purified on an ion exchange column (Mono-Q, Amersham Pharmacia Biotech, Orsay, France). They can be stored at –80°C for subsequent use or tetramerized by mixing with fluorescent avidin or streptavidin at a molar ratio of 0.8:4. This ratio must be adjusted carefully for each mix, so that tetramers only are obtained and not a mix of monomers, dimers, trimers, and tetramers. Optimal conditions for each preparation are determined by checking the end product on a gel filtration column. The fluorescent tetramer can be stored for several months at 4°C and used to label antigen-specific T cells. For class II MHC-peptide complexes, the yield of the above method is so poor that alternative strategies have been developed. They rely on the transfection of the two MHC-peptide multimers: tools of choice for detecting and sorting antigen-specific T-cells