Tetramer-blocking assay for defining antigen-specific cytotoxic T lymphocytes using peptide-MHC tetramer.

Peptide-MHC tetramers have been engineered to allow accurate detection of antigen-specific cytotoxic C lymphocytes (CTL) by flow cytometry. Here, we propose a novel use for peptide-MHC tetramers in the specific and sensitive analysis of the cytotoxic function of antigen-specific CTL by blocking MHC-restricted antigen-specific cytotoxicity. We found that pretreatment of ovalbumin (OVA)-specific CD8(+) CTL (OT-1 CTL), derived from OT-1 T-cell receptor (TCR)-transgenic mice, with OVA(257-264) peptide-H-2K(b) tetramer caused a marked inhibition of the cytotoxicity against OVA-expressing EG-7 tumor cells. OVA(257-264) peptide-H-2K(b) tetramer did not block the cytotoxicity mediated by 2C mouse (H-2(b))-derived CD8(+) CTL, which recognize allo (H-2L(d)) antigens. Moreover, OT-I CTL activity was not inhibited by an irrelevant HBV(208-216) peptide-H-2K(b) tetramer. These results indicate that the blocking of CTL activity with peptide-MHC tetramer was caused by interference with the interaction between the TCR and H-2K(b)-OVA(257-264) peptide complex, but not with the CD8-MHC class I interaction. The blocking activity of OVA(257-264) peptide-H-2K(b) tetramer was reversible because OT-I CTL pretreated with the tetramer recovered their cytotoxicity after culturing with interleukin-2 for 24 h. The same results were also demonstrated in freshly isolated, in vivo-primed OT-1 CTL sorted by the tetramer. These results demonstrate that peptide-MHC tetramer is a useful tool for defining MHC-restricted antigen-specific CTL function. Moreover, our finding implies that the measurement of CTL activity immediately after tetramer-guided sorting is not a suitable method for evaluating the function of in vivo-induced tetramer-positive CTL. We believe that the tetramer-blocking assay presented here will be useful for functionally monitor the induction of MHC-restricted antigen-specific CTL during vaccination therapy against tumor and infectious diseases.