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Departments of
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Surgery and Science and
Cardiovascular Surgery, and
Division of Pathophysiological and Experimental Pathology, Department of Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
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Abstract |
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, and IFN--inducible protein-10, significantly reduced the number of microvessels and inhibited the growth of HCC, compared with HCC-transferred control pCAGGS plasmid. The inhibition of tumor growth in immunosuppressed mice was comparable with that of mIL-12 gene therapy in immunocompetent mice. Intratumoral mIL-12 gene therapy enhanced lymphocytic infiltration into the tumor and elicited the MH134-specific CTL response even under FK506. The dose of FK506 was sufficient to prevent the rejection of distant allogenic skin grafts (BALB/c mice, H-2d) and tumors, B7-p815 (H-2d) used as transplants, during mIL-12 gene therapy against MH134. Ab-mediated depletion studies suggested that the inhibition of tumor growth, neovascularization, and spontaneous lung metastasis by mIL-12 was dependent almost entirely on NK cells and partially on T cells. These results suggest that intratumoral mIL-12 gene therapy is a potent effective strategy not only to treat recurrences of HCC in liver transplantation, but also to treat solid malignant tumors in immunosuppressed patients with transplanted organ. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The risk factors for skin cancer incidence in organ transplantation include high dose and long-term administration of immunosuppressants (8). Therefore, many prophylactic studies, which include sun protection, chemoprophylaxis, and reducing immunosuppressants, were reported and these techniques have succeeded in decreasing the incidence of tumors (8). In addition to this, in liver transplantation, pre-, intra-, and postoperative chemotherapy have been performed after liver transplantation for the prevention of recurrent HCC (9). However, it has been shown that the survival of the recurrent cases after transplant with a tumor size larger than 7 cm and with vascular invasion was significantly poor despite the systemic chemotherapy (6). Then, once such malignancies occur, passive treatment, such as reducing the dose of immunosuppressants, which may put recipients at risk of rejection, in addition to conventional therapy for each malignancy, is selected. Therefore, to treat malignancy in immunosuppressed organ-transplant recipients, we need to develop a new active strategy, which suppresses the local tumor growth and has a tumor vaccination effect without affecting graft function.
IL-12 p70, that consists of a p35 and a p40 subunit, exerts a variety of immunomodulatory antitumor effects, including induction of IFN- secretion from T cell and NK (10, 11), promotes maturation of CTL (12), and induces antiangiogenic effects (13). The antiangiogenic effect of IL-12 is mediated by IFN-, which in turn induces the production of the chemokine IFN-inducible protein-10 (IP-10) that, in addition to acting as a chemoattractant for lymphocytes, has a powerful inhibitory effect on the proliferation and differentiation of endothelial cells (14). Moreover, IL-12-stimulated NK cells are cytotoxic for activated endothelium thus contributing to block the formation of new tumoral vessels (15). It has recently been demonstrated that local or systemic treatment with rIL-12 protein mediates profound antitumor effects in vivo, causing regression of established tumors and their distant metastases (16). However, systemic administration of IL-12 protein has caused dose-dependent toxicity in mice (17), and this was also found in human trials (18). Therefore, recent investigations focus on the local IL-12 gene expression for the treatment of various tumors. We also reported that electroporation-mediated gene transfer of murine IL-12 (mIL-12) was found to be effective for established HCC as well as distant HCC (19).
Today, the gene therapy using IL-12 is being used in clinical applications, however, whether or not such a gene therapy can be used to treat malignancy after organ transplantation has not yet been investigated. A significant problem with such a treatment strategy is that a nonspecific immunological effect may result, causing the rejection of the transplanted graft, because IL-12 activates a T cell immune response from the organ transplantation recipients.
In this study, we investigated the efficacy of intratumoral mIL-12 gene transfer for s.c. HCC in mice, which were immunosuppressed by FK506, a calcineurin inhibitor as well as carcinogenic immunosuppressant (20). We show that intratumoral mIL-12 gene therapy has a beneficial antitumor effect mediated by NK cells and T cells even in mice immunosuppressed by FK506. We also found that the treatment did not induce the rejection of both the allogenic skin and allogenic tumor.
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Materials and Methods |
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B7-P815 (H-2d) (21) was kindly provided from Dr. L. L. Lanier (Department of Immunology, DNAX Research Institute for Cellular and Molecular Biology, Palo Alto, CA). MH134 (H-2k), a mouse hepatocellular carcinoma cell line induced using carbon tetrachloride in C3H mice, was obtained from the Institute of Development, Aging and Cancer (Tohoku University, Tohoku, Japan). X5563 (H-2k), plasmacytoma derived from C3H stain, was obtained from the Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation (Kyushu University, Fukuoka, Japan). These cell lines were maintained in RPMI 1640 (Sigma-Aldrich, St. Louis, MO) medium supplemented with 20 mM HEPES, 0.2% sodium bicarbonate, 50 µM 2-ME, 10 µg/ml gentamicin sodium, and 10% heat-inactivated FBS (ICN Biomedicals, Aurora, OH).
Animals
Eight-week-old female C3H/HEN Crj mice (H-2k) and BALB/cAnNCrj mice (H-2d) were purchased from Charles River Laboratories (Tokyo, Japan) and were maintained under specific pathogen-free conditions in the animal facility at the Kyushu University Medical School. These experiments were approved by the Kyushu University Institutional Animal Care and Use Committee and conformed to all the guidelines outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy for Sciences and published by the National Institutes of Health.
Plasmid DNA
mIL-12 plasmid vector, designated pCAGGS-mIL-12 for IL-12-gene therapy, and empty plasmid vector, designated pCAGGS for control vector, were used. Details including the construction were previously described (19, 22).
Murine tumor models
An established s.c. HCC model in mice, which was previously described (19), was used. MH134 cells (2.5 x 106) were injected s.c. in the right flank of the C3H mice. The HCCs developed about 7 days later. At the time when the tumor volume reached 
0.5 cm3, 100 µg of DNA solutions (control pCAGGS or pCAGGS-mIL-12) were injected into the established MH134 and electroporation was performed as previously described (19). From 1 day before the DNA injection, the mice were treated with 100 µl of PBS (i.p.; Invitrogen Life Technologies, Carlsbad, CA) or FK506 (3 mg/kg; FR900506, Fujisawa Pharmaceutical, Osaka, Japan) daily. The appropriate dose of FK506 was used to control rejection which was determined using a titration study as described in Results. For 16 days after gene therapy, the tumor volumes were calculated according to the following formula: V = A x B2/2 (cm3), where A is the largest diameter (cm), and B is the smallest diameter (cm). On day 20 after gene therapy, the lungs of the mice were removed and spontaneous metastatic lesions were macroscopically and microscopically assessed.
In another experiment, 1 x 107 B7-P815 cells in the left flank and 2.5 x 106 MH134 cells in the right flank were simultaneously injected. After both tumors were established, treatment with FK506 (3 mg/kg) or 100 µl of PBS and intratumoral gene therapy against MH134 were performed.
ELISA for mIL-12 and mIFN-
Frozen specimens of the resected tumor were homogenized in an extraction buffer on ice and assayed for total murine IL-12 (mIL-12) (Pierce, Rockford, IL) and mIFN- (BioSource International, Camarillo, CA) using an ELISA kit, according to the manufacturer’s instructions. Each cytokine concentration was divided by the total protein concentration.
Histopathological and immunohistochemical analysis
The tumorous tissue from the s.c. HCC and the lung were resected, fixed in 10% buffered formalin, and embedded in paraffin. The paraffin-embedded sections were stained with H&E for histopathological analysis. The sections of s.c. HCC were stained with biotinylated anti-mouse CD31 mAbs (BD Pharmingen, San Diego, CA) followed by streptavidin-peroxidase and a diaminobenzidine-reaction system (Vector Laboratories, Burlingame, CA) for immunohistochemical assessment of tumor microvessels at the time when the tumor volume reached almost 1 cm3 after the gene transfer, as previously described (19). The CD31-positive microvessels of s.c. HCC within the hot spot area (mean microvessel density (mean MVD)) were expressed as counts per high power field (HPF) according to the Weidner et al. (23, 24) method.
Flow cytometric analysis of tumor-infiltrating lymphocytes (TILs)
The TILs from tumorous tissue of s.c. HCC were prepared under a modified protocol as previously described (19, 25). In brief, the resected tumor specimens were minced with a scalpel into pieces smaller than 1 mm2 and underwent enzymatic treatment in a collagenase solution (Worthington Biochemical, Lakewood, NJ). The cells obtained were then resuspended in 5 ml of 45% Percoll (Sigma-Aldrich) and layered on 5 ml of 75% Percoll. The discontinuous density gradient solution was centrifuged at 2500 rpm at room temperature, the TILs were collected from the interface layer and the viable cell number of TILs was counted using a standard trypan-blue system. The cells were stained with the following various Abs and analyzed by a FACSCalibur flow cytometer with the CellQuest program (BD Biosciences, San Jose, CA) as previously described above (19). The biotinylated anti-CD45 Ab (30F 11.1,), which was detected with streptavidin-allophycocyanin, was used for pan-lymphocyte gating. PE-conjugated anti-CD3
Ab (145-2C11), anti-CD4 Ab (H129.19), anti-CD8 Ab (53-6.7), anti-CD56 (DX5) Ab, and anti-CD11C Ab (HL3), and FITC-conjugated anti-CD3 Ab (145-2C11), anti-B220 Ab (rat IgG2a
), pan-NK cells mAb (rat anti-mouse CD49b), anti-Mac-1 Ab (M1/70.15), and anti-mouse F4/80 (MCA479F) were used.
Assay system for secondary CTL response
Assay for secondary CTL response was performed as previously described (26, 27). In brief, 5 x 106 splenic cells from the mice with s.c. HCC at the right flank, into which pCAGGS-mIL-12 or pCAGGS had been transferred 14 days before, were cultivated with 1 x 105 mitomycin C (100 µg/ml) treated tumor cells for 5 days. After 5 days, the effector cells were harvested and the 51Cr release assay was performed using corresponding tumor target cells. The percentage of specific lysis was calculated as follows: percent-specific lysis = (experimental release – spontaneous release)/(maximum release – spontaneous release). X5563 plasmacytoma derived from C3H stain was used as a third party target.
RT-PCR for the detection of IP-10
The total mRNA was extracted from the tumor tissues, into which gene transfer had been performed 7 days before, and synthesis of cDNA was performed, as previously described (28). The PCR was performed in the GeneAmp PCR System 2400 (PerkinElmer, Wellesley, MA) for 30 cycles (94°C, 30 s; 67°C, 30 s; 72°C, 60 s). The primer sequences were as follows: murine IP-10 (mIP-10) sense: ACC ATG AAC CCA AGT GCT GCC GTC, mIP-10 antisense: GCT TCA CTC CAG TTA AGG AGC CCT; GAPDH sense: GCC ACC CAG AAG ACT GTG GAT GGC, GAPDH antisense: CAT GTA GGC CAT GAG GTC CAC CAC. The PCR products of IP-10 and GAPDH were fragments of 312 and 447 bp in length, respectively.
Ab-mediated depletion studies of NK cells and T cells
For in vivo depletion studies of NK cells, 100 µg of anti-asialoGM1 antiserum (ASGM1; Wako Bioproducts, Tokyo, Japan) were administered i.p. 2 days before gene therapy, and subsequently once every 3 days afterward for an additional 16 days (six times in total). For in vivo depletion studies of T cells, 280 µg of anti-TCR
Ab (H57–597) (29) were administered i.v. through the tail vein of the mice on days 2 and 0 before the commencement of gene therapy. It was confirmed using FACS analysis that administration of these Abs resulted in the depletion of >94% of the pan-NK marker+ cells or of >99% of CD3+CD4+ and CD3+CD8+ cells.
Skin grafting
Skin grafting was performed using modified methods as previously described (30). Briefly, square full-thickness skin grafts (1 cm2) were prepared from the trunk skin of BALB/c mice. Graft beds (1 cm2) were prepared on the left flank of the C3H mice. The grafts were fixed to the graft bed with eight interrupted sutures of 6-0 Proline thread (Johnson and Johnson, North Ryde, Australia) and were covered with protective tape. The first inspection was conducted on the sixth day, followed by daily inspection. Grafts were considered as rejected at the time of complete sloughing or when they formed a dry scar.
Statistical analysis
Statistical evaluations of numerical variables in both groups were conducted using the Mann-Whitney U test. Differences in tumor growth were statistically analyzed using the repeated measure ANOVA test. Significance was defined as p < 0.05.
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Results |
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In current clinical transplantation, calcineurin inhibitor, FK506, is a useful and efficient immunosuppressant to control allograft rejection (31). At first, to obtain immunosuppressed recipient C3H (H-2k) mice, we determined an appropriate dose of FK506 to control rejection for the allogenic and highly immunogenic B7-P815 (mastocytoma, H-2d) cell (21) in an allogenic tumor inoculation model. Recipient C3H mice, which had been i.p. administered with 100 µl of FK506 (1 or 3 mg/kg) or 100 µl of PBS every day from 1 day before tumor inoculation, were s.c. inoculated with 3 x 106 B7-P815 cells. All untreated immunocompetent mice rejected the B7-P815 cells (data not shown). Under treatment of FK506 at the dose of 1 mg/kg, but not at the dose of 3 mg/kg, the recipient mice rejected the B7-P815 cells (Fig. 1 and data not shown). This observation was consistent with experiments using 5 x 106 and 1 x 107 B7-P815 cells (Fig. 1, and data not shown). Furthermore, we confirmed that recipient C3H mice, which were administered with 3 mg/kg FK506 every day from 1 day before skin grafting, could not reject the fully allogenic skin graft from BALB/c (H-2d) mice (data not shown). Therefore, we used the 3 mg/kg i.p. FK506 in all subsequent experiments as a sufficient treatment for immunosuppression of C3H mice.
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Because IL-12 has an immunological and nonimmunological antitumor effect (10, 11, 12, 13), it is expected that intratumoral gene transfer of mIL-12 could result in suppression of tumor growth even under immunosuppression. Initially, according to the optimized electroporation method for intratumoral gene transfer as previously described in our report (19), we transferred the mIL-12-gene into established syngenic HCCs that were s.c. inoculated in the immunosuppressed or nonimmunosuppressed mice. A higher expression of intratumoral mIL-12 was detected in the pCAGGS-mIL-12-transferred MH134, as compared with that in the control pCAGGS-transferred MH134, in cases of nonimmunosuppressed mice on day 7 after gene transfer (Fig. 2A). A similar elevation of intratumoral mIL-12 expression on day 7 after gene transfer was observed in cases of mice which were immunosuppressed by FK506 (Fig. 2A). On day 14 after gene transfer, the mIL-12 expression decreased in the pCAGGS-mIL-12-transferred MH134 on the nonimmunosuppressed mice, but the high expression of mIL-12 was maintained in the pCAGGS-mIL-12-transferred MH134 on immunosuppressed mice (data not shown). Furthermore, as an assay for biological activity of mIL-12, we examined up-regulation of intratumoral IFN-. The intratumoral mIFN- levels in pCAGGS-mIL-12-transferred MH134 on both nonimmunosuppressed and immunosuppressed mice increased on day 7 after transfer, as compared with those of the control pCAGGS-transferred MH134 (Fig. 2B). As correlated with the continuous high expression of mIL-12, the high intratumoral mIFN- level in pCAGGS-mIL-12-transferred MH134 on immunosuppressed mice was maintained on day 14, compared with those in the other three groups (data not shown). Therefore, biologically active mIL-12 could be also expressed by electroporation using pCAGGS-mIL-12 in the established tumor in vivo even if the mice were under immunosuppression by FK506.
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As previously reported, under no immunosuppressive therapy, the growth of pCAGGS-mIL-12-transferred MH134 in in vivo was significantly inhibited, compared with control pCAGGS-transferred MH134 (p < 0.01, Fig. 3). The growth of pCAGGS-mIL-12-transferred MH134 on mice immunosuppressed by FK506 was also significantly inhibited, compared with that of the control pCAGGS-transferred MH134 in nonimmunosuppressed or immunosuppressed mice (p < 0.01, Fig. 3). In this mIL-12 gene transfer system under immunosuppression, the mice burdened with pCAGGS-mIL-12-transferred MH134 did not reject the distant allogenic B7-P815, which was simultaneously inoculated with the MH134 (data not shown).
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Fourteen days after treatment, histopathological analysis of s.c. HCC stained with H&E revealed that the amounts of viable tumor cells were reduced and necrotic areas were increased in pCAGGS-mIL-12-transferred MH134, compared with the control pCAGGS-transferred MH134 in nonimmunosuppressed and immunosuppressed mice (Fig. 4). Furthermore, even in immunosuppressed mice, a higher number of chronic inflammatory cells was microscopically observed in pCAGGS-mIL-12-transferred MH134 than in the control pCAGGS-transferred MH134 (Fig. 4). These observations were confirmed by a flow cytometric analysis for TILs: the numbers of all analyzed lymphocytes, namely Mac-1+ cells (pCAGGS-mIL-12-transferred HCC vs control pCAGGS-transferred HCC: 11.7 x 103/cm3 of tumor vs 2.5 x 103/cm3 of tumor), NK cells (3.3 x 103/cm3 vs 1.3 x 103/cm3), CD4+ T cells (4.3 x 103/cm3 vs 1.1 x 103/cm3), CD8+ T cells (6.0 x 103/cm3 vs 2.3 x 103/cm3), and B cells (5.6 x 103/cm3 vs 1.3 x 103/cm3) increased in mIL-12 gene-transferred MH134 in immunosuppressed mice.
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Because the amounts of histopathological necrosis by mIL-12 gene therapy were increased, we assessed the number of microvessels stained with an anti-mouse CD31 mAb within the HCC and in the tissue surrounding the HCC (Fig. 5, A–D). In both nonimmunosuppressed mice and immunosuppressed mice, mean MVD in pCAGGS-mIL-12-transferred MH134 (mean ± SE = 5.1 ± 0.4/HPF and 4.0 ± 0.5/HPF) were significantly fewer than in the control pCAGGS-transferred MH134 (13.1 ± 1.6/HPF and 9.5 ± 1.1/HPF) at the time when the tumor volume reached almost 1 cm3 after the gene transfer (Fig. 5E; p < 0.0001). Immunosuppression by FK506 had no effect on the mIL12-mediated antiangiogenic effect as compared with pCAGGS-mIL-12-transferred MH134 in nonimmunosuppressed mice (Fig. 5E). Because IP-10, one of the CXC chemokines, is a secondary mediator for antiangiogenic effect in the IL-12-IFN- pathway in vivo (13, 32), we investigated the IP-10 gene expression to research the relationship between histopathological findings of tumoral neovascularization and intratumoral IL-12 gene therapy. IP-10 gene expressions were strongly detected in pCAGGS-mIL-12-transferred MH134 in both nonimmunosuppressed mice and immunosuppressed mice on day 7 after gene transfer, but were faintly detected in control pCAGGS-transferred MH134 in nonimmunosuppressed mice and were not detected in control pCAGGS-transferred MH134 in immunosuppressed mice. (Fig. 5F)
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In general, CTL plays an important role in suppressing tumor progression (32) and is used to vaccinate individuals against various tumors (33). Therefore it was very interesting to investigate how intratumoral mIL-12 gene therapy affects the systemic CTL response in mice which were sufficiently immunosuppressed by FK506. Secondary CTL response, which was generated from spleen cells of immunosuppressed mice with control pCAGGS-transferred MH134 day 14 after gene transfer, was not detected at all, while CTL response, which was generated from spleen cells of nonimmunosuppressed mice with control pCAGGS-transferred MH134, was marginally detected. In contrast, independently of whether the host mice were immunosuppressed or not, the CTL response could be detected from the spleen of mice which were treated with intratumoral mIL-12 gene therapy (Fig. 6). In additional experiments, it was confirmed that such augmented cytotoxic responses are MH134-specific, because the generated CTL had no killing directed toward the third party plasmacytoma, X5563, derived from the C3H stain (Table I).
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It was interesting to find the mechanisms that worked in the inhibition of tumor growth using the IL-12 gene therapy under immunosuppression. In general, IL-12 is a potent cytokine that has immunological antitumor effects mediated by NK cells and T cells (10, 11, 12, 13, 14, 15, 16) in immunocompetent hosts. Therefore, Ab-mediated depletion studies of NK cells or T cells were performed. Under immunosuppressive therapy, depletion of NK cells or T cells had little effect on the tumor growth of s.c. HCC in mice with control pCAGGS-transferred MH134. In contrast, in a case of NK cell depletion by an anti-asialoGM1 Ab, the inhibition of tumor growth by IL-12 gene therapy almost entirely disappeared (p < 0.01 compared with the tumor growth in immunosuppressed mice without Ab treatment, Fig. 7). Moreover, in a case of T cell depletion by the anti-TCR Ab, the inhibition of tumor growth by IL-12 gene therapy was partially recovered (p < 0.01, Fig. 7). These findings suggested that, even under immunosuppression, NK cells and T cells played a critical role in IL-12-intratumoral gene therapy.
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We investigated the antimetastatic effects of electroporation-mediated gene therapy using mIL-12 plasmid DNA under immunosuppression. The immunosuppressed mice were sacrificed to examine the spontaneous metastasis at the site of the lungs on day 20 after gene therapy. Only one metastatic lesion in one lung was observed among the mice with pCAGGS-mIL-12-transferred MH134. In contrast, multiple metastatic lesions were observed in all the lungs of the mice with control pCAGGS-transferred MH134 (Table II). The IL-12-meditated antimetastatic effect was abrogated completely through the administration of an anti-asialoGM1 Ab and partially by the administration of an anti-TCR Ab (Table II). These findings suggested that IL-12 treatment under immunosuppression inhibited spontaneous lung metastases through NK cell and T cell-mediated immune response.
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In the above experiments, we administered FK506 1 day before IL-12 gene therapy. To exclude the inadequate duration of FK506 administration, we performed IL-12 gene therapy on day 7 after the initial FK506 administration. In the experimental setting, we simultaneously performed fully allogenic skin grafting and investigated whether IL-12 gene therapy disturbed the maintenance of the skin graft by FK506. A fully allogenic skin graft of BALB/c (H-2d) was transplanted on the left flank of C3H (H-2k) mice and MH134 (H-2k) cells were simultaneously injected in the right flank of the same C3H mice. From 1 day before the skin grafting and tumor injection, the mice were treated with FK506 (3 mg/kg) every day. After 7 days of the skin graft and tumor injection, the intratumoral gene therapy against MH134 was performed. Immunocompetent C3H mice rejected the fully allogenic skin grafts within 12 days. In contrast, the allogenic skin grafts were maintained for 30 days during FK506 administration both in the mice with pCAGGS-mIL-12-transferred MH134 (n = 3) and in the mice with control pCAGGS-transferred MH134 (n = 3) (Fig. 9). Also, in this case, this IL-12 gene therapy had a similar antitumor effect (Fig. 9 and data not shown) as that observed in the original protocol (Fig. 3). Therefore, we found that IL-12 gene therapy showed a stable antitumoral effect regardless of the duration of FK 506 administration, but did not disturb the maintenance of the skin graft by FK506.
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Discussion |
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Previous reports showed that the IL-12 therapy had an antitumor effect independent of immunological compartments in some experimental settings using SCID or SCID-beige mice (37, 38). However, the immunological compartment dependency of the local antitumor and antimetastatic effects differ among experimental settings, such as the kinds of tumor, the vector used, the administration route, and the dose of IL-12 protein or vectors. In B16 melanoma, systemic rIL-12 administration induces a local antitumor effect dependent on CD8+ T cells (16) and an antimetastatic effect dependent NK cells (39). In contrast, in mammary adenocarcinoma, systemic rIL-12 administration induces a local antitumor effect and an antimetastatic effect dependent on CD8+ T cells and partially on NK cells (40). IL-12-producing neuroblastoma induces a CD8+ T cell-dependent local antitumoral effect and an antimetastatic effect. The high IL-12-producing cells show a NK- or T cell-independent antiangiogenic effect induced by IP-10, although the final rejection in immunocompetent mice is totally dependent on T cells (28, 41). In prostate cancer, adenoviral-mediated IL-12 gene therapy had an antimetastatic effect in partial dependency on NK cells (42). In bladder carcinoma and colon cancer, adenoviral-mediated IL-12 gene therapy had a T cell-dependent local antitumor effect (43, 44). Concerning the dependency of immune cells in the antiangiogenic effect induced by IL-12 (45), there are also contrasting reports (32, 46). Therefore, it is important to investigate the mechanism of the antitumoral effect in individual experiments. Especially, in our experiments using a widely used immunosuppressant, FK506, which has a biological action not only on T cells, but also on NK cells, macrophages, and fibroblasts (47, 48, 49), it is very interesting to see whether the mechanism of the antitumoral effect of electroporation mediated IL-12 gene therapy is dependent on immunological components or not. We previously reported that electroporation-mediated IL-12 gene therapies for HCC were found to have an antitumor effect against mIL-12-tranferred HCC, distant HCC, and spontaneous lung metastasis (19). Although, in the previous study, the precise mechanism was not elucidated, we interpreted the effect as the result of the cooperation of three mechanisms; namely, the suppression of the neovascularization of HCC, generation of tumor-specific CTL, and enhancement of infiltration of various lymphocytes by local and systemic elevation of bioactive IL-12. In this study, the production of IP-10 by the IL-12-IFN- pathway and lymphocytic infiltration were almost entirely unaffected by FK506 in vivo. Therefore, even under an immunosuppressed state, the tumor growth inhibition was efficiently induced by IP-10, which has a direct antiangiogenetic effect (50), and the antitumor effect of infiltrated lymphocytes. We also found that the antitumor effect by IL-12 gene therapy was dependent largely on NK cells and partially on T cells. Another interesting observation was that the effect of antineovascularization of the tumor was also dependent on the two cellular components. In several recent reports, NK cells and T cells are important mediators for tumor-angiogenesis inhibition (46, 51) depending on IFN- production (51). In the current study, noteworthy points were that, even under immunosuppression, not only NK cells but also T cells contributed to the effect of antineovascularization of the tumor using IL-12 gene therapy. Therefore, even in sufficient immunosuppressive mice, the three mechanisms functioned well to a comparable extent with the three mechanisms functioning in immunocompetent mice.
We previously demonstrated the high and long-lasting production of IL-12 in the mice which had been treated with intratumoral electroporation-mediated IL-12 gene transfer (19). In the present study using FK506, the intratumoral IL-12 production was higher and longer lasting in the immunosuppressed mice than that of immunocompetent mice (Fig. 2A and data not shown). The precise mechanism is not clear. It is unlikely that this mechanism was due to more viable tumor cells, which probably produced IL-12 by electroporation-mediated gene transfer, in immunosuppressed mice than in nonimmunosuppressed mice, because the pCAGGS-mIL-12-transferred tumor volume, necrotic area, and number of tumor neovascularizations were comparable between nonimmunosuppressed mice and immunosuppressed mice. Considering a report suggesting that long-lasting IL-12 production by injection of IL-12-encoding plasmid DNA due to an autocrine or paracrine positive feedback mechanism whereby several cell population including NK cells are involved (52), long-lasting IL-12 production in our present study may also be due to the positive feedback mechanism by which the production of endogenous IL-12 is actually produced by host macrophages and dendritic cells. Moreover, Conboy et al. (48) reported that FK506, a calcineurin inhibitor, acts in macrophages and fibroblasts to augment cytokines, IL-12 and TNF-, and this effect is paradoxical compared with that in naive T cells. In addition, Hortelano et al. (47) reported that FK506 has a protective effect on the apoptosis of activated macrophages. Therefore, the higher and longer-lasting production of IL-12 in the tumors of IL-12-treated immunosuppressed animals may be caused by the augmentation of endogenous IL-12 production of macrophages by FK506.
Surprisingly, intratumoral IL-12 gene therapy not only generated the tumor-specific CTL in immunosuppressed mice, but also generated higher CTL induction in immunosuppressed mice than that found in the immunocompetent mice. It is unlikely that this is due to an insufficient dose of FK506, because the dose of FK506 was enough to prolong allogenic skin graft survival (53) and the host could not eradicate both the allogenic skin graft and the highly immunogenic allogenic tumor, B7-P815 (Fig. 9, data not shown). Because FK506 prevents apoptosis of activated T cells and is not able to block the proliferation of activated T cells (54) (but is able to block priming of naive T cells), it is possible that once T cells are primed to resist apoptosis and suppression of the effector function under FK506-administration, this then leads to the accumulation of tumor-specific T cells in FK506-injected mice. In addition, IL-12 enhances CTL generation and IFN- production as well as increases perforin production which is independent of IL-2-production (55). Therefore, these mechanisms may cooperate to augment CTL response in IL-12-treated immunosuppressed mice.
Although it is believed that the priming of T cells to tumors occurs in the regional lymph nodes, a recent report suggested that the priming can occur in at a tumor site in a particular setting (56). Fernandez et al. (57) suggested that the possible site of T cell expansion was the IL-12 gene-transferred tumor itself, because public CD8+ T cell frequency in the tumor site and blood is augmented more than that in spleen and lymph nodes. Therefore, we hypothesize that the priming of T cells to MH134 could occur in the IL-12-induced tumor site where the neovascularization was suppressed and the concentration of the immunosuppressant decreased, as a result, the T cells were primed and expanded. This hypothesis is strongly supported by the findings that augmentation of IFN- production and existence of CD8+ T cells at the site of IL-12 gene-transferred tumor were not affected by treatment using FK506 at all. Also MH134-specific T cells were primed but T cells against the highly immunogenic fully allogenic skin grafts were not primed in immunosuppressed mice (Figs. 6 and 9), suggesting that immunosuppression is sufficient in the lymph nodes even under systemic elevation of IL-12, because priming of T cells to allogenic skin grafts occurs in the lymph nodes (58). The direct evidence of T cell priming in tumor sites could not be obtained in this study. To prove the hypothesis, it is necessary to elucidate the dominant peptide epitope of tumor-specific Ags in MH134 or to obtain tumor-specific Ag-reactive TCR transgenic mice. However, because we do not have the materials at present, this investigation should be done in a future study.
Before this new strategy can be applied to treat malignant tumors in clinical organ transplantation, there is an important point that we must clarify. The point is whether this intratumoral IL-12 gene therapy is effective in other immunosuppressants, including cyclophosphamide, mycophenolate mofetil, steroids, and rapamycin, which have been favorably used to control rejection at present. Matsue et al. (59) reported that FK506 or cyclosporin A, which were both calcineurin inhibitors, more effectively suppress the priming of the T cell, including IFN- production, and dendritic cells in the T cell-dendritic cell direction in vitro than rapamycin and steroids. Lui et al. (60) reported that cyclosporin A more effectively suppresses IFN- production of T cells at the priming in vivo than mycophenolate mofetil. As for cyclophosphamide, Tsung et al. (61) clearly show that the administration of cyclophosphamide together with IL-12 protein therapy used against sarcoma tumors elevated the cure rate of established tumor-burdened mice and this effect is dependent on IFN- produced by T cells (62). From these reports, it is possible that intratumoral IL-12 gene therapy may be effective to treat malignant tumors in transplant recipients, who are immunosuppressed by other immunosuppressants. In fact, under administration of such a strong immunosuppressant, FK506, the intratumoral IL-12 gene transfer resulted in vigorous IFN- production in the tumor associated with lymphocyte infiltration to a comparable level or rather a relatively enhanced level of IL-12 gene-transferred tumor in immunocompetent mice (Fig. 2).
In conclusion, to treat the incidence of fatal solid malignant tumors and the recurrence of these tumors in immunocompromised transplant recipients, it is possible that our intratumoral IL-12 gene therapy can become a new effective strategy without affecting allograft and can contribute to the improvement of long-term recipient survival.
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1 This study was supported in part by a Grant-in-Aid for General Research from the Ministry of Education, Culture, Sports and Technology, Japan (13671243 and 13357011). 
2 Address correspondence and reprint requests to Dr. Noboru Harada, Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan or Dr. Shinji Okano, Department of Pathology, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan. E-mail addresses: nharada{at}surg2.med.kyushu-u.ac.jp or okap{at}surg2.med.kyushu-u.ac.jp
3 Abbreviations used in this paper: HCC, hepatocellular carcinoma; IP-10, IFN--inducible protein-10; m, murine; mean MVD, mean microvessel density; HPF, high power field; TIL, tumor-infiltrating lymphocyte; RFA, radiofrequency ablation; MCT, microwave coagulation therapy.
Received for publication February 5, 2004. Accepted for publication September 21, 2004.
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; n = 3), 5 x 106 (
; n = 3), and 1 x 107 (
; n = 3) B7-P815 cells. The tumor growth was monitored every day. The data are indicated as mean tumor volume ± SE in each group at indicated postinoculated days. Several symbols are uncertain due to pile. The data are representative of two independent experiments.
, pCAGGS-mIL-12, n = 3) or 3 mg/kg FK506 (
, control pCAGGS, n = 3; 
, pCAGGS-mIL-12, n = 3) daily from the day before gene transfer. The tumors were resected on day 7 (A and B) after gene transfer and protein levels of intratumoral mIL-12 and mIFN-
, control pCAGGS, n = 5; 
, pCAGGS-mIL-12, n = 5) or 3 mg/kg FK506 (
