T cells specific for HPV16 E7 epitopes in patients with squamous cell carcinoma of the oropharynx Thomas K. Hoffmann 1 * , Christian Arsov 1 , Kerstin Schirlau 1 , Murat Bas 1 , Ulrike Friebe-Hoffmann 2 , Jens Peter Klussmann 3 , Kathrin Scheckenbach 1 , Vera Balz 1 , Henning Bier 1 and Theresa L. Whiteside 4 1 Department of Otorhinolaryngology, University of Duesseldorf, Germany 2 Department of Obstetrics and Gynecology, University of Duesseldorf, Germany 3 Department of Otorhinolaryngology, University of Cologne, Germany 4 Hillman Cancer Center, University of Pittsburgh, PA, USA Squamous cell carcinomas of the oropharynx (SCCO) are often infected with oncogenic human papilloma virus (HPV) subtype 16. To determine the frequency of T cells specific for human leukocyte antigen (HLA)-A2.1 restricted HPV16 E7 protein-derived epito- pes, tetramer analysis was performed using peripheral blood lym- phocytes of 20 HLA-A2.1 1 patients and 20 HLA-A2.1 1 healthy individuals. Tetramers specific for 3 HPV16 peptides (E7 11–20 , E7 82–90 and E7 86–93) , an influenza matrix peptide (a model recall antigen) or an HIV reverse transcriptase peptide (a model novel antigen) were used in multicolor flow analysis. The HPV-specific T-cell frequencies were correlated with the HPV16 E7 and p16 status in tumor sections. In vitro stimulation (IVS) with autologous dendritic cells (DC) pulsed with HPV16 E7 epitopes was per- formed to demonstrate proliferation and antitumor activity of the HPV-responsive T cells. Frequencies of CD8 1 T cells specific for HPV16 E7 peptides were not significantly different in patients with SCCO relative to normal donors. However, patients with tumors expressing HPV16 E7 (60%) and p16 (50%) had an increased frequency (p < 0.05) of T cells specific for the E7 11–20 epitope compared to those with tumors negative for both markers. HPV16 E7 11–20 and HPV16 E7 86–93 specific T cells were expand- able upon IVS with cognate peptide-pulsed DC and were reactive against peptide-pulsed targets or, in case of the E7 11–20 epitope- specific T cells, against HPV16 E7 expressing CaSki cell line. Thus, in patients with HPV16 1 SCCO, precursor T cells specific for E7 11–20 epitope are present (1/3,947) in the circulation, are responsive to stimulation with the cognate viral peptide and recog- nize in vitro HPV16 E7 1 tumor cells. Further studies have to eluci- date why those T cells are unable to eliminate the tumor in vivo and this might also allow for finding potential strategies that will increase the chances of developing a future HPV-based vaccine in patients with SCCO. ' 2005 Wiley-Liss, Inc. Key words: HPV; specific T cells; head and neck cancer; tetramer Squamous cell carcinomas (SCC) represent the most common type of head and neck cancer, accounting for 6% of all new cancer cases. 1 Typical risk factors include alcohol and nicotine consump- tion. However, a significant proportion of the patients do not have these risk factors, and recent studies have suggested an association of SCC to viral pathogens such as high risk (oncogenic) human papilloma virus (HPV) types, particularly HPV16 or 18. These viruses are frequently found in squamous carcinoma of the head and neck. 2–11 HPV16 infections have been observed in approxi- mately one half of squamous cell carcinomas of the oropharynx (SCCO). 7,10–12 The HPV16 1 SCCO have a good prognosis and are not associated with conventional risk factors, suggesting they may represent a separate tumor entity. 3,7,10,11 The HPV-derived oncoproteins E6 and E7 are mainly responsi- ble for both the onset and maintenance of malignant transforma- tion through inactivation of the p53 and retinoblastoma (Rb) tumor suppressor genes, respectively. 13,14 Inhibition of Rb protein (pRb) by the HPV16 E7 oncogene product leads to upregulation of p16 via a loss of negative control of pRb expression. Thus, HPV16 E7 1 SCCO coexpress p16 in nearly 90% of cases, and p16 has been suggested as a surrogate marker for the HPV status in SCCO. 15 Since infections with HPV16 lead to expression of foreign pro- teins in the tumor, i.e., the HPV16 E7 oncoprotein, it could be expected that T-cell responses specific for these proteins develop and can be detected in peripheral circulation, similar to T-cell responses detected upon infection with the influenza virus. To investigate T-cell responses against HPV-derived peptides/epito- pes, we used fluorochrome labeled tetrameric peptide–MHC class I complexes (tetramers) in multicolor flow cytometry. Tetramers allow for direct identification of antigen-specific T cells in the peripheral circulation. 16,17 To generate HPV16 E7-specific te- tramers, immunogenic peptides had to be selected. To date, only a few human leukocyte antigen (HLA)-A2.1 restricted HPV16 E7- derived peptides, HPV16 E7 11–20 , HPV16 E7 82–90 and HPV16 E7 86–93 , have been shown to be immunogenic in cervical carcino- mas. 18–25 We, therefore, selected these peptides for the production of tetramers to be used for the quantification of HPV16 E7-spe- cific T cells in the peripheral blood of SCCO patients and normal controls. In this manuscript, we report on the frequency of tetramer 1 T cells in the peripheral circulation of SCCO patients with HPV 1 and HPV 2 tumors, and on the in vitro capability of these T cells to differentiate into antitumor effector T cells. Material and methods Peripheral blood mononuclear cells Peripheral blood samples or leukapheresis products were obtained from 20 HLA-A2.1 1 SCCHN patients and 20 HLA- A2.1 1 healthy donors. Leukapheresis products were obtained from the Institute of Transfusion Medicine, D€ usseldorf. The mean age of the patients (16 male and 4 female) was 49.4 years and that of normal controls (14 male and 6 female) was 50.2 years. None of the female individuals had a history of CIN. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation over Lympho Sep 1 lymphocyte separation medium (ICN, Aurora, Ohio) in Leucosep 1 Tubes (Greiner, Solingen, Germany). The study was approved by the local ethics committee at the Univer- sity of D€ usseldorf, and informed consent was obtained from each Grant sponsor: The Deutsche Krebshilfe (T.K.H.); Grant number: 70- 2976-Ho I; Grant sponsor: The National Institutes of Health; Grant num- ber: PO-1 DE12321 (T.L.W.). *Correspondence to: Department of Otorhinolaryngology, Heinrich- Heine-University D€ usseldorf, Moorenstr. 5, D-40225 D€ usseldorf, Germany. Fax: 149-211-811-8880. E-mail: tkhoffmann@uni-duesseldorf.de Received 1 June 2005; Accepted after revision 24 August 2005 DOI 10.1002/ijc.21565 Published online 11 November 2005 in Wiley InterScience (www. interscience.wiley.com). Abbreviations: Ab, antibody; APC, antigen presenting cell; CTL, cyto- toxic T lymphocyte; DC, dendritic cell; ELISPOT, enzyme linked immu- nospot; GM-CSF, granulocyte macrophage colony stimulating factor; HLA, human leukocyte antigen; HPV, human papilloma virus; IL, inter- leukin; IVS, in vitro sensitization; mAb, monoclonal antibody; MHC, major histocompatibility complex; PBMC, peripheral blood mononuclear cells; SCCO, squamous cell carcinoma of the oropharynx. Int. J. Cancer: 118, 1984–1991 (2006) ' 2005 Wiley-Liss, Inc. Publication of the International Union Against Cancer