Elsevier

Clinical Immunology

Volume 114, Issue 2, February 2005, Pages 119-129
Clinical Immunology

Preservation and redirection of HPV16E7-specific T cell receptors for immunotherapy of cervical cancer

https://doi.org/10.1016/j.clim.2004.11.005Get rights and content

Abstract

Human papilloma virus (HPV) type 16 infections of the genital tract are associated with the development of cervical cancer (CxCa) in women. HPV16-derived oncoproteins E6 and E7 are expressed constitutively in these lesions and might therefore be attractive candidates for T-cell-mediated adoptive immunotherapy. However, the low precursor frequency of HPV16E7-specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer. To overcome this problem, we have isolated T cell receptor (TCR) genes from four different HPV16E7-specific healthy donor and patient-derived human cytotoxic T lymphocyte (CTL) clones. We examined whether genetic engineering of peripheral blood-derived CD8+ T cells in order to express HPV16E711–20-specific TCRs is feasible for adoptive transfer purposes. Reporter cells (Jurkat/MA) carrying a transgenic TCR were shown to bind relevant but not irrelevant tetramers. Moreover, these TCR-transgenic Jurkat/MA cells showed reactivity towards relevant target cells, indicating proper functional activity of the TCRs isolated from already available T cell clones. We next introduced an HPV16E711–20-specific TCR into blood-derived, CD8+ recipient T cells. Transgenic CTL clones stained positive for tetramers presenting the relevant HPV16E711–20 epitope and biological activity of the TCR in transduced CTL was confirmed by lytic activity and by interferon (IFN)-γ secretion upon antigen-specific stimulation. Importantly, we show recognition of the endogenously processed and HLA-A2 presented HPV16E711–20 CTL epitope by A9-TCR-transgenic T cells. Collectively, our data indicate that HPV16E7 TCR gene transfer is feasible as an alternative strategy to generate human HPV16E7-specific T cells for the treatment of patients suffering from cervical cancer and other HPV16-induced malignancies.

Introduction

Cervical cancer (CxCa) is the second leading cause of cancer-related death among women worldwide. Infection with the sexually transmitted human papilloma virus (HPV) is proposed to be associated with cervical cancer [1] because HPV DNA is detected in more than 99% of all tumors of the uterine cervix [2]. Vaccination strategies against HPV are currently based on the induction of HPV-specific, virus-neutralizing antibodies that reduce viral load and infection, thereby preventing the development of premalignant lesions and cervical cancer. The immunogens used for prophylactic vaccines consist of the late proteins, L1 and L2, which are structural elements of the virus. From the work of Koutsky et al. [3], it is clear that these prophylactic vaccines can indeed prevent the appearance of premalignant lesions. While prophylactic vaccines are useful in preventing HPV infection, they are useless in the treatment of existing, HPV-induced premalignant lesions and cervical cancer. The increased incidence and progression of HPV infections in immunosuppressed individuals suggest that T-cell-mediated immune responses may be important for the control and eradication of HPV-associated cervical neoplasia [4].

Therapeutic vaccines have been designed to prime antigen-specific T cell responses directed against virus-infected cells. The late proteins can be excluded from therapeutic vaccines because they are not expressed in CxCa or its precursor lesions. The early HPV proteins E6 and E7 are responsible for malignant transformation of HPV-infected cells. The constitutive expression of these proteins is necessary for the maintenance of a transformed phenotype and is therefore considered an ideal target for T-cell-mediated immunotherapy [5]. Therapeutic vaccines consisting of E6 and/or E7 have been tested in patients and have proven to be safe and effective against benign warts, however have had limited therapeutic effect so far in cases of cervical cancer [5]. Tumor-infiltrating lymphocytes directed against HPV16E7 have been found in cervical cancers; therefore, these cells might be attractive for adoptive T cell transfer [6], [7].

Recent clinical studies have shown regression of human melanoma upon adoptive transfer of tumor-specific cytotoxic T lymphocyte (CTL) [8], [9]. In the case of melanoma, the frequencies of melanoma antigen-specific CTL are usually high within tumor-infiltrating lymphocytes and also in peripheral blood. However, in the case of cervical carcinoma HPV16E7-specific CTL are relatively rare, hampering the isolation of tumor-specific CTL for T-cell-mediated adoptive transfer [10], [11]. To circumvent this problem, we have used T cell receptor (TCR) gene transfer, which allows the generation of high numbers of HPV-specific CTL. Previous reports have indicated that TCR gene transfer can result in the successful redirection of antitumor reactivity [12], [13], [14], and that the avidity and peptide fine specificity are preserved following TCR gene transfer into human CTL [15], [16]. Additionally, Kessels et al. [17] showed antigen-specific expansion and antitumor reactivity of TCR-transduced CTL in vivo in a murine tumor model.

In this study, we describe TCR gene transfer of a number of different TCRs, specific for the HPV16E711–20 epitope, isolated from different CTL clones. TCR-transduced T cells showed MHC restriction and specific reactivity against the endogenously processed and HLA-A2 presented HPV16E711–20 epitope. Our data indicate that TCR gene transfer might be an alternative strategy to generate HPV16E7-specific, CxCa-reactive T cells.

Section snippets

Cell lines and CTL culture

The HPV16-positive, HLA-A2-negative CxCa cell line SiHa (American Type Culture Collection, ATCC, Manassas, VA) and the HPV16-positive, HLA-A2-positive CxCa cell line SiHa-A2 (SiHa transfected with HLA-A2.1, kindly provided by Dr. S. Man, University of Wales College of Medicine, Cardiff, UK) were cultured in keratinocyte serum-free medium (Life technologies, Paisley, UK) supplemented with 5% (v/v) fetal calf serum (FCS; Perbio, Helsingborg, Sweden), 20–30 μg/ml bovine pituitary extract (Life

Original HPV16E7-specific CTL clones and validation of tetramer staining

We investigated whether we could preserve the specificity of several different CxCa-reactive CTL clones by isolating their TCR genes. The four HPV16E711–20-specific CTL clones used in this study were isolated after several rounds of in vitro stimulation of purified CD8β+ peripheral blood-derived T cells using mature dendritic cells exogenously loaded with the HPV16E711–20 peptide, followed by tetramer guided sorting and limiting dilution cloning. CTL clone A9 was isolated from a healthy donor

Discussion

Previously, adoptive transfer of specific T cells has been used successfully to generate T cell immunity in patients suffering from malignant melanoma [8], [9] and in immunocompromised patients suffering from viral infections [27], [28]. Adoptive immunotherapy employing HPV16-specific CTL might represent an attractive tool in cases of cervical cancer and other HPV16-induced malignancies. Validated tetramers containing the relevant CTL epitope can be used to isolate HPV16-specific CTL as has

Acknowledgments

The authors wish to thank Dr. TNM Schumacher from the Netherlands Cancer Institute, Amsterdam, for a set of TCR primers; Dr. S Man from the University of Wales College of Medicine, Cardiff, UK, for the cell line SiHa-A2; and the Maurits and Anna de Kock Foundation for financial support in the purchase of an HPLC.

This study was financially supported by grant 901-10-124 from the Netherlands Organization for Scientific Research and grant VUMC2001-2503 from the Dutch Cancer Society.

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