Sensitive Detection of Human Papillomavirus Type 16 E7-Specific T Cells
Cervical cancer is the second most common gynecological cancer amongst women world-wide. Despite optimized protocols, standard treatments still face several disadvantages. Therefore, research aims at the development of immune-based strategies using tumor antigen-loaded dendritic cells for the induction of cellular anti-tumor immunity.
An
Published 26 October 2006
Abstract
Background
Cervical cancer is the second most common gynecological cancer amongst women world-wide. Despite optimized protocols, standard treatments still face several disadvantages. Therefore, research aims at the development of immune-based strategies using tumor antigen-loaded dendritic cells for the induction of cellular anti-tumor immunity.
Results
In this study, we used dendritic cells loaded with the HLA-A2-restricted HPV type 16 E711–20 peptide in order to induce an in vitro CD8+ T cell response. For this purpose, peptide-pulsed dendritic cells were co-cultured with autologous CD8+ T cells. After 5 weekly stimulations with peptide-pulsed mature dendritic cells, cultured T cells were analyzed for antigen specificity by an IFN-γ ELISPOT assay. Using this ELISPOT assay, we were able to detect E7-specific IFN-γ-secreting CD8+ T cells in 5/5 healthy donors.
Conclusion
We show that peptide-pulsed mature dendritic cells are able to stimulate a HPV type 16 E7 peptide-specific immune response in vitro. These experiments describe an efficient culture protocol for antigen-specific T cells for use in pre-clinical vaccination research and confirm the need for sensitive T cell assays for detection of tumor-specific immune responses in vitro.
Background
Cervical cancer contributes to approximately 12% of the global cancer burden in women and represents the second most frequent gynecological malignancy in the world [1,2]. At an early stage, cervical cancer is treated by means of surgery and radiotherapy [3]. In a more advanced stage, one uses a combination therapy of cisplatinum-containing chemotherapy and radiotherapy [4]. Despite technological progress in conventional treatment modalities, more than 35% of patients develop a metastasizing malignancy with poor results after treatment. An additional disadvantage of radio- and chemotherapy is a pronounced and long-lasting negative effect on the immune system. For these reasons, current research aims at the development of new and more efficient strategies. In this context, dendritic cells (DC) loaded with tumor antigens for activation and/or expansion of tumor-specific T cells are currently the subject of intensive research in the field of cellular immunotherapy [5-7].
One of the major risk factors for the development of cervical cancer is infection with human papillomavirus (HPV). More than 20 oncogenic HPV genotypes have been characterized, while HPV type 16 (HPV-16) and type 18 (HPV-18) are the most prevalent in cervical cancer [8]. In HPV-16 positive tumors, the E7 oncoprotein is constitutively expressed in cervical tumor cells [9] and is responsible for transformation of these cells [10]. Moreover, HPV-16 E7-specific cytotoxic T lymphocytes (CTL) have been demonstrated in the peripheral blood, the lymph nodes and the tumor tissue of HPV-16-positive cervical carcinoma patients [11,12]. An effective HPV-specific cellular immune response can be generated after active immunization [13-16]. For these reasons, the HPV-16 E7 protein is a target of choice for the development of a specific immune therapy directed against cervical cancer.
Stimulation of the immune system against specific tumor antigens might become a suitable secondary (or even primary) therapy to treat cancer. Because tumor cells do not efficiently function as antigen-presenting cells (APC) for the activation of CTL, a vigorous immune response is generally absent or defective. To overcome this problem, transfer of tumor antigens from tumor cells to professional APC, like DC, might be a valuable strategy [17].
DC are the most efficient antigen-capturing and -presenting cells of the immune system and are potent inducers of (primary) immune responses directed against tumors and viral antigens [18]. In their immature state, DC are skilled in antigen uptake by means of endocytosis and phagocytosis. After uptake, antigens are processed by DC to peptide fragments which are bound to major histocompatibility complex (MHC) class I and II molecules. After transport to the plasma membrane, these peptide-MHC complexes can be recognized by a T cell receptor (TCR) with high specificity for the antigenic peptide-MHC complex. If antigen uptake and presentation is associated with danger signals, provided by microbial components such as lipopolysaccharide (LPS) [19], DC are activated and an effective immune response can be induced.
In this study, we used in vitro cultured mature DC loaded with an HLA-A*0201-restricted HPV-16 E7 peptide for the in vitro activation of antigen-specific IFN-γ-producing CD8+ T cells. After several rounds of in vitro stimulation of CD8+ T cells using peptide-pulsed mature DC, the presence of HPV-16 E7 peptide-specific T cells was monitored using an IFN-γ enzyme-linked immunospot (ELISPOT) assay.
Results
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