Elsevier

Human Immunology

Volume 68, Issue 5, May 2007, Pages 334-341
Human Immunology

α-Fetoprotein and Interleukin-18 Gene–Modified Dendritic Cells Effectively Stimulate Specific Type-1 CD4- and CD8-Mediated T-Cell Response from Hepatocellular Carcinoma Patients in Vitro

https://doi.org/10.1016/j.humimm.2007.01.008Get rights and content

Abstract

The T-helper 1 (Th1) immune reaction is most important in dendritic cell (DC)–based immunotherapy. Interleukin (IL)–18, a Th1-biasing cytokine, plays a pivotal role in inducing cytotoxic T lymphocyte (CTL) responses. In this study, we analyzed whether dendritic cells (DCs) from patients with hepatocellular carcinoma (HCC) can be transduced with the IL-18 gene and/or α-fetoprotein (AFP) gene, and we examined whether vaccinations using these genetically engineered DC can induce stronger therapeutic antitumor immunity. The results showed that DC transfected with AdIL-18/AFP can expressed IL-18 and AFP by reverse transcriptase–polymerase chain reaction and enzyme-linked immunoassay. Compared with those before transfection, the expressions of membrane molecules were increased dramatically. Specific T cells generated by DC transfected with AdIL-18/AFP recognized HLA-matched HepG2 cell lines specifically. Most importantly, The cytotoxic activity of CTLs against HepG2 with DC expressing AFP(AFP-DC) was significantly augmented by co-transduction with the IL-18 gene. Administration with such vaccine also significantly increased the production of interleukin-12p70 and interferon-γ. These results indicate that a vaccination therapy using DC co-transduced with the TAA gene and IL-18 genes is effective strategy for immunotherapy in terms of the activation of DCs, CD4+ T, cells and CD8+ T cells, and may be useful in the clinical application of a cancer vaccine therapy.

Introduction

Hepatocellular carcinoma (HCC) is one of the most common malignancies in Asian countries [1], and the number of patients with HCC is expected to increase rapidly in the world with the spread of the hepatitis C virus (HCV). Recent progress in imaging techniques has enabled the diagnosis of small HCC, and advances in treatment modalities have resulted in the increased survival of patients with HCC. The long-term prognosis, however, is still disappointing because of the high frequency of HCC recurrence; and none of the existing therapies for HCC has shown any promise in this regard. Therefore, novel strategies to prevent proliferation of malignant cells are urgently needed. Recent advances in the basic understanding of immunology and tumor biology have sparked renewed interest in active immunotherapy as a treatment for various cancers [2]. A promising approach may be the prophylactic vaccination directed against tumor-associated antigen (TAA).

Dendritic cells (DC) play a crucial role in the initiation of an immune response [3]. They have the ability take up antigen efficiently. After processing, they present the antigen on their surface in association with MHC molecules, and stimulated naive T cells to proliferation and differentiation via their antigen-specific receptors [4]. Because of these immunostimulating properties, the DC may be effective for the prevention of carcinogenesis, as well as for the treatment of cancers. Recently, Zhang et al. [5] reported that DCs transfected with the total RNA, which were extracted from the HCC cell line, induced specific anti-HCC T-cell responses with autologous DCs transfected with total tumor cell RNA. However, mRNA is not easy to treat because of their low stability. Another clinical trial, using DCs pulsed with α-fetoprotein (AFP), showed the generation of AFP-specific T-cell responses in patients with AFP-positive HCC [6, 7]. However, transduction of DCs with tumor-associated antigen (TAA)–encoding transgene offers several potential advantages over peptide pulsing. The gene modifications of DCs with the TAA gene have the potential to present various known and unknown TAA epitopes, and the endogenous processing and presentation of TAA peptides may be more efficient for cell surface presentation than the exogenous loading of synthetic TAA peptides. In this study, adenoviral vectors were selected, as we and others have found adenovirus (Ad) to be a highly efficient and reproducible method of gene transfer into DCs.

The goal of DC-based therapeutic vaccines is to induce effective tumor-specific immunity, but the issue remains how can we address the limitation of the vaccine and achieve the goal. It is well recognized that the activation of the helper arm is essential to overcome immunosuppression and to induce tumor-specific cytotoxic T lymphocyte (CTLs) [8, 9], but there are predominant expressions of Th2 cytokines in tumors [10]. Therefore the development of a protocol to induce Th1 response may be required to develop more efficient immunotherapy. Interleukin (IL)-18, a well identified Th1-biasing cytokine, mediates many important biologic functions including augmentation of interferon (IFN)–γ production, stimulation of T-cell proliferation, and induction of CTL [11, 12]. Sato et al. [13] also reported that Th1 cytokine conditioned DC, termed DC1 and induced by culture of cells in the presence of IFN-γ and IL-12, most efficiently supported Th1-dominant immunity suitable for CTL generation, whereas the contrary results were observed after Th2 cytokine conditioned DC stimulation. Therefore we hypothesized that co-transfection of IL-18 gene and AFP gene into DC would increase the production of Th1 cytokines (IFN-γ and IL-12), enhance the AFP-specific CTL response against the HCC cells, thus profoundly potentiate DC-based vaccine efficacy. In this study we tried the strategy using IL-18 and AFP gene–co-transfected DC as vaccine to induce antitumor response against HCC cells. The results demonstrate that vaccination with IL-18 and AFP gene–co-transfected DC can significantly induce antitumor responses.

Section snippets

Construction and Production of Recombinant Adenovirus Encoding AFP and IL-18

The Ad-AFP and Ad-IL-18 vector was made using the AdEasy system (Quantum Biotech, Montreal, Canada). Briefly, the fragment of the AFP and IL-18 gene (derived from plasmid pEGFP-AFP and pCR3.1-IL-18, kindly provided by Dr. Zhi-Qiang Yao from the Catholic University of Leuven, Belgium) was cloned into the pTrack plasmid containing the CMV promoter. To generate the infectious recombinant adenoviral plasmid, after Pme I linearized pTrack-CMV-AFP vector or pTrack-CMV-IL-18, they were co-transformed

Ad-Mediated IL-18 and/or AFP Genetic Modification of DCs

The DCs derived from peripheral blood mononuclear cells were cultured in the previously defined medium containing GM-CSF and IL-4. Flow-cytometric analysis was performed 48 hours after transduction with AdGFP (at MOIs of 50–200) to determine the transduction efficiency and the impact of Ad infection on the DCs phenotypes. The DCs proved amenable to the in vitro Ad-mediated gene transfer and its efficiency was increased in an MOI-dependent manner. An MOI of 200 accomplished transgene expression

Discussion

Recent advances in the basic understanding of immunology and tumor biology have sparked renewed interest in active immunotherapy as a treatment for various cancers. One of the promising approaches is to design vaccine using DCs as the vehicle to deliver cancer antigens for an effective induction of T-cell antitumor immunity. To enhance loading of DCs with TAA in vitro and to increase further the efficacy of the DCs vaccines various techniques for delivery of the priming antigen have been

References (21)

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D.-Y.C. and J.-Y.Y. contributed equally to this work.

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