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Intratumoral coinjection of two adenoviral vectors expressing functional interleukin-18 and inducible protein-10, respectively, synergizes to facilitate regression of established tumors

Abstract

We have constructed two recombinant adenoviral vectors AdVIP-10 and AdVIL-18 expressing the functional chemokine IFN-γ inducible protein (IP)-10 and cytokine interleukin (IL)-18, respectively. Injection of either AdVIP-10 or AdVIL-18 subcutaneously into tumor nodules derived from the J558 murine myeloma cell line delayed some tumor growth but it was not curative in all cases. Coinjection of these two vectors at the same tumor nodule not only significantly suppressed the tumor growth, but also cured established tumors in 8 of 10 (80% tumor free) mice. The latter treatment stimulated T-cell infiltration into tumors in association with tumor necrosis formation, induced a type 1 immune response and induced the activation of J558 tumor–specific cytotoxic T lymphocytes. Moreover, the antitumor activity of IP-10 and IL-18 combined gene therapy was significantly diminished in mice with depletion of either CD4+ (50% tumor free) or CD8+ (40% tumor free) T cells, and completely lost (0% tumor free) in T cell–deficient nude and IFN-γ knockout mice, indicating the critical roles of T cells and IFN-γ in this therapeutical model. Taken together, the findings of this study demonstrate that the combined use of two adenoviral vectors expressing IP-10 and IL-18, respectively, synergize to facilitate regression of established tumors. These observations also suggest the potential use of double-recombinant adenoviral vectors expressing chemokines and immunomodulatory cytokines in cancer gene therapy.

Main

Interleukin (IL)-18 is a cytokine that was originally designated as gamma interferon (IFN-γ)–inducing factor (IGIF).1 This cytokine strongly induces IFN-γ production in spleen cells and enhances the expression and function of Fas ligand on T cells.2,3 Furthermore, it induces the production of granulocyte–macrophage colony-stimulating factor (GM-CSF).4 The in vitro expression of IL-2 receptor by T cells is enhanced by IL-18, as is the production of IL-2. IL-18 also enhances the production of Th1-type cytokines, which are associated with antitumor cytotoxic T-cell responses,5 and plays some roles in synergy with IL-12, particularly in the induction of IFN-γ.1,6 The use of IL-18 in the treatment of cancer has not been thoroughly investigated. Recently, Osaki et al demonstrated that administration of IL-18 before and after challenge with mouse melanoma cells significantly suppressed tumor growth and reduced the number of mice developing tumors from 60% to 20%.7 More recently, it has been shown that IL-18 can induce antitumor immune responses that are mediated by both CD4+ and CD8+ T cells and associated with IFN-γ production.8,9

IFN-γ inducible protein-10 (IP-10)10 is a chemokine that belongs to the CXC chemokine family known to stimulate the IP-10 receptor CXCR3.11 IP-10, which is produced by activated monocytes, fibroblasts, endothelial cells, and keratinocytes can induce chemotaxis of activated T cells and inhibit angiogenesis.12 IP-10 binds to a seven-transmembrane G protein–coupled receptor, CXCR3, expressed on activated T cells, leading to chemotaxis.11 Stably transfected tumor cell lines expressing IP-10 were rejected through an immune system–mediated mechanism.13 Furthermore, it has been recently shown that IP-10 also displays its ability to impair tumoral angiogenesis.14 Therefore, IP-10 has been involved in the antitumor immune responses by recruitment of T cells to the malignancies and the nonimmune responses by its antiangiogenesis effect. These data suggest that both IL-18 and IP-10 have potent antitumor effects and are potent candidates for transgene therapy of cancer.

Adenoviral vectors have been extensively used for transimmunogene delivery into tumors in cancer gene therapy.15,16,17 In this study, we constructed two adenoviral vectors AdVIL-18 and AdVIP-10 expressing IL-18 and IP-10 molecules, respectively, and investigated the antitumor effects by intratumoral coinjection of these two adenoviral vectors into J558 tumors established in BALB/c mice.

Materials and methods

Antibodies, cell lines, and mice

Rat monoclonal antibodies Gk1.5 (anti-CD4) and 3.155 (anti-CD8) were purified by affinity chromatography from the respective hybridoma ascites. These two hybridoma cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD). The biotin-conjugated anti-mouse H-2Kd, Iad, CD3, CD4, CD8, CD25, CD28, CD40L, CD69, Fas, and FasL antibodies as well as the phycoerythrin (PE)-conjugated anti-mouse IL-4, IFN-γ, and perforin antibodies were obtained from Pharmingen (Mississauga, Ontario, Canada). The fluorescein isothiocyanate (FITC)-conjugated avidin was obtained from Bio/Can Scientific (Mississauga, Ontario, Canada). J558 and SP2/0 are poorly immunogenic mouse myeloma cell lines obtained from ATCC, and were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS), penicillin, and streptomycin (100 μg/mL). Female BALB/c mice and T cell–deficient nude as well as IFN-γ knockout (KO) mice on BALB/c background (4–6 weeks old) were obtained from Charles River (St. Constant, Quebec, Canada) and The Jackson Laboratory (Bar Harbor, ME), respectively, and maintained in the animal facility at the Saskatoon Cancer Center.

Recombinant adenoviral vectors

A 1-kb cDNA fragment coding for the full open reading frame of mouse IP-10 gene was cloned by reverse transcription–polymerase chain reaction (RT-PCR) from a cDNA library of monocytes using the TaqI polymerase. The monocytes were collected by peritoneal lavage of mice given an intraperitoneal (i.p.) injection of 1.5 mL of thioglycollate broth (Becton Dickinson, San Jose, CA) 4 days before harvest.17 Two primers specific for the mouse IP-10 gene were used, namely, the sense primer (5′-ATGAA CCCAA GTGCT GCCGT C-3′) and the antisense primer (5′-TTAAG GAGCC CTTTT AGACC TTT-3′). The cloned cDNA fragment was ligated into the pCR2.1 vector (Invitrogene, Carlsbed, CA) to form pCR2.1-IP-10. The IP-10 sequence was verified by the dideoxy nucleotide sequencing method. The cDNA fragment of IP-10 (XbaI/HindIII) from the pCR2.1-IP-10 vector was further ligated into the pLpA plasmid to form the adenoviral vector pLpA-IP-10. The cDNA clone encoding IL-18 was generously provided by Dr Hua Yu, University of South Florida. The IL-18 cDNA insert (SalI and XbaI) was ligated into the plasmid pLpA to form the adenoviral vector pLpA-IL-18. The construction of recombinant adenoviruses AdVIP-10 and AdVIL-18 from pLpA-IP-10/pLpA-IL-18 and pJM17 vectors was conducted as previously described.17 The adenoviruses AdVLacZ expressing the E. coli β-galactosidase and AdVpLpA (i.e., with no gene insert) used as the marker and the control adenovirus, respectively, in this study were previously constructed in our laboratory.17 These E1-deleted replication-deficient recombinant adenoviruses under the control of the cytomegalovirus (CMV) early/immediate promoter/enhancer were amplified in 293 cell line (adenoviral E1 transformed human embryonic kidney cells), purified by cesium chloride ultracentrifugation and stored at −80°C.

Adenoviral transfection in vitro and in vivo

To test the susceptibility of J558 tumor cells to adenoviral infection, serial dilutions of AdVLacZ stock (2×1010 pfu/mL) were added to J558 cells seeded in triplicate in 96-well plates (1×105 cells/well) to form different multiplicities of infection (MOIs). The cells were incubated with the adenovirus in 293 serum-free medium (GIBCO, Gaithersburg, MD) for 2 hours at 37°C, then the media was replaced with DMEM/10% FCS and the cells incubated for an additional 24 hours at 37°C. To assess β-galactosidase expression16 the cells were fixed in formaldehyde/glutaraldehyde, then stained and counterstained with X-gal and nuclear fast red, respectively. The proportions of positive (i.e., blue-staining) cells were determined from triplicate wells and taken as the percentage of transduction. Control J558 cells transfected with AdVpLpA did not exhibit any intrinsic β-galactosidase activity or false-positive staining. We observed a dose-dependent response to the infecting dose of adenovirus, with maximal staining (70%) at an MOI of ≥200. Therefore, an MOI of 200 was selected for transfection of J558 cells with AdVIP-10 and AdVIL-18 in this study. For transfection of J558 cells with AdVIP-10 and AdVIL-18 at 200 MOI, following viral adsorption for 1 hour at 37°C in 24-well culture plates, the culture medium was replaced with DMEM/10% FCS and the cells (0.1×106 cells/well) incubated for another 24 hours at 37°C. The J558 tumor cells transfected with AdVIP-10 (termed J558/AdVIP-10) and AdVIL-18 (termed J558/AdVIL-18) and their culture supernatants were then harvested for examination of IP-10 and IL-18 expression using the reverse transcription polymerase chain reaction (RT-PCR) and functional analysis of secreted IP-10 and IL-18, respectively.

The ability of AdVLacZ to infect J558 tumors in vivo was also investigated. Briefly, 0.5×106 J558 cells were subcutaneously (s.c.) injected into each athymic nude mouse. Twelve days later, when the J558 tumors were 8 mm in diameter, bolus 50-μl aliquots of AdVLacZ virus (2×109 plaque-forming units [PFU] or 5-fold dilutions thereof) were injected into the J558 tumors. One day after viral injection, each J558 tumor was removed and cut into three approximately equal sections. These tumor tissues were mounted in tissue-freezing medium (Triangle Biomedical Sciences, Durham, NC) and snap-frozen by immersion in 2-methylbutane (JT Baker, Phillipsburg, NJ), that had been chilled over liquid nitrogen. Frozen 6-μm sections were obtained for the analysis of β-galactosidase expression.17 To accomplish this, the tissue sections of transfected J558 tumors were fixed in phosphate-buffered saline (PBS) containing 37% formaldehyde and 25% glutaraldehyde, stained with X-gal, and then counterstained with nuclear fast red (Poly Scientific, Bay Shore, NY). The mean proportion of blue-staining J558 cells from triplicate wells or from three tumor sections were taken as the percentage of transfection. We observed a dose-dependent response to the adenovirus, with maximum staining (40% of cells) at 2×109 PFU AdVLacZ. Thereafter, 2×109 PFU was selected as the optimal dose for delivery of AdVIP-10/AdVIL-18 to tumors in vivo.

RT-PCR analysis

Naı¨ve splenic T lymphocytes were nylon wool purified from BALB/c mice as previously described18 (>99% purity as determined using the anti-CD3 antibody by flow cytometry, data not shown). T cells were activated in DMEM, 10% FCS, IL-2 (20 U/mL), and Con A (2 μg/mL) for 4 days. Total RNA was obtained from the activated T lymphocytes, the wild-type J558, and the transfected J558/AdVIP-10 and J558/AdVIL-18 cells. The first-strand cDNA synthesis for the RT-PCR was performed with 5 μg of RNA using a commercial kit (Stratagene, La Jolla, CA), following the manufacturer's instructions. The PCR primers were specific for IP-10 (the sense primer, 5′-ATGAA CCCAA GTGCT GCCGT C-3′; the antisense primer 5′-TTAAG GAGCC CTTTT AGACC TTT-3′), IL-18 (the sense primer, 5′-CGTTG ACAAA AGACA GCGTG-3′; the antisense primer, 5′-CGTTG ACAAA AGACA GCGTG-3′), chemokine (IP-10) receptor CXCR3 (the sense primer, 5′-CCTAC GATTA TGGG AAAC GAG-3′; the antisense primer, 5′-TGATT CTCTC CGTGA AGATG ACG-3′, Ref.11), and glyceraldehyde phosphate dehydrogenase (GAPDH: sense primer, 5′-CAGGT TGTCT CCTGC GACTT-3′; antisense primer, 5′-CTTGC TCAGT GTCCT TGCTG-3′). The PCR conditions comprised 1 cycle at 94°C (5 minutes), 54°C (1 minute), and 72°C (1 minute) and 25 cycles at 94°C (1 minute), 55°C (1 minute), and 72°C (1 minute). All PCR reaction products were resolved on 1% agarose gels with ethidium bromide staining.

Chemotaxis assay

The chemotactic response of activated T lymphocytes to IP-1012 was examined using modified Boyden microchemotaxis chambers (Neuroprobe, Gaithersburg, MD) and polyvinyl pyrrolidone-free 5-μm pore-size polycarbonate membranes, essentially as described.18 Recombinant IP-10 (R&D Systems, Minneapolis, MN), diluted in DMEM/0.1% bovine serum albumin (BSA) to 0.1–1000 ng/mL, was added to triplicate lower chambers of the wells, and 105 activated T lymphocytes in DMEM/BSA were added to the upper chambers. After incubation for 2 hours at 37°C, the cells that had not migrated into the membranes were wiped from the upper surfaces of the membranes, which were then fixed in 70% methanol and stained using a Diff-Quik kit (American Scientific Products, McGraw Hill, IL). The lymphocytes that were associated with the membranes were enumerated by direct counting in a blinded fashion of at least nine 40× objective fields per well. The results are expressed as the mean number of cells/40× field (±SEM).

Functional activity of IL-18

To test the functional activity of IL-18, 3×106 splenocytes in 1 mL DMEM containing 10% FCS, anti-CD3 antibody (2 μg/mL) were added to each well of a 24-well plate, which contained 1 mL of culture supernatant from J558/AdVpLpA and J558/AdV-IL-18 cells, respectively. The cells were incubated at 37°C for 24 hours. Quantitation of secreted IFN-γ from cultured splenocytes was carried out in an enzyme-linked immunosorbent assay (ELISA) by using the IFN-γ ELISA kit (Endogene, Woburn, MA). The results were normalized to the known standard curves.

Animal studies

BALB/c mice (10 per group) were inoculated s.c. in their right thighs with 0.5×106 viable J558 tumor cells. Ten days later, when tumors reached 4–5 mm in diameter, different recombinant adenoviruses (AdVIP-10, AdVIL-18, AdVpLpA, or a mixture of AdVIP-10 and AdVIL-18) at 2×109 PFU consisting of 1×109 PFU of each virus were intratumorally injected in a volume of 10–20 μl. Tumor growth was monitored once every 2 days by measuring two perpendicular tumor diameters using a precision caliper. Animal showing severe distress or with tumors that exceeded 1.5 cm in diameter were sacrificed for ethical reasons according to the institutional guidelines. Animals with tumor regression were monitored for a total of 60 days. To induce bilateral tumors, 0.5×106 viable J558 tumor cells were injected s.c. into BALB/c mice (six per group) at both right and left thighs. To study the protective immunity, mice with tumor regression were rechallenged with s.c. injection of 0.5×106 J558 or SP2/0 tumor cells 2 months subsequent to the tumor regression.

To study the immune mechanisms involved in J558 tumor regression with adenovirus-mediated gene transfer, we investigated tumor growth or tumor regression in T-cell subset depleted, T cell–deficient nude and IFN-γ–deficient KO mice, respectively. T-cell subset depletion experiments were performed in vivo as described.19 Briefly, BALB/c mice (eight per group) were i.p. injected with anti-CD4 and anti-CD8 antibodies, respectively, at 0.5 mg per mouse 2 days before, on the day of and 3 and 6 days subsequent to the intratumoral injection of adenovirus. A total of four injections was given to each mouse. As a control, one group of mice was injected with the same amount of irrelevant isotype-matched rat antibodies, respectively. The depletion of CD4+ and CD8+ subsets of T cells in spleens were >95% by flow cytometric analysis as previously described9 (data not shown). Mice were monitored for tumor progression or regression as described above.

Histology

Tumor nodules or tissues at the injection sites were removed for histological analysis 1, 3, and 7 days subsequent to the coinjection of AdVIP-10 and AdVIL-18. J558 tumor nodules or tissues at the tumor inoculation site were fixed in 10% formaldehyde and embedded in paraffin. Sections of 6- to 7-μm thickness were stained with hematoxylin–eosin according to the standard procedures.

Phenotypic characterization of activated T and J558 tumor cells

Mouse spleens were removed for preparation of single-cell suspensions 2 weeks after treatment of AdVIP-10 and AdVIL-18. Red cells were lysed using 0.84% ammonium chloride. Splenocytes (5×106) were cocultured with 2×105 irradiated J558 cells in each well of a 24-well plate. Four days subsequently, the activated lymphocytes were purified from the cultures using Ficoll-Paque density gradient centrifugation and analyzed by flow cytometry. Briefly, these T cells were incubated for 1 hour on ice with the biotin-conjugated rat anti-mouse CD3, CD25, CD28, CD40L, CD69 and FasL antibodies (5 μg/mL), washed with PBS, then incubated for another 1 hour on ice with FITC-conjugated avidin. After another three washes with PBS, the cells were analyzed by flow cytometry. Isotype-matched irrelevant antibodies were used as controls. To examine the intracellular expression of cytokines, these activated T cells were processed using a commercial kit (Cytofix/CytoPerm Plus with GolgiPlug; Pharmingen), stained with RE-conjugated anti–IL-4, IFN-γ and perforin antibodies according to the manufacturer's protocols and analyzed by flow cytometry. For phenotypic characterization of SP2/0 tumor cells, these tumor cells were stained with the anti–H-2Kd, Iad, and Fas antibodies and analyzed by flow cytometry as described above.

Cytotoxicity assay

The activated T cells were further used as effector cells in chromium release assays. Target cells included J558 and SP2/0 tumor cells, which were radiolabeled with [51Cr]chromate. Ten thousand labeled target cells per well were mixed in triplicate with effector cells at various effector/target cell ratios, either as is or with anti–H-2Kd and anti-Iad antibodies (15 μg/mL) added, respectively, and were incubated for 6 hours. Percentage of specific lysis was calculated as: 100×[(experimental CPM−spontaneous CPM)/(maximal CPM−spontaneous CPM)]. Spontaneous count per minute (CPM) released in the absence of effector cells was less than 10% of specific lysis. The maximal CPM was released by adding 1% Triton X-100 to wells in the experiment.

Results

Functional IP-10 secreted by transfected J558/AdVIP-10 tumor cells

To examine IP-10 expression of transfected J558/AdVIP-10 cells, RNA extracted from J558/AdVIP-10 cells was subjected to RT-PCR analysis. As shown in Figure 1A, IP-10 expression was found in AdVIP-10–transfected J558/AdVIP-10, but not in the control adenovirus-transfected J558/AdVpLpA cells. To evaluate whether the secreted IP-10 is functional, the culture supernatant of J558/AdVIP-10, which contained IP-10 was subjected to chemotaxis analysis. To validate the IP-10–mediated T-cell response, we first demonstrated using RT-PCR that highly purified mouse activated T cells expressed substantial levels of the IP-10 receptor CXCR3 whereas J558 tumor cells did not (Fig 1B). We then assessed the activated T-cell chemotactic properties of recombinant IP-10, as well that of the culture supernatants of J558/AdVIP-10 tumor cells. Our data confirmed that both the recombinant IP-10 and the J558/AdVIP-10 supernatant (but not the control J558/AdVpLpA one) were able to chemoattract the activated T cells in a dose-dependent fashion (Fig 2).

Figure 1
figure1

Expression of chemokine IP-10, IL-18, and chemokine receptor CXCR3. A: RNA was obtained from wild-type J558 and the transfected J558/AdVIP-10 or J558/AdVIL-18 cells for examination of IP-10 or IL-18 expression. B: RNA was obtained from naı¨ve and activated T cells for examination of CXCR4 expression. The first-strand cDNA was synthesized from RNA using the reverse transcriptase, and the PCRs were conducted using sets of primers for IP-10, CXCR4, and the control “housekeeping” gene GAPDH, respectively.

Figure 2
figure2

IP-10 is a specific chemoattractant for activated T cells. A: Chemotaxis assays were performed using naı¨ve and activated T cells in response to recombinant IP-10 (0–1000 ng/mL). After a 2-hour chemoattraction period, the number of activated T lymphocytes that were associated with the membranes was determined by direct counting and the results expressed as the mean number of cells/40× field (±SEM). Chemotaxis assays were also performed using activated T cells in response to aqueous supernatants of J558/AdVpLpA and J558/AdVIP-10 cells. One representative experiment of two is shown.

Functional IL-18 secreted by transfected J558/AdVIL-18 tumor cells

To examine IL-18 expression of transfected J558/AdVIL-18 cells, RNA extracted from J558/AdVIL-18 cells was subjected to RT-PCR analysis. As shown in Figure 1A, IL-18 expression was found in AdVIL-18–transfected J558/AdVIL-18, but not in the control adenovirus-transfected J558/AdVpLpA cells. To evaluate whether the IL-18 is functional, the culture supernatant of J558/AdVIL-18 was added to the splenocyte culture. One day following incubation, the IFN-γ production in splenocyte culture with J558/AdVIL-18 supernatant was estimated to be 80 U/mL compared to the 20 U/mL seen in splenocyte culture with J558/AdVpLpA supernatant. These data indicate that IL-18 secreted by J558/AdVIL-18 cells is functional and able to significantly enhance IFN-γ production of splenocytes in vitro.

Intratumoral coinjection of AdVIP-10 and AdVIL-18 displayed synergistic antitumor effects

To explore whether AdVIP-10 or AdVIL-18 had therapeutic effects against tumors, J558 tumor cells were s.c. injected into syngeneic BALB/c mice. On day 10, tumor nodules ranging 4–5 mm in diameter were injected with 2×109 PFU of AdVIP-10, AdVIL-18, and the control adenovirus AdVpLpA. As shown in Figure 3, all tumors were lethal in the control group of mice. Tumor-bearing mice injected with AdVpLpA died within 4 weeks after tumor inoculation. Although either AdVIP-10 or AdVIL-18 treatment alone did not cure any mice, a significant delay of tumor growth was noted. However, no significant difference was found between these two treatment groups. To study the potent synergistic antitumor effects derived from a combinational treatment of AdVIP-10 and AdVIL-18, we then conducted coinjection of AdVIP-10 (1×109 PFU) and AdVIL-18 (1×109 PFU) into tumors simultaneously. As shown in Figure 3, the combinational treatment of AdVIP-10 with AdVIL-18 resulted in complete tumor regression in 8 of 10 (80%) mice. The tumor growth in the other 20% of mice was dramatically retarded. These data clearly indicate the synergistic antitumor effects derived from the combination treatment of AdVIP-10 and AdVIL-18.

Figure 3
figure3

Combination of intratumoral AdVIP-10 and AdVIL-18. Groups of mice bearing J558 tumor nodules inoculated 10 days before treatment onset were intratumorally injected with the indicated adenovirus (2×109 PFU). Individual evolution of the tumor diameter is shown, and the fraction of mice completely rejecting their tumors is indicated. One representative experiment of two is shown. We performed a statistic analysis on the combined data from two independent experiments by using the χ2 (chi-squared) test. We found that the mouse survival rates (tumor-free mice/total treated mice) in CD4+ and CD8+ T cell–depleted groups are significantly lower than that in the immune competent mouse group (P<.05).

The synergistic effect required coinjection of AdVIP-10 and AdVIL-18 in the same tumor nodule

To ascertain as to whether adenoviral gene transfer of IP-10 and IL-18 required expression of the therapeutic transgenes in the same tumor nodule for induction of the synergistic effects, we conducted experiments in which two tumor nodules were simultaneously generated by s.c. inoculation of J558 tumor cells into opposite thighs of the same mouse. As shown in Table 1, the combination treatment of AdVIP-10 and AdVIL-18 led to tumor nodule regression at the treated sites in five of six mice. However, treatment of mice with AdVIP-10 and AdVIL-18 at each thigh of the same mouse, respectively, did not cure any mice although the tumor growth in these mice was also delayed compared with that in mice treated with AdVpLpA injection. Their tumor growth curves are similar to those shown in Figure 3 (data not shown). These results indicate the requirement of coexpression of IP-10 and IL-18 in the same tumor nodule for induction of the synergistic antitumor effects.

Table 1 Tumor regression of bilateral s.c. J558 tumors upon AdVIP-10 and AdVIL-18 intratumoral treatment

IFN-γ played a critical role in the antitumor effect of combinational treatment

Because the antitumor immunity of both IP-10 and IL-18 is dependent on the induction of IFN-γ production, we further conducted an experiment to study the potent role of IFN-γ using the IFN-γ KO mice in this therapeutic tumor model. As shown in Figure 5 intratumoral treatment of AdVIP-10 and AdVIL-18 did not cure any tumor-bearing IFN-γ KO mice. Tumors grew aggressively. All eight mice died within 18 days after adenovirus injection, indicating the critical role of IFN-γ in tumor regression derived from the treatment of AdVIP-10 and AdVIL-18.

Figure 5
figure5

T-cell and IFN-γ requirement for tumor rejection with AdVIP-10 and AdVIL-18. J558 tumor cells were s.c. injected to groups of mice depleted of CD4+ and CD8+ T cells, and to groups of T cell–deficient nude mice and IFN-γ KO mice. When J558 tumor nodules reached 4–5 mm in diameter, tumor-bearing mice were intratumorally injected with the indicated adenovirus (2×109 PFU). Individual evolution of the tumor diameter is shown, and the fraction of mice completely rejecting their tumors is indicated. A group of mice receiving the polyclonal rat IgG rejected their tumor on treatment of AdVIP-10 and IL-18 as fast as those in mice not receiving antibody treatment (data not shown). One representative experiment of two is shown.

Both CD4+ and CD8+ T cells were involved in the antitumor efficacy of combined administration of AdVIP-10 and AdVIL-18

To study the cellular immune responses, tumor nodules or tissues at the injection sites were removed for histological analysis 12 hours or 2 and 6 days subsequent to the coinjection of AdVIP-10 and AdVIL-18. As shown in Figure 4A, the wild-type J558 tumors grew aggressively with many mitosis. Six hours postinjection, a few areas of tumor displayed a significant amount of lymphocyte infiltration associated with some isolated apoptotic tumor cells (Fig 4B). Twelve hours subsequent to the treatment, small foci of necrosis were observed in association with many lymphocytes in tumors (Fig 4C). Two days postinjection, multiple areas of necrosis occurred in the tumors (Fig 4D). By 6 days postinjection the tumor cells were no longer detectable, although numerous lymphocytes, macrophages, and fibroblasts were present (Fig 4E). These data suggest that T cells may be involved in the tumor necrosis formation leading to the complete tumor regression derived from the coinjection of AdVIP-10 and IL-18.

Figure 4
figure4

Lymphocyte infiltration into J558 tumors after coinjection of AdVIP-10 and AdVIL-18. BALB/c mice were s.c. injected with J558 tumor cells. Ten days later, when tumors reached 4–5 mm in diameter, each tumor was treated by coinjection of AdVIP-10 and AdVIL-18. At varying times thereafter injection site biopsies were processed for histologic assessment of the tissue responses. A: Section from a 10-day J558 tumor growing in a BALB/c mouse, showing brisk mitotic activity (arrows) without lymphocyte infiltration. B–E: Sections taken from tumors at 6 and 12 hours or 2 and 6 days subsequent to the coinjection of AdVIP-10 and AdVIL-18 into J558 tumors. B: Six hours subsequent to the treatment, J558 tumors displayed significant lymphocyte infiltration (arrows) and isolated apoptotic tumor cells (arrow heads). C: Twelve hours subsequent to the treatment, there were small foci of necrosis (center) associated with many lymphocytes (arrows). D: Areas of significant necrosis (arrows) appeared on day 2. E: Six days subsequent to the adenovirus injection, tumor cells were no longer detectable, with the injections sites comprising only lymphocytes, macrophages and fibroblasts. Sections were stained with hematoxylin and eosin. Magnification: ×240 for (A), (B), (C), and (E) and ×120 for (D).

To confirm the potent involvement of T cells in the antitumor immune responses, we further conducted the therapeutic experiments in CD4+ and CD8+ T cell–depleted or T cell–deficient mice. We found that 50% and 40% of CD4+ and CD8+ T cell–depleted mice became tumor free after intratumoral coinjection of AdVIP-10 and AdVIL-18 (Fig 5). The mouse survival rates in CD4+ and CD8+ T cell–depleted groups are significantly lower than that in the immune competent mouse group after the treatment (P<.05). These data suggest that both CD4+ and CD8+ T cells are involved in mediating the antitumor immune responses of combined treatment of AdVIP-10 and AdVIL-18. Furthermore, in T cell–deficient BALB/c nude mice, intratumoral treatment resulted in lethal tumor progression in all eight nude mice (Fig 5), further confirming the absolute T-cell requirement.

Protective immunity was against the wild-type J558 tumors

To examine whether the tumor regression of combination treatment results in the induction of protective immunity against the wild-type J558 tumor, eight mice that experienced tumor regression were rechallenged in a similar fashion using the parental J558 or SP2/0 tumor cells. We found that no J558 tumor growth was found in mice with tumor regression after the combination treatment, although the tumor growth of an irrelevant SP2/0 tumor cell line occurred in all these immunized mice (data not shown). These data illustrate that mice with tumor regression after the combination treatment of AdVIP-10 and AdVIL-18 induced a protective immune response specifically against the parental J558 tumor, but not against the irrelevant SP2/0 tumor.

Intratumoral coinjection of AdVIP-10 and AdVIL-18 induced type 1 antitumor immune responses

To elucidate the immune mechanism involved in tumor regression, the activated T cells were prepared by coculturing splenocytes of the mice with tumor regression with irradiated J558 tumor cells for 4 days. These T cells were purified with Ficoll-Paque gradient (Pharmacia Biotech, Uppsala, Sweden), and then subjected to phenotypic characterization by flow cytometry. As shown in Figure 6 (upper panel), essentially all of these cells were CD3+ and CD28+; they uniformly displayed high-level expression of the two T-cell activation markers, CD25 (IL-2R) and CD69, compared to low-level expression of these molecules in naı¨ve T cells, indicating they were indeed activated T cells. In addition, these activated T cells also displayed the costimulatory molecule CD40L and high level of FasL, compared to T cells from naı¨ve mice. Furthermore, following in vitro J558 cell challenge, these T cells stained positively for intracellular IFN-γ, but not for IL-4 (Fig 6, lower panel), indicating that the intratumoral treatment of AdVIP-10 and AdVIL-18 induced type 1 antitumor immune responses. In addition, these activated T cells also expressed significant amounts of intracellular perforin.

Figure 6
figure6

Phenotypic analysis of J558 tumor–specific T cells. Upper panel: Splenic lymphocytes from mice with tumor regression after treatment of AdVIP-10 and AdVIL-18 were stimulated in vitro for 4 days with irradiated J558 cells (activated T cells), then immunostained for assessment of multiple markers by flow cytometry using specific antibodies. Unstimulated T cells from naı¨ve mice were used as negative control cells (naı¨ve T cells). Cell surface CD3, CD25, CD28, CD40L, CD69, and FasL were detected using marker specific primary antibodies (solid lines) or isotype-matched irrelevant control antibodies (dotted lines) and FITC-conjugated secondary antibodies. Lower panel: The activated T cells were permeabilized and stained for intracellular IL-4, IFN-γ, and perforin using a commercial kit and PE-conjugated antibodies (solid lines). Unpermeablized T cells were used as negative control cells (dotted lines).

Cytotoxic T lymphocytes mediated the antitumor immune responses

Because the phenotypic markers associated with these activated T cells were consistent with cytotoxic T lymphocytes (CTLs), we tested their cytolytic activities. We first examined the phenotypic characterization of J558 tumor cells. As shown in Figure 7, the wild-type J558 tumor cells displayed major histocompatibility complex (MHC) class I (H-2Kd) antigen, but neither MHC class II (Iad) antigen nor Fas molecule. We then conducted chromium release assays using activated T and 51Cr-labeled J558 tumor cells as effector and target cells, respectively. As shown in Figure 8, the activated T cells from mice with tumor regression showed a specific killing activity (48% specific killing at an effector:target cell ratio of 50) for J558 tumor cells, but not for the irrelevant SP2/0 tumor cells (0% specific killing at an effector:target cell ratio of 50). Nearly 80% of CTL-mediated killing could be blocked using the anti–H-2Kd, but not the anti-Iad, antibodies (data not shown). On the contrary, T cells from mice inoculated with irradiated J558 tumor cells showed little killing activity (4% specific killing at an effector:target cell ratio of 50) for J558 tumor cells. These results indicate that mice with complete tumor regression after the treatment developed a strong J558-specific immune response mediated by CD8+ CTLs.

Figure 7
figure7

Phenotypic analysis of the wild-type J558 tumor cells. J558 tumor cells were stained with anti–H-2Kd, Iad and Fas antibodies (solid lines) or isotype-matched irrelevant control antibodies (dotted lines), followed by FITC-anti-mouse antibody and analyzed by flow cytometry.

Figure 8
figure8

Cytotoxicity assay. The activated T cells (black squares), as described in Figure 6, were used as effector cells in chromium release assays, in which 51Cr-labeled J558 tumor cells were used as target cells. To confirm that T-cell cytotoxicity was J558 tumor–specific, we also included SP2/0 cells (black circles) as a target control. Activated T cells from mice inoculated with irradiated J558 tumor cells were used as a effector control (open circles) in chromium release assays, in which 51Cr-labeled J558 tumor cells were used as target cells. Each point represents the mean of triplicates. One representative experiment of two is shown.

Discussion

In cancer therapy, the successful manipulation of the immune system requires a deep understanding of the endogenous immune response to neoplastic cells and, more specifically, comprehending why natural antitumor responses are relatively ineffective. The current paradigm holds that tumor cells are poorly immunogenic. Bearing this in mind, much work has been directed at enhancing the immunogenicity of cancer cells. Adenoviral vectors have been extensively used in cancer gene therapy for transgene delivery into tumors to increase the immunogenicity of tumors.15,16,17 Although these approaches are effective in induction of protective immunity against certain tumors, their therapeutic benefit is still limited because they can only either delay tumor growth or regress some of the tumors in early stages. To improve the therapeutic efficacy, the combinational use of two immunogene-expressing adenoviral vectors was reported. Putzer et al have demonstrated that IL-12 and B7-1 costimulatory molecule expressed by an adenovirus vector acted synergistically to facilitate tumor regression in a transgenic PyMidT animal model.20 Recently, Emtage et al have shown that adenoviral vectors expressing lymphotactin and IL-12 enhanced tumor regression in murine breast cancer models.21 More recently, Narvaiza et al have reported that intratumoral coinjection of two adenoviruses, one encoding the chemokine IP-10 and another encoding cytokine IL-12, resulted in marked antitumor synergy.22

In this study, we constructed two recombinant adenoviral vectors AdVIL-18 and AdVIP-10 expressing the functional IL-18 and IP-10 molecules, respectively, and investigated the antitumor effects by intratumoral injection of these two adenoviral vectors into established J558 tumors in animal models. Our data showed that treatment of either AdVIP-10 or AdVIL-18 alone cannot cure any established tumors though the delay of tumor growth in these treated mice was noted. Interestingly, treatment of coinjection of AdVIP-10 and AdVIL-18 not only significantly suppressed the tumor growth, but also cured established tumors in 8 of 10 (80%) tumor-bearing mice, indicating a synergized antitumor immunity. However, the synergy of chemokine IP-10 and cytokine IL-18 only took place when both recombinant adenoviruses were given to the same nodule, but not when identical doses were injected into distant tumor nodules. This is in agreement with the so-called “attraction and activation” hypothesis by Paillard,23 which predicts the necessity of colocalization of immunostimulatory and chemoattractant factors as previously seen for chemokine lymphotactin and cytokine IL-2.24

In this study, we also showed that the coinjection of AdVIP-10 and AdVIL-18 induced activation of tumor-specific T cells with a dominant expression of IFN-γ; this pattern of cytokine expression is consistent with the type 1 immune response usually associated with antitumor immunity.25 These activated T cells displayed efficient tumor-specific killing activity (48% specific killing at an effector: target cell ratio of 50) for J558 tumor cells in vitro. CTLs can directly eradicate tumor cells through cognate interactions that may involve either perforin or Fas-mediated lytic mechanisms.26 In the present study, the lymphotoxin perforin but not Fas/FasL interaction mediates the CTL activity because these activated T cells uniformly displayed high level expression of FasL and perforin, whereas the wild-type J558 tumor cells do not express Fas molecule.

IP-10 has two potential antitumor effects, antiangiogenesis and immunodulation, similar to what has been described for IL-12.27 Recently, IP-10 was reported necessary for effector T-cell trafficking and function and host survival in Toxoplasma gondii infection.28 Neutralization of IP-10 inhibited the influx of T cells into spleens and livers of mice infected by T. gondii, resulting in a simultaneous 3-log increase in the parasite burden and a significant decrease in survival. In the present study, we demonstrated that significant amounts of lymphocytes infiltrated into tumors in association with tumor necrosis formation leading to complete tumor regression after the combined treatment of AdVIP-10 and AdVIL-18. We also demonstrated that the number of tumor-free mice was decreased to 50% and 40% in CD4+ and CD8+ T cell–depleted mice, respectively, after the treatment. The mouse survival rates in CD4+ and CD8+ T cell–depleted groups are significantly lower than that in the immune competent mouse group after the treatment (P<.05). The above results indicate that both CD4+ and CD8+ T cells were involved in the antitumor efficacy of combined administration of AdVIP-10 and AdVIL-18. The regression of J558 tumor in some of the CD4+ T cell–depleted mice could be attributed to the cytotoxic effect of CD8+ cytotoxic T cells directly stimulated by IL-18. The regression of J558 tumor in some of the CD8+ T cell–depleted mice could be caused by the development of CD4+ cytotoxic T cells induced by IL-18.7 In addition, our data also showed that, in T cell–deficient BALB/c nude mice, intratumoral treatment resulted in lethal tumor progression in all eight nude mice, further confirming the absolute T-cell requirement in this therapeutic model.

IFN-γ, a cytokine secreted by activated T cells and natural killer cells, has multiple immunoregulatory effects on various cell types,29 including the capacity to stimulate the activation of CTLs,30 natural killer cells,31 and macrophages32,33 and to induce/enhance the expression of class II MHC antigens.34 Through these effects, IFN-γ plays a central role in promoting innate and adaptive mechanisms of host defense including tumor immunity.35 In fact, this notion was supported recently by the observations that IL-12–induced tumor regression is blocked by neutralizing IFN-γ produced after IL-12 injections.36,37 Tumor rejection involves a number of processes: sensitization/activation of effector T cells with tumor antigens in lymphoid organs; migration of T cells together with other effector cells to tumor masses; and tumor cell attack by these tumor-infiltrating effectors. To study the role of IFN-γ, we grew J558 tumors in IFN-γ KO mice and treated these tumors with intratumoral coinjection of AdVIP-10 and AdVIL-18. Our data showed that all tumors grew aggressively in these IFN-γ KO mice after the treatment, indicating the critical role of IFN-γ in this antitumor therapeutic model. Previous studies38,39 have suggested that IFN-γ plays a role in some of the processes such as an intratumoral antitumor effector mechanism. More recently, Nakajima et al have demonstrated another critical role of IFN-γ in the process of inducing T-cell migration into tumors.40 In their report, T cells with capacity to reject tumor cells failed to migrate to tumor sites in IFN-γ–deficient mice. Therefore, IFN-γ may play two roles in this therapeutic model, the recruitment of CTLs into tumor sites and the local antitumor effect.

Taken together, the findings of this study demonstrate that the combined use of two adenoviral vectors expressing IP-10 and IL-18, respectively, synergize to facilitate regression of established tumors. These observations also suggest the potential use of double-recombinant adenoviral vectors expressing chemokines and immunomodulatory cytokines in cancer gene therapy.

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Acknowledgements

This study was supported by a research grant (ROP-15151) from the Canadian Institute of Health Research. We thank Dr H Tabel for his useful comments on this manuscript and Mr X Bi for his technical support in this study.

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Correspondence to Jim Xiang.

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Liu, Y., Huang, H., Saxena, A. et al. Intratumoral coinjection of two adenoviral vectors expressing functional interleukin-18 and inducible protein-10, respectively, synergizes to facilitate regression of established tumors. Cancer Gene Ther 9, 533–542 (2002). https://doi.org/10.1038/sj.cgt.7700466

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Keywords

  • adenoviral vector
  • IL-18
  • IP-10
  • cancer gene therapy

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