Cell-based gene therapy after cytokine gene transfer is being investigated for autologous and allogeneic vaccination in cancer therapy. Here we show that mice vaccinated with 3–5 × 106interleukin 12(IL-12) gene-transduced CT26 colon cancer cells developed a long-lasting antitumor immune memory able to reject not only parental cells but also syngeneic, LM3 mammary, and MCE fibrosarcoma tumorigenic cells. In contrast, mice vaccinated with 0.5–1 × 106 CT26 cells transduced with pBabe neo IL-12 retrovirus cells (CT26-IL12) were only able to reject parental cells. An increase in the total circulating levels of IgG2a and a clear shift toward a systemic Th1 response developed,regardless of the amount of injected CT26-IL12 cells. On the contrary,a strong increase in anti-CT26-specific IgG2a levels was observed only when 3–5 × 106 CT26-IL12 cells were injected. Immunocompetent mice vaccinated with 3–5 × 106 CT26-IL12 cells developed local nodules for a few days,which then ceased growing. These nodules comprised mainly blood vessels, suggesting that an angiogenic process was taking place. CD8+ T cells were responsible for the anti-LM3 tumor cell memory, whereas CD4+T cells were not involved. Splenocytes and lymphocytes obtained from mice immunized against CT26 cells were able to kill LM3 cells in vitro. Adoptive transfer of lymphocytes obtained from animals immunized against CT26 colon cancer cells suppressed LM3 mammary tumor growth in tumor-bearing mice. The present studies raised the possibility of isolating CTL clones and identifying CTL epitopes shared by different tumor cell types, which can be a target for cancer therapy.

IL8-12 is a heterodimeric cytokine that stimulates NK cells(1, 2, 3), promotes maturation of CTLs(4, 5), and induces IFN-γ production, stressing its role as an efficient molecule for the initiation of a Th1 response (1, 2). However, in the presence of IL-4, IL-12 may also favor the generation of IL-4-producing T cells from Th0 cells (6, 7) and the exacerbation of an established Th2 response (8, 9). IL-12 enhances the humoral immunity through the recruitment of new B-cell clones through a mechanism involving IFN-γ (10, 11). It was also shown that IL-12 has anti-angiogenic properties that may contribute to its antitumor effect (12).

Local and systemic treatment with IL-12 led to the eradication of primary tumors or metastatic lesions (13, 14). However,the toxicity observed after systemic administration of IL-12 supported the development of delivery mechanisms providing relatively high levels locally. Indeed, murine tumor cells genetically modified to produce IL-12 were able to induce antitumor responses with no major toxic effects (15).

In the studies described here, we show for the first time that mice vaccinated with CT26 colon carcinoma cells engineered to express IL-12 developed a long-lasting antitumor immune memory able to reject a challenge not only with parental cells but also with syngeneic LM3 mammary tumor cells and MCE fibrosarcoma cells. This memory rests on CD8+ T cells correlated with a shift toward a CT26-specific Th1 response and involved a T-cell-mediated pro-angiogenic process. Moreover, adoptive transfer of lymphocytes from lymph nodes of mice vaccinated with CT26 cells producing IL-12 suppressed mammary tumor growth in tumor-bearing mice.

Vector Construction, Transfection of Packaging Cells, and Transduction of Tumor Cells.

The full-length cDNA corresponding to murine p35 and p40 subunits of IL-12 (kindly provided by Maurice Gately, Hoffmann-La Roche, Inc.) were cloned in the pBabe retroviral vector (16). The p35 subunit was cut with NcoI, blunt-ended with Klenow, and cut with EcoRI. The NcoI/blunt-EcoRI fragment containing p35 was inserted into pBabe Neo linearized with BamHI, blunt-ended with Klenow, and digested with EcoRI. To insert the p40 subunit downstream of the encephalomyocarditis virus internal ribosome entry site, p40 was cut with EcoRI, blunt-ended, and cut with NcoI. This fragment was cloned in pCITE vector (Novagen, Madison, WI) cut previously with NcoI-EcoRV. This NcoI site within pCITE contains the ATG corresponding to the translation start site. The IRES-p40 fusion was excised with EcoRI-SalI and cloned into pBabe Neo-p35 cut previously with EcoRI-SalI. The resulting vector was named pBabe neo IL-12. Twenty μg of pBabe neo IL-12 was transfected into GP+env Am12 cells. Geneticin (Life Technologies, Inc.,Rockville, MD) was added up to a concentration of 1.5 mg/ml, and resistant cells were used to generate helper-free, virus-containing supernatants. CT26 cells were transduced by exposure to undiluted supernatant. Two days later, cells were split and selected in Geneticin up to a concentration of 1.5 mg/ml. Cells transduced with pBabe neo vector without insert were used as controls.

Cell Lines.

The CT26 mouse colon carcinoma cell line (17) was obtained from the M. D. Anderson Cancer Research Center (Houston, TX). The LM3 mammary tumor cell line is derived from a mammary tumor which appeared spontaneously in BALB/c mice (18). Both cell types were routinely maintained in DMEM containing 10% (vol/vol) FCS, 2 mm glutamine, 2.5 units/ml penicillin, and 2.5 μg/ml streptomycin. The amphotropic GP+envAm12 packaging cell line was maintained in DMEM + 10% newborn calf serum as described(19). The LB T-cell lymphoma and the MCE fibrosarcoma were kindly provided by Dr. R. A. Ruggiero, Buenos Aires(20). All of the cell lines were kept free of Mycoplasma and were routinely tested with the MYCOTECT Kit(Life Technologies, Inc.).

Assessment of IL-12 Production and IgGs Levels.

A capture ELISA using antimouse IL-12 (gA5 capture/5C3 detect mAbs, gifts from Dr. M. Gately, Hoffman-La Roche, Nutley, NJ)was used for quantification of IL-12 production. After overnight incubation at 4°C, plates were blocked at room temperature with BSA and washed with 0.05% Tween-PBS. Cell-conditioned media and IL-12 control samples were added on the plates and incubated overnight at 4°C. After washes with Tween-PBS, plates were incubated overnight at 4°C with biotinylated anti-IL-12 antibody. IL-12 levels were calculated using standard curves of murine IL-12.

Total levels of circulating and anti-CT26-specific IgG2a and IgG1 were performed as described previously (20).

Reverse Transcription-PCR Analysis and T Cell Proliferation Assay.

For the detection of AH1 mRNA, total RNA from the different cell lines was obtained with TRIzol (Life Technologies, Inc.) and reverse transcribed. The 5′ and 3′ primers for detecting AH1 transcript were synthesized as described (21). As a control, peptides corresponding to two different genes were used: one corresponding to the CPD1 cDNA (GenBank accession no. V89345) and the other corresponding to the Fru-1,6-biphosphatase cDNA (EC 3.1.3.11). To establish the specific response against AH1 peptide, BALB/c mice were injected with 3 × 106 CT26-neo or CT26-IL12 cells and challenged with parental cells 2 weeks later. After 1 week, spleen cells were obtained from both types of mice and naive mice were grown in RPMI containing 5% heat-inactivated FBS, l-glutamine, 2-ME, and antibiotics. Cells were incubated for 4 days with the different peptides pulsed with 1 μCi of[3H] thymidine for 7 h and counted(22).

Immunohistochemical Studies.

The sites of injection of tumor cells were removed, fixed, and embedded in paraffin. Immunohistochemical studies were performed on 5-μm sections. Sections were preheated in a microwave oven for 12 min in the presence of citrate buffer. Sections were washed, passed through graded alcohols, and incubated with H2O2 in methanol to eliminate endogenous peroxidase. Then, they were incubated with rat mAb antimouse granulocytes (Ly-6G, PharMingen, San Diego, CA) overnight at 4°C (final concentration, 2.5 μg/ml), and then incubated with biotin-labeled goat antirat antisera (Jackson Immunoresearch Lab, West Grove, PA). After washing, sections were incubated with ABC Vectastain ABC Elite reagent (Vector Laboratories, Burlingame, CA). Staining was developed with diaminobenzidine and sections were counterstained with hematoxilin. For frozen sections, the site of tumor cell injection was included in OCT (Miles, Elkhart, IN). Seven-μm sections were incubated overnight with either rat antimouse antimacrophages antibody(F4/80; Serotec, Oxford, United Kingdom; 1/50 final dilution) or rat antimouse anti-CD34 antibody (MEC 14.7; Serotec; 1/20 final dilution). Sections were developed as described previously for paraffin embedded tissues.

Statistical Analysis.

The significance of differences was determined by using the Student’s t test, one-way ANOVA, and Tukey-Kramer Multiple Comparison Tests.

In Vivo Studies.

None of the BALB/c mice that received injections of 2 × 105 to 1 × 106 CT26-IL12 cells developed tumors (Table 1). Mice that received injections of 3 or 5 × 106 CT26-IL12 cells showed palpable nodules between days 7 and 15 after injection, after which the nodules ceased growing and regressed (Fig. 1, A and B). Surprisingly, these nodules containing a central necrotic area were composed of blood vessels, active fibroblasts, and an immune infiltrate composed mainly of neutrophils(Fig. 2, B–D). Macrophages and mast cells were scarcely seen (not shown). Blood vessels and the presence of macrophages was confirmed by using specific antibodies (not shown). By contrast, all of the mice injected with CT26-neo cells showed large tumor masses (Fig. 2). Only a few neutrophils and vessels were seen (Fig. 2,A). By using the technique of everted skin(23), we confirmed that injection of CT26-IL12 cells induced neovascularization compared with CT26-neo cells (2.41 ± 0.6 versus 1.74 ± 0.43 vessels/mm2 skin; P < 0.001; Fig. 2, E and F). In addition, all of the athymic nude mice injected with 3 × 106 CT26-neo cells developed tumors. But only 50% of the nude mice injected with CT26-IL12 cells developed tumors,with great delay, suggesting the involvement of both T cells and non-T cells in the initial rejection of CT26-IL12 cells (Fig. 1 C). Histological analysis of the site of injection of CT26-IL12 cells and of the tumors formed after injection with CT26-IL12 cells showed no evidence of neovessel formation (not shown).

Antitumor Immune Memory.

All of the mice that received injections of CT26-IL12 cells were able to reject a contralateral challenge with parental cells even when challenged with 3 × 106 cells(Table 1). Mice were also able to reject a second challenge with parental cells performed 8 weeks after the first injection (Table 1).

To evaluate whether the immune memory might induce cross-protection against non-organ-related tumor cells, mice that rejected CT26-IL12 cells were challenged with three different nonimmunogenic, syngeneic, non-organ-related tumor cell types. None of the mice that rejected 2 or 5 × 105 CT26-IL12 cells was able to reject a challenge with 3 × 105 LM3 mammary tumor cells (Table 1). However, 60–90% of mice that rejected 3 or 5 × 106 CT26-IL12 cell growth rejected 3 × 105 LM3 cells injection when challenged 3–9 weeks later (Table 1). In addition, 75%of mice that rejected a first injection of 3 × 106 CT26-IL12 cells and a double challenge with parental CT26 cells rejected a challenge with LM3 cells performed 4 months later (Table 1). Moreover, 50% (12 of 24) of mice vaccinated with 3 × 106 CT26-IL12 cells that rejected a challenge with parental cells 2 weeks later were able to reject a challenge with tumorigenic inocula of MCE fibrosarcoma cells(Table 1). Moreover, 10 of 12 of these mice rejected a second challenge with MCE cells performed 4 weeks later. Under the same conditions,these mice were unable to reject a challenge with LB T lymphoma cells. All of the control mice injected with LM3, MCE, and LB cells developed tumors and were not protected against a challenge with parental cells(not shown).

Levels of Circulating and CT26-specific IgG1 and IgG2a Subclasses.

Vaccination of mice with CT26-IL12 cells induced in all cases a strong increase in IgG2a circulating levels, leading to a Th1 systemic status regardless of the amount of injected cells (Table 2). Vaccination with 0.5 and 1.0 × 106 CT26-IL12 cells led to moderate increases both in IgG1 and IgG2a anti-CT26-specific levels. On the contrary,vaccination with 3 and 5 × 106CT26-IL12 cells induced a 5- to 10-fold increase in anti-CT26-specific IgG2a levels and decreased levels or no change in anti-CT26-specific IgG1, showing a clear shift toward a Th1-dominated response (Table 2). None of the serum samples obtained from these mice recognized LM3 cells regardless of whether the samples corresponded to mice that did or did not reject the LM3 challenge (not shown).

Characterization of the Anti-LM3 Immune Response.

Depletion of CD8+ T cells abrogated the anti-LM3 immune memory, whereas depletion of CD4+ T cells had no effect (Table 3). Interestingly, arrested CT26-IL12 tumors resumed growth 1 week after the depletion of CD8+ cells, suggesting the existence of remaining viable tumor cells (Table 3).

A significant increase in anti-CT26 CTL activity was observed with spleen cells obtained from mice vaccinated with 3 × 106 CT26-IL12 cells. This cytolytic activity was able to lyse both parental and LM3 cells (Table 4). The CTL activity against LM3 cells was even more evident when lymphocytes obtained from the draining lymph nodes were used (Table 4). No difference was observed when sera from the immunized animals was used as an adjuvant added in the assay (not shown).

The previously described immunodominant MHC class I-restricted AH1 peptide (21) does not seem to be the target of the CTLs derived from mice vaccinated with CT26-IL12 cells. Reverse transcription-PCR studies demonstrated that CT26 and LM3 cells as well as B16 murine melanoma cells expressed the MuLV env antigen mRNA from which AH1 is derived (not shown). A 6- to 7-fold increase in the stimulation index of spleen cells obtained from mice vaccinated with CT26-neo cells was observed when stimulated to proliferate in response to AH1 peptides (Fig. 3). Under similar conditions, no increase in the stimulation index was observed with spleen cells obtained from mice vaccinated with CT26-IL12 cells (Fig. 3).

In Vivo Adoptive Transfer Experiments.

To assess the in vivo effect of immune cells, we adoptively transferred spleen cells and lymphocytes, draining the site of tumor cell injection into mice bearing 1-day-old LM3 tumors. In an initial experiment, spleen cells obtained from mice vaccinated with CT26-IL12 cells (Sp-IL12) were able to delay LM3 tumor growth, compared with spleen cells obtained from control mice vaccinated with CT26-neo cells (Sp-neo; not shown). In a second experiment, Sp-IL12 cells restimulated in vitro with mitomycin C-treated CT26-cells signifi- cantly delayed LM3 growth in 5 of 12 mice, whereas Sp-neo cells had no effect (Fig. 4, A and B). Moreover, LM3 primary tumors ceased growing, and lung metastases did not develop in two of the mice after adoptive transfer of Sp-IL12 cells (Fig. 4, A and B, and Table 5). Adoptive transfer of Ly-neo cells had no effect on LM3 primary tumor growth but partially inhibited the development of large metastatic nodules. On the contrary, a complete suppression of LM3 primary tumor growth and the absence of lung metastases was observed in mice adoptively transferred with Ly-IL12 cells (Fig. 4, C and D, and Table 5).

The present study provides for the first time evidence that mice vaccinated with CT26 colon cancer cells producing IL-12 developed an immune-mediated, antitumor cross-protection that enabled them to reject not only a challenge with parental cells but also with syngeneic mammary tumor cells and MCE fibrosarcoma cells. No antitumor cross-tolerance developed against T lymphoma cells. Adoptive transfer of spleen cells and lymphocytes obtained from animals vaccinated with CT26-IL12 cells suppressed mammary tumor growth and metastases development in tumor-bearing mice.

The antitumor activity of IL-12 after either the injection of the recombinant protein or the gene transfer has been reported in different murine models (24). But, Phase II clinical trials aimed at treating cancer patients with the recombinant protein were halted because of the high toxicity of IL-12 (25). Studies in rodents and squirrel monkeys demonstrated splenomegaly,pulmonary edema, myelosuppression, and leukopenia as major toxic effects (26, 27). The use of a predose for desensitizing the host to the toxic effects of IL-12 failed to improve its antitumor efficacy (28). But local production of IL-12 after gene expression by tumor cells or fibroblasts inhibited tumor growth(24) with no accompanying splenomegaly, although NK cell activity was significantly induced (29). Therefore, the assessment of IL-12 antitumor effect after gene transfer is of potential clinical interest, because local production of IL-12 appears less toxic than IL-12 protein therapy.

The present study demonstrated that mice vaccinated with high doses of CT26-IL12 cells developed an immune-mediated, long-lasting antitumor cross-protection able to reject up to three challenges with parental and non-organ-related tumor cells even 4 months after the initial injection. This CT26-IL12-mediated immune memory was strong enough to induce the development of CTL activity able to eliminate mammary tumor cells in vitro and in vivo. Previous studies have shown that CTLs obtained from mice vaccinated with C26 cells producing IL-12 were able to kill other colon cancer cell lines derived from C26 cells (30). However, no previous evidence appeared in the literature regarding the capacity of immunized animals to generate CTL activity able to recognize and eliminate non-organ-related tumor cells. Indeed, mice immune to SCK mammary carcinoma cells expressing the costimulatory molecule B7 and after administration of IL-12 were unable to reject syngeneic SaI sarcoma cells (31). Mice immune to MB49 bladder cancer or to MCA207 sarcoma after expression of IL-12 were unable to reject syngeneic MC38 sarcoma(32) and B16 melanoma cells (15),respectively.

Only mice vaccinated with 3 or 5 × 106 CT26-IL12 cells showed a strong shift toward a CT26-specific Th1 response and were immunized not only against parental cells but also against LM3 and MCE malignant cells. Previous studies have shown that systemic administration of IL-12 or IL-12 gene transfer can induce a Th1 differentiation pattern, which seems to be largely dependent on the induction of IFNγby NK cells (33). IFNγ production was highly dependent on continued administration of IL-12, and serum IFNγ levels decreased markedly 48 h after stopping IL-12 (34). In addition,IL-12 was shown to induce the production of the Th2 cytokine IL-10 as a control mechanism to stop an ongoing Th1 response (35, 36)and to exacerbate an established Th2 response (9, 36),suggesting that IL-12 may stimulate both a Th1 and a Th2 response. The present data suggests that vaccination with a high number of CT26 cells producing sustained amounts of IL-12 might support the establishment of a long-term Th1 response and prevent the appearance of a Th2 response. The fact that CD8+ T cell depletion led to regrowth of CT26-IL12 dormant tumor cells indicates that the continuous production of IL-12 by viable CT26-IL12 cells might support the recruitment of immune cells, the strong bias toward a long term-Th1 response, and the induction of antitumor cross protection.

The present data also supports recent findings demonstrating that the target of CTLs obtained from animals carrying C26 tumors and cured after vaccination with IL-12-transduced cells is not the AH1 immunodominant peptide (37). These studies confirmed previous evidence that IL-12 may modulate the immunodominance of T cell epitopes (37, 38). Unlike IL-10, whose expression in engineered CT26 cells up-regulated MHC class I expression(19), IL-12 production by CT26 cells down-regulated MHC Class I (not shown), possibly favoring an initial non-T cell antitumor response. A major role for NK cells and macrophages in the primary antitumor response after IL-12 expression is supported by previous evidence from the literature (39, 40, 41) and our own data with nude mice. Tumor antigens shared by the different cell types might be the target of CD8+ T cells that appeared to be responsible for the cross-protection because depletion of CD4+ cells before the challenge had no effect. In a previous study, CD4+ T cells were shown to play a key role in the rejection of parental cells after vaccination of mice with B16 cells producing GM-CSF (42). CTLs might play an important role in the CT26 model because B16 cells constitutively express FasL, which may generate an immunoprivileged zone(42). Alternatively, the main role of GM-CSF is the differentiation of bone marrow progenitors to antigen presenting cells(APC), which may act by cross priming CD8+ T cell through the activation of CD4+ T cells (43); whereas IL-12 can act both by up-regulating MHC expression in APC via IFNγ expression by infiltrating NK or T cells, or act directly by stimulating Th1 differentiation and CTL activity (4, 5).

The anti-angiogenic property of IL-12 was well documented in different in vitro and in vivo studies (14, 44). Only recently, the severe tumor hypoxia-dependent apoptosis that appeared after IL-12 dependent inhibition of tumor cell-induced angiogenesis suggested that inhibition of angiogenesis might affect tumor growth (44). The IL-12-dependent angiogenesis inhibition seems to be mediated by IFN-γ and IP-10, which by itself was also shown to be chemotactic for monocytes and T cells(45). An IFNγ/IP-10 dependent mechanism seems to mediate the IL-12-induced infiltration of activated macrophages to different organs (46). IP-10 gene transfer of two different cell lines led to tumor cell rejection via a T-cell-dependent mechanism (47). The present data shows the development of a transient CT26-IL12-dependent pro-angiogenic process. Whether this pro-angiogenic process is an IL-12-mediated direct effect or mediated by an intermediate compound is still unknown, but it might actively contribute to the support of the initial recruitment of immune cells to the site of tumor cell injection. It is conceivable to assume that the overall IL-12/IFNγ/IP-10 effect as angiostatic and chemotactic factors for immune cells might occur in sequential steps. In an initial stage, an IL-12-mediated pro-angiogenic scenario can be envisioned to allow the immune cells to infiltrate the tumor mass. The IFNγ/IP-10-mediated angiostatic effect induced by IL-12 will occur as a secondary event once the immune cells have reached the tumor mass and started to produce angiostatic cytokines. The histology of the site of CT26-IL12 injection demonstrated mainly the presence of neutrophils. T cells were also suggested to be involved in this angiogenic process because of the absence of angiogenesis in studies of nude mice. Although tumor cells were not visualized in histological sections,CT26-IL12 tumor growth after depletion of CD8+ T cells demonstrated the presence of viable CT26-IL12 tumor cells in the angiogenic nodules. Sustained production of angiogenic factors like IL-8, MGSA, and other ELR-CXC chemokines might be produced either by neutrophils, by endothelial cells, or by the tumor cells themselves (48). Moreover, T lymphocytes from tumor-bearing mice with the participation of tumor cells were shown to trigger an angiogenic cascade driven by oxygen derivatives (49, 50). Therefore, the different cell types that constitute a tumor might contribute to the development of a localized, transient angiogenic process that is able to support the initial recruitment of immune cells.

The identification of tumor-associated antigens and CTL epitopes shared by many patients’ tumors led to the initiation of clinical protocols involving treatment with allogeneic tumor cell vaccines(51). Most of the tumor antigens and CTL epitopes were obtained from human melanoma samples, whereas the identification of such kinds of molecules from other malignant tissues was less frequent(51). Recently, Kayaga et al.(52)have shown that whole allogeneic tumor cell vaccines expressing GM-CSF can be used successfully in a melanoma murine model. The present studies raised the possibility of identifying tumor-associated antigens and CTL epitopes shared by different tumor types that also can be a target for the production of vaccines with multiple antigens or for adoptive transfer of CTLs.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

      
1

Supported in part by grants from Rene Barón Foundation, The National Council for Scientific and Technological Research (CONICET), and a joint grant from Antorchas Foundation and the British Council, Argentina. E. C. and O. L. P. belong to the Research Career of CONICET. S. A. is a fellow of the University of Buenos Aires, and M. B. is a fellow of CONICET.

                                          
8

The abbreviations used are: IL, interleukin; NK,natural killerCT26-neo, CT26 cells transduced with pBabe neo retrovirus; CT26-IL12, CT26 cells transduced with pBabe neo IL12 retrovirus; GM-CSF, granulocyte/macrophage-colony stimulating factor; mAb, monoclonal antibody; Sp-neo, Sp-IL12, Ly-neo, and Ly-IL12:spleen cells and lymphocytes obtained from mice vaccinated either with CT26-neo or CT26-IL12 cells and adoptively transferred to mice carrying LM3 tumors.

Table 1

Tumor development and induction of cross immune memory after injection of CT26 cells expressing IL-12

No. of cellsaInjected cellsPrimary tumor developmentFirst challenge with CT26 cellsbSecond challenge with CT26 cellsbChallenge with non-organ-related cellsc
2× 105 CT26-neo 5 /5d    
 CT26-IL12 0 /5    5/5 (2 wk)e 
5× 105 CT26-neo 16 /16    
 CT26-IL12 0 /7 0/7 (4 wk)   
 CT26-IL12 0 /5    5/5 (3 wk) 
1× 106 CT26-neo 10 /10    
 CT26-IL12 0 /10 0/10 (4 wk)   
3× 106 CT26-neo 16 /16    
 CT26-IL12 0 /8 0/8 (3 wk) 0/8 (8 wk)  2/8 (12 wk) 
 CT26-IL12 0 /10    1/8 (3 wk) 
 CT26-IL12 0 /10 0/8f (3 wk)   3/10 (9 wk) 
5× 106 CT26-neo 7 /7    
 CT26-IL12 0 /7    3/7 (7 wk) 
3× 106 CT26-neo 10 /10    
 CT26-IL12 0 /24 0/24 (2 wk)  12/24 (4 wk)g 
 CT26-IL12 0 /14 0/14 (2 wk)  14/14 (4 wk)h 
No. of cellsaInjected cellsPrimary tumor developmentFirst challenge with CT26 cellsbSecond challenge with CT26 cellsbChallenge with non-organ-related cellsc
2× 105 CT26-neo 5 /5d    
 CT26-IL12 0 /5    5/5 (2 wk)e 
5× 105 CT26-neo 16 /16    
 CT26-IL12 0 /7 0/7 (4 wk)   
 CT26-IL12 0 /5    5/5 (3 wk) 
1× 106 CT26-neo 10 /10    
 CT26-IL12 0 /10 0/10 (4 wk)   
3× 106 CT26-neo 16 /16    
 CT26-IL12 0 /8 0/8 (3 wk) 0/8 (8 wk)  2/8 (12 wk) 
 CT26-IL12 0 /10    1/8 (3 wk) 
 CT26-IL12 0 /10 0/8f (3 wk)   3/10 (9 wk) 
5× 106 CT26-neo 7 /7    
 CT26-IL12 0 /7    3/7 (7 wk) 
3× 106 CT26-neo 10 /10    
 CT26-IL12 0 /24 0/24 (2 wk)  12/24 (4 wk)g 
 CT26-IL12 0 /14 0/14 (2 wk)  14/14 (4 wk)h 
a

8–12-week-old male Balb/c mice (H-2d; Angel H. Roffo Institute, Buenos Aires) were injected with different amounts of cells.

b

At the indicated times, mice were challenged in the opposite flank with 5 × 105CT26 (10 times the minimum tumorigenic dose).

c

At the indicated times, mice were challenged in the opposite flank with 3 × 105LM3 cells (six times the tumorigenic inocula).

d

No. of mice that developed tumors per total mice receiving injections.

e

Time at which mice were challenged.

f

No. of mice challenged with 3 × 106 CT26 cells.

g

At the indicated times, mice were challenged in the opposite flank with 1 mm3 fragment of MCE fibrosarcoma.

h

At the indicated times, mice were challenged in the opposite flank with 1 × 105LB T lymphoma cells.

Fig. 1.

In vivo growth of CT26-IL12 and CT26-neo cells. BALB/c mice were given s.c. injections of 3 × 106 (A) and 5 × 106 (B) CT26-IL12 cells producing 0.21 ng/ml per 105 cells/24 h IL-12, and control CT26-neo cells. C, BALB/c nu/nu mice received injections of 3 × 106 CT26-IL12 cells. Tumor growth was evaluated by measuring the two perpendicular diameters with calipers. A representative experiment is shown.

Fig. 1.

In vivo growth of CT26-IL12 and CT26-neo cells. BALB/c mice were given s.c. injections of 3 × 106 (A) and 5 × 106 (B) CT26-IL12 cells producing 0.21 ng/ml per 105 cells/24 h IL-12, and control CT26-neo cells. C, BALB/c nu/nu mice received injections of 3 × 106 CT26-IL12 cells. Tumor growth was evaluated by measuring the two perpendicular diameters with calipers. A representative experiment is shown.

Close modal
Fig. 2.

Immunohistochemical studies of paraffin-embedded tissue sections corresponding to the site of injection of CT26-neo cells(A) showing a tumor mass (T) with few infiltrating neutrophils (arrow) and CT26-IL12 cells(B–D) showing a high number of vessels(arrow, V), and infiltrating neutrophils(arrow). See the erythrocytes inside the vessels. The microphotographs correspond to samples obtained 12 days after tumor cell injection. A, ×630; BD, ×1000. E and F correspond to photographs from everted skin obtained after injection of CT26-neo (E) and CT26-IL12(F) cells. The black spot corresponds to black ink injected together with the cells. See the difference in the density of blood vessels (arrows).

Fig. 2.

Immunohistochemical studies of paraffin-embedded tissue sections corresponding to the site of injection of CT26-neo cells(A) showing a tumor mass (T) with few infiltrating neutrophils (arrow) and CT26-IL12 cells(B–D) showing a high number of vessels(arrow, V), and infiltrating neutrophils(arrow). See the erythrocytes inside the vessels. The microphotographs correspond to samples obtained 12 days after tumor cell injection. A, ×630; BD, ×1000. E and F correspond to photographs from everted skin obtained after injection of CT26-neo (E) and CT26-IL12(F) cells. The black spot corresponds to black ink injected together with the cells. See the difference in the density of blood vessels (arrows).

Close modal
Table 2

Levels of total serum and CT26-specific IgG2a and IgG1 subclasses,after mice received injections of tumor cells expressing or not expressing IL-12

Assessment of total levels of circulating and anti-CT26 specific IgGs was performed essentially as described (19).

No. of cellsSerumTotal IgG2aTotal IgG1IgG2a:IgG1Anti-CT26 IgG2aAnti-CT26 IgG1IgG2a: IgG1
 Normal 102 ± 1.1a 6.7 ± 1.8 15.3b ND ND  
2× 105 CT26-Neo 100 ± 1.3 7.1 ± 1.3 14.2 ND ND  
 CT26-IL12 306 ± 8.9 4.5 ± 1.0 68.0 ND ND  
 Normal 34 ± 1.0 1.8 ± 1.0 18.9  
5× 105 CT26-Neo 46 ± 2.0 4.8 ± 2.1 9.6 21 ± 3.8c 34 ± 3.1 0.62 
 CT26-IL12 166 ± 10 2.1 ± 2.1 75.5 63 ± 1.9 64 ± 5.6 0.98 
 Normal 39 ± 1.4 6.5 ± 0.1 6.0  
1× 106 CT26-Neo 131 ± 11 17.2 ± 1.1 7.6 20 ± 4.1 21 ± 8.1 0.95 
 CT26-IL12 409 ± 11 11.2 ± 2.8 36.5 83 ± 1.8 32 ± 1.6 2.59 
 Normal 34.1 ± 1.1 1.8 ± 0.9 18.8  
3× 106 CT26-Neo 46 ± 1.8 3.0 ± 1.5 15.4 18 ± 2.9 41 ± 2.3 0.43 
 CT26-IL12 142 ± 18 3.6 ± 2.1 39.5 200 ± 5.1 3 ± 3.7 66.7 
 Normal 39 ± 1.4 6.5 ± 0.1 6.0  
5× 106 CT26-Neo 146 ± 45 11.9 ± 4.3 12.2 25 ± 3.1 5 ± 6.9 
 CT26-IL12 475 ± 1.4 10.4 ± 1.7 45.6 212 ± 3.2 3 ± 5.2 70.7 
No. of cellsSerumTotal IgG2aTotal IgG1IgG2a:IgG1Anti-CT26 IgG2aAnti-CT26 IgG1IgG2a: IgG1
 Normal 102 ± 1.1a 6.7 ± 1.8 15.3b ND ND  
2× 105 CT26-Neo 100 ± 1.3 7.1 ± 1.3 14.2 ND ND  
 CT26-IL12 306 ± 8.9 4.5 ± 1.0 68.0 ND ND  
 Normal 34 ± 1.0 1.8 ± 1.0 18.9  
5× 105 CT26-Neo 46 ± 2.0 4.8 ± 2.1 9.6 21 ± 3.8c 34 ± 3.1 0.62 
 CT26-IL12 166 ± 10 2.1 ± 2.1 75.5 63 ± 1.9 64 ± 5.6 0.98 
 Normal 39 ± 1.4 6.5 ± 0.1 6.0  
1× 106 CT26-Neo 131 ± 11 17.2 ± 1.1 7.6 20 ± 4.1 21 ± 8.1 0.95 
 CT26-IL12 409 ± 11 11.2 ± 2.8 36.5 83 ± 1.8 32 ± 1.6 2.59 
 Normal 34.1 ± 1.1 1.8 ± 0.9 18.8  
3× 106 CT26-Neo 46 ± 1.8 3.0 ± 1.5 15.4 18 ± 2.9 41 ± 2.3 0.43 
 CT26-IL12 142 ± 18 3.6 ± 2.1 39.5 200 ± 5.1 3 ± 3.7 66.7 
 Normal 39 ± 1.4 6.5 ± 0.1 6.0  
5× 106 CT26-Neo 146 ± 45 11.9 ± 4.3 12.2 25 ± 3.1 5 ± 6.9 
 CT26-IL12 475 ± 1.4 10.4 ± 1.7 45.6 212 ± 3.2 3 ± 5.2 70.7 
a

Data are expressed as μg/ml(mean ± SD).

b

Ratio in arbitrary units. Each value was obtained as an average of 8–10 mice.

c

Data are expressed as ng of bound Ig/106 cells (mean ± SD).

Table 3

Involvement of CD4+ and CD8+ T cells in the antitumor memory against LM3 mammary tumor cells

Balb/c mice received injections of 3 × 106CT26-IL12 cells. Twelve days later, depletion of T lymphocyte subsets was started by injecting 0.5 mg in 0.3 ml of PBS of mAb YTS 191.1 for CD4+ cells and YTS 169.4 for CD8+ cells (50). Two days later, mice were challenged with 3 × 105 LM3 cells. The same amount of antibody was injected each 7 days for 2 months. Antibodies were prepared as described (19).

Antibody usedTumor growth
CT26-IL12LM3
None 0 /5a  
None  5 /5b 
Anti-CD8+ 6 /9 7 /9 
Anti-CD4+ 0 /9 0 /9 
Antibody usedTumor growth
CT26-IL12LM3
None 0 /5a  
None  5 /5b 
Anti-CD8+ 6 /9 7 /9 
Anti-CD4+ 0 /9 0 /9 
a

Mice that developed tumors/total mice receiving injections.

b

LM3 cells injected at the same time as the challenge as a control of tumorigenicity.

Table 4

Anti-CT26 and anti-LM3 cytotoxic activity in splenocytes and lymphocytes obtained from mice receiving injections of CT26-IL12 and CT26-neo cells

Balb/c mice received injections of 3 × 106CT26-neo or CT26-IL12 cells. Eighteen to 20 days later, mice were challenged with 5 × 105 CT26 cells; 3 days later, spleen cells and lymphocytes from draining lymph nodes were harvested and incubated for 4 days at 37°C and 5% CO2 in the presence of 5 μg/ml Concanavalin A. Target cells labeled 45 min at 37°C with Cr were incubated for 48 h with spleen cells or lymphocytes. Supernatants were harvested at the end for cpm determination. For each group, immune cells were obtained from pools of six animals. These results correspond to one experiment of two with similar results. Each point was performed in triplicate.

InjectionsE:T ratio% lysis on target cells
SplenocytesLymphocytes
CT26LM3CT26LM3
Saline 100:1 9.8 11.5 7.2 
 50:1 10 nd 
CT26-neo 100:1 1.6 21.9 28.3 
 50:1 nd 
CT26-IL12 100:1 62.8 49.2 57.5 21 
 50:1 17.9 21.5 24.3 nd 
InjectionsE:T ratio% lysis on target cells
SplenocytesLymphocytes
CT26LM3CT26LM3
Saline 100:1 9.8 11.5 7.2 
 50:1 10 nd 
CT26-neo 100:1 1.6 21.9 28.3 
 50:1 nd 
CT26-IL12 100:1 62.8 49.2 57.5 21 
 50:1 17.9 21.5 24.3 nd 
Fig. 3.

BALB/c mice received injections of 3 × 106 CT26-neo or CT26-IL12 cells and were challenged 2 weeks later with parental cells. Spleen cells were obtained from both types of mice and naive mice as described in “Materials and Methods.” Data were expressed as stimulation index(S.I) in arbitrary units. The stimulation index is defined as the mean of the experimental wells divided by the mean of the control wells, without antigen (22). Each point was performed in triplicate. One representative experiment of four with similar results is shown, using as control peptide the one derived from CPD1 cDNA.

Fig. 3.

BALB/c mice received injections of 3 × 106 CT26-neo or CT26-IL12 cells and were challenged 2 weeks later with parental cells. Spleen cells were obtained from both types of mice and naive mice as described in “Materials and Methods.” Data were expressed as stimulation index(S.I) in arbitrary units. The stimulation index is defined as the mean of the experimental wells divided by the mean of the control wells, without antigen (22). Each point was performed in triplicate. One representative experiment of four with similar results is shown, using as control peptide the one derived from CPD1 cDNA.

Close modal
Fig. 4.

Treatment of LM3 mammary tumors by adoptive transfer of splenocytes and lymphocytes. Male Balb/c mice, 7–9 weeks of age, received injections of 3 × 106CT26-neo and CT26-IL12 cells and challenged with 5 × 105 CT26 cells 20 days later. Two days later, spleen cells were obtained separately from each group and pooled. Similarly,lymphocytes obtained from lymph nodes draining the site of tumor cell injection were separately obtained from each group and pooled. Cells were incubated for 48 h with 2 μg/ml Concanavalin A in the presence of mytomicin C-treated CT26 cells, and 1 ml containing 50 × 106 cells was injected i.p. in female Balb/c mice bearing 1-day-old mammary tumors after injection of 5 × 105 LM3 cells. Splenocytes and lymphocytes obtained from mice vaccinated with CT26-neo cells(A and C) and CT26-IL12 cells(B and D) were adoptively transferred to tumor-bearing mice as described. The control corresponds to mice that received injections of LM3 cells alone (E). ∗, ∗∗:mice in which adoptive transfer of Sp-IL12 totally suppressed LM3 tumor growth. Tumor growth in each individual mouse is shown.

Fig. 4.

Treatment of LM3 mammary tumors by adoptive transfer of splenocytes and lymphocytes. Male Balb/c mice, 7–9 weeks of age, received injections of 3 × 106CT26-neo and CT26-IL12 cells and challenged with 5 × 105 CT26 cells 20 days later. Two days later, spleen cells were obtained separately from each group and pooled. Similarly,lymphocytes obtained from lymph nodes draining the site of tumor cell injection were separately obtained from each group and pooled. Cells were incubated for 48 h with 2 μg/ml Concanavalin A in the presence of mytomicin C-treated CT26 cells, and 1 ml containing 50 × 106 cells was injected i.p. in female Balb/c mice bearing 1-day-old mammary tumors after injection of 5 × 105 LM3 cells. Splenocytes and lymphocytes obtained from mice vaccinated with CT26-neo cells(A and C) and CT26-IL12 cells(B and D) were adoptively transferred to tumor-bearing mice as described. The control corresponds to mice that received injections of LM3 cells alone (E). ∗, ∗∗:mice in which adoptive transfer of Sp-IL12 totally suppressed LM3 tumor growth. Tumor growth in each individual mouse is shown.

Close modal
Table 5

Lung metastatases development after adoptive transfer of spleen cells and lymphocytes to LM3 tumor-bearing mice

Mice were sacrificed when LM3 tumors from the different groups reached an average size of 1.5 cm3. The experiment was ended when the remaining Sp-neo→LM3 mice also reached an average size of 1.5 cm3 (day 39). Lung metastases were histologically analyzed at autopsy.

Injected cellsDays after adoptive transfer
273039
LM3   2/2 (50% > 0.1 cm2)a 3/3 (60% > 0.1 cm2
Sp-neo→LM3 3/4 (70% > 0.1 cm24/4 (50% > 0.1 cm24/4 (50% > 0.1 cm2
Sp-IL12→LM3 4/4 (22% > 0.1 cm23/3 (40% > 0.1 cm23/5 (28% > 0.1 cm2
Ly-neo→LM3   3/3 (0% > 0.1 cm2)  
Ly-IL12→LM3   0/3 
Injected cellsDays after adoptive transfer
273039
LM3   2/2 (50% > 0.1 cm2)a 3/3 (60% > 0.1 cm2
Sp-neo→LM3 3/4 (70% > 0.1 cm24/4 (50% > 0.1 cm24/4 (50% > 0.1 cm2
Sp-IL12→LM3 4/4 (22% > 0.1 cm23/3 (40% > 0.1 cm23/5 (28% > 0.1 cm2
Ly-neo→LM3   3/3 (0% > 0.1 cm2)  
Ly-IL12→LM3   0/3 
a

Mice bearing metastases/total mice. Between parentheses, percentage of metastatic nodules larger than 0.1 cm2. The number of metastatic nodules varied from 0 to 10,depending on the treatment and the time of sacrifice. For more details see Fig. 2.

We thank Liliana Alonso for helping to type the manuscript,Cecilia Rotondaro for performing the immunohistochemistry, and Fabio Fraga, head of the Animal Facility of Fundacion Campomar, for technical support.

1
Trinchieri G. Interleukin-12: a cytokine produced by antigen-presenting cells with immunoregulatory functions in the generation of T-helper cells type 1 and cytotoxic lymphocytes.
Blood
,
84
:
4008
-4027,  
1994
.
2
Kobayashi M., Fitz L., Ryan M., Hewick M., Clarck S. C., Chan S., Loudon R., Sherman F., Perussia B., Trinchieri G. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biological effects on human lymphocytes.
J. Exp. Med.
,
17
:
827
-845,  
1989
.
3
Chan S. H., Perussia B., Gupta J. W., Kobayashi M., Pospisil M., Young D., Wolf S. F., Young D., Clarck S. C., Trinchieri G. Induction of Interferon-γ production by NK cell stimulatory factor (NKSF): characterization of the responder cells and synergy with other inducers.
J. Exp. Med.
,
173
:
869
-879,  
1991
.
4
Gubler U., Chua A. O., Schoenhaut D. S., Dwyer C. M., McComas W., Motyka R., Nabavi N., Wolytzki A. G., Quinn P. M., Familletti P. C., Gately M. K. Coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor.
Proc. Natl. Acad. Sci. USA
,
88
:
4143
-4147,  
1991
.
5
Wolf S. F., Temple P. A., Kobayashi M., Young D., Dicig M., Lowe L., Dzialo R., Fitl L., Ferenz C., Hewick R. M., Kelleher K., Herrmann S. H., Clarck S. C., Azzoni L., Chan S. H., Trinchieri G., Perussia B. Cloning for cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biological effects on T and natural killer cells.
J. Immunol.
,
146
:
3074
-3081,  
1991
.
6
Schmitt E., Hoehn O., Gremann T., Rude E. Differential effects of interleukin 12 on the development of naive mouse CD4+ T cells.
Eur. J. Immunol.
,
24
:
343
-347,  
1994
.
7
Wu C. Y., Demeure C. E., Gately M., Podlaski F., Yssei H., Kiniwa M., Delespesse G. J. In vitro maturation of human neonatal CD4 T lymphocytes: induction of IL-4-producing cells after long-term culture in the presence of IL-4 plus either IL-2 or IL-12.
J. Immunol.
,
152
:
1141
-1153,  
1994
.
8
Wang Z. E., Zheng S., Corry D. B., Dalton D. K., Seder R. A., Reinner S. L., Locksley R. M. Interferon γ-independent effects of interleukin 12 administration during acute or established infection due to Leishmania major.
Proc. Natl. Acad. Sci. USA
,
91
:
12932
-12936,  
1994
.
9
Wynn T. A., Jankovic S., Hieny K., Zioncheck K., Jardieu P., Cheever A. W., Sher A. J. IL-12 exacerbates rather than suppresses T helper 2-dependent pathology in the absence of exogenous IFNγ.
J. Immunol.
,
154
:
3999
-4009,  
1995
.
10
Metzger D. W., Buchanan J. M., Collins J. T., Lester L. T., Murray K. S., Van Cleave V. H., Vogel L. A., Dunnick W. A. Enhancement of humoral immunity by interleukin-12.
Ann. NY Acad. Sci.
,
795
:
100
-115,  
1996
.
11
Metzger D. W., McNutt R. M., Collins J. T., Buchanan J. M., Van Cleave V. H., Dunnick W. A. Interleukin-12 acts as an adjuvant for humoral immunity through interferon γ-dependent and -independent mechanisms.
Eur. J. Immunol.
,
27
:
1958
-1965,  
1997
.
12
Voest E. E., Kenyon B. M., O’Reilly M. S., Truit G., D’Amato R. J., Folkman J. Inhibition of angiogenesis in vivo by interleukin 12.
J. Natl. Cancer Inst.
,
87
:
581
-586,  
1995
.
13
Brunda M. J., Luistro L., Warrier R. R., Wright R. B., Hubbard B. R., Nurphy M., Wolf S. F., Gately M. K. Antitumor and antimetastatic activity of interleukin 12 against murine tumors.
J. Exp. Med.
,
178
:
1223
-1230,  
1994
.
14
Nastala C. L., Edington H., McKinney T., Tahara H., Nalesnik M., Brunda M., Gately M., Wolf S., Schreiber R., Storkus W., Lotze M. Recombinant IL-12 administration induces tumor regression in association with IFN-γ production.
J. Immunol.
,
153
:
1697
-1706,  
1994
.
15
Tahara H., Zitvogel L., Storkus W. J., Zeh H. J., III, McKinney T. G., Schreiber R. D., Gubler U., Robbins P. D., Lotze M. T. Effective eradication of established murine tumors with IL-12 gene therapy using a polycistronic retroviral vector.
J. Immunol.
,
154
:
6466
-6474,  
1995
.
16
Morgenstern J. P., Land H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line.
Nucleic Acids Res.
,
18
:
3587
-3596,  
1990
.
17
Corbett T. H., Griswald D. D., Roberts E. J., Peckham J. C., Schabel F. M. Tumor induction relationship in development of transplantable cancers of the colon in mice for chemotherapy assays with a note on carcinogen structure.
Cancer Res.
,
35
:
2434
-2439,  
1975
.
18
Aguirre Ghiso J. A., Diament M., D’Elia I., Bal de Kier Jofffe E., Klein S. Effect of in vivo culture of murine mammary adenocarcinoma cells on tumor and metastatic growth.
Tumor Biol.
,
18
:
41
-52,  
1997
.
19
Adris S. K., Klein S., Jasnis M. A., Chuluyan E., Ledda M. F., Bravo A. I., Carbone C., Chernajovsky Y., Podhajcer O. L. IL-10 expression by CT26 colon carcinoma cells inhibits their malignant phenotype and induces a T cell-mediated tumor rejection in the context of a systemic Th2 response.
Gene Ther.
,
6
:
1705
-1712,  
1999
.
20
Franco M., Bustuoabad O. D., di Gianni P. D., Goldman A., Pasqualini C. D., Ruggiero R. A. A serum-mediated mechanism for concomitant resistance shared by immunogenic and non-immunogenic murine tumors.
Br. J. Cancer
,
74
:
178
-186,  
1996
.
21
Huang A. Y. C., Gulden P. H., Woods A. S., Thomas M. C., Tong C. D., Wang W., Engelhard V. H., Pasternack G., Cotter R., Hunt D., Pardoll D. M., Jaffee E. M. The immunodominant major histocompatibility complex class I-restricted antigen of a murine colon tumor derives from an endogenous retroviral gene product.
Proc. Natl. Acad. Sci. USA
,
93
:
9730
-9735,  
1996
.
22
Disis M. L., Gralow J. R., Bernhard H., Hand S. L., Rubin W. D., Cheever M. A. Peptide-based, but not whole protein, vaccines, elicit immunity to HER-2/neu, an oncogenic self-protein.
J. Immunol.
,
156
:
3151
-3158,  
1996
.
23
Jain R. K., Schlenger K., Hockel M., Yuan F. Quantitative angiogenesis assays: progress and problems.
Nat. Med.
,
3
:
1203
-1208,  
1997
.
24
Tahara H., Lotze M. T. Antitumor effects of interleukin-12 (IL-12): applications for the immunotherapy and gene therapy of cancer.
Gene Ther.
,
2
:
96
-106,  
1995
.
25
Cohen J. IL-12 deaths: explanation and a puzzle.
Science (Washington DC)
,
270
:
908
1995
.
26
Car B. D., Eng V. M., Schnyder B., LeHir M., Shakhov A. N., Woerly G., Huang S., Aguet M., Anderson T. D., Ryffel B. Role of interferon-γ in interleukin 12-induced pathology in mice.
Am. J. Pathol.
,
147
:
1693
-1707,  
1995
.
27
Sarmiento U. M., Riley J. H., Knack P. A., Lipman J. M., Becker J. M., Gately M. K., Chizzonite R., Anderson T. D. Biologic effects of recombinant human interleukin 12 in squirrel monkeys (Scuireus samiri).
Lab. Investig.
,
71
:
862
-874,  
1994
.
28
Coughlin C. M., Wysocka M., Trinchieri G., Lee W. M. F. The effect of interleukin 12 desensitization on the antitumor efficacy of recombinant interleukin 12.
Cancer Res.
,
57
:
2460
-2467,  
1997
.
29
Tan J., Newton C. A., Djeu J. Y., Gutsch D. E., Chang A. E., Yang N. S., Klein T. W., Hua Y. Injection of complementary DNA encoding interleukin-12 inhibits tumor establishment at a distant site in a murine renal carcinoma model.
Cancer Res.
,
5
:
3399
-3403,  
1996
.
30
Rodolfo M., Zilocchi C., Melani C., Cappetti B., Arioli I., Parmiani G., Colombo M. P. Immunotherapy of experimental metastases by vaccination with interleukin gene-transduced adenocarcinoma cells sharing tumor-associated antigens.
J. Immunol.
,
157
:
5536
-5542,  
1996
.
31
Coughlin C. M., Wysocka M., Kurzawa H. L., Lee W. M. F., Trinchieri G., Eck S. L. B7–1 and interleukin 12 synergistically induce effective antitumor therapy.
Cancer Res.
,
55
:
4980
-4987,  
1995
.
32
Chen L., Chen D., Block E., O’Donnell M., Kufe D. W., Clinton S. K. Eradication of murine bladder carcinoma by intratumor injection of a bicistronic adenoviral vector carrying cDNAs for the IL-12 heterodimer and its inhibition by the IL-12 p40 subunit homodimer.
J. Immunol.
,
159
:
351
-359,  
1997
.
33
McKnight A. J., Zimmer G. J., Fogelman I., Wolf S. F., Abbas A. K. Effects of IL-12 on helper T cell-dependent immune responses in vivo.
J. Immunol.
,
152
:
2172
-2179,  
1994
.
34
Rempel J. D., Wang M. D. , and Hay Glass, K.
T. In vivo IL-12 administration induces profound but transient commitment to T helper cell type 1-associated patterns of cytokine and antibody production. J. Immunol.
,
159
:
1490
-1496,  
1997
.
35
Meyaard L., Hovenkamp E., Otto S. A., Miedema F. IL-12-induced IL-10 production by human T cells as a negative feedback for IL-12-induced immune responses.
J. Immunol.
,
156
:
2776
-2782,  
1996
.
36
Jeannin P., Delneste Y., Seveso M., Life P., Bonnefoy J-Y. IL-12 synergizes with IL-2 and other stimuli in inducing IL-10 production by human T cells.
J. Immunol.
,
156
:
3159
-3165,  
1996
.
37
Rodolfo M., Zilocchi C., Capetti B., Parmiani G., Melani C., Colomobo M. P. Cytotoxic T lymphocytes response against non-immunoselected tumor antigens predicts the outcome of gene therapy with IL-12-transduced tumor cell vaccine.
Gene Ther.
,
6
:
865
-872,  
1999
.
38
Eberl G., Kessler B., Eberl L. P., Brunda M. J., Valmori D., Corradin G. Immunodominance of cytotoxic T lymphocyte epitopes co-injected in vivo and modulation by interleukin-12.
Eur. J. Immunol.
,
26
:
2709
-2716,  
1996
.
39
Tsung K., Meko J. B., Peplinski G. R., Tsung Y. L., Norton J. A. IL-12 induces T helper 1-directed antitumor response.
J. Immunol.
,
158
:
3359
-3365,  
1997
.
40
Cui J., Shin T., Kawano T., Sato H., Kondo E., Toura I., Kaneko Y., Koseki H., Kanno M., Taniguchi M. Requirement for VαNKT cells in IL-12-mediated rejection of tumors.
Science (Washington DC)
,
278
:
1623
-1626,  
1997
.
41
Kawamura T., Takeda K., Mendiratta S. K., Kawamura H., Van Kaer L., Yagita H., Abo T., Okamura K. Cutting edge: critical role of NK1+T cells in IL-12-induced immune responses in vivo.
J. Immunol.
,
160
:
16
-19,  
1998
.
42
Gee M. S., Koch C. J., Evans S. M., Jenkins W. T., Pletcher C. H., Jr., Moore J. S., Koblish H. K., Lee J., Lord E. M., Trinchieri G., Lee W. M. F. Hypoxia-mediated apoptosis from angiogenesis inhibition underlies tumor control by recombinant interleukin 12.
Cancer Res.
,
59
:
4882
-4889,  
1999
.
43
Sgadari C., Angiolillo A. L., Tosato G. Inhibition of angiogenesis by interleukin-12 is mediated by the interferon-inducible protein 10.
Blood
,
87
:
3877
-3882,  
1996
.
44
Luster A. D., Leder P. IP-10, a C-X-C chemokine, elicits a potent thymus dependent antitumor response in vivo.
J. Exp. Med.
,
178
:
1057
-1065,  
1993
.
45
Colville-Nash P. R., Willoughby D. A. Growth factors in angiogenesis: current interest and therapeutic potential.
Mol. Med. Today
,
1
:
14
-23,  
1997
.
46
Tannenbaum C. S., Wicker N., Armstrong D., Tubbs R., Finke J., Bukowski R. M., Hamilton T. A. Cytokine and chemokine expression in tumors of mice receiving systemic therapy with IL-12.
J. Immunol.
,
156
:
693
-699,  
1996
.
47
Hung K., Hayashi R., Lafond-Walker A., Lowenstein C., Pardoll D., Levitzky H. The central role of CD4+ T cells in the antitumor immune response.
J. Exp. Med.
,
188
:
2357
-2368,  
1998
.
48
Lu Z., Yuan L., Zhou X., Sotomayor E., Levitzky H. I., Pardoll D. M. CD40-independent pathways of T cell help for priming of CD8+ cytotoxic lymphocytes.
J. Exp. Med.
,
191
:
541
-550,  
2000
.
49
Monte M., Davel L. E. , and Sacerdote de Lustig, E.
Hydrogen peroxide is involved in lymphocyte activation mechanisms to induce angiogenesis. Eur. J. Cancer
,
33
:
676
-682,  
1997
.
50
Szabrowski T., Nathan C. Production of large amount of hydrogen peroxide by human tumor cells.
Cancer Res.
,
51
:
794
-798,  
1991
.
51
Rosenberg S. A. Development of cancer immunotherapies based on the identification of the genes encoding cancer regression antigens.
J. Natl. Cancer Inst.
,
88
:
1635
-1644,  
1996
.
52
Kayaga J., Souberbielle B. E., Shekh N., Morrow W. J. W., Scott-Taylor T., Vile R., Dalgleish A. G. Antitumor activity against B16–F10 melanoma with a GM-CSF secreting allogeneic tumor cell vaccine.
Gene Ther.
,
6
:
1475
-1481,  
1999
.