[CANCER RESEARCH 60, 365–371, January 15, 2000] 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 1 Sarah J. Youde, 2,3 P. R. Rod Dunbar, 3,4 Eve M. L. Evans, Alison N. Fiander, Leszek K. Borysiewicz, Vincenzo Cerundolo, and Stephen Man 5 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.] ABSTRACT 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 ne- oplasia (CIN3) are strongly associated with infection by human papillo- mavirus (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 flu- orogenic 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 con- structed 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 periph- eral blood mononuclear cells from CIN3 patients. Furthermore, the tet- ramer 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. INTRODUCTION CaCx 6 is the second most common cause of cancer in women world wide (1), and with premalignant CIN3 is associated with HPV infec- tion (2). The DNA of HPVs, particularly types 16 and 18, are found in 95% of CaCx patients (3). The E6 and E7 proteins of the virus can immortalize cells in vitro, and these proteins are consistently retained and expressed in cervical tumor cells (4, 5). Animal models suggest that HPV-specific CD8+ CTLs may be therapeutic against HPV-transformed tumor cells (6 –9). There is also circumstantial evidence in humans that cell-mediated immunity, including CD8 + CTLs, are important in controlling papillomavirus infection (10 –13). However, it has been difficult to detect human CTLs against HPV (14 –16). Recently several groups, using peptides (17, 18), tumor cells (19), soluble proteins (20), or recombinant adenoviruses (21, 22), have demonstrated that human CTLs against HPV 16 restimulate PBMCs from patients in vitro. The use of different patient groups (CaCx or CIN3) and different techniques for in vitro restimulation of HPV- specific CTLs have led to detection rates of 0 – 80%. Even when the same HLA-A*0201-restricted peptide epitope (HPV 16 E7 11–20 ) has been used to restimulate CTLs, there has been considerable variation in detection of CTLs among patients (15, 17, 18). Despite this vari- ation, HPV-specific CTLs cannot be detected in the PBMCs of normal controls unless dendritic cells are used as antigen-presenting cells in vitro (18). Collectively, these studies suggest that memory CTLs against HPV can be detected in patients but that they are at low frequencies compared with systemic CTLs against viruses such as influenza A. The variation in CTL responses detected may reflect either an individual variation in response against HPV or the effi- ciency of in vitro restimulation/detection methods (23). There is a need to increase the sensitivity of HPV-specific CTL detection both in blood and at sites of disease for immune monitoring in epidemiolog- ical and clinical trial studies. Novel technologies such as ELISPOT assays, intracellular cytokine staining, and fluorogenic MHC-peptide complexes (tetramers) have been used to greatly increase the sensitivity of antigen-specific CD8 + T-cell detection (24 –28). The CD8 + T-cell frequencies obtained with these techniques suggest that conventional techniques may greatly underrepresent the actual number of antigen-specific CD8 + T-cell effectors (23, 25). The use of tetramers is particularly attractive because it allows direct quantitation of antigen-specific T cells from blood or sites of disease without the need for in vitro restimulation. Furthermore, the CD8 + T cells detected by tetramers are largely cytotoxic T-cell effectors (25, 29). To investigate whether tetramers could be used to identify HPV-specific CTLs, we constructed a tetramer containing HLA-A*0201 complexed to the best studied HPV CTL epitope, HPV 16 E7 11–20 . In this report, we describe the use of this HPV tetramer to quantify HPV-specific CTLs in peripheral blood and as a tool to isolate high-affinity CTLs capable of killing HPV 16 + tumor cells. MATERIALS AND METHODS Media. RPMI 1640 (Life Technologies Inc., Gaithersburg, MD) was al- ways used with the following additions: 0.02 M HEPES (Sigma-Aldrich Co. Ltd., Irvine, United Kingdom), 2 mML-glutamine (Life Technologies), 100 units/ml penicillin and 100 g/ml streptomycin (Life Technologies). For the culture of T cells, this medium was supplemented with 10% pooled human AB serum (National Blood Transfusion Service, Pontyclun, Wales) and known as RAB. Cell Lines. The C1R-A2 cell line, a transfectant expressing the HLA A*0201 allele (30) was maintained in RPMI 1640 containing 10% FCS (Life Received 7/27/99; accepted 11/12/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from the Medical Research Council, HEFC(W), The Cancer Research Campaign, and Ligue National Contre le Cancer. 2 To whom requests for reprints should be addressed, at University of Wales College of Medicine, Tenovus Building, Heath Park, Cardiff CF4 4XX, United Kingdom. Phone: 44-1222-745004; Fax: 44-1222-745003. 3 These authors contributed equally to this work. 4 P. R. Dunbar is the Girdlers Research Fellow at Green College, University of Oxford. 5 S. Man is a Royal Society University Research Fellow. 6 The abbreviations used are: CaCx, cervical cancer; CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus; CD, cluster of differentiation; PBMC, peripheral blood mononuclear cell; RAB, RPMI 1640 supplemented with 10% pooled human AB serum; PE, phycoerythrin; IL-2, interleukin 2; FACS, fluorescence-activated cell sorting; TCR, T-cell receptor; CTL, cytotoxic T lymphocyte. 365 Research. on June 29, 2015. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from