Intratumoral delivery of IL-18 naked DNA induces T-cell activation and Th1 response in a mouse hepatic cancer model
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The novel cytokine, interleukin (IL)-18, is a strong interferon-γ inducer and costimulatory factor in Th1 cell activation. IL-18 triggers IFN-γ production and enhances cytolytic activity in both T and NK cells. However, the exact mechanism of antitumor action of IL-18 remains to be clarified. To determine the effects of IL-18 plasmid DNA on hepatic cancer in mice, CT26 murine colon adenocarcinoma cells were established in mouse liver.
Plasmid vectors encoding IL-18 were transferred directly into the liver 7 days after tumor injection to restrict IL-18 expression within the tumor site. The IL-18 protein level was increased in the liver 4 days after plasmid injection, and a marked antitumoral effect was observed at day 7. Antitumor effects were evaluated by measuring tumor regression, immune cell population, and IFN-γ production.
The IL-18 plasmid controlled the growth of hepatic tumors and proliferation of splenic immune cells. Moreover, treatment of CT26 tumors with the IL-18 plasmid significantly enhanced the population of the effector T and NK cells in the spleen and peripheral blood. In spleen, the population of CD4+CD62Low cells was augmented in response to IL-18 on day 7. These results are consistent with the increase in CD4+ T cells secreting IFN-γ, but not CD8+ T cells. The marked reduction of tumor growth in tumor-bearing mice was associated with the maintenance of IFN-γ production in spleen in response to IL-18. These antitumoral effects were maintained until 14 days after plasmid injection.
Our results suggest that direct plasmid DNA transfer of IL-18 with no accompanying reagents to augment transfection efficiency may be useful in tumor immunotherapy.
KeywordsIntratumoral Injection Immune Cell Population CT26 Tumor Ammonium Chloride Solution Costimulatory Factor
Effective eradication of established tumors and generation of a lasting systemic immune response with a simple gene delivery system are important goals for cancer gene immunotherapy . Cytokines are the most extensively studied immunostimulatory agents in cancer gene therapy . Interferon-γ-inducing factor (IL-18) is a recently characterized murine and human cytokine. The murine IL-18 gene encodes a precursor protein of 192 amino acids, which is processed to a mature protein containing 157 residues . This cytokine, produced by Kupffer cells, is a potent inducer of IFN-γ production by T cells and a costimulatory factor for T cell activation [4, 5]. Accumulating evidence that IL-18 is a multifunctional cytokine that shares several biological activities with IL-12 has led to a series of studies on its effects on T and NK cells . Analogous to IL-12, IL-18 stimulates T cell proliferation and NK cell activity . The finding that IL-18 stimulates the differentiation of Th1 cells that produce cytokines necessary for the development of cell-mediated immune responses suggests a prominent role in defense against tumors . Similarly, high IFN-γ levels are induced by IL-18 in splenic-derived CD4 T cells in the presence of B cells or adherent cells . Furthermore, IL-18-transfected tumor cell vaccines and local delivery of the IL-18 gene as naked DNA via a gene gun or viral vectors has been extensively investigated. Numerous animal studies show that IL-18 has potent antitumor effects, but induces side-effects upon systemic administration . Direct intratumoral DNA administration is reliable and reproducible, and may limit the need for systemic cytokine administration [9, 10, 11]. Here, we report the effects of direct intratumoral injection of a nonviral plasmid vector encoding murine IL-18 DNA in established CT26 liver tumors .
Animals and cell lines
BALB/c (H-2d) mice (6 to 8 weeks old) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). All mice were housed in specific pathogen-free conditions, in accordance with institutional guidelines. CT26 tumor cells were maintained in RPMI 1640 cell culture medium (Biowhittaker, Walkersville, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml streptomycin, and 100 μg/ml penicillin.
The 11 kb mIL-18 DNA expression plasmid vector, pCEP4-mIL18, was constructed using a CMV early enhancer/promoter/EBNA-1-based pCEP4 plasmid vector (Invitrogen, San Diego, CA) with an ampicillin selection gene. Plasmid DNA was purified in the absence of ethidium bromide using a commercial column chromatography method, according to the manufacturer's protocol (Qiagen, Chatsworth, CA).
Tumor model and therapeutic protocol
CT26, an undifferentiated murine adenocarcinoma, was induced by rectal injection of N-nitroso-N-methylurethane in BALB/c mice. Colorectal cancer is often metastatic, the most common site of metastasis being the liver. CT26 cells were suspended for implantation at 1 × 105 cells/50 μl saline. Colon carcinoma was established in the left lateral lobe of 6- to 8-week-old male BALB/c mice by an abdominal operation. Treatments began 7 days after a defined solid tumor growth was identified within the injected lobe. For intratumoral injection, naked DNA (10, 25, and 50 μg) was diluted in 50 μl saline, and injected into the parenchyma of the lower surface of the left liver lobe via insulin syringes (31 gauge, 0.8 inch needles; Becton Dickinson, Franklin Lake, NJ). Mice were sacrificed at 1, 4, 7, and 14 days after DNA treatment, and tumor growth monitored by measuring liver weight .
Flow cytometric analysis
Total nuclear cells in the peripheral blood were isolated by erythrocyte lysis with ammonium chloride solution (PharM Lyse, Becton Dickinson). Single spleen cell suspensions were obtained by teasing apart spleen tissue and disaggregating cells through a 70 μm mesh. Briefly, splenocytes and PBMCs were incubated with PE-, Cy5, or FITC-conjugated Abs, and the corresponding isotypes (purchased from Pharmingen, San Diego, CA, USA). Anti-mouse NK (clone DX5), -CD3, -CD4, -CD8, -Cd62L, -CD69, -CD19, and -CD11b Abs were used to stain the populations of NK cells, macrophages, T-cells, and B-cells in the spleen. Samples were analyzed for surface phenotypes using FACS Vantage (Becton-Dickinson, San Jose, CA, USA) and CellQuest software (Becton-Dickinson Labware).
Flow cytometric assessment of intracellular cytokine production
For parallel evaluation of intracellular cytokines, surface staining was initially performed for CT26 lysate-stimulated splenocytes from each group. Cells were fixed with 4% paraformaldehyde solution for 20 min, treated with permeabilization buffer (0.1% saponin, 1% FBS in PBS) for 5 min at RT in the dark, and washed. Intracellular staining was performed by incubation of mAb to IFN-γ with cell pellets for 20 min at RT in the dark. Cells were washed, and measured using a flow cytometer.
Assessment of cytokines (IL-18, IFN-γ)
We obtained total liver protein by homogenization of frozen tissue in extraction buffer containing 1% Triton X-100, 10 mM Tris-HCL (pH 7.6), 5 mM EDTA, 50 mM NaCl, 30 mM Na4P2O7, 50 mM NaF, 200 μM Na3VO4, 2 mM PMSF, 5 μg/ml aprotinin, 1 μg/ml pepstatin A, and 2 μg of leupeptin. The suspension was centrifuged at 14,000 r.p.m. for 20 min at 4°, and the supernatant stored at -70°. Supernatant fractions were analyzed with the IL-18 ELISA kit (MBL, Nagoya, Japan). IFN-γ concentrations in the splenocyte culture supernatants were determined with specific ELISA performed according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). The absolute cytokine levels were calculated by comparison to assay performance in the presence of known quantities of recombinant cytokine standards.
An unpaired t-test was employed to compare tumor weight, quantitative cytokine production, and flow cytometry analyses. All analyses were performed with SigmaPlot 2000. P < 0.05 was considered significant.
Levels of transgene expression within the treated tumor site
In vivokinetic studies of the immune cell population in spleen
Regression of established CT26 hepatic tumors after direct intratumoral mIL-18 gene transfer
Regression phenotypic changes of immune effector cells in spleen and blood samples
Frequency of CD4+CD62Low, CD8+CD62Low, DX5+, CD19+cells in spleen and blood: major subsets
17.75 ± 0.92
0.997 ± 0.05
3.39 ± 1.26
44.51 ± 3.40
17.62 ± 2.15
5.93 ± 0.96
15.45 ± 1.19
28.97 ± 3.52
30.05 ± 1.85a
1.995 ± 0.34
1.73 ± 0.08
41.57 ± 1.09
30.58 ± 4.59a
9.88 ± 0.94
18.4 ± 2.91
36.49 ± 5.95
DNA vaccination of mice resulted in broad immune responses, characterized by the activation of B cells, helper CD4+ and cytotoxic CD8+ T cells . Based on these results, we propose that mIL-18 overexpression in the tumor site induces phenotypic changes in the T cell and B cell subsets, and protects against tumor development by stimulating IFN-γ production by CD4+ T cells.
Intratumoral injection of mIL-18 plasmid DNA elevates IFN-γ production by splenocytes
Recent trials using cytokine genes show that intratumoral IL-18 gene transfer is a feasible procedure, but exerts only mild antitumor effects . Gene transfer with increased mIL-18 doses may enhance antitumor efficacy. In this study, we validate the antitumoral efficacy of mIL-18 overexpression in CT26 tumor-bearing mice by using plasmid vectors to transfer the mouse IL-18 gene alone. Our data show marked inhibition of tumor growth and significant phenotypic changes of immune cells, suggesting that mIL-18 intratumoral plasmid transfer may be developed as an effective alternative therapy for cancer treatment with no significant side-effects.
This research was supported by grants from the IN-SUNG Foundation for Medical Research, Seoul, Korea
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