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. 2004 Jan;111(1):27–34. doi: 10.1111/j.1365-2567.2003.01784.x

Efficient induction of T helper type 1-mediated immune responses in antigen-primed mice by anti-CD3 single-chain Fv/interleukin-18 fusion DNA

E J Kim *, D Cho ‡, T S Kim *,†
PMCID: PMC1782390  PMID: 14678196

Abstract

Two types of T helper (Th) cells – Th1 and Th2 – play different roles in protection and immunopathology. The Th1 cell-mediated immune response plays an important role in inducing the host defence against intracellular bacteria and also in cancer immunotherapy. To effectively induce Th1 immune responses, we constructed a mammalian expression plasmid (pAnti-CD3sFv/IL-18) carrying a fusion gene in which anti-CD3 single-chain Fv (sFv) cDNA, the smallest unit of antibody recognizing the CD3 epsilon moiety of the T-cell receptor, was covalently linked to mature interleukin (IL)-18 cDNA. Intramuscular injection of ovalbumin (OVA)-sensitized BALB/c mice with pAnti-CD3sFv/IL-18 DNA efficiently increased the production of both OVA-specific interferon-γ and anti-OVA immunoglobulin G2a, compared to injection with pAnti-CD3sFv DNA. In addition, pAnti-CD3sFv/IL-18 was more efficient than a mixture of pAnti-CD3sFv + pIL-18 in inducing OVA-specific, Th1 immune responses and also in inhibiting OVA-specific, IL-4 production. These studies indicate that vaccination with pAnti-CD3sFv/IL-18 fusion DNA efficiently induces the Th1 immune response in antigen-sensitized mice.

Introduction

The T-helper lymphocyte is responsible for orchestrating the appropriate immune response to a wide variety of pathogens. The recognition of the polarized T-helper (Th) cell subsets Th1 and Th2 has led to an understanding of the role of these cells in co-ordinating a variety of immune responses.1,2 Th1 cells selectively secrete interferon-γ (IFN-γ), interleukin (IL)-2 and tumour necrosis factor-β (TNF-β), and regulate cell-mediated immunity characterized by the production of complement-fixing and opsonizing antibodies such as immunoglobulin G2a (IgG2a). Th2 cells produce IL-4, IL-5, IL-6 and IL-10, which are involved in the development of humoral immune responses, including expression of immunoglobulin G1 (IgG1) and immunoglobulin E (IgE) isotypes. These two Th subsets regulate each others' function through the antagonistic activity of their respective cytokines.

Increasing evidence indicates that the outcome of many diseases is determined by the balance between Th1- and Th2-mediated immune responses.3,4 Polarized Th1-and Th2-type responses play different roles in protection, Th1 being effective in the defence against intracellular pathogens and Th2 against intestinal nematodes. Moreover, Th1 and Th2 subsets are responsible for different types of immunopathological reactions. Th1 responses predominate in organ-specific autoimmune disorders, acute allograft rejection, recurrent abortions and in some chronic inflammatory disorders. In contrast, Th2 responses predominate in Omenn's syndrome, transplantation tolerance, chronic graft-versus-host disease and allergic diseases.

A number of factors determine which Th subsets predominate in an immune response.5 These include the physical form of the antigen, as well as the density and affinity of the peptide ligand, costimulatory signals provided by antigen-presenting cells (APCs), hormones released into the microenvironment and genetic background of the T cells.6,7 Most importantly, the cytokine microenvironment in which the initial antigen priming occurs is responsible for influencing the induction of either Th1- or Th2-mediated immune responses. IFN-γ and IL-12 induce Th1-mediated responses, while IL-4 drives the response into Th2-mediated immune responses.

IL-18 is an 18 300 molecular-weight cytokine that was first identified as an IFN-γ-inducing factor (IGIF) by its ability to induce IFN-γ production in mice with endotoxin shock.8 IL-18 is initially produced in a biologically inactive precursor form and, after cleavage with IL-1β-converting enzyme (ICE; caspase-1), a bioactive, mature IL-18 is secreted.9 In collaboration with IL-12, IL-18 is known to stimulate Th1-mediated immune responses, which play a critical role in host defences against several infectious micro-organisms (including intracellular bacteria, fungi and protozoa) through the induction of IFN-γ, and also in tumour immunotherapy by its potent capacity to augment the cytotoxic activity of natural killer (NK) and T cells in vivo.10,11 IL-18 enhances IFN-γ production by non-adherent splenocytes and established Th1 clones in the presence of anti-CD3, concanavalin A, IL-2, IL-12, or antigen plus APCs.12 In contrast, recent studies demonstrate a convincing role for IL-18 in the induction of Th2 cytokines by NK cells, mast cells and basophils, indicating that IL-18 stimulates both Th1 and Th2 responses, depending on its cytokine milieu.13

A number of reports have shown that nucleic acids (including DNA) can be used as a vaccine, as expression plasmids inoculated into muscle are effectively taken up by myofibres and the encoded genes are expressed.14,15 In particular, DNA vaccines are a promising new approach for generating all types of desired immunity, such as cytotoxic T lymphocytes, T helper cells and antibodies, whilst being a technology that has the potential for global usage in terms of manufacturing ease, broad population administration and safety. Recent studies have demonstrated that bacterial DNA and oligonucleotides (CpG DNA) can stimulate immune responses and have potential for use as novel agents to enhance immunogenicity.16 The breadth of applications for DNA vaccines thus ranges from prophylactic vaccines to immunotherapy for infectious diseases, cancer, and autoimmune and allergic diseases.17

In this study, as a way to skew immune responses towards antigen-specific, Th1 responses by DNA-based immunization, we constructed a mammalian expression plasmid that contained a fusion gene of the anti-CD3 single-chain Fv cDNA (sFv) and murine mature IL-18 cDNA, linked by a spacer of six amino acid-encoded nucleotides (pAnti-CD3sFv/IL-18). Previously, we reported that the covalent linkage of IL-2 and anti-CD3sFv confined the effect of IL-2 to CD3+ T cells, leading to the selective protection of CD3+ T cells from dexamethasone-induced apoptosis. Furthermore, the anti-CD3sFv/IL-2 protein strongly increased the CD3+ T-cell population in spleen cells, as demonstrated by both in vitro and in vivo models.18

In this report, our results show that pAnti-CD3sFv/IL-18 DNA increases the production of both OVA-specific IFN-γ and anti-OVA IgG2a in OVA-sensitized mice, thereby leading to the efficient induction of Th1 immune responses in an antigen-specific manner.

Materials and methods

Materials, cells and mice

Murine recombinant IFN-γ and recombinant IL-4 were obtained from Genzyme Co. (Cambridge, MA). Ovalbumin (OVA) was obtained from ICN Biomedicals (Irvine, CA). Anti-OVA IgG1 and IgG2a monoclonal antibodies (mAbs) were used as standards for each isotype-specific enzyme-linked immunosorbent assay (ELISA). Rabbit anti-OVA serum was purchased from Cappel Co. (Durham, NC). Polyclonal mouse anti-OVA were prepared from the sera of immunized mice after repeated injections of OVA in complete Freunds adjuvant, followed by OVA in incomplete Freunds adjuvant. Horseradish peroxidase (HRP)-labelled goat anti-mouse IgG1 or IgG2a were purchased from Southern Biotechnology Associates Inc. (Birmingham, AL). HeLa cells and spleen cells were grown in Dulbecco's modified Eagle's minimal essential medium (DMEM) containing 10% fetal bovine serum and antibiotics. HeLa cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD). Female, 6- to 8-week-old BALB/c (H-2d) mice were purchased from Daehan Animal Co., Ltd (Seoul, Korea). The mice were maintained and treated according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

Construction of an expression plasmid carrying an anti-CD3sFv/IL-18 gene

A mammalian expression plasmid (donated from Dr M. E. Reff),19 containing an SV40 origin of replication and designed for expression of immunoglobulin genes, was modified to eliminate most of the immunoglobulin-coding regions as well as the neomycin-resistance gene. The plasmid pAnti-CD3sFv/IL-18 was constructed by first inserting the anti-CD3sFv cDNA in-frame with the human immunoglobulin κ leader sequence, to permit secretion of the translated protein. Then, the anti-CD3sFv cDNA was cloned by polymerase chain reaction (PCR) from an anti-CD3sFv-containing plasmid (obtained from Dr B. R. Blazar, University of Minnesota, Minneapolis, MN) using primers carrying the desired restriction sites. Thereafter, the mature IL-18 cDNA was inserted downstream of the anti-CD3sFv cDNA, separated by a spacer encoding six amino acid residues (SSGGGG) (Fig. 1a). The mature IL-18 cDNAs were also cloned by PCR from previously cloned cDNA constructs (obtained from Dr D. S. Lim, Daejon, Korea) using primers containing the desired restriction sites. The base sequences of primers used in this study are as follows: anti-CD3sFv primers (DraIII-anti-CD3sFv 5′-GCGCCACGATGTGATGACATCCAGATGACCCAG-3′; anti-CD3sFv-XhoI 5′-CCCGCTCGAGCCTCCGGAGACGGTGACCATGGT-3′); and IL-18 primers (XhoI-IL-18 5′-CCGCTCGAGCGGAAACTTTGGCCGACTT-3′; IL-18stop-BglII 5′-GAAGATCTCTAACTTTGATGTAAGTT-3′). As controls, the plasmids pAnti-CD3sFv and pIL-18 were constructed by PCR using primers containing a stop codon at each 3′ primer of both genes: anti-CD3sFv primers (DraIII-anti-CD3sFv 5′-GCGCCACGATGTGATGACATCCAGATGACCCAG-3′; anti-CD3sFvstop-BamHI 5′-CGCCGGATCCCTACCTCCGGAGACGGTGACCATGGT-3′); and IL-18 primers (DraIII-IL-18 5′-TTTCCACGATGTGAAACTTTGGCCGACTT-3′; IL-18stop-BglII 5′-GAAGATCTCTAACTTTGATGTAAGTT-3′). The constructed plasmids were, respectively, electroporated into Escherichia coli and purified from large-scale cultures by alkaline lysis and caesium chloride density-gradient centrifugation. The endotoxin levels of the purified plasmids were <20 endotoxin unit (EU)/mg of DNA, as detected by the limulus amebocyte lysate (LAL) assay kit (BioWhittaker, Walkersville, MD).

Figure 1.

Figure 1

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Expression of a recombinant anti-CD3sFv/interleukin-18 (IL-18) protein by HeLa cells transfected with pAnti-CD3sFv/IL-18 DNA. (a) Schematic drawing of expression plasmids carrying an anti-CD3sFv gene or an anti-CD3sFv/IL-18 fusion gene. (b) Western blot analysis of the anti-CD3sFv/IL-18 protein expressed by the transfected HeLa cells. The recombinant anti-CD3sFv and anti-CD3sFv/IL-18 proteins, purified by immunoprecipitation, were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), immunoblotted, and detected by anti-IL-18 monoclonal antibody (mAb). Lane M represents Coomassie blue-stained molecular weight markers. (c) IL-18 bioactivity of recombinant proteins produced by HeLa cells transfected with pAnti-CD3sFv/IL-18 DNA or pAnti-CD3sFv DNA. The culture supernatants were titred in the presence of either anti-mIL-18 mAb or isotype-control mAb. The values shown represent the mean ± standard deviation (SD) of triplicate determinations. *P < 0.01, relative to any other groups.

Transfection, sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblot analysis

HeLa cells were transfected with pAnti-CD3sFv/IL-18 or pAnti-CD3sFv DNA using the Superfect transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The culture supernatants from the transfected cells were harvested after 3 days, and the secreted proteins were immunoprecipitated by using CD3-conjugated Sepharose 4B resin (Sigma Chemical Co., St Louis, MO). The resins were washed three times with 0.1% Tween-20 in Tris buffer (pH 8.0), and the precipitated proteins were analysed by SDS–PAGE in a Mini-Protein II gel apparatus (Bio-Rad, Richmond, CA) using gradient gels. Afterwards, proteins were transferred onto nitrocellulose membrane by semidry electroblotting, as previously described.20 The blots were probed with mouse anti-IL-18 mAb, washed and exposed to HRP-labelled anti-mouse IgG. The blots were then developed with the ECL system (Amersham, Arlington Heights, IL) according to the manufacturer's protocol.

Sensitization of mice with OVA and intramuscular injection of plasmid DNA

Mice were injected intraperitoneally (i.p.) with 200 µg of OVA adsorbed on 5 mg of Al(OH)3 (alum) adjuvant, followed by one additional injection with 200 µg of OVA alone. One week later, mice were injected intramuscularly (i.m.), with varying concentrations of each plasmid DNA in 100 µl of 0.85% normal saline, into each quadricep muscle by using a 26-gauge insulin syringe. The quadricep muscles were visualized by making a 1-cm incision in the skin with a microdissecting scissor. The injection depth of the needle was adjusted to 2 mm by using a steel collar, as previously described.21

In vitro stimulation of splenic CD4+ T cells

Spleens from mice were aseptically removed and single-cell suspensions were prepared as previously described.22 CD4+ T cells were purified by negative selection using magnetic antibody cell sorting (MACS) with a cocktail of biotinylated anti-mouse CD8, I-A, B220, and Mac-1 antibodies (Miltenyi, Sunnyvale, CA), as previously described.20 More than 98% of the isolated cells were CD4+ T cells, as analysed by cytofluorometry. For cytokine assay, 5 × 105 spleen CD4+ T cells were distributed into each well of 96-well plates and incubated in vitro with 100 µg of an antigen (OVA) for 4 days, after which the levels of IFN-γ and IL-4 in culture supernatants were determined by ELISA.

Cytokine assays

The quantities of IFN-γ and IL-4 in culture supernatants were determined by sandwich ELISA using mAbs specific for mouse IFN-γ and IL-4 (Genzyme Co.) as previously described.23 The mAbs for coating the plates and detecting the cytokines were as follows: for IFN-γ, rat anti-mouse IFN-γ (HB170) and biotinylated rat anti-mouse IFN-γ (XMG1.2); for IL-4, rat anti-mouse IL-4 (BVD4) and biotinylated anti-mouse IL-4 (BVD6). The lower limit of detection was 125 pg/ml for IFN-γ and 3 pg/ml for IL-4. Recombinant mouse IFN-γ and IL-4 (Genzyme Co.) were used as standards. The biological activity of IL-18 produced by the transfectants was established by determining its ability to stimulate IFN-γ production in spleen cells in vitro, as previously described.24 In brief, 2 × 106 spleen cells were cultured in 2 ml of cell-culture medium in 12-well plates in the presence of the transfectants' supernatants, and the IFN-γ levels in the supernatants were determined by IFN-γ-specific ELISA.

Determination of the anti-OVA Ig2a isotype

Blood samples were obtained from mice during the course of the experiments, and the concentrations of OVA-specific IgG2a in the sera were measured by ELISA. Briefly, ELISA plates were coated with 100 µl of 5 µg/ml/well of OVA. After coating, serial dilutions of sera were added to the plates, which were then incubated overnight at 4°. After washing, HRP-labelled anti-mouse IgG2a was added and the plates were incubated for 2 hr at room temperature. After additional washing, o-phenylenediamine (OPD) substrate was added and the plates were incubated for 10 min after which the absorbance (A) of each well was determined at 492 nm in a Kinetic Microplate Reader (Molecular Devices, Sunnyvale, CA).

Statistical analyses

The Student's t-test and one-way analysis of variance (anova) followed by the Bonferroni method were used to determine the statistical significance of differences between the values of experimental and control groups. P-values of < 0.05 were considered statistically significant.

Results

Anti-CD3sFv/IL-18 fusion protein expressed in HeLa cells is biologically active

The expression plasmid carrying an anti-CD3sFv/IL-18 fusion gene (pAnti-CD3sFv/IL-18; Fig. 1a) was constructed as described in the Materials and methods. The pAnti-CD3sFv/IL-18 expression plasmid was tested, by transient transfection of HeLa cells, for its ability to produce recombinant anti-CD3sFv/IL-18 fusion protein. As a control, the plasmid was also constructed to carry an anti-CD3sFv gene (pAnti-CD3sFv) and the recombinant anti-CD3sFv protein was expressed in HeLa cells. The recombinant anti-CD3sFv and anti-CD3sFv/IL-18 proteins purified by CD3 immunoprecipitation were characterized by Western blot analysis using an anti-IL-18 mAb. As shown in Fig. 1(b), Western blot analysis showed a band that corresponds to the expected size (≈48 000 molecular weight) for an anti-CD3sFv/IL-18 protein. The anti-CD3sFv/IL-18 protein was immunoblotted with an anti-mIL-18 mAb, while the anti-CD3sFv protein was not.

To further determine whether anti-CD3sFv/IL-18 protein secreted by the transfected HeLa cells retained bioactivity for IL-18, the ability of anti-CD3sFv/IL-18 protein to induce IFN-γ production in spleen cells was determined. As shown in Fig. 1(c), the culture supernatants of HeLa cells transfected with pAnti-CD3sFv/IL-18 DNA enhanced IFN-γ production by mouse spleen cells, which was blocked by anti-IL-18 antibody, indicating that an IL-18 component in the anti-CD3sFv/IL-18 fusion protein is biologically active. In contrast, the culture supernatants from HeLa cells transfected with the pAnti-CD3sFv control DNA did not enhance IFN-γ production by spleen cells.

Intramuscular injection of pAnti-CD3sFv/IL-18 DNA increased production of OVA-specific IgG2a in OVA-sensitized mice

To determine whether vaccination with pAnti-CD3sFv/IL-18 DNA efficiently induces Th1-mediated immune responses in an antigen-specific manner, BALB/c mice were injected i.p. with OVA/alum, as described in the Materials and Methods, and then immunized i.m. with varying amounts (0–500 µg) of pAnti-CD3sFv/IL-18, pAnti-CD3sFv, or empty vector (control). Mice were bled 2 weeks after the injection, and the levels of anti-OVA IgG2a in the sera were determined. As indicated in Fig. 2, injection with pAnti-CD3sFv/IL-18 induced a significant level of anti-OVA IgG2a at injection doses of 100, 200 and 500 μg. Importantly, the levels of anti-OVA IgG2a in the mice injected with pAnti-CD3sFv/IL-18 DNA were significantly higher than those in the mice injected with pAnti-CD3sFv DNA.

Figure 2.

Figure 2

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Production of anti-ovalbumin (OVA) IgG2a in OVA-sensitized mice injected with pAnti-CD3sFv/interleukin-18 (IL-18) DNA. BALB/c mice (five per group) were injected with OVA/alum, as described in the Materials and methods, and then injected intramuscularly with varying amounts of pAnti-CD3sFv/IL-18, pAnti-CD3sFv DNA, or empty vector (control). The mice were bled 2 weeks after injection and the levels of anti-OVA IgG2a were determined by enzyme-linked immunosorbent assay (ELISA). The values shown represent the mean ± standard error (SE) of five mice. The experiment was repeated twice, with similar results obtained on each occasion. *P < 0.001, relative to a group injected with pAnti-CD3sFv DNA.

pAnti-CD3sFv/IL-18 DNA induced and maintained Th1 responses in OVA-sensitized mice

We next used a time-course study to investigate the production of anti-OVA IgG2a after injection with pAnti-CD3sFv/IL-18 DNA. The mice were sensitized with OVA/alum and then injected i.m. with 100 µg of pAnti-CD3sFv/IL-18 or pAnti-CD3sFv, and were bled at 1, 2, 4 and 6 weeks after injection. The levels of anti-OVA IgG2a in the sera were determined. As shown in Fig. 3, anti-OVA IgG2a was detected, even at the week-1 time-point after injection, and the levels were sustained for the entire 8-week observation period. Mice injected with pAnti-CD3sFv/IL-18 DNA maintained higher levels of anti-OVA IgG2a during the 6-week observation period than the mice injected with pAnti-CD3sFv DNA.

Figure 3.

Figure 3

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Sustained production of anti-ovalbumin (OVA) IgG2a in OVA-sensitized mice injected with pAnti-CD3sFv/interleukin-18 (IL-18) DNA. BALB/c mice (five per group) were injected with OVA/alum and then injected intramuscularly with 100 µg of pAnti-CD3sFv/IL-18, pAnti-CD3sFv DNA, or empty vector (control). The mice were bled 1, 2, 4 and 6 weeks after injection and the blood samples were used to determine the levels of anti-OVA IgG2a in the sera. The values shown represent the mean ± standard error (SE) of five mice. The experiment was repeated twice, with similar results obtained on each occasion. *P < 0.001, relative to a group injected with pAnti-CD3sFv DNA.

We also examined whether vaccination with pAnti-CD3sFv/IL-18 DNA increased the production of IFN-γ, a Th1 cytokine, in OVA-sensitized BALB/c mice. Mice were injected with OVA/alum and then immunized i.m. with either pAnti-CD3sFv/IL-18 DNA (100 µg) or pAnti-CD3sFv DNA (100 µg), and the spleens from the immunized mice were removed at 1, 2, 4 and 6 weeks after the injection, and splenic CD4+ T cells from each treatment were stimulated in vitro with 100 µg of OVA. IFN-γ production in culture supernatants was determined by ELISA. As shown in Fig. 4, splenic CD4+ T cells from the mice injected with pAnti-CD3sFv/IL-18 DNA produced significantly higher levels of IFN-γ than those from the mice injected with pAnti-CD3sFv DNA when the cells were stimulated in vitro with OVA.

Figure 4.

Figure 4

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Ovalbumin (OVA)-specific, interferon-γ (IFN-γ) production in splenic CD4+ T cells from OVA-sensitized mice injected with pAnti-CD3sFv/interleukin-18 (IL-18) DNA. BALB/c mice (three per group) were injected with OVA/alum and then injected intramuscularly with 100 µg of pAnti-CD3sFv/IL-18, pAnti-CD3sFv DNA, or empty vector (control). The mice were killed 1, 2, 4 or 6 weeks after injection and splenic CD4+ T cells were prepared and stimulated in vitro with 100 µg of OVA for 4 days. The levels of interferon-γ (IFN-γ) in the culture supernatants were analysed by enzyme-linked immunosorbent assay (ELISA). The values shown represent the mean ± standard error (SE) (n = 3). *P < 0.01, relative to a group injected with pAnti-CD3sFv DNA.

pAnti-CD3sFv/IL-18 DNA was more efficient than a mixture of pAnti-CD3sFv DNA + pIL-18 DNA in inducing Th1 immune responses

To investigate the importance of the direct linkage between anti-CD3sFv and IL-18 in the pAnti-CD3sFv/IL-18 plasmid, we included a group of mice injected with a mixture of pAnti-CD3sFv DNA and pIL-18 DNA. BALB/c mice were injected with OVA/alum and then immunized i.m. with 100 µg of pAnti-CD3sFv or pAnti-CD3sFv/IL-18 DNA, or with a simple mixture of pAnti-CD3sFv DNA (100 µg) + pIL-18 DNA (100 µg). Two weeks later, the levels of IFN-γ produced by the splenic CD4+ T cells were determined after stimulation in vitro with 100 µg of OVA. Five mice of each group were bled 2 weeks after the injection and the levels of anti-OVA IgG2a in the sera were determined. As indicated in Fig. 5(a), the levels of anti-OVA IgG2a were significantly higher in mice injected with pAnti-CD3sFv/IL-18 DNA alone than in those injected with pAnti-CD3sFv DNA alone, or with a mixture of pAnti-CD3sFv DNA + pIL-18 DNA. Furthermore, injection with pAnti-CD3sFv/IL-18 DNA also increased OVA-specific, IFN-γ production to a significantly higher level in CD4+ T cells than injection with pAnti-CD3sFv DNA, or with a mixture of pAnti-CD3sFv DNA and pIL-18 DNA (Fig. 5b).

Figure 5.

Figure 5

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pAnti-CD3sFv/interleukin-18 (IL-18) DNA is more efficient than a simple mixture of pAnti-CD3sFv DNA + pIL-18 DNA in inducing T helper 1 (Th1) immune responses. BALB/c mice were injected with ovalbumin (OVA)/alum and then injected intramuscularly with 100 µg of pAnti-CD3sFv/IL-18, 100 µg of pAnti-CD3sFv DNA, or with a mixture of 100 µg of pAnti-CD3sFv DNA + 100 µg of pIL-18 DNA. (a) Two weeks after injection, the levels of anti-OVA IgG2a in the sera were determined. The values shown represent the mean ± standard error (SE) of five mice. The data are representative of two separate experiments. *P < 0.001, relative to a group injected with a mixture of pAnti-CD3sFv DNA + pIL-18 DNA. (b) The levels of interferon-γ (IFN-γ) produced from CD4+ T cells were determined after in vitro stimulation with 100 µg of OVA. The values represent the mean ± SE (n = 3). **P < 0.001, relative to a group injected with a mixture of pAnti-CD3sFv DNA + pIL-18 DNA.

Therefore, pAnti-CD3sFv/IL-18 DNA was more efficient than a simple mixture of pAnti-CD3sFv DNA and pIL-18 DNA in inducing antigen-specific, Th1 immune responses.

pAnti-CD3sFv/IL-18 DNA inhibited OVA-specific, IL-4 production in CD4+ T cells

To determine whether vaccination with pAnti-CD3sFv/IL-18 DNA inhibited the OVA-specific, Th2 immune response, BALB/c mice were injected with OVA/alum and then immunized i.m. with varying amounts of pAnti-CD3sFv/IL-18 DNA or pAnti-CD3sFv DNA. Two weeks after injection, the levels of IL-4 produced by splenic CD4+ T cells were determined after in vitro stimulation with 100 µg of OVA. As indicated in Fig. 6(a), the levels of IL-4 production in the mice immunized with pAnti-CD3sFv/IL-18 DNA were significantly lower than those in the mice injected with pAnti-CD3sFv DNA at doses of 50, 100 and 200 µg. In addition, pAnti-CD3sFv/IL-18 DNA is more efficient than a mixture of pAnti-CD3sFv DNA and pIL-18 DNA in inhibiting IL-4 production (Fig. 6b).

Figure 6.

Figure 6

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pAnti-CD3sFv/interleukin-18 (IL-18) DNA is more efficient than a simple mixture of pAnti-CD3sFv DNA + pIL-18 DNA in inhibiting interleukin-4 (IL-4) production. (a) BALB/c mice (five per group) were injected with ovalbumin (OVA)/alum and then injected intramuscularly with varying amounts of pAnti-CD3sFv/IL-18, pAnti-CD3sFv DNA, or empty vector (control). Two weeks after injection, the production of IL-4 from splenic CD4+ T cells was determined after in vitro stimulation with 100 µg of OVA. The values shown represent the mean ± SE (n = 3). *P < 0.001, relative to a group injected with pAnti-CD3sFv DNA. (b) BALB/c mice were injected intramuscularly with 100 µg of pAnti-CD3sFv/IL-18, pAnti-CD3sFv DNA, or with a mixture of 100 µg of pAnti-CD3sFv DNA + 100 µg of pIL-18 DNA. The values shown represent the mean ± SE (n = 3). **P < 0.01, relative to a group injected with a mixture of pAnti-CD3sFv DNA + pIL-18 DNA.

Therefore, the pAnti-CD3sFv/IL-18 DNA was more efficient than a simple mixture of pAnti-CD3sFv DNA and pIL-18 DNA in inhibiting antigen-specific IL-4 production.

Discussion

In this report we have shown that the linkage between IL-18 and anti-CD3sFv genes confined the effect of IL-18 to CD3+ T cells, leading to the induction of Th1 immune responses in an antigen-specific manner in OVA-sensitized mice, when the mice were injected i.m. with the DNA construct pAnti-CD3sFv/IL-18. The induced Th1 response was characterized by high titres of OVA-specific IgG2a in sera, and by high levels of IFN-γ production from the OVA-specific CD4+ T cells. In contrast, injection with a simple mixture of pIL-18 DNA and pAnti-CD3sFv DNA also increased the production of OVA-specific IFN-γ and anti-OVA IgG2a; however, these increased levels were significantly lower than those stimulated by injection with pAnti-CD3sFv/IL-18 DNA. Thus, the Th1 effects of IL-18 were efficiently confined to the OVA-specific T cells by the linkage of IL-18 to the anti-CD3sFv. The pAnti-CD3sFv/IL-18 DNA also efficiently induced OVA-specific, Th1-mediated immune responses in C57BL/c mice (H-2d), indicating that these phenomena were not strain-specific (data not shown). Furthermore, the anti-CD3sFv/IL-18 DNA inhibited OVA-specific, IL-4 production from CD4+ T cells in OVA-sensitized mice (Fig. 6). The levels of anti-OVA IgE in sera were significantly lowered in mice injected with the pAnti-CD3sFv/IL-18 DNA although the levels of anti-OVA IgG1 were not significantly different among the mice injected with pAnti-CD3sFv/IL-18, pAnti-CD3sFv, or a mixture of pAnti-CD3sFv and pIL-18 (data not shown).

DNA immunization has been proven to be effective in eliciting protective immune responses to a variety of pathogens, from viruses to parasites. It seems that efficient uptake of the plasmid vector, without any carrier molecule, is a result of the intrinsic characteristics of striated muscles.25 Whitten & Yokoyama reported that proteins expressed by DNA vaccines induce both local and systemic immune responses.26 Our data suggested that secretion of the intact fusion protein by resident tissues occurred because we were able to confirm expression of the OVA/IL-18 protein by the HeLa cells transfected with pAnti-CD3sFv/IL-18 DNA and the pAnti-CD3sFv/IL-18 DNA vaccination demonstrated in vivo the biological effects of IL-18. Furthermore, several factors, including the identity of the antigen and the route of DNA administration, have been suggested to determine the types of immune response following DNA immunization.27,28 In the present study, we demonstrated that the direct linkage of a Th1 cytokine, IL-18, to an anti-CD3sFv could efficiently direct the immune responses towards a Th1 profile in antigen-sensitized mice.

Although the best-known function of IL-18 thus far is to induce IFN-γ production by T cells and NK cells, IL-18 has not demonstrated IFN-γ-inducing activity, to date, when used as the sole source of stimulation. Murine IL-18 enhances IFN-γ production by non-adherent splenocytes and established Th1 clones in the presence of anti-CD3, concanavalin A, IL-2, IL-12, or antigen plus APCs.12 In particular, IL-18 and IL-12 exhibit a marked synergism in IFN-γ induction in T cells because IL-12 up-regulates expression of the IL-18 receptor on cells producing IFN-γ and IL-18 also up-regulates expression of the IL-12 receptor β2 subunit.29,30 Recent studies have shown that the secretion of mature IL-18 by IL-18 gene-inserted tumour cells, or vaccination with mature IL-18 cDNA, induced a Th1 type immune response in mice, leading to the induction of immunoprotective responses against cancer and infectious micro-organisms, respectively.31,32 We also found that i.m. injection with an expression plasmid encoding mature IL-18 cDNA induced persistent resistance in vivo to Mycobacterium avium infection in genetically susceptible BALB/6 mice.33

The reason why pAnti-CD3sFv/IL-18 DNA induces higher levels of an OVA-specific, Th1 response compared with the mixture of pAnti-CD3sFv DNA + pIL-18 DNA, is not clear. One possibility is that the expressed anti-CD3sFv/IL-18 protein may efficiently induce OVA-specific, Th1 immune responses by increasing the in vivo half-life of IL-18 activity of the anti-CD3sFv/IL-18 fusion protein compared with free recombinant IL-18. Gillies et al. reported that either IL-2 or granulocyte–macrophage colony-stimulating factor (GM-CSF) fused to antibodies had a prolonged half-life in serum compared with IL-2 or GM-CSF alone.34 However, our co-immunization experiments demonstrated much more absolute dependence on fusing the cytokine to the antigen for inducing antigen-specific, Th1 immune responses. Immunization with the mixture of pAnti-CD3sFv and pIL-18 also induced OVA-specific, Th1-like immune responses, which, however, were significantly less efficient than those induced with the pAnti-CD3sFv/IL-18 DNA vaccination. Tsuji-Takayama et al. demonstrated that IL-18, combined with anti-CD3 mAb, induced IFN-γ production by murine Th1 cells, while neither IL-18 or anti-CD3 mAb alone induced IFN-γ production.35 Therefore, the linkage between anti-CD3sFv and IL-18 allows the Th1 effect of IL-18 to localize sites in immune organs where OVA-specific T cells are being activated. For example, the pAnti-CD3sFv/IL-18 fusion DNA may be more efficient than a mixture of pAnti-CD3sFv + pIL-18 in stimulating OVA-specific Th1 cells to produce IFN-γ. The presence of cytokines in the microenvironment of antigen-specific immune responses confers advantages in T-cell immunobiology and cancer therapy, as direct injection of cytokine gene-containing vectors into tumour cells, and injection of cells secreting specific cytokines, have been shown to result in tumour regression and acquisition of systemic anti-tumour immunity.36,37 In addition, further experiments should be carried out to determine whether vaccination with pAnti-CD3sFv/IL-18 DNA can convert the established Th2 immune responses and also whether combinations of pAnti-CD3sFv/IL-18 DNA and pAnti-CD3sFv/IL-12 DNA synergistically induce antigen-specific, Th1 immune responses. IL-18 synergizes with IL-12 for IFN-γ production and the induction of Th1-mediated responses.38,39

In conclusion, the pAnti-CD3sFv/IL-18 fusion DNA is more efficient than a simple mixture of pAnti-CD3sFv DNA and pIL-18 DNA in inducing OVA-specific, Th1-mediated immune responses and in inhibiting OVA-specific, IL-4 production. These studies suggest that the direct linkage of IL-18 to anti-CD3sFv can confine the Th1 effect of IL-18 to OVA-specific T cells and therefore an antigen/IL-18 plasmid may be beneficial in the treatment of diseases caused by undesired Th2 responses, including certain parasitic infections and allergic diseases.

Acknowledgments

We thank Drs B. R. Blazar, M. E. Reff and D. S. Lim for providing valuable reagents, and Dr S. H. Choi for helpful discussion. This work was supported by a grant from the KRF made in the program year of 1998 (1998–019-F00043).

Abbreviations

APCs

antigen-presenting cells

ELISA

enzyme-linked immunosorbent assay

HRP

horseradish peroxidase

IFN-γ

interferon-γ

IgG2a

immunoglobulin G2a isotype

IL

interleukin

i.m.

intramuscular

i.p.

intraperitoneal

mAb

monoclonal antibody

NK

natural killer

OVA

ovalbumin

PCR

polymerase chain reaction

sFv

single-chain Fv

SDS–PAGE

sodium dodecyl sulphate–polyacrylamide gel electrophoresis

Th

T helper.

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