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Use of Fluorogenic Histocompatibility Leukocyte Antigen-A*0201/HPV 16 E7 Peptide Complexes to Isolate Rare Human Cytotoxic T-Lymphocyte- recognizing Endogenous Human Papillomavirus Antigens¹

Department of Medicine, University of Wales College of Medicine, Cardiff CF4 4XX, United Kingdom [S. J. Y., E. M. L., L. K. B., S. M.]; Department of Obstetrics and Gynaecology, Llandough Hospital, Penarth, CF64 2XX, United Kingdom [A. N. F.]; Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, United Kingdom [P. R. D., V. C.]

Cervical cancer (CaCx) is the second most common female malignancy worldwide and remains a clinical problem despite improvements in early detection and therapy. CaCx and preinvasive cervical intraepithelial neoplasia (CIN3) are strongly associated with infection by human papillomavirus (HPV), particularly types 16 and 18. Two nonstructural viral proteins, E6 and E7, are constitutively expressed in cervical tumors and are crucial for the maintenance of the transformed phenotype. These proteins thus provide attractive targets for immunotherapy of CaCx mediated by CD8+ CTLs. However, reliable detection and generation of HPV-specific CTLs in humans has been difficult. Recently, soluble fluorogenic MHC-peptide complexes (tetramers) have greatly increased the sensitivity of antiviral and antitumor CTL detection. To examine the feasibility of this approach for detecting HPV-specific CTLs, we constructed 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 E7_11–20 CTL lines derived from CaCx patients stained highly with the tetramer. 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). However, short-term in vitro stimulation with the HPV 16 E7_11–20 peptide expanded CD8⁺tetramer⁺ cells to a greater extent in the peripheral blood 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 were able to lyse both peptide-pulsed targets and targets expressing endogenously processed HPV antigens. This tetramer may therefore be useful for selecting rare high-affinity HPV-specific CTLs for the immunotherapy of CaCx.

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