Use of fluorogenic histocompatibility leukocyte antigen-A*0201/HPV 16 E7 peptide complexes to isolate rare human cytotoxic T-lymphocyte-recognizing endogenous human papillomavirus antigens

Cervical cancer (CaCx) is the second most common female malignancy worldwid e and remains a clinical problem despite improvements in early detection an d therapy, CaCx and preinvasive cervical intraepithelial neoplasia (CIN3) a re strongly associated with infection by human papillomavirus (HPV), partic ularly types 16 and 18, Two nonstructural viral proteins, E6 and E7, are co nstitutively expressed in cervical tumors and are crucial for the maintenan ce of the transformed phenotype. These proteins thus provide attractive tar gets for immunotherapy of CaCx mediated by CD8+ CTLs. However, reliable det ection and generation of HPV-specific CTLs in humans has been difficult. Re cently, soluble fluorogenic MHC-peptide complexes (tetramers) have greatly increased the sensitivity of antiviral and antitumor CTL detection. To exam ine the feasibility of this approach for detecting HPV-specific CTLs, we co nstructed a tetramer consisting of HLA-A*0201 and the best studied HPV CTL peptide epitope, HPV 16 E7(11-20). Between 2 and 12% of short-term HPV 16 E 7(11-20) CTL lines derived from CaCx patients stained highly with the tetra mer. Direct ex vivo staining of peripheral blood mononuclear cells revealed CD8(+) tetramer(+) high cells at low frequencies in both CIN3 patients (1 of 1,260 to 1 of 19,073) and normal controls (1 of 1,855 to 1 of 42,004). H owever, short-term in vitro stimulation with the HPV 16 E7(11-20) peptide e xpanded CD8(+) tetramer(+) cells to a greater extent in the peripheral bloo d mononuclear cells from CIN3 patients. Furthermore, the tetramer provided a powerful tool to isolate polyclonal and clonal peptide-specific CTLs from an established HPV 16 E7(11-20)-specific CTL line. These purified CTLs wer e able to lyse both peptide-pulsed targets and targets expressing endogenou sly processed HPV antigens, This tetramer may therefore be useful for selec ting rare high-affinity HPV-specific CTLs for the immunotherapy of CaCx.