Peptide-MHC class I tetrameric complexes display exquisite ligand specificity

The production of synthetic MHC-peptide tetramers has revolutionized cellul ar immunology by revealing enormous CD8(+) T cell expansions specific for p eptides from various pathogens. A feature of these reagents, essential for their staining function, is that they bind T tells with relatively high avi dity. This could, theoretically, promote cross-reactivity with irrelevant T cells leading to overestimates of epitope-specific T cell numbers. Therefo re, we have investigated the fine specificity of CTL staining with these re agents for comparison with functional data. Using a panel of CTL clones wit h distinct fine specificity patterns for analogs of an HLA-B8-binding EBV e pitope, together with B8 tetramers incorporating these peptides, we show a very good correlation between tetramer staining and peptide activity in cyt otoxicity assays. Significant staining only occurred with tetramers that in corporate strong stimulatory agonist peptides and not weak agonists that ar e unlikely to induce full T cell activation at physiological levels of pres entation. In almost every case where a peptide analog had >10-fold less act ivity than the optimal EBV peptide in cytotoxicity assays, the correspondin g tetramer stained with >10-fold less intensity than the EBV epitope tetram er, Furthermore, by examining an EBV-specific clonotypic T cell expansion i n EBV-exposed individuals, we show similar fine specificity in tetramer sta ining of fresh peripheral T cells. Collectively, our data demonstrate the e xquisite specificity of class I MHC-peptide tetramers, underlining their ac curacy in quantifying only those T cells capable of recognizing the low lev els of cell surface peptide presented after endogenous Ag processing.