Interferon-α and interleukin-12 gene therapy of cancer: interferon-α induces tumor-specific immune responses while interleukin-12 stimulates non-specific killing

Abstract

Cytokine gene therapy is applied in clinical studies of tumors, and IFN-α and IL-12 are widely used for cancer immunotherapy. Using a poorly immunogenic murine colorectal cancer cell line, MC38, we compared antitumor effects of IFN-α and IL-12. Transduced MC38 cell lines expressing IFN-α or IL-12 (MC38-IFNα or MC38-IL12, respectively) were established using retroviral vectors. Transduction of IFN-α or IL-12 gene to MC38 cells significantly reduced tumorigenicity in immunocompetent mice. When tumor-free mice initially injected with MC38-IFNα or MC38-IL12 cells were reinjected contralaterally with wild-type MC38 cells (MC38-WT) after 35 days, 7 of 12 or 2 of 12 mice rejected MC38-WT cells, respectively. In therapy-model mice with established tumor derived from MC38-WT cells, inoculation of gene-transduced cells significantly suppressed growth of the tumor in MC38-IFNα-inoculated groups, but not in the IL-12-inoculated group. Immunohistologic and flow cytometric analyses showed marked infiltration of CD8⁺ cells in wild-type tumors of mice inoculated with IFN-α-expressing cells. Leukocyte-depletion experiments implicated CD8⁺ T cells in tumor rejection induced by IFN-α-transduction; both CD8⁺ T cells and natural killer cells were implicated in the more modest antitumor effect from IL-12 expression. To investigate induction of tumor-specific immune responses, we stimulated splenocytes from tumor-free mice twice in vitro with genetically modified MC38 cells. In vitro stimulations with MC38-IFNα cells induced definite MC38-specific lysis, but not stimulations with MC38-IL-12 cells. Injecting combination of MC38-IFNα and MC38-IL-12 cells caused an additive antitumor effect in the therapy model. These data suggested that IFN-α induces cytotoxic T lymphocytes and elicits long-lasting tumor-specific immunity, whereas IL-12 seems to stimulate non-specific killing. With additional refinements, combined IFN-α and IL-12 gene therapy might warrant clinical trials.

Introduction

Cellular immune response is considered to be impaired in patients with advanced malignant tumors, which escape immune surveillance by several mechanisms [2, 23]. To overcome immune suppression or immune escape in patients with malignant tumors, therapy aimed at improving cellular immune responses is needed. In several murine studies, cytokine gene transduction of tumor cells induced potent antitumor immune responses without systemic toxicity. Subcutaneous injection of transduced cells can induce local inflammation at the site of injection, associated with inflammatory cells including activated natural killer (NK) cells, macrophages, dendritic cells, and T lymphocytes [4, 20]. Based on these phenomena, clinical vaccination with tumor cells carrying transduced cytokine genes may be an attractive and feasible model of gene therapy.

A heterodimeric cytokine, interleukin (IL)-12, initially was originally characterized as an NK cell stimulatory factor [15] and as a cytotoxic lymphocyte maturation factor [29]. IL-12 gene therapy has been studied both in clinical trials and in treatment of experimental tumors. IL-12, produced mainly by dendritic cells and macrophages, can stimulate both growth and function in NK cells and tumor-specific T cells [7, 26, 32]; these effects result in T-helper (Th)1-type immune responses that reduce tumor growth. In addition, IL-12 has an antiangiogenic effect that contributes to tumor regression. Various studies have reported antitumor effects of IL-12 delivery using IL-12 gene-transduced tumor cells [37], fibroblasts [36], or dendritic cells [22].

Interferon (IFN), first identified as an inhibitor of viral infection in 1957 [12], was found to be heterogenous, representing two groups of peptides: type-I IFNs (later designated IFN-α and IFN-β) and type-II IFN (later designated IFN-γ). Effects of IFN-α include not only induction of an antiviral response, but also suppression of cell growth and inhibition of angiogenesis [24]. In addition, IFN-α increases expression of major histocompatibility complex (MHC) class I on cell surface. Type-I IFNs also induce the production of other IFN-α/β [14, 16] as well as IL-15 [5, 35], and additionally enhance proliferation of Th1 lymphocytes [1]. IFN-α and IFN-β modify maturation of dendritic cells, which play a crucial regulatory role in the immune system [13, 28]. Finally, previous studies have emphasized the importance of IFN-α in specific immune responses against tumors [6, 34].

In a murine model, we previously demonstrated that tumorigenicity of a poorly immunogenic colorectal cancer cell line was reduced when the tumor cells were transduced with the IFN-α gene [10]. Furthermore, we also showed that IFN-α induced generation of tumor-specific CD8⁺ cytotoxic T lymphocytes (CTL), and that IFN-α expressed by tumor cells promotes survival of a tumor-specific CTL line by preventing apoptosis [11]. Since type-I IFNs mediate a variety of immunoregulatory effects, IFN-α is used widely in treatment of patients with such malignant tumors as melanoma, renal cancer, and leukemia.

Several studies have suggested that a Th1-type response plays a critical role in antitumor effects of therapy in experimental models. Since both IFN-α and IL-12 favor proliferation of Th1 lymphocytes, a combination of both IFN-α and IL-12 gene therapy might be expected to elicit a particularly potent immune response against a tumor. Interestingly, type-I IFNs are potent inhibitors of IL-12 production by human monocytes [3]. Additionally, IFN-α has been found to block NK cell production of IFN-γ in response to IL-12 [21]. On the other hand, type-I IFNs induce a high-affinity form of the IL-12 receptor on human T cells [8, 27]. Recently, Mendiratta et al. have reported that combined IFN-α and IL-12 gene therapy synergistically increased antitumor response against colon and renal cell carcinoma [19].

Fuller understanding of mechanisms related to cytokine gene therapy is needed for effective and safe clinical application. To compare the efficacy and to clarify the mechanism of Th1-type cytokines, IFN-α and IL-12, in gene therapy for cancer, we established colorectal cancer cell lines transduced with a murine IFN-α or IL-12 gene and observed the changes caused by transduction on tumorigenicity compared with non-transduced parental tumor cells. Then we evaluated in vivo effects against established parental cell tumors after inoculation with the cytokine-expressing tumor cell lines, using immunohistologic staining of tumors, flow cytometric analysis of tumor-infiltrating lymphocytes (TIL), and induction of tumor-specific T lymphocytes from splenocytes of immune mice.

Materials and methods

Mice

Female C57BL/6 (B6) mice 6–8 weeks old were purchased from Saitama Experimental Animals Supply (Saitama, Japan) for use in experiments at ages from 8–12 weeks. Mice were maintained in an animal care facility at Showa University.

Cell lines, culture medium, and reagents

The MC38 murine colorectal adenocarcinoma cell line [31] and YAC-1 lymphoma cells (both of B6 mouse origin) were provided by M.T. Lotze (University of Pittsburgh) and H. Tahara (University of Tokyo), respectively. YAC-1 cells were used as target cells for assessing non-specific killing in cytolytic assays. Cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES buffer, 1 mM Minimum Essential Medium sodium pyruvate, 0.1 mM Minimum Essential Medium non-essential amino acids (complete medium, CM) in a humidified incubator with 5% CO₂ in air at 37°C. All cell culture reagents were purchased from Life Technologies (Gaithersburg, Maryland).

Retroviral transduction and genetically modified tumor cell lines

Tumor cells were transduced using retroviral vectors according to standard protocols [25] and selected for antibiotic resistance in CM containing 0.75 mg/ml G418 (Sigma, St. Louis, Mo.). Construction of the retroviral vector, TFG-murine IL-12-neo, has been described in previous studies [30]. Retroviral culture supernatants for IL-12 were provided by Y. Nishioka (Tokushima University). The MC38 cell line producing IFN-α was established as described previously [10]. Expression of murine IL-12 and IFN-α was confirmed by enzyme-linked immunosorbent assay (ELISA) using commercially available kits according to the manufacturers' instructions (mouse IL-12 p70 ELISA, Endogen, Woburn, Mass; mouse IFN-α ELISA, PBL Biomedical Laboratories, New Brunswick, N.J.). MC38 cells expressing the neomycin-resistance gene following retroviral transduction with MFG-Neo (MC38-Neo) were used as control cells [10].

Determination of in vitro growth of wild-type and genetically modified tumor cells

Wild-type MC38 (MC38-WT), MC38-Neo, murine IL-12-transduced MC38 (MC38-IL12), or murine IFN-α-transduced MC38 (MC38-IFNα) cells (1×10⁵ cells) were incubated in CM in 6-well-plate. Nine wells were prepared for each tumor cell line. Three wells were harvested every 4 days, and cell numbers in each well were determined microscopically.

Tumor establishment models

To investigate tumor establishment, B6 mice were injected subcutaneously (s.c.) in the right flank with 1×10⁵ MC38-WT, MC38-Neo, MC38-IFNα, or MC38-IL12 cells. Tumor size was measured twice a week using vernier calipers. This evaluation was performed in a blinded fashion. Tumor-free mice in establishment models were rechallenged with 3×10⁵ wild-type tumor cells 35 days after the initial challenge. Mice with ulcerated tumors or tumors larger than 20 mm in diameter were killed. Results are reported as mean tumor area (square millimeters) plus standard error (SE). Experiments of tumor establishment were performed twice.

Antibody-mediated depletion of leukocytes in vivo

To determine the role of the immune system in reduction of in vivo tumor growth in the establishment model, CD4⁺ T cells, CD8⁺ T cells, or NK cells were depleted by an antibody method. Culture medium from hybridomas producing the following antibodies was used at appropriate dilutions/concentrations: anti-CD4 (GK1.5, TIB207; American Type Culture Collection; ATCC, Manassas, Va.) and anti-CD8 (2.43, TIB210; ATCC). For depletion of NK cells, anti-asialo-GM1 was obtained from WAKO (Osaka, Japan). All antibody doses and treatment regimens were determined in preliminary studies using the same lots of antibody employed for the experiments. Treatment was confirmed to completely delete the desired cell population for the entire duration of the study, as determined by flow cytometric analysis (data not shown).

Therapeutic models

To evaluate use of cytokine-expressing tumor cells in treating established tumors, we measured the size of established MC38-WT tumors in mice before and after treatment with genetically modified MC38 cells as previously described [10]. In brief, firstly, B6 mice were injected in the right flank with MC38-WT cells (1×10⁵ cells s.c.). Injections of 1×10⁵ MC38-Neo, MC38-IFNα, or MC38-IL-12 cells followed in the contralateral (left) flank every 3 days starting at day 7, when tumors had reached a tumor area of 16–30 mm². When modified cell lines were co-injected, 5×10⁴ cells of each type were admixed. Each experiment involved six mice per group. Tumor size was measured twice a week using vernier calipers.

Immunohistologic analysis

B6 mice were injected (s.c.) in the right flank with 1×10⁵ MC38-WT cells. On days 7, 10, and 13, 1×10⁵ genetically modified MC38 cells were inoculated in the left flank. Tumor tissues were harvested 3 days after inoculation of genetically modified tumor (16 days after WT inoculation), and were immediately embedded in optimal clotting temperature (OCT) compound (Tissue Tek, Elkhart, Ind.) and frozen. Serial 5-μm sections were exposed to antibodies to CD8α (Nippon Becton Dickinson, Tokyo, Japan). Otherwise, for granulocyte detection in tumor tissues, B6 mice were injected s.c. with 1×10⁵ genetically modified MC38 cells around the established wild-type tumor 7 days after 1×10⁵ MC38-WT inoculation. Tumor tissues were harvested 3 days after inoculation of genetically modified tumor (10 days after WT inoculation), and serial 5-μm sections were exposed to anti-Gr-1 antibody (Nippon Becton Dickinson). Rat IgG2a (Nippon Becton Dickinson) was used as control antibody. Immunostaining was completed with a Vectastain ABC kit (Vector, Burlingame, Calif.). Immunoreactive cells were counted in ten fields using light microscopy (×400) in a blinded fashion.

Analysis of tumor-infiltrating lymphocytes and splenocytes

B6 mice were injected (s.c.) in the right flank with 1×10⁵ MC38-WT cells. On days 7, 10, and 13, 1×10⁵ genetically modified MC38 cells were inoculated in the left flank. Tumor tissues and spleens were harvested 3 days after this third inoculation (16 days after WT inoculation). Splenocytes were harvested using a standard method. The TIL were separated from tumor tissue as previously reported [24] with some modifications. In brief, tumor fragments were incubated in a mixture of 30 U/ml hyaluronidase (Sigma), and 500 U/ml DNAse (Sigma) in Hank's balanced salt solution (HBSS; Life Technologies), at ambient temperatures for 3 h. The cell suspension was strained through a grid and washed three times with HBSS. Lymphocytes were separated from tumor cells by centrifugation at 2000 rpm for 20 min using a density separation medium (Lympholyte-M; Nycograde Pharma, Oslo, Norway). Lymphocytes were harvested and washed three times with CM. Flow cytometric analyses were performed using FACScan (Becton Dickinson) to analyze the phenotype of splenocytes and TIL of mice treated with genetically modified tumor cells. Monoclonal antibodies used in this analysis were fluorecein isothiocyanate (FITC)-conjugated anti-CD4, CD8, or NK1.1 antibody (Becton Dickinson).

Induction of tumor-specific CTL

MC38-specific CTL was induced as reported previously with some modifications [11]. Briefly, mice were rendered MC38-immune by initial injection with 1×10⁵ non-irradiated MC38-IFNα cells on day 0. Subsequently, tumor-free animals received challenges of 3×10⁵ MC38-WT cells on day 35 and with 1×10⁶ WT cells on day 70. Splenocytes (3×10⁶ cells/ml) from these immune mice were harvested on day 77 and stimulated with irradiated (10,000 rad) MC38-Neo, MC38-IFNα, or MC38-IL12 tumor cells (3×10⁵ cells/ml). Seven days later, responder cells (1×10⁶ cells/ml) were restimulated with the same type of irradiated tumor cell (10,000 rad, 1×10⁵ cells/ml) and irradiated syngeneic naive splenocytes (3000 rad, 1×10⁶ cells/ml) in the presence of 50 IU/ml recombinant mouse IL-2 (Becton Dickinson). Cytolytic assays were performed 5 days after the last stimulation using the responder cells as effector cells.

Cytolytic assays

Cytolytic assay was performed essentially as previously described [11]. Tumor-stimulated effector cells were assessed for cytolytic activity against MC38-WT and YAC-1 cells in triplicate in 4-h ⁵¹Cr-release assays. Target cells (1×10⁶ cells/ml) were labeled with 100 μCi of Na₂ ⁵¹CrO₄ (Amersham Pharmacia Biotech, Tokyo, Japan) for 1 h at 37°C. Labeled cells were washed and resuspended. Target cells (1×10⁴) and various numbers of effector cells at indicated effector to target ratios (E:T) were plated in 200 μl of CM in each well of 96-well round-bottom plates. ⁵¹Cr-release was measured after a 4-h incubation at 37°C. Percentage lysis was determined using the formula: (release in assay minus spontaneous release) × 100/(maximum release minus spontaneous release). Maximum release was determined by lysis of labeled target cells with 1% Triton X-100. Spontaneous release was measured by incubating target cells in the absence of effector cells, and was less than 15% of maximum release.

Statistical analyses

Significance was assessed by Student's t test. The difference between groups was considered significant when the p value was lower than 0.05.

Results

IFN-α or IL-12 transduction of tumor cells does not affect growth in vitro

MC38 tumor cells were transduced retrovirally with a murine IFN-α or IL-12 gene. After antibiotic selection with G418, production of murine IFN-α or IL-12 by the cells was confirmed with ELISA; MC38-IL12 and MC38-IFNα produced 4.0±0.8 and 157.0±4.2 ng/10⁶ cells/48 h of the respective cytokine. Compared with a previously established IFN-α-transduced MC38 cell line [10], the MC38 cells transduced in the present study produced almost twice as much IFN-α as the previously described cells (157.0±4.2 vs 80.3±15.9 ng/10⁶ cells/48 h). WT, Neo-, murine IFN-α-, or murine IL-12-transduced MC38 cells were seeded at 1×10⁵ cells/wells in 6-well plates, and cells per well were counted every 3 days in triplicate. The growth rate did not differ significantly between MC38-WT, MC38-Neo, MC38-IFNα, and MC38-IL12 cells (data not shown).

Transduction of a murine IFN-α or IL-12 gene reduces tumorigenicity of MC38 cells

Syngeneic immunocompetent B6 mice were inoculated s.c. with 1×10⁵ WT or genetically modified MC38 tumor cells and tumor growth was measured. As shown in Fig. 1A, transduction of IFN-α or IL-12 significantly reduced growth of the inoculated cells compared with MC38-WT or MC38-Neo in this poorly immunogenic tumor model (IFN-α; p=0.0092 vs WT; p=0.0041 vs Neo, or IL-12; p=0.0399 vs WT; p=0.0253 vs Neo).

Fig. 1.

A IFN-α- or IL-12-transduction reduces tumorigenicity of MC38 tumor cells. MC38-WT, MC38-Neo, MC38-IFNα, or MC38-IL-12 cells were inoculated (1×10⁵ cells subcutaneously) into six B6 mice per group. Tumor size was measured twice a week in a blinded fashion. Mice with tumors exceeding 20 mm in diameter were killed. Results are reported as mean tumor area (square millimeters) plus standard error for each group. Experiments were performed twice with similar results. B Tumor-free mice after injection of genetically modified MC38 tumor cells and subsequent injection of wild-type tumor cells. Tumor-free mice challenged with 3×10⁵ wild-type tumor cells contralaterally at day 35 after injection of modified cells. Results are reported as the number of tumor-free mice at day 35 (following first injection) and day 70 (35 days following second injection). Six mice per group were injected and the experiment was performed twice

Figure 1B shows total numbers of tumor-free mice in two separate experiments on day 35 (first inoculation) or day 70 (second inoculation). Of 12 mice inoculated with MC38-IFNα, 9 were tumor free on day 35. Of these 9 mice, 7 rejected a subsequent tumor rechallenge with 3×10⁵ MC38-WT tumor cells on day 70. In contrast, IL-12 transduction reduced tumorigenicity of MC38 tumor cell less efficiently; of 12 mice inoculated with MC38-IL12, only 4 were tumor free on day 35. Of the 4 tumor-free mice, 2 rejected a subsequent tumor rechallenge on day 70. These findings suggest that IFN-α transduction reduce tumorigenicity of the MC38 tumor cells more efficiently than IL-12 transduction, and that IFN-α induces potent, long-lasting immune responses.

Repeated therapeutic inoculations with IFN-α-transduced MC38 tumor cells suppress growth of established MC38 tumors on the contralateral flank

We next examined the therapeutic effects of immunization using tumor cells genetically modified to produce IFN-α or IL-12 in the MC38 tumor model. Mice bearing established MC38-WT tumors were treated by repeated injections of cytokine gene-transduced tumor cells in the contralateral flank starting on day 7. Mice were injected every 3 days with MC38-IFNα or MC38-IL12 tumor cells. Repeated injections of MC38-IFNα cells suppressed outgrowth of the established MC38 tumor, compared with repeated injections of MC38-Neo (p=0.0088; Fig. 2). Although the mean tumor size of mice treated with MC38-IL-12 was also decreased, the suppressive effect of IL-12 treatment on established tumor growth did not reach significance (p=0.0563).

Fig. 2.

IFN-α transduction of MC38 tumor cells suppresses the growth of wild-type tumor established in the contralateral flank. Animals were inoculated with MC38-WT (1×10⁵ cells) in the right flank on day 0. On day 7, established MC38 (16–30 mm² in size) were treated three times, with Neo-, IFN-α-, or IL-12-expressing MC38 cells (10⁵ cells), in the contralateral (left) flank, every third day starting 7 days after wild-type tumor injection. Six mice were studied in each group. Results are reported as mean tumor area (square millimeters) plus standard error. Significance at 95% confidence limits is indicated. This experiment was performed twice with similar results

Immunohistologic analysis of CD8⁺ cells infiltrating established wild-type tumors in mice treated with IFN-α

To examine how IFN-α and IL-12 gene therapy inhibit growth of established MC38 tumors, we immunohistochemically analyzed the leukocytic infiltrate of established tumors after contralateral injection of cytokine-expressing cells. After mice were killed 3 days after the third treatment inoculation, tumor samples were harvested and examined for CD8⁺ cells. Wild-type tumors from mice treated with MC38-IFNα showed marked infiltration with CD8⁺ cells (Table 1), whereas only a few CD8⁺ cells could be detected in wild-type tumors of mice treated with MC38-Neo or MC38-IL-12.

Table 1.

Immunohistologic analysis of CD8⁺ T-cell infiltration in established wild-type tumors treated with genetically modified MC38 cells

Treatment	No. of positive CD8+ cells
MC38-Neo	12.2±7.9
MC38-IFNα	38.4±8.6
MC38-IL12	13.5±4.6

Cells/field (×400)

Immunoreactive cells were counted in ten microscopic fields (×400) without knowledge of the experimental group. Results are reported as mean number of positive cells±SE

These results suggest that immunization with MC38-IFNα leads to induction of cellular antitumor immunity associated with infiltrating CD8⁺ cells.

CD8⁺ T cells are responsible for IFN-α-induced tumor rejection, whereas both CD8⁺ T cells and NK cells contribute to IL-12-induced rejection

To characterize actions of the immune system in reduction of in vivo tumor growth in the MC38 tumor model, immunocompetent B6 mice were depleted of CD4⁺ T cells, CD8⁺ T cells, or NK cells using antibodies (Fig. 3). Depletion of CD8⁺ T cells prevented tumor reduction induced by transduction of either IFN-α or IL-12, whereas depletion of NK cells prevented only the reduction induced by IL-12. Depletion of CD4⁺ T cells did not affect tumor growth reduction from MC38-IFNα or MC38-IL12. These findings suggest that CD8⁺ T cells may be solely responsible for tumor regression induced by transduction of IFN-α, whereas both CD8⁺ T cells and NK cells contribute to reduction of tumorigenicity by IL-12 transduction.

Fig. 3.

CD8⁺ cells mediate tumor regression of IFN-α gene therapy, whereas CD8⁺ and NK1.1⁺ cells mediate regression in IL-12 gene therapy. Animals were depleted of CD4⁺ T cells, CD8⁺ T cells, or NK cells using antibodies. Anti-CD4 (open circles) or anti-CD8 antibody (squares) was injected intraperitoneally every 3 days, or anti-asialo-GM1 antibody (triangles) was injected intravenously (control; solid circles). Three days after antibodies were injected, 1×10⁵ MC38-Neo (upper panel), -IFNα (middle panel), or -IL12 cells (lower panel) were inoculated (subcutaneously) into six B6 mice per group. Results are reported as mean tumor size (square millimeters) plus standard error for each group

Treatment with both IFN-α-transduced cells and IL-12-transduced cells: additive effects on established wild-type tumors

Combination gene therapy was performed against established wild-type tumor to model this approach in possible clinical treatment. Tumor-bearing mice were injected contralaterally with 1×10⁵ cells of MC38-Neo; 5×10⁴ cells each of MC38-Neo and MC38-IFNα; MC38-Neo and MC38-IL12; or MC38-IFNα and MC38-IL12. No significant difference was detected in established tumor growth between MC38-Neo (1×10⁵ cells/mouse)-treated mice and mice treated with combinations of genetically modified tumor cells (5×10⁴ cells each), except for the combination of MC38-IFNα with MC38-IL12 (Table 2). Outgrowth of established wild-type tumor was significantly suppressed on day 28 by repeated therapeutic inoculations of both MC38-IFNα and MC38-IL12 (p=0.0287). Furthermore, 1 of 12 mice treated with MC38-IFNα and MC38-IL12 was tumor free at day 35.

Table 2.

Size of established wild-type tumors treated with combinations of genetically modified MC38 cells

Treatment	Tumor size (mm2; day 26)
Neo	248.7±38.7*
Neo+IFNα	150.3±40.6
Neo+IL12	166.7±55.5
IFNα+IL12	123.8±29.9*

*p=0.0287

Animals were inoculated with 10⁵ of MC38-WT cells in the right flank on day 0. On day 7, mice with established MC38 tumors (16–30 mm² in size) were injected in the left flank with 1×10⁵ MC38-Neo cells or 5×10⁴ cells of each of MC38-Neo and -IFNα, MC38-Neo and -IL12, or MC38-IFNα and -IL12. Each experiment included six mice per group, and tumor size was measured using vernier calipers. Results are reported as mean tumor area±SE

CD8⁺ cells and NK1.1⁺cells in established tumors and spleens of mice treated with MC38-IFNα and/or MC38-IL12

Table 3 shows results of flow cytometric analyses of immune cells infiltrating an established wild-type tumor, or immune cells in the spleen of mice treated with MC38-IFNα and/or MC38-IL12 tumor cells. In established tumors, more CD8⁺ T cells infiltrated in the MC38-IFNα-treated group than in the MC38-Neo- or MC38-IL-12-treated group. On the other hand, many NK1.1⁺ cells were detected in the IL-12-treated group. Furthermore, in mice treated with IFN-α and IL-12 in combination, both CD8⁺ cells and NK1.1⁺ cells were increased in the tumor compared with Neo-treated mice.

Table 3.

Population of tumor-infiltrating lymphocytes or splenocytes positive for CD4, CD8, and NK1.1 antibody. TIL tumor-infiltrating lymphocytes

Treatment	Positivity for markers
	CD4	CD8	NK1.1
TIL (%)
Neo	8.9	18.2	14.6
IFNα	3.7	37.7	13.8
IL12	3.3	25.3	24.4
IFNα+IL12	5.4	35.1	33.3
Splenocytes (%)
Neo	13.3	9.5	1.2
IFNα	13.2	15.4	0.7
IL12	13.5	11.9	11.3
IFNα+IL12	15.0	13.2	10.1

Spleen and tumor tissue were harvested 3 days after inoculation of genetically modified tumor cells (13 days after inoculation of wild-type cells). Mononuclear cells in the spleen or tumor were isolated using a gradient centrifugation method. CD4, CD8, or NK1.1 expression on the surface of these cells was analyzed by flow cytometry. Results are given as the percentage of positive cells using each monoclonal antibody

In the spleen, changes essentially paralleled those in TIL. IFN-α increased CD8⁺ cells, whereas IL-12 increased NK1.1⁺ cells.

These results support the impression that IL-12 has an additive effect on in vivo antitumor immune response to IFN-α, involving stimulation of NK cells.

Tumor-specific cytolysis was detected when splenocytes of hyper-immunized mouse were stimulated by IFN-α-transduced tumor cells

To investigate the effects of cytokine gene transduction on propagation of tumor-specific CTL, we sought to induce MC38-specific CTL using genetically modified MC38 cells and splenocytes of hyper-immunized mice. Immune mice were made by injection of 1×10⁵ MC38-IFNα on day 0, followed by injections of 3×10⁵ MC38-WT on day 35 and 1×10⁶ MC38-WT on day 70. Splenocytes of hyper-immunized mice were harvested on day 77 and were stimulated twice weekly in vitro with genetically modified tumor cells. Cytolytic assays against MC38 and YAC-1 cells, which are sensitive for NK cells, were performed 7 days after the second stimulation. As shown in Fig. 4, effector cells stimulated with MC38-IFNα were highly specific for MC38-WT (53.1% vs MC38, 8.8% vs YAC-1; E:T=40; Fig. 4B). When MC38-IL-12 cells were used as stimulator cells, cytolysis was non-specifically similar with MC38 and YAC-1 cells as targets (31.2 vs MC38, 32.7 vs YAC-1; E:T=40; Fig. 4C); thus, tumor-specific cytolysis was clearly observed when hyper-immune splenocytes were stimulated in vitro with MC38-IFNα, but not with MC38-IL-12.

Fig. 4A–C.

Stimulation with MC38-IFNα, but not with MC38-IL12, induces potent tumor-specific cytolysis. Immune mice received injections of 1×10⁵ MC38-IFNα on day 0, 3×10⁵ MC38-WT on day 35, and 1×10⁶ MC38-WT on day 70. Splenocytes of the hyper-immunized mice were harvested on day 77 and were stimulated weekly for 2 weeks in vitro with MC38-Neo (A), MC38-IFNα (B), or -IL12 (C). Cytolytic assay against MC38 or YAC-1 cells was performed 5 days after the second stimulation. Results are reported as mean percentage of cytotoxicity plus standard deviation. This experiment was performed twice with similar results

Discussion

The murine colorectal cancer cell line MC38 [31] has been used as a model of poorly immunogenic tumors. We previously reported that IFN-α-producing MC38 cells showed reduced tumorigenicity [10], and that the IFN-α expression elicited MC38-specific CD8⁺ CTL. IFN-α promoted survival of a MC38-specific CTL line by preventing activation-induced cell death [11]. In the present study, transduction of murine colorectal cancer cells with IL-12 or IFN-α genes reduced tumorigenicity in this poorly immunogenic murine colorectal cancer cell model. Although cell growth in MC38-WT, MC38-Neo, MC38-IFNα, or MC38-IL12 tumors did not differ in vitro, growth of IFN-α- or IL-12-producing MC38 tumors was clearly suppressed in in vivo tumor establishment model. These results suggest that these cytokines do not directly suppress growth of tumor cells, but do so indirectly through induction of immune responses against tumor cells in immunocompetent hosts. Our experiments with depletion of CD4⁺, CD8⁺, or asialo-GM1⁺ cells suggest that different mechanisms contribute to antitumor effects of IFN-α and IL-12 gene therapy of cancer. Furthermore, we demonstrated therapeutic effects of repeatedly injected IFN-α-transduced tumor cells against established tumors, and observed additive effects with a combination of IFN-α and IL-12. Immunohistologic analyses of established tumors in mice treated with genetically modified tumor cells, as well as flow cytometric analyses of TIL and splenocytes from these mice, supported observations that IFN-α stimulates CD8⁺ cells, and that IL-12 activates NK cells. In addition, tumor-specific CTL could be induced when splenocytes from hyper-immunized mice were stimulated with IFN-α-transduced tumor cells, but not with IL-12- or control gene-transduced tumor cells. These findings suggest that IFN-α effectively induces specific immune response against a poorly immunogenic tumor, whereas IL-12 mainly stimulates non-specific antitumor responses.

Tumor-based vaccination in combination with cytokine gene therapy is considered to have the advantage that paracrine cytokine delivery by genetically modified tumor cells reduces systemic toxicity and could be used clinically irrespective of any detectable tumor-associated antigen (TAA). Furthermore, cytokine gene transduction of tumor cells could upregulate TAA together with MHC class-I molecules on the surface of transduced cells. We confirmed by flow cytometry that MHC class-I molecules on both IFN-α- and IL-12-transduced tumor cells were upregulated compared with wild-type or control gene-transduced cells (data not shown). Clinical disadvantages of this mode of treatment include requirement of a relatively large volume of resected tumor and difficulty of establishing appropriate tumor cell lines that produce sufficient amounts of cytokine.

We observed a considerable quantitative difference in cytokine production between the genetically modified tumor cell lines, MC38-IFNα, and MC38-IL12. To produce biologically active IL-12, simultaneous transduction of mammalian cells with two different genes is required [9]. Therefore, differences of cytokine production may be related to the complexity of the retroviral construct; DFG-murine IFN-α-Neo retroviral vector has two components, whereas TFG-murine-IL-12-Neo consists of three parts: IL-12 p40; IL-12 p35; and the neomycin resistance gene. Okada et al. compared therapeutic effects of four kinds of cytokine gene therapy on a rat glioma model using IL-4, IL-12, granulocyte macrophage colony stimulating factor, and IFN-α gene-transduced tumor cells [24]. When they immunized rats with IL-12 gene-transduced glioma cells estimated to produce 2–3 ng/day, no therapeutic benefit was observed. In the present study, we treated mice with 1×10⁵ of genetically modified tumor cells, which were estimated to produce only 0.2 ng/day of cytokine at the inoculation site. Since higher doses of IL-12 may improve the antitumor effect, further developments of vectors enabling transfectants to produce IL-12 at higher concentrations might show better results. If the lower production of IL-12 by genetically modified tumor cells in our study depends on complexity of the retroviral vector, the same phenomenon would occur in the clinical application, and production of IL-12 would be much less than that of IFN-α. As a preclinical study, we performed experiments using IFN-α and IL-12 gene-transduced tumor cell lines which could produce the highest amount of each cytokine among the established gene-transduced cell lines because we expected the most effective antitumor activities.

We sought to elicit tumor-specific CTL to compare abilities of genetically modified tumor cells to propagate tumor-specific immune responses in hyper-immunized mice, as previously described [11]. Our previous study established that MC38-specific CD8⁺ CTL exist in the spleen of such mice, and Mehrotra et al. suggested that IL-12 promotes selective maturation of CD8⁺ T cells into CTL [18]. In this study, we performed induction of CTL from hyper-immunized mice initially inoculated with MC38-IFNα cells because we could hardly obtain tumor-free mice initially injected with MC38-IL12 cells. Almost all mice had obvious tumors after the second injection of MC38-WT cells (77 days after MC38-IL12 inoculation). When we tried to induce tumor-specific CTL from the mice without development of tumor, we could detect no specific cytolytic response against MC38 cells (data not shown). In the present CTL assay, we did not observe any specific killing when hyper-immunized splenocytes were stimulated with MC38-IL12, although CD8⁺ cell depletion reduced antitumor effects of IL-12 transduction. Moreover, non-specific cytolysis was decreased when splenocytes from hyper-immunized mice were stimulated with MC38-IL12 (Fig. 4). Other experiments in this study, however, indicated that IL-12 enhanced non-specific killing; treatment with MC38-IL-12 resulted in infiltration of NK cells. This apparent discrepancy may have been caused by an insufficient dose of IL-12 or differences between in vivo and in vitro experiments. Further studies are needed to address this issue.

In our therapy model, we treated animals by injection of genetically modified tumor cells in the flank contralateral to the established wild-type tumor. Using this model, we evaluated the influence of cytokine gene therapy on systemic induction of an antitumor immune response. Interestingly, phenotypic analyses of splenocytes as well as TIL from mice injected with IFN-α- and IL-12-transduced tumor cells demonstrated enhanced contralateral tumor infiltration of CD8⁺ and NK1.1⁺ cells, respectively, indicating not only a local but also a systemic immune response. Because IL-12 is known as a potent antitumor cytokine inducing Th1-type responses in vivo, we investigated mechanism in IL-12 gene therapy of cancer in comparison with IFN-α gene therapy, and raised the possibility of using IFN-α in combination with IL-12 expression. Although suppressive effects of IFN-α in combination with IL-12 on growth of the established tumor were observed in the therapy model (Table 2), we could not see any reduction in the size of the parental MC38 tumors except in one mouse. Far better results would be needed for clinical application; this might require combination of IFN-α and IL-12 with other cytokines, adjuvants, or dendritic cells, which induce potent antitumor immune responses [17, 33]. The additive effect of combined Th1-type cytokine gene therapy that we demonstrated is thought to result from combining different antitumor mechanisms of IFN-α and IL-12 gene therapy; IFN-α elicits tumor-specific CTL, whereas IL-12 seems to stimulate non-specific killing. Mechanisms of antitumor immune responses induced by a cytokine gene therapy are complex and much further study of combined cytokine gene therapies is required prior to conclusive clinical trials.