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The Journal of Immunology, 2004, 172: 7368-7376.
Copyright © 2004 by The American Association of Immunologists

IL-12 Pretreatments Enhance IFN-{alpha}-Induced Janus Kinase-STAT Signaling and Potentiate the Antitumor Effects of IFN-{alpha} in a Murine Model of Malignant Melanoma1

Gregory B. Lesinski*, Brian Badgwell{dagger}, Jason Zimmerer*, Tim Crespin§, Yan Hu{dagger}, Gerard Abood{dagger} and William E. Carson, III2,*,{dagger},{ddagger}

* Departments of Human Cancer Genetics, {dagger} Surgery and {ddagger} Medical Microbiology, Virology and Immunology, Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, Ohio State University, Columbus, OH 43210; and § Primetrics, Inc., Hilliard, OH 43026


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IFN-{alpha} 2b (IFN-{alpha}) has been used to treat patients with metastatic malignant melanoma and patients rendered disease-free via surgery but at high risk for recurrence. We hypothesized that IL-12 pretreatments would result in endogenous IFN-{gamma} production, and that this, in turn, would up-regulate levels of Janus kinase-STAT signaling intermediates and lead to increased expression of genes regulated by IFN-{alpha}. Treatment of PBMCs with IL-12 stimulated a significant and dose-dependent production of IFN-{gamma}. Pretreatment of PBMCs and tumor cells with IFN-{gamma}-containing supernatants from IL-12-stimulated PBMCs led to up-regulation of STAT1, STAT2, and IFN regulatory factor 9 (IRF9) and potentiated IFN-{alpha}-induced STAT signaling within PBMCs and tumor cells. These effects were abrogated by neutralization of IFN-{gamma} in the PBMC supernatants with an anti-IFN-{gamma} Ab. Pretreatment of HT144 melanoma cells and PBMCs with IFN-{gamma} or IFN-{gamma}-containing supernatants enhanced the actions of IFN-{alpha} at the transcriptional level, as measured by real-time RT PCR analysis of the IFN-stimulated gene 15. Experiments in wild-type C57BL/6 and IFN-{gamma} receptor knockout (B6.129S7-Ifngrtm1Agt) mice demonstrated that a regimen of IL-12 pretreatment, followed by IFN-{alpha}, could cure mice of i.p. B16F1 melanoma tumors (p < 0.007), whereas mice treated with either agent alone or PBS succumbed to fatal tumor burden. However, this treatment regimen did not significantly prolong the survival of IFN-{gamma}-deficient (B6.129S7-Ifngtm1Ts) mice compared with mice treated with IFN-{alpha} alone. These results suggest that the response to IFN-{alpha} immunotherapy can be significantly enhanced by IL-12 pretreatment, and this effect is dependent upon endogenous IFN-{gamma} production and its actions on melanoma cells.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The treatment of malignant melanoma has undergone significant changes over the past 2 decades with the introduction of high dose cytokine therapy, biochemotherapeutic regimens, novel vaccination strategies, and sentinel lymph node biopsy techniques (1, 2, 3, 4, 5). Despite these advances, the therapeutic options for patients with metastatic disease remain limited, and the overall prognosis is poor. An analysis of trials using recombinant IFN-{alpha} in patients with metastatic malignant melanoma reveals overall response rates ranging from 10 to 20%, which is comparable to the results achieved with high dose IL-2 or chemotherapy (6, 7, 8, 9). IFN-{alpha} has also been used as an adjuvant in surgically treated patients who are at risk for recurrence; however, its effects in this setting appear to be limited to a distinct subgroup of patients (10, 11, 12, 13, 14). The efficacy of IFN-{alpha} therapy in malignant melanoma appears to be dependent on the administration of relatively high dosages for prolonged periods of time (10, 15). Unfortunately, at these concentrations, toxicities requiring dose reduction or treatment delays are frequently seen.

Sensitization of tumors and immune effectors to the actions of IFN-{alpha} might allow for lower doses to be used with less toxicity. Cells can be sensitized to IFN-{alpha} by pretreatment with compounds that up-regulate levels of critical signaling intermediates. Previous work by our group and others has shown that pretreatment of IFN-{alpha}-responsive melanoma cell lines with IFN-{gamma} resulted in enhanced responsiveness to low doses of IFN-{alpha} via the increased expression of STAT1 and the p48 DNA-binding protein known as IFN-responsive factor 9 (IRF9)3 (16, 17, 18). Expression of the IFN-{alpha} receptor is also up-regulated in the T98G human neuroblastoma cell line after exposure to IFN-{gamma} (19). Of note, previous studies by Lehtonen et al. (20) have shown that IFN-{alpha}-induced gene regulation in PBMCs could also be achieved via IFN-{gamma} pretreatment, a finding that confirms the broad applicability of this technique. Therefore, sensitization of the patient to IFN-{alpha} via IFN-{gamma} pretreatment might enable clinicians to administer lower amounts of the cytokine and thereby avoid the toxicities that are routinely encountered with this regimen. IL-12 is known to induce the secretion of IFN-{gamma} from T and NK cells and to promote the maturation and activation of Th1 cells (21). Based on these data, we considered the use of IL-12 as a means of inducing the endogenous production of IFN-{gamma}. We hypothesized that administration of IL-12 might result in a more sustained release of IFN-{gamma} than would be observed after a simple injection of the recombinant cytokine.

Previous studies have demonstrated a role for STAT1-dependent signaling within host tissues to mediate the anti-tumor effects of IFN-{alpha} (22). STAT1 is also required for the generation of antitumor cytolytic T cells in response to an IL-12-based vaccine (23). Although the antiproliferative effects of IFN-{alpha} on tumor cells have been well characterized (24), our results demonstrated that it is the actions of IFN-{alpha} on host innate immune effectors and not a direct effect that drives the antitumor activity of IFN-{alpha}. In contrast, the actions of endogenously produced IFN-{gamma} have been shown to play a key role in tumor immunosurveillance and can prevent the development of tumors in susceptible mice via its ability to enhance tumor immunogenicity (25). Therefore, it appears as though the antitumor effects of the type I and II IFNs are mediated via their effects on both tumor cells and host immune effectors. Consequently, we have examined the combined effects of IL-12 and IFN-{alpha} on signal transduction and gene expression in both tumor and immune cells.

In the present report we demonstrate that IL-12 can be used to sensitize both tumor cells and immune effectors to low dose IFN-{alpha} at the level of signal transduction and gene regulation. In addition, we provide evidence that IL-12 can potentiate the antitumor effects of IFN-{alpha} in a murine model of malignant melanoma. Additional experiments in genetically altered mice revealed that the antitumor effects of IL-12 pretreatment were dependent on the endogenous production of IFN-{gamma} and its direct actions on tumor cells. These experiments demonstrate that manipulations designed to enhance cytokine signal transduction can lead to improved efficacy in vivo.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and cytokines

The HT144 (human) and B16F1 (murine) cell lines were obtained from American Type Culture Collection (Manassas, VA). The 1259 MEL, 18105 MEL, 1106 MEL, MEL 39, and 1174 MEL human melanoma cell lines were gifts from Dr. S. Ferrone (Roswell Park Cancer Institute, Buffalo, NY). Cell lines were cultured in RPMI 1640 with 10% FBS, antibiotics, and antimycotics. Recombinant human IFN-{alpha} (sp. act., 2 x 108 IU/mg) was purchased from Schering-Plough (Nutley, NJ). Recombinant human IFN-{gamma} (sp. act., 3 x 107 U/mg) was obtained from Genentech (San Francisco, CA). Murine IFN-{alpha} was purchased from Lee Biomolecular Research (San Diego, CA). Murine IL-12 was provided by Genetics Institute (Cambridge, MA). All cytokines were resuspended in PBS supplemented with 0.1% human albumin (Sigma-Aldrich, St. Louis, MO).

Stimulation of PBMCs with IL-12 to generate IFN-{gamma}-containing culture supernatants

PBMCs were isolated from source leukocytes (American Red Cross, Columbus, OH) via density gradient centrifugation with Ficoll-Paque Plus (Pharmacia Biotech, Uppsala, Sweden). PBMCs were cultured in 12-well plates in RPMI 1640 supplemented with 10% human AB serum and various concentrations of IL-12 (0.1, 1, and 10 ng/ml). Supernatants were harvested at 48 or 72 h and snap-frozen at –70° C. Supernatants from IL-12-stimulated PBMCs (which contained IFN-{gamma}) were combined with fresh medium in a 1:1 ratio and subsequently applied to tumor cells or freshly isolated PBMCs in culture. In this manner we hoped to closely simulate the cytokine milieu that might confront tumor cells and immune effectors after the in vivo administration of IL-12.

IFN-{gamma} ELISA

Culture supernatants or serum samples were thawed at 37°C and centrifuged briefly before analysis by ELISA. Samples were analyzed in duplicate for IFN-{gamma} levels using a commercially available, paired Ab ELISA system (Endogen, Rockford, IL) (26). OD was measured on a PerkinElmer bioassay reader (Shelton, CT).

Cell proliferation assay

HT144 cells (2 x 105 cells/well) were plated in 96-well culture plates for 6 h, after which the cells were washed briefly with PBS and cultured for an additional 18 h with supernatants from IL-12-stimulated or PBS-stimulated PBMCs. Supernatants were diluted 1/1 in fresh completed medium before addition to the cells. After this culture period the cells were washed twice with PBS and resuspended in fresh medium supplemented with varying doses of IFN-{alpha} (101–105 U/ml) or PBS for 24 h. Cells were pulsed with 1 mCi of [3H]thymidine during the final 12 h of culture and harvested as previously described (27).

Immunoblot analysis and flow cytometric analysis

The HT144 melanoma cell line and PBMCs were cultured in supernatants from PBS-treated and IL-12-treated (10 ng/ml) PBMCs for 24, 48, and 72 h. Cell lysates from these cultures were quantitated, equally loaded according to protein content, separated by SDS-PAGE, and transferred to nitrocellulose. Nitrocellulose sheets were incubated with Abs to STAT1, STAT2, Janus kinase 1 (Jak1), IRF9, (BD Transduction Laboratories, Franklin Lakes, NJ), tyrosine kinase 2 (Tyk2; Cell Signaling Technology, Beverly, MA), and the IFN-{alpha} receptor, IFNAR (Santa Cruz Biotechnology, Santa Cruz, CA), for 1 h at room temperature, followed by incubation with the appropriate HRP-conjugated secondary antibody. Immune complexes were detected via the enhanced chemiluminescence reaction (Pharmacia Biotech). Fresh PBMCs or HT144 cells were also treated overnight with supernatants from IL-12- or PBS-treated PBMCs and examined for levels of STAT1 via intracellular flow cytometry as previously described (28). Briefly, cells were harvested from culture, washed, and permeabilized with Fix and Perm reagent (Caltag, Burlingame, CA). Cells were then incubated with a murine anti-STAT1 Ab (Transduction Laboratories, San Diego, CA) for 15 min, washed, stained with an anti-murine FITC-conjugated secondary Ab, and analyzed on a Coulter Elite flow cytometer (Fullerton, CA).

EMSA

Whole-cell extracts (5 x 106 cells) were prepared as previously described (29) from HT144 melanoma cells and PBMCs. A double-stranded Sis-inducible element (SIE) oligonucleotide with affinity for activated human STAT proteins (5'-GATCCGATTCCGGGAATCA-3') (30) was end-labeled using T4 kinase and [32P]dATP. Whole-cell extracts (5 µ g) were preincubated in 20 µg of reaction buffer (50 mM HEPES (pH 7.9), 250 mM KCl, 5 mM EDTA, 25 mM MgCl2, 50% glycerol, 25 mM DTT, and 5 mg/ml BSA) containing 5 µg of poly(dI-dC) for 15 min on ice. Labeled probe (20,000 cpm) was then added, and the binding reaction was continued for 15 min at room temperature. Ten microliters of the reaction mixture was loaded onto a 4% polyacrylamide gel in 0.5x Tris-borate EDTA buffer and electrophoresed at 120 V. The radioactive pattern was visualized by autoradiography after exposure to film overnight.

Real-time RT-PCR

Real-time PCR was used to quantitate levels of IFN-stimulated gene 15 (ISG-15) mRNA in HT144 melanoma cells or PBMCs. Cells were treated with PBS or IFN-{gamma} (10 ng/ml) for 18 h, then washed and treated with either PBS or varying concentrations of IFN-{alpha} for 4 h. In a separate set of experiments, HT144 cells or PBMCs were pretreated with supernatants from PBS- or IL-12-stimulated PBMCs, then washed and treated with either PBS or varying concentrations of IFN-{alpha} for 4 h. Total RNA was isolated using the RNeasy RNA Isolation Kit (Qiagen, Valencia, CA) and quantitated via the RiboGreen RNA Quantitation Kit (Molecular Probes, Eugene, OR). RT was performed using 2 µg of cellular RNA and random hexamers (PerkinElmer, Norwalk, CT) as primers for first-strand synthesis as previously described (31). The resulting cDNA (2 µl) was used as a template for real-time PCR using predesigned primer/probe sets for the ISG-15 gene (Assays On Demand; Applied Biosystems, Foster City, CA) and 2x TaqMan Universal PCR Master Mix (Applied Biosystems) according to the manufacturer’s recommendations as previously described (32). Predesigned primer/probe sets for human {beta}-actin were used as an internal control in each reaction well (Applied Biosystems). Real-time PCR data were analyzed using Sequence Detector software (version 1.6; Applied Biosystems).

Murine tumor models

An i.p. model of murine malignant melanoma was used to determine whether IL-12 pretreatment could enhance the antitumor effects of IFN-{alpha} in the setting of advanced disease. Female C57BL/6, B6.129S7-Ifngrtm1Agt (IFN-{gamma} receptor-deficient), or B6.129S7-Ifngtm1Ts (IFN-{gamma}-deficient) mice (The Jackson Laboratory, Bar Harbor, ME) were injected i.p. on day 0 with 1 x 106 B16F1 malignant melanoma cells. Beginning the next day, animals received i.p. injections of PBS, IL-12, IFN-{alpha}, or IL-12 plus IFN-{alpha}. IFN-{alpha} was administered at a dose of 2 x 104 U daily, and IL-12 was administered at a dose of 300 ng, either daily or three times per week. In mice that received both cytokines, IL-12 was administered 12 h before injection of IFN-{alpha}. All challenge studies used a minimum of five mice per treatment group.

Statistical analysis

Statistical analyses compared mean survival intervals across treatment groups using ANOVAs with planned comparisons. Planned comparisons were single-degree F tests with Welch’s adjustment for heterogeneity of variance, with p < 0.05 considered statistically significant. This alternative to survival analysis was chosen based on the modest sample sizes and the fact that the few censored cases were in the combined treatment group. We calculated mean survival intervals per experimental group on the presumption that censored cases died immediately after the last observation interval. This presumption makes our analyses conservative because it underestimates survival in the combined treatment group.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-12 induces endogenous production of IFN-{gamma} in vitro

We conducted a series of in vitro experiments using normal donor PBMCs to confirm that pretreatment with IL-12 would induce endogenous production of IFN-{gamma}. After treatment of donor PBMCs with recombinant human IL-12, a dose-dependent production of IFN-{gamma} was observed at 72 h (Fig. 1A). Peak production of IFN-{gamma} occurred at 72 h after exposure to IL-12, but detectable levels were obtained within 8 h (data not shown). Cell density also affected IFN-{gamma} production by PBMCs, as the highest production of IFN-{gamma} was observed when cells were plated at a density of 4 x 106 cells/ml (Fig. 1B). This cell density was used in the production of IFN-{gamma}-containing supernatants for use in subsequent experiments.



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  		FIGURE 1. Effects of IL-12 on PBMC production of IFN-{gamma}. A, Normal donor PBMCs were isolated from peripheral blood and cultured in vitro with IL-12 (0.1, 1, and 10 ng/ml) or medium alone. Culture supernatants were harvested at 72 h and analyzed for IFN-{gamma} production by ELISA. B, PBMCs were stimulated with PBS or 5, 10, or 20 ng/ml IL-12 for 72 h at 1 x 106, 2 x 106, and 4 x 106 cells/ml. Culture supernatants were analyzed for IFN-{gamma} content by ELISA. These results are representative of data from three individual experiments.

 
Antiproliferative effects of IFN-{alpha} are enhanced by pretreatment of melanoma cells with IFN-{gamma}-containing supernatants from IL-12-stimulated PBMCs

IFN-{alpha}-sensitive HT144 melanoma cells were cultured overnight in IFN-{gamma}-containing supernatants from IL-12-stimulated or PBS-stimulated (control) PBMCs. This in vitro model was used to simulate the cytokine milieu that would confront immune effectors and tumor cells in a patient receiving IL-12 pretreatment along with IFN-{alpha}. After these pretreatments, melanoma cells were washed and cultured with varying concentrations of IFN-{alpha} (101–105 U/ml) for 24 h. Cell proliferation was measured via the incorporation of [methyl-3H]thymidine during the final 12 h of culture (Fig. 2). Pretreatment of cells with IL-12-stimulated supernatants produced a significant decrease in thymidine incorporation compared with cells pretreated with PBS-stimulated supernatants (p ≤ 0.039 for each dose level, by Wilcoxon signed ranks test for two related samples).



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  		FIGURE 2. Antiproliferative effects of IFN-{alpha} after IL-12 pretreatment in a human melanoma cell line. HT144 melanoma cells were cultured overnight in supernatants from IL-12-stimulated PBMCs that contained high levels of IFN-{gamma} (1280 pg/ml). As a control, a separate aliquot of melanoma cells was treated with supernatants that had not been stimulated with IL-12 and contained no IFN-{gamma}. After these pretreatments, melanoma cells were washed and cultured with varying concentrations of IFN-{alpha} (101–105 U/ml) or PBS for a period of 24 h. Cell proliferation was measured via the incorporation of [methyl-3H]thymidine during the final 12 h of culture.

 
Levels of Jak-STAT signaling intermediates in melanoma cells and normal PBMCs are up-regulated by pretreatment with IFN-{gamma}-containing supernatants

To identify the effects of IL-12 pretreatment on the host immune system and melanoma cells, we analyzed levels of STAT1, STAT2, IRF9, Jak1, Tyk2, and IFNAR in donor PBMCs and HT144 melanoma cells by immunoblot analysis after exposure to supernatants from PBMCs stimulated with PBS or IL-12. Pretreatment of both PBMCs and the HT144 melanoma cell line with supernatants from IL-12-treated PBMCs for 12 h resulted in increased levels of STAT1, STAT2, and IRF9 (Fig. 3, A and B). Levels of Tyk2, Jak1, and the IFNAR were not affected by pretreatment with IFN-{gamma}-containing supernatants (Fig. 3, C and D). Flow cytometry was used to confirm the ability of IL-12-treated PBMC supernatants to up-regulate Jak-STAT signaling intermediates. As predicted, pretreatment of both PBMCs and six different human melanoma cell lines (HT144, 1259 MEL, 18105 MEL, 1106 MEL, MEL 39, and 1174 MEL) for 72 h with IFN-{gamma}-containing supernatants resulted in increased levels of STAT1, as detected by flow cytometry (Fig. 3, E and F, and data not shown).



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  		FIGURE 3. Up-regulation of Jak-STAT signaling intermediates by pretreatment with IFN-{gamma}-containing supernatants. Cell lysates were prepared from PBMCs (A) and HT144 cells (B) cultured in supernatants from PBS-treated and IL-12-treated PBMCs. Immunoblot analysis showed that pretreatment with IFN-{gamma}-containing supernatants from IL-12-stimulated PBMCs up-regulated levels of STAT1, STAT2, and IRF9 proteins. No up-regulation of Jak1, Tyk2, or IFNAR was observed in either PBMCs (C) or HT144 cells (D). Flow cytometric analysis of STAT1 protein within PBMCs (E) and HT144 cells (F) was used to confirm our findings. Cells were harvested and stained with an anti-STAT1 or an appropriate isotype control Ab after incubation in supernatants from IL-12- or PBS-stimulated PBMCs for 72 h. Data shown are representative of three individual experiments. Similar results were observed in the 1259 MEL, 18105 MEL, 1106 MEL, MEL 39, and 1174 MEL human melanoma cell lines (data not shown).

 
Pretreatment with IL-12 supernatants enhances IFN-{alpha}-induced signal transduction

An EMSA was performed to examine the effects of supernatants from PBS- and IL-12-stimulated PBMCs on Jak-STAT signaling in PBMC and HT144 cells. In these experiments PBMCs were treated with supernatants for 18 h and then washed before stimulation with PBS or human recombinant IFN-{alpha} (at concentrations of 102–104 U/ml). PBMCs treated with control supernatants demonstrated DNA binding activity only in response to 104 U/ml IFN-{alpha} (Fig. 4A). In contrast, pretreatment with IFN-{gamma}-containing supernatants led to enhanced binding to the SIE probe in response to IFN-{alpha} at 102 and 103 U/ml (a 100-fold increase in sensitivity).



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  		FIGURE 4. Pretreatment of HT144s and PBMCs with IL-12-stimulated supernatants enhances IFN-{alpha}-induced STAT signaling. A, PBMCs were incubated for 18 h in PBS- or IL-12-stimulated supernatants. Cells were then washed and stimulated with PBS or IFN-{alpha} (102–104 U/ml) for 15 min. Whole-cell extracts were prepared and used in an EMSA with a DNA probe specific for activated murine STAT proteins (SIE). B, The HT144 melanoma cell line was pretreated with IFN-{gamma} or supernatants from IL-12-treated PBMCs, washed, then stimulated for 15 min with 103 U/ml IFN-{alpha}. EMSA of cell lysates confirmed that pretreatment of HT144 cells with supernatants from IL-12-stimulated PBMCs enhanced binding to the SIE probe. Pretreatment of PBMC supernatants with an anti-IFN-{gamma} Ab resulted in DNA-binding activity similar to that in cells stimulated with IFN-{alpha} alone.

 
We next tested whether IL-12 pretreatment resulted in similar effects on tumor cells. HT144 melanoma cells were treated with supernatants from IL-12-stimulated PBMCs for 18 h before stimulation with 103 U/ml recombinant human IFN-{alpha} (Fig. 4B). After treatment, cells were harvested, washed, and rested for 1 h before use in a gel-shift assay with the high affinity, SIE-radiolabeled DNA probe. In the absence of cytokine treatment, no significant DNA binding activity was observed (lane 1), whereas nonpretreated cells generated modest levels of DNA binding in response to treatment with 103 U/ml IFN-{alpha} (lane 2). As shown previously, treatment of melanoma cells with IFN-{gamma} resulted in enhancement of IFN-{alpha}-induced binding to the SIE probe (lane 4). A similar degree of binding was achieved by simple overnight culture of melanoma cells in IFN-{gamma}-containing PBMC supernatants before the addition of IFN-{alpha} (lane 6). The role of IFN-{gamma} in this phenomenon was confirmed by using an anti-IFN-{gamma} Ab to neutralize IFN-{gamma} in the PBMC supernatants before their addition to melanoma cultures. This manipulation resulted in a return of the IFN-{alpha}-induced binding to baseline levels (lane 8). To control for nonspecific Ab interactions, an isotype control Ab was included in the binding reaction corresponding to lane 7. These experiments confirmed that IFN-{gamma} in the supernatants of IL-12-treated PBMCs can sensitize melanoma cells and PBMCs to the effects of low dose IFN-{alpha} at the level of signal transduction.

Analysis of IFN-{alpha}-mediated gene regulation via real-time PCR

We hypothesized that IFN-{gamma} production in response to IL-12 pretreatment would enhance STAT1 signaling after exposure to low concentrations of IFN-{alpha}, which, in turn, would lead to increased transcription of IFN-{alpha}-responsive genes. ISG-15 has been used extensively as a marker for IFN-{alpha} activation in other systems, and we have shown that its expression is STAT1 dependent (33, 34, 35, 36). Although the biologic function of ISG-15 has not been fully elucidated, it is known to be a ubiquitin homologue, and it may also exert immunomodulatory effects on NK cells and other effector cells (37). HT144 cells were treated overnight with PBS or IFN-{gamma} (10 ng/ml). Cells were then washed and treated with various concentrations of IFN-{alpha} or PBS for 4 h. Analysis of transcripts by real-time PCR demonstrated that ISG-15 transcript was strongly regulated by IFN-{alpha} and that pretreatment with IFN-{gamma} enhanced this effect (Fig. 5A). We obtained comparable results when PBMCs received IFN-{alpha} after IFN-{gamma} pretreatment (Fig. 5B). Pretreatment of HT144 cells and PBMCs with IL-12-stimulated supernatants also enhanced the production of ISG-15 transcript in response to IFN-{alpha} compared with cells that were pretreated with PBS-stimulated supernatants (Fig. 5, C and D).



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  		FIGURE 5. IL-12 pretreatment led to increased transcription of IFN-{alpha}-responsive genes. Analysis of the IFN-responsive gene, ISG-15, by real-time RT-PCR indicated that IFN-{gamma} pretreatment increased ISG-15 transcript levels after stimulation with IFN-{alpha} (102–104 U/ml). Cells were treated with PBS, 10 ng/ml IFN-{gamma}, or 100 ng/ml IFN-{gamma} for 18 h and then treated with varying concentrations of IFN-{alpha} (101–105 U/ml). This effect was observed in both HT144 melanoma cells (A) and PBMCs (B) from a normal healthy donor. Increased levels of ISG-15 transcript were also detected after pretreatment of HT144 melanoma cells (C) or PBMCs (D) for 18 h with supernatants from IL-12-stimulated PBMCs before a 4-h treatment with IFN-{alpha}. All gene expression levels were relative to {beta}-actin expression. Pretreatment of PBMCs with PBS did not elicit this effect. Gene expression data obtained from PBMCs are representative of three normal donors.

 
IL-12 pretreatment prolongs survival in a murine model of malignant melanoma

The effects of IL-12 pretreatment were studied in a mouse model of malignant melanoma in which B16F1 cells (1 x 106) were injected i.p. into C57BL/6 mice (n = 10 mice/group) (38). Preliminary studies indicated that maximal serum levels of IFN-{gamma} were achieved 12–18 h after i.p. injection of murine IL-12 (39, 40). One day after tumor challenge, mice received i.p. pretreatment of murine IL-12 (300 ng/day) 12 h before the administration of IFN-{alpha} (2 x 104 U/day). Control mice received IL-12 pretreatment alone (IL-12 and PBS), IFN-{alpha} alone (PBS and IFN-{alpha}), or PBS alone (PBS and PBS) according to the same treatment regimen. As expected, treatment of wild-type mice with IFN-{alpha} alone (PBS and IFN-{alpha}) led to a significant improvement in survival compared with treatment with PBS alone (Fig. 6A; p < 0.01). Daily administration of IL-12 alone (IL-12 and PBS) resulted in only a modest enhancement of survival that did not meet statistical significance (p = 0.3). However, daily pretreatment of mice with IL-12 before the administration of IFN-{alpha} (IL-12 and IFN-{alpha}) resulted in prolonged survival, and 60% of mice were cured of their tumor (Fig. 6A; p = 0.005). Similar results were obtained when IL-12 pretreatments were administered three times per week before daily IFN-{alpha} (2 x 104 U/day), indicating the broad applicability of this approach (Fig. 6B). Interestingly, mice receiving IL-12 and IFN-{alpha} according to the second regimen displayed a higher cure rate (87%) and greater overall survival compared with mice pretreated daily (60%). Of note, a significant increase in the survival of mice receiving IL-12 alone was also notable when pretreatments were administered three times per week (p = 0.007). Based on these data, subsequent experiments used a regimen of three times weekly IL-12 pretreatments.



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  		FIGURE 6. IL-12 pretreatment, followed by IFN-{alpha} administration, enhanced the survival of wild-type mice in a murine model of malignant melanoma. A, C57BL/6 mice (n = 10 mice/group) were injected i.p. with 106 B16F1 cells on day 0. Beginning on day 1, mice received daily injections of PBS or IL-12 (a.m.) and PBS or IFN-{alpha} (p.m.). Compared with negative controls (PBS and PBS), survival was significantly prolonged after IFN-{alpha} treatment alone (PBS + IFN-{alpha}; p < 0.01), but not after IL-12 pretreatment alone (IL-12 + PBS; p = 0.3). Survival of all IL-12-pretreated, IFN-{alpha}-treated mice (IL-12 + IFN-{alpha}) was significantly prolonged (p = 0.005). A 60% cure rate was observed with this regimen. B, Administration of IL-12 pretreatment three times per week on alternating days (n = 8 mice/group) resulted in significantly enhanced survival of tumor-bearing mice treated with IL-12 alone (IL-12 + PBS; p = 0.007) and IFN-{alpha} alone (PBS + IFN-{alpha}; p = 0.01). The survival of IL-12-pretreated, IFN-{alpha}-treated mice (IL-12 + IFN-{alpha}) was significantly prolonged (p < 0.0001) compared with IL-12 alone or IFN-{alpha} alone. This treatment regimen resulted in an 87% cure rate of mice bearing B16F1 melanoma tumors. Data shown are representative of three individual experiments.

 
Host-derived IFN-{gamma} is required to mediate the enhanced antitumor activity of IL-12

We next tested whether the enhanced survival in response to IL-12 pretreatment was dependent on IFN-{gamma} production by host tissues. B16F1 melanoma cells (1 x 106) were injected i.p. into B6.129S7-Ifngtm1Ts (GKO) or B6.129S7-Ifngrtm1Agt (GRKO) mice (n = 5/group). Interestingly, pretreatment with IL-12 (three times per week), followed by daily IFN-{alpha} administration, did not significantly enhance the survival of GKO mice compared with that of mice treated with IFN-{alpha} or IL-12 alone (Fig. 7; p = 0.31). However, treatment of GRKO mice with IL-12 and IFN-{alpha} according to the same regimen resulted in enhanced production of IFN-{gamma} (serum IFN-{gamma} levels were 532 ± 203 pg/ml at 24 h after IL-12 administration) and tumor clearance within 100% of mice (Fig. 8). These data suggested that host production of IFN-{gamma} and its direct on tumor cells (rather than on host tissues) after IL-12 pretreatment was necessary for the control of tumor growth in this experimental model. As expected, both strains of mice exhibited prolonged survival after treatment with exogenous IFN-{alpha} alone (PBS and IFN-{alpha}), confirming our earlier findings that the antitumor effects of IFN-{alpha} alone are independent of host IFN-{gamma} production (22).



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  		FIGURE 7. IL-12 pretreatment followed by IFN-{alpha} administration does not enhance the survival of GKO mice in a murine model of malignant melanoma. GKO mice (n = 5 mice/group) were injected i.p. with 106 B16F1 cells on day 0. Beginning on day 1, mice received injections of PBS or IL-12 (a.m.) and PBS or IFN-{alpha} (p.m.) three times per week on alternating days and daily treatments with IFN-{alpha} or PBS. As expected, the survival of mice receiving IFN-{alpha} within any regimen (PBS + IFN-{alpha} or IL-12 + IFN-{alpha}) was prolonged compared with that of negative controls (PBS + PBS). Pretreatment with IL-12 followed by IFN-{alpha} (IL-12 + IFN-{alpha}) did not cure or significantly enhance the survival of GKO mice bearing B16F1 melanoma tumors (p = 0.31) compared with mice receiving IFN-{alpha} alone (PBS and IFN-{alpha}).

 


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  		FIGURE 8. IL-12 pretreatment followed by IFN-{alpha} administration resulted in elimination of B16F1 tumors in GRKO mice. GRKO mice (n = 5 mice/group) were injected i.p. with 106 B16F1 cells on day 0. Beginning on day 1, mice received injections of PBS or IL-12 (a.m.) and PBS or IFN-{alpha} (p.m.), three times per week on alternating days, and daily treatment with IFN-{alpha} or PBS. Compared with negative controls (PBS + PBS), survival was significantly prolonged after IFN-{alpha} treatment alone (PBS + IFN-{alpha}; p = 0.01). All B16F1 tumors were effectively eliminated in GRKO mice after IL-12 pretreatment followed by IFN-{alpha} treatment (IL-12 + IFN-{alpha}; p < 0.0001).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present report we demonstrate that IL-12 pretreatment can enhance the antitumor effects of IFN-{alpha}. In vitro experiments with freshly isolated PBMCs and an IFN-{alpha}-sensitive human melanoma cell line (HT144) revealed that pretreatment with supernatants from IL-12-stimulated PBMCs up-regulated critical Jak-STAT signaling intermediates. An increase in STAT1 expression was confirmed by immunoblot and intracellular flow cytometry. Intracellular signaling in response to lower concentrations of IFN-{alpha} (103 U/ml) was also enhanced by these pretreatments (as measured by EMSA). This led to increased transcription of ISG-15, a gene that is highly responsive to IFN-{alpha}. Importantly, these effects translated into elimination of B16F1 tumors in an animal model of malignant melanoma in which mice received IL-12 pretreatment in combination with IFN-{alpha}. This treatment regimen also eliminated tumors in mice lacking the IFN-{gamma} receptor, but was not effective in mice incapable of producing IFN-{gamma}. These findings show that pretreatment with IL-12 can induce the endogenous production of IFN-{gamma} and thereby augment the responsiveness of both tumor cells and host immune cells to the effects of low dose IFN-{alpha}. Thus, enhancement of IFN-{alpha}-stimulated signal transduction and gene expression translated into improved survival in vivo.

There are significant data to suggest that host tumor surveillance is dependent on endogenously produced IFN-{gamma} and its ability to exert a direct antitumor effect. Studies by Kaplan et al. (25) indicated that the key target of IFN-{gamma} action is the transformed cell itself, and that IFN-{gamma} acted to enhance the recognition of the transformed cell by the immune system. Interestingly, our studies in GRKO and GKO mice suggested that the efficacy of the IL-12/IFN-{alpha} cytokine combination was also dependent on the direct effects of endogenously produced IFN-{gamma} on tumor cells. Although the role of immunotherapeutic agents was not examined, data from a study by Kakuta et al. (41) demonstrated that endogenous IFN-{gamma} prevented B16 melanoma metastasis to the lungs and liver of wild-type mice. They also showed decreased survival and increased tumor deposits in the lungs and liver of GKO, but not GRKO, mice, much as we did. Collectively these data suggest that endogenously produced IFN-{gamma} may enhance immune-mediated tumor rejection. Recent studies from our group have shown that the antitumor effects of exogenously administered IFN-{alpha} on transplanted B16 melanomas were dependent on STAT1 signal transduction within host NK cells (22). Thus, the present study supports the concept that endogenously produced IFN-{gamma} and exogenously administered IFN-{alpha} can exert complementary and synergistic antitumor effects via their actions on tumor cells and host immune effectors, respectively. IL-12 exerts dose-dependent antitumor effects in mice with established tumors. In addition to promoting the regression of primary tumors in the majority of animals and inhibiting the formation of metastases, mice treated with murine IL-12 have been shown to develop a long-lasting, protective, antitumor immune response (42). In contrast, IL-12 has demonstrated modest activity as a single agent in phase I clinical trials (42, 43). However, these studies have confirmed the ability of IL-12 to induce the endogenous production of IFN-{gamma}, which is of theoretical importance in the generation of antitumor immunity.

A significant amount of information regarding the influence of type I and II IFNs on host immunity has been obtained from studies involving viral infection (44). However, many of these cytokine combinations have only recently been investigated for the purposes of tumor immunotherapy. One important function of type I IFNs is their ability to facilitate the expression of other IFN-stimulated genes. For example, IFN-{beta} and IFN-{alpha} 4 are induced first in cells that are the target of viral infection and subsequently bind to IFN receptors on neighboring cells, which leads to activation of STAT1 and induction of IRF7. Recent studies have also shown that type I IFN produced endogenously during viral infection can activate alternative signaling pathways (e.g., STAT4), leading to increased IFN-{gamma} production (44). Thus, it is possible that the ability of IFN-{gamma} to induce STAT1, STAT2, and IRF9 expression reflects the need for the host cells to respond to additional signals from activated immune effectors to achieve full resistance to viral infection or augment the antitumor response (45).

Our data clearly demonstrate that IL-12 is a potent inducer of IFN-{gamma} and that only low levels of IFN-{gamma} are needed for there to be distinct effects on IFN-{alpha}-induced Jak-STAT signaling in both immune effectors and human melanoma cells. In addition to its ability to induce IFN-{gamma} production, IL-12 is also known to have direct, IFN-{gamma}-independent effects on tumor cells (43, 44, 45, 46). Furthermore, in tumors expressing the IL-12R{beta}1, IL-12 can act directly on tumor cells to activate NF-{kappa}B and enhance IFN-{gamma}-mediated STAT1 phosphorylation (47). The use of IL-12-stimulated culture supernatants as a source of IFN-{gamma} was based on a previous observation that subthreshold concentrations of IFN-{gamma} increased the sensitivity of macrophages to IFN-{alpha} without inducing feedback inhibition (48). Other studies have reported that combinations of IL-12 and IFN-{alpha} can induce tumor rejection when administered intratumorally; however, no mechanism of action was proposed. For example, in a study by Mendiratta et al. (49), intratumoral injections of plasmids encoding IFN-{alpha} and IL-12 into mice resulted in 100 and 50% rejection of s.c. renal cell and colon cell carcinomas, respectively. They also demonstrated that combination gene therapy induced resistance to subsequent tumor challenge (49). Similar results were obtained by Li et al. (50), who determined that direct injection of murine IL-12 and IFN-{alpha} cytokine genes into mice bearing floor of mouth squamous cell carcinoma tumors led to inhibition of tumor growth and increased activation of NK cells and CD8+ T cells. Dabrowska et al. (51) have shown that intratumoral injection of murine IL-12 enhanced the actions of recombinant human IFN-A/D in a murine tumor model. However, in their study IL-12 was not administered as a pretreatment to IFN-{alpha}, which might explain why complete tumor eradication was not achieved as was observed in the present report. Of note, the combination of IL-12 and IFN-{alpha} has also been shown to enhance Ab-dependent cell cytotoxicity mediated by a murine mAb 17-1A (which targets the tumor-associated Ag epithelial cell adhesion molecule) to a greater extent than either cytokine alone (52). These studies demonstrate how combination therapy using IL-12 and IFN-{alpha} may be useful for a number of malignancies; however, they do not offer a mechanism for the apparent synergy between the two cytokines. The present work suggests that an IFN-{gamma}-dependent up-regulation of signaling molecules may be the underlying mechanism responsible for the success of this cytokine combination. The present report, to the best of our understanding, represents the first instance where enhancement of cytokine signal transduction and downstream gene regulation has been shown to correlate with improved survival.

To date, concurrent administration of IFN-{gamma} and IFN-{alpha} in patients with advanced malignancy has been well tolerated in phase I and II clinical trials (53, 54, 55, 56, 57, 58). We hypothesize that IL-12 pretreatment can stimulate the production of endogenous IFN-{gamma} and augment the responsiveness to IFN-{alpha} in the clinical setting. In addition to its ability to induce IFN-{gamma} production, IL-12 has antiangiogenic properties and exerts direct effects on tumor cells that may contribute to its antitumor activity in vivo. Direct administration of IFN-{gamma} is a less attractive alternative, in that the serum half-lives of this cytokine after i.v. and s.c. administration are only 38 min and 5.9 h, respectively (Physician’s Desk Reference; www.pdr.net). Significant toxicity was encountered in an early clinical trial when a test dose was omitted from a high dose regimen of IL-12 (42). However, in a subsequent study of single-agent IL-12 in patients with malignant melanoma and renal cell carcinoma, it was demonstrated that a test dose was not necessary if the cytokine was given on a twice weekly schedule (59). Importantly, the revised regimen was well tolerated, and there were no further episodes of drug-related toxicity. Data from the present study support the use of IL-12 pretreatment as a means of inducing the endogenous production of IFN-{gamma}, which could up-regulate levels of Jak-STAT signaling intermediates in tumor cells and PBMCs and render them more sensitive to the effects of low dose IFN-{alpha}.

In conclusion, we have shown that IL-12 administration resulted in the endogenous production of IFN-{gamma}, which, in turn, up-regulated levels of Jak-STAT signaling intermediates in PBMCs and tumor cells. This led to increased sensitivity to low dose IFN-{alpha} and an enhanced antitumor response. These findings suggest that pretreatment with IL-12 may augment the antitumor actions of IFN-{alpha} in patients with malignant melanoma and allow for the administration of lower, less toxic doses of IFN-{alpha}. This hypothesis is currently being tested in a national phase II trial in which IL-12 pretreatment is followed by low dose IFN-{alpha}.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Research Grants CA84402 and P30CA16058, The Valvano Foundation for Cancer Research Award, and an Ohio State University Department of Surgery Clinical Science Seed Grant (to W.E.C.). G.B.L. is a National Research Scientist Award T32 Fellow (5T32CA90223-02). B.B. is supported by an Ohio State University General Surgery Resident Research Award. Back

2 Address correspondence and reprint requests to Dr. William E. Carson III, Division of Surgical Oncology, Department of Surgery, Ohio State University, N924 Doan Hall, 410 West 10th Avenue, Columbus, OH 43210. E-mail address: carson-1{at}medctr.osu.edu Back

3 Abbreviations used in this paper; IRF9, IFN-responsive factor 9; IFNAR, IFN-{alpha} receptor; Jak, Janus kinase; SIE, Sis-inducible element; Tyk, tyrosine kinase; ISG-15, IFN-stimulated gene 15. Back

Received for publication December 4, 2003. Accepted for publication April 12, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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