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The Journal of Immunology, 2007, 178: 2278-2286.
Copyright © 2007 by The American Association of Immunologists, Inc.

CXC Chemokine Ligand 9/Monokine Induced by IFN-{gamma} Production by Tumor Cells Is Critical for T Cell-Mediated Suppression of Cutaneous Tumors1

Anton V. Gorbachev2,3,*, Hirohito Kobayashi3,*, Daisuke Kudo*, Charles S. Tannenbaum*, James H. Finke*, Suyu Shu{dagger}, Joshua M. Farber{ddagger} and Robert L. Fairchild*

* Department of Immunology and {dagger} Center for Surgery Research, Cleveland Clinic, Cleveland, OH 44195, and {ddagger} Laboratory of Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The role of tumor-produced chemokines in the growth of malignancies remains poorly understood. We retrieved an in vivo growing MCA205 fibrosarcoma and isolated tumor cell clones that produce both CXCL9/monokine induced by IFN-{gamma} (Mig) and CXCL10/IFN-{gamma}-inducible protein 10 following stimulation with IFN-{gamma} and clones that produce IFN-{gamma}-inducible protein 10 but not Mig. The Mig-deficient variants grew more aggressively as cutaneous tumors in wild-type mice than the Mig-producing tumor cells. The growth of Mig-expressing, but not Mig-deficient, tumor cells was suppressed by NK and T cell activity. Transduction of Mig-negative variants to generate constitutive tumor cell production of Mig resulted in T cell-dependent rejection of the tumors and in induction of protective tumor-specific CD8+ T cell responses to Mig-deficient tumors. The results indicate a critical role for tumor-derived Mig in T cell-mediated responses to cutaneous fibrosarcomas and suggest the loss of Mig expression as a mechanism used by tumor cells to evade these responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The chemokines CXCL9/monokine induced by IFN-{gamma} (Mig)4 and CXCL10/IFN-{gamma}-inducible protein 10 (IP-10) are critical components of many immune responses as chemoattractants of activated NK and Th1 cells into sites of inflammation (1, 2, 3, 4). Both of these chemokines mediate this activity through binding to CXCR3 expressed on activated Th1 and NK cells (5, 6). Mig and IP-10 also influence the behavior of nonimmune cells that express a CXCR3. Both chemokines inhibit endothelial cell proliferation through interfering with the function of receptors for the growth factors basic fibroblast growth factor and vascular endothelial growth factor resulting in the inhibition of angiogenesis (7). Mig expression is induced specifically by type I and type II IFNs whereas IP-10 is also induced by other stimuli such as TNF-{alpha} and LPS (8, 9, 10). Moreover, differences in tissue expression patterns of Mig and IP-10 during the course of viral and parasitic infections are observed (8). These studies suggest that in addition to chemoattraction of Ag-activated T cells, IP-10 and Mig may exert other nonredundant functions in vivo.

The potential role of IP-10 and Mig in the suppression of malignancies has focused primarily on the chemotactic and angiostatic functions of these chemokines. Delivery of Mig or IP-10 into tumors by injection of protein or by genetic engineering to express the chemokines has been shown to suppress tumor growth through inhibition of angiogenesis (11, 12, 13, 14). In murine cancer models, intratumor delivery of immunotherapeutic agents such as IL-2, IL-10, IL-12, or CCL21 correlates with increased expression of Mig and/or IP-10 and with increased T cell infiltration into the tumor (15, 16, 17, 18). Clinical studies of renal carcinoma patients have also indicated that intratumor expression of Mig and IP-10 correlates with increased CD8+ T cell infiltration, decreased tumor size, and with rare recurrence of tumors after surgical resection (19). These studies suggest that the chemotactic functions of Mig and IP-10 for activated T cells may be as critical as the angiostatic functions of these chemokines to inhibit tumor growth. What remains unclear from these studies is the source of the Mig and IP-10 produced during tumor growth as well as potential mechanisms that tumors use to resist the suppressive effects mediated by these chemokines.

During studies investigating the expression of CXCR3- and CCR5-binding chemokines during growth of methylcholanthrene (MCA)-induced fibrosarcomas in mice we observed decreases in intratumor Mig expression as tumor growth progressed. This observation raised the possibility that the decrease in Mig expression contributed to the tumorigenicity of the tumor. To begin to investigate this possibility, a growing solid tumor was resected and individual clones were generated and tested for the induction of Mig and IP-10 expression. In the current study, we show that the in vivo growth of Mig-deficient tumor cells is significantly increased and is not suppressed by NK or T cells when compared with tumor cells expressing Mig. Restoration of Mig production in the Mig-deficient variants reverses the high tumorigenicity of these tumor cells and results in T cell-mediated suppression of tumor growth in immunocompetent mice. Furthermore, mice that reject the constitutively expressing Mig tumor become resistant to rechallenge with the highly tumorigenic Mig-deficient tumor cells. These results highlight the role of tumor-produced Mig in tumor-specific T cell responses that suppress tumor growth.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6 mice and RAG1–/– mice on the C57BL/6 background were purchased from The Jackson Laboratory. The generation and characterization of the Mig–/– mice on the C57BL/6 background has been reported (20). Adult female mice 8–10 wk old were used throughout these studies. All animal experiments were performed according to the National Institutes of Health Guides for the Care and Use of Laboratory Animals and all protocols were approved by the Institutional Animal Care Use Committee of The Cleveland Clinic.

Antibodies

Biotinylated mAb AF6-88.5 (anti-H-2Kb), mAb 28-14-8 (anti-H-2Db), streptavidin-FITC, FITC-labeled anti-CD45 mAb, and PE-labeled anti-CD4 and anti-CD8 mAb were purchased from BD Pharmingen. Allophycocyanin-labeled rat isotype control Ab and anti-CXCR3 mAb and polyclonal goat anti-mouse Mig Abs were purchased from R&D Systems. Biotin-conjugated rabbit Abs specific for goat IgG were purchased from Vector Laboratories.

Mice were injected on 3 consecutive days with 200 µg of anti-NK1.1 mAb PK136 (BioExpress) to deplete NK/NK T cells or with 200 µg each of anti-CD4 mAb GK1.5 and anti-CD8 mAbs YTS169 and TIB105 (BioExpress) to deplete CD4+ and CD8+ T cells. This treatment resulted in >90% depletion of the targeted cell population as assessed by flow cytometry of treated sentinel mice. Control mice were injected with the same doses of control rat IgG (Sigma-Aldrich) and this treatment did not have an effect on the presence of NK or T cell populations.

Tumor growth in vitro and in vivo

Cells of the murine fibrosarcoma MCA205 were grown in RPMI 1640 culture medium supplemented with 10% FCS, 2 mM L-glutamine, and 1% antibiotic/antimycotic solution containing penicillin G, streptomycin, and amphotericin B (Invitrogen Life Technologies).

For induction of solid tumors, syngeneic C57BL6 mice were injected intradermally with 3 x 105 tumor cells. Tumor sizes were measured in two dimensions using a caliper every 48 h beginning on day +7 postinjection. Tumor size was estimated according to the formula: (smallest diameter)2 x (longest diameter) as previously described (15).

Generation of Mig-expressing and Mig-deficient MCA205 tumor cell clones

The parental MCA205 mouse fibrosarcoma was grown intradermally in mice and the solid tumor was retrieved. A tumor cell suspension was prepared by digestion of the solid tumor with a mixture of 0.1% collagenase, 0.01% DNase and 2.5 U/ml hyaluronidase (Sigma-Aldrich). The tumor cells were seeded in 96-well culture plates at 0.3 cells/well in complete RPMI 1640 culture medium with 10% FCS. The wells were observed daily under a light microscope and randomly selected single colonies of tumor cells were harvested and cultured in 6-well plates with complete RPMI 1640 medium. At the time of 90% cell confluence, 10 ng/ml mouse recombinant IFN-{gamma} (R&D Systems) was added to the culture. Total RNA was extracted from tumor cells using TRIzol Reagent (Invitrogen Life Technologies) at 6, 12, and 24 h after culture with rIFN-{gamma} and the expression of chemokine mRNA was tested by RNase protection assay (RPA).

RNase protection assay

The template cDNA for murine Mig was provided by C. S. Tannenbaum. Template cDNAs for murine IP-10 and GAPDH were purchased from BD Pharmingen. The isolation of whole cell RNA, generation of 32P-labeled probes, and the RPA were performed as previously described (21). Densitometry using gel scanner Storm 840 (Molecular Dynamics) was used to measure the chemokine and GAPDH signals for each sample of the blot. The chemokine signals for each sample were then normalized by expressing the density of the chemokine signal as a ratio of the GAPDH signal.

Analyses of Mig and IP-10 protein production

Two selected MCA205 cell clones, MCA205-4 and MCA205-10, were expanded in 75 cm2 tissue-culture flasks in serum- and protein-free hybridoma medium, HL-1 (BioWhittaker) supplemented with 2 mM L-glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin, 50 µg/ml gentamicin, and 0.5 µg/ml fungizone (Invitrogen Life Technologies) and were either left untreated or stimulated with 10 ng/ml rIFN-{gamma}. Supernatants from the MCA205-4 and MCA205-10 clones cultured with or without rIFN-{gamma} for 72 h were tested using Mig and IP-10-specific ELISA kits following the manufacturer’s instructions (R&D Systems).

To test intratumor production of chemokines, tumors were excised on day 8 and tissue homogenates were prepared in the presence of protease inhibitors (10 µg/ml PMSF, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 100 µg/ml chymostatin). Following standardization of protein levels to 2 mg of total protein/ml, aliquots of each tissue sample were tested in triplicate by ELISA to quantitate levels of chemokine protein and were expressed as a ratio of protein concentration to the weight of the tumor sample.

Flow cytometry

To compare MHC class I expression by MCA205 tumor cell clones, cells were cultured with or without 10 ng/ml rIFN-{gamma}. After 30 h, the cells were harvested, washed with staining buffer (Dulbecco’s PBS with 2% FCS/0.2% NaN3) and 5 x 105 cell aliquots were incubated for 20 min on ice in 100 µl of rat serum (Rockland) diluted 1/1000 in the staining buffer. Then cells were washed and stained with biotinylated anti-mouse H-2Kb and H-2Db mAb followed by streptavidin-FITC. Stained cells were washed five times, resuspended in staining buffer and analyzed by two-color flow cytometry.

To assess the frequency of CXCR3-expressing T cells infiltrating tumors, tumors were excised on day 14 posttransplant and cell suspensions were prepared by digestion in 0.1% collagenase, 0.01% DNase, and 2.5 U/ml hyaluronidase (Sigma-Aldrich) for 1 h at room temperature (RT). The suspensions were filtered through a nylon mesh, washed, and stained with FITC-labeled anti-CD45 mAb, PE-labeled anti-CD4, or anti-CD8 mAb and allophycocyanin-labeled anti-CXCR3 mAb. Stained cells were washed five times, resuspended in staining buffer and analyzed by three-color flow cytometry. CD45+ cells were first gated and then CXCR3 expression on the CD4+ and CD8+ T cells was assessed.

Immunohistology

For immunohistology, intradermal tumors were excised, fixed with 10% buffered formalin, and paraffin-embedded sections were cut at 8-µm thickness and mounted onto slides. Slides were immersed in PBS for 10 min and in 0.03% H2O2 for 10 min to eliminate endogenous peroxidase activity. To block nonspecific binding, slides were incubated for 30 min with normal rabbit serum. The slides were then incubated with polyclonal goat anti-Mig Ab, diluted 1/100, in a humid chamber at 4°C overnight. After three washes in PBS, slides were incubated for 1 h at RT with biotinylated rabbit anti-goat IgG diluted 1/300 in PBS with 10% normal rabbit serum, washed three times with PBS, and incubated with streptavidin-HRP (DakoCytomation) for 30 min at RT. The substrate-chromagen solution 3,3'-diaminobenzidine (DAB) was prepared using the DAB Peroxidase Substrate kit (Vector Laboratories) following the manufacturer’s protocol and was applied to the slides and incubated for 3–7 min. After a final wash in H2O, the slides were counterstained with hematoxylin, rinsed, and dehydrated in ethyl alcohol and then in CitriSolv solution (Fisher Scientific). The slides were mounted in Cytoseal 60 medium (Richard-Allan Scientific), viewed with a light microscope, and images were captured using Image Pro Plus (Media Cybernetics).

Tumor-specific ELISPOT assays

ELISPOT assays to enumerate IFN-{gamma}-producing cells were performed as previously described (22). Briefly, ELISPOT plates (Unifilter 350; Polyfiltronics) were coated with 100 µl of 4 µg/ml anti-IFN-{gamma} mAb R26A2 and incubated overnight at 4°C. The plates were blocked with 1% BSA in PBS for 90 min at 37°C and washed four times with PBS. Lymph node cells were prepared from naive mice or from tumor-challenged mice on day 8 after tumor inoculation and CD4+ and CD8+ T cells were separated by negative selection with anti-CD8 or anti-CD4 mAb-coated magnetic beads (Dynal Biotech) and used as responder cells. Syngeneic spleen cells from naive mice or MCA205 tumor cells were treated with 50 µg/ml mitomycin C, and used as stimulator cells. Responder CD4+ or CD8+ T were resuspended in serum-free HL-1 medium (BioWhittaker) and cultured at 5 x 105 cells/well with 5 x 105 stimulator cells/well 24 h at 37°C in 5% CO2. In all experiments, responder cells cultured with syngeneic splenocytes and responder cells from naive mice cultured with MCA205 tumor cells were used as negative controls. After 24 h, cells were removed from the culture wells by extensive washing with PBS and then PBS/0.2% Tween 20. Biotinylated anti-IFN-{gamma} mAb XMG1.2 (2 µg/ml) was added, and the plate was incubated overnight at 4°C. The wells were washed three times with PBS/0.2% Tween 20 and incubated with antibiotin alkaline phosphatase conjugate. After 2 h at room temperature, the wells were washed with PBS and nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl substrate (Kirkegaard & Perry Laboratories) was added. The resulting spots were counted on an ImmunoSpot Series 1 Analyzer (Cellular Technology) that was designed to detect spots with predetermined criteria on size, shape, and colorimetric density. The results are expressed as the mean number of spots detected in triplicate wells after subtraction of spots from negative control wells containing T cells from the same experimental group cultured with syngeneic spleen cells (typically less than five spots per well).

Generation of tumor cells constitutively producing Mig

GP+E cell lines transduced with pBABE puromycin retroviral vector with or without a Mig cDNA insert were grown in complete RPMI 1640 medium with 5 µg/ml puromycin to prepare supernatants containing the retroviral vectors. MCA205-4 cells were infected with supernatants containing either Mig+ or Mig– retroviral vectors in the presence of Polybrene by overnight culture at 37°C, 5% CO2. The cells were washed and cultured in RPMI 1640 containing 5 µg/ml puromycin and puromycin-resistant cell clones overexpressing Mig (Mig-transduced) or control vector (vector-transduced) were selected and expanded. Culture supernatants from Mig and vector transductants were tested for Mig production by ELISA.

Statistical analysis

Statistical analysis to assess differences between experimental groups was performed using the Student t test. Differences were considered significant when p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Isolation of tumor cells deficient in production of CXCL9/Mig but not CXCL10/IP-10 from tumors growing in vivo

During preliminary studies, we observed high mRNA expression of CXCL9/Mig induced in the lungs of mice bearing lung metastasis of MCA205 fibrosarcoma that decreased at later times of tumor growth (H. Kobayshi, manuscript in preparation). One potential explanation for this observation is the outgrowth of tumor cells deficient in the expression of Mig. To test whether Mig-deficient cells were present in the tumors growing in vivo, a cell suspension was prepared by enzyme digestion of growing solid tumors and the cells were subcloned by limiting dilution. Eight single-cell clones were randomly selected and tested for the expression of Mig and IP-10 mRNA following stimulation with IFN-{gamma}. Without IFN-{gamma} stimulation the expression of IP-10 and Mig mRNA was low to undetectable in all tested cell clones. Seven of eight clones expressed low to background levels of Mig mRNA (Fig. 1A, clones 2–8, 11, and 13), while only one clone (clone 10) expressed high levels of Mig mRNA in response to IFN-{gamma} stimulation. In contrast to Mig expression, IP-10 mRNA was induced in all of these tumor cell clones by IFN-{gamma} (data not shown). These results were confirmed by comparing the production of Mig and IP-10 protein by the selected Mig-deficient (MCA205-4) vs Mig-expressing (MCA205-10) tumor cell clones using Mig- and IP-10-specific ELISA (Fig. 1B). The results suggested a high frequency of tumor cells deficient in Mig, but not IP-10, production are present in MCA205 tumors growing in vivo. To further investigate whether Mig-deficient tumor cells were present at a high frequency in the parental MCA205 cell line, equal numbers of parental MCA205, Mig-producing MCA205-10 and Mig-deficient MCA205-4 cells were stimulated in vitro with IFN-{gamma} and culture supernatants were tested for Mig production. Without IFN-{gamma} stimulation none of the tumor cells produced Mig (data not shown). Following IFN-{gamma} stimulation, the parental MCA205 cells produced similar amounts of Mig as the Mig-expressing MCA205-10 tumor clone, while the Mig-deficient MCA205-4 tumor clone did not (Fig. 1C). Furthermore, IFN-{gamma} stimulation of cocultured Mig-deficient and Mig-producing tumor cells resulted in half the production of Mig when compared with cultures of the Mig-expressing clone or parental MCA205 cells. The results suggest that Mig-producing cells predominate the MCA205 parental cell line and suggest that outgrowth of Mig-deficient tumor cells occurs during the in vivo growth of the tumors.


Figure 1
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  		FIGURE 1. Mig and IP-10 mRNA expression and protein production in cell clones isolated from solid MCA205 tumors growing in vivo. A, Solid tumors of parental MCA205 cells were excised, digested, and the cells were subcloned by limiting dilution. Single-cell colonies were randomly selected, expanded, and cultured with 10 ng/ml rIFN-{gamma} ({blacksquare}) or without rIFN-{gamma} stimulation ({square}). After 24 h, cells were lysed and whole cell RNA was isolated. Aliquots (10 µg) of total RNA from each cell clone were tested for levels of Mig mRNA expression by RPA. The results are expressed as the ratio of chemokine/GAPDH mRNA signal. B, Supernatants from tumor cell clones MCA205-10 ({square}) or MCA205-4 ({blacksquare}) cultured with rIFN-{gamma} for 48 h were tested for IP-10 and Mig production by ELISA. C, A total of 2 x 105 cell aliquots of parental MCA205 cells, Mig-producing clone MCA205-10 (Mig+), Mig-deficient clone MCA205-4 (Mig–), or a mixture of Mig+ and Mig– cells (1 x 105 cells of each) were cultured with rIFN-{gamma} for 48 h. Supernatants were tested for Mig production by ELISA. *, p < 0.05. D, A total of 3 x 105 cell aliquots of Mig-producing ({square}) or Mig-deficient ({blacksquare}) tumor cells were injected intradermally into mice. Tumors were excised on day 8 posttransplantation, tissue homogenates were prepared and tested for IP-10 and Mig protein levels by ELISA. Homogenates of the skin of naive mice (Figure 1) were used as a negative control. The results are expressed as mean concentration of chemokine per mg of excised tumor or skin weight.

 
Decreased Mig production in the tumors induced by Mig-deficient tumor cells

The production of IP-10 and Mig during the growth of the Mig-expressing MCA205-10 and Mig-deficient MCA205-4 tumor clones in vivo was tested. Tissue homogenates of solid tumors were prepared and tested for IP-10 and Mig protein by ELISA. Homogenates from the skin of naive mice not bearing tumors contained low levels of IP-10, while no Mig was detected. IP-10 production was increased to similar levels in the tumors induced by Mig-expressing and Mig-deficient tumor clones. In contrast, Mig production in Mig-deficient tumors was present but considerably lower than that in Mig-producing tumors (Fig. 1D). To define the source of Mig produced in the tumor microenvironment, C57BL/6 wild-type and B6.Mig–/– mice were used as recipients of Mig-deficient MCA205-4 cells or Mig-expressing MCA205-10 cells. Growing solid tumors were excised 8 days later, paraffin sections were prepared and stained with Mig-specific Ab. Low amounts of Mig protein were observed in Mig-deficient tumors growing in wild-type hosts and were not detected in B6.Mig–/– recipients of these tumors indicating that tumor-infiltrating host cells were the source of Mig during this tumor growth (Fig. 2, A vs B). Production of Mig was abundant in the tumors induced by Mig-producing cells in wild-type recipients (Fig. 2C) and remained detectable in B6.Mig–/– recipients (Fig. 2D). These results further confirmed the absence of Mig production by Mig-deficient tumor variants growing in vivo and indicated that Mig is produced by host-derived cells as well as by the tumor during growth of the Mig-producing tumor cells.


Figure 2
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  		FIGURE 2. Mig production by Mig-deficient and Mig-expressing tumor clones growing in vivo. Solid tumors were induced by intradermal transplantation of Mig-deficient MCA205-4 cells (A and B) or Mig-expressing MCA205-10 cells (C and D) into wild-type (A and C) or Mig–/– (B and D) recipient mice. Tumors were excised on day +15 posttransplantation and prepared paraffin sections were stained with Mig-specific goat polyclonal Ab plus secondary rabbit anti-goat peroxidase-labeled Ab. Areas of intensive Mig production are indicated by arrows. Magnification, x50 with x200 inserts in upper right corner.

 
Mig-deficient tumor cells are more tumorigenic than Mig-expressing tumor cells

Next, the in vivo growth of the Mig-deficient vs Mig-producing tumor clones was compared by injection of these tumor variants into mice to induce cutaneous tumors. Growth of solid tumors induced by intradermal injection of the Mig-deficient tumor cells was significantly increased when compared with the Mig-producing tumor cells (Fig. 3A). Furthermore, the in vivo growth of Mig-producing tumor cells was significantly increased when the tumor-bearing hosts were treated with neutralizing Mig antiserum (Fig. 3B). The results indicated increased growth of MCA205 tumors when Mig production is low or neutralized. To begin to test the mechanism of the increased tumorigenicity of Mig-deficient tumor variants, the induction of tumor-specific CD4+ and CD8+ T cells producing IFN-{gamma} in recipient mice challenged with Mig-deficient or Mig-producing tumor cells was compared by ELISPOT assay. Skin draining lymph nodes from naive mice did not have T cells producing IFN-{gamma} during culture with MCA205 tumor cells (data not shown). The numbers of CD4+ T and CD8+ T cells producing IFN-{gamma} during culture with the tumor cells (vs syngeneic spleen cells) were low to undetectable on day 8 posttransplant in the tumor-draining (inguinal) lymph nodes of mice bearing Mig-deficient MCA205-4 tumors. In contrast, mice bearing the Mig-producing MCA205-10 tumors had high numbers of tumor-specific CD4+ T cells and low numbers of CD8+ T cells producing IFN-{gamma} at the indicated time point (Fig. 3C; day 8: Mig+ vs Mig– groups). On day 14 posttransplant, CD4+ and CD8+ T cells producing IFN-{gamma} were detected in tumor-draining lymph nodes of both groups, however, the numbers of IFN-{gamma}-producing CD8+ T cells were moderately increased in mice bearing Mig-producing tumors vs Mig-deficient tumor recipients (Fig. 3C, day 14). To test whether the increased induction of tumor-specific T cells correlated with T cell recruitment into tumors, tumor cell suspensions were prepared on days 8 and 14 posttransplant and analyzed for the frequency of CD4+ and CD8+ T cells by flow cytometry. To separate infiltrating leukocytes from tumor cells, the CD45+ cell population was gated (Fig. 4A) and then analyzed for CD4+ and CD8+ T cells expressing CXCR3 (Fig. 4B). Low numbers of T cells were detected in both Mig-producing and Mig-deficient tumors on day 8 posttransplant (not shown). On day 14 posttransplant, the presence of both CD4+ and CD8+ T cells was increased in Mig-producing tumors when compared with the Mig-deficient variant. Furthermore, the frequencies of CD4+ and CD8+ T cells expressing CXCR3 were strikingly increased in Mig-producing tumors, indicating augmented recruitment of activated T cells into these tumors (Fig. 4B). The results indicated that the induction of IFN-{gamma} producing CD4+ T cells in response to tumor growth is delayed and intratumor T cell infiltration is low in recipients bearing Mig-deficient tumors.


Figure 3
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  		FIGURE 3. Tumor growth and activation of tumor-specific T cells induced by Mig-expressing vs Mig-deficient tumor cells. A, Mig-expressing MCA205-10 ({diamond}) or Mig-deficient MCA205-4 ({blacksquare}) tumor cells were injected intradermally into groups of five C57BL/6 mice and tumor size was measured on the indicated days. The results indicate the mean tumor size for each recipient group. *, p < 0.05. B, Mig-expressing MCA205-10 tumor cells were injected intradermally into groups of five C57BL/6 mice and the recipients were treated by i.p. injections of 500 µl of control rabbit sera ({square}) or Mig-specific antisera ({blacksquare}) every 48 h after transplantation. Tumor size was measured on the indicated days. The results indicate the mean tumor size for each recipient group. *, p < 0.05. C, Mig-expressing MCA205-10 or Mig-deficient MCA205-4 tumor cells were injected intradermally into C57BL/6 mice. On days 8 and 14 posttransplant, CD4+ and CD8+ T cell-enriched cell suspensions were prepared from the lymph nodes of the tumor recipients or from naive mice using negative selection and ELISPOT assay was used to enumerate tumor-specific IFN-{gamma}-producing cells. The mean number ± SEM of IFN-{gamma}-producing CD4+ ({square}) and CD8+ T cells ({blacksquare}) per 5 x 105 responder cells in triplicate cultures minus the number of spots from control cultures with syngeneic spleen cell stimulators for two individual mice are shown.

 

Figure 4
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  		FIGURE 4. Recruitment of CXCR3-expressing T cells into Mig-deficient and Mig-producing tumors. Tumors were excised on day 14 posttransplant and digested in a collagenase, DNase and hyaluronidase mixture. Cell suspensions obtained were washed and stained with anti-CD45, anti-CD4 or anti-CD8, and anti-CXCR3 mAb. The tumor-infiltrating leukocyte cell population was gated as CD45+ cells (A) and then analyzed for the frequencies of CD4+ and CD8+ T cells that were positively stained by anti-CXCR3 mAb (B, gate R3) and were negative when stained with isotype control IgG (data not shown). The numbers in the dot plots indicate the mean percentage ± SEM of CXCR3+ T cells in 50,000 cell aliquots analyzed for three tumors in each group.

 
In vitro growth and class I MHC expression of Mig-deficient and Mig-expressing tumor cells

To test whether the increased tumorigenicity of Mig-deficient vs Mig-producing tumor cells was mediated by inherent differences in the ability to proliferate, the in vitro growth of these tumor variants was compared. When equivalent aliquots of Mig-deficient and Mig-producing tumor cells were placed in separate culture wells, similar numbers of cells were counted after 48, 72, and 96 h of culture (Fig. 5A). Similar proliferation of Mig-deficient and Mig-producing tumor cells labeled with CFSE and cultured for 72 h was also observed (data not shown).


Figure 5
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  		FIGURE 5. Proliferation and class I MHC expression of Mig-deficient and Mig-expressing tumor cells in vitro. A, Aliquots of Mig-deficient MCA205-4 ({blacksquare}) and Mig-expressing MCA205-10 ({square}) tumor cells were cultured in triplicate for 48, 72, and 96 h. Cells were detached by 0.5% EDTA/trypsin and viable cells counted using trypan blue exclusion. The results are expressed as mean total cell number after the indicated time of culture. B, Mig-deficient MCA205-4 and Mig-producing MCA205-10 tumor cells were left untreated (solid line) or cultured with rIFN-{gamma} (filled histogram). After 24 h, the cells were washed and stained with anti-class I MHC Kb or Db mAb. Tumor cells incubated with isotype control Ab is shown as a negative control (dotted line).

 
Loss of class I MHC expression is a common mechanism used by tumors to evade host immune responses and may also contribute to increased tumorigenicity (23). Equal numbers of Mig-deficient or Mig-expressing tumor cells were cultured for 48 h with or without IFN-{gamma} and class I MHC expression on the cells was tested by flow cytometry. On nonstimulated cells the expression of H-2Kb and H-2Db (Fig. 5B) was detectable on both tumor clones. IFN-{gamma} stimulation resulted in up-regulation of class I MHC expression to similar levels on both Mig-deficient and Mig-expressing tumor cells. Therefore, an increased ability to proliferate, a loss of class I MHC expression or unresponsiveness to IFN-{gamma} signaling are not likely mechanisms mediating the increased tumorigenicity of the Mig-deficient tumor cells.

In vivo growth of Mig-expressing tumor cells is negatively regulated by NK and T cells

Activated NK/NK T cell and T cell populations express CXCR3, the receptor binding Mig (5, 6, 24). Because the results suggested that tumor-derived Mig negatively regulates the in vivo growth of the MCA205 fibrosarcoma, the potential contribution of NK/NK T and T cells to this regulation was tested. Tumors induced by Mig-deficient MCA205-4 cells in control IgG-treated wild-type recipient mice grew significantly faster than tumors induced by Mig-producing MCA205-10 cells (Fig. 6, A vs B, {square}). However, growth of the Mig-producing tumor cells was comparable to that of Mig-deficient variants when host mice were depleted of NK and NK T cells, whereas this depletion had no significant effect on the growth of the Mig-deficient cells (Fig. 6, A vs B, {blacksquare}). Likewise, the growth of MCA205-10 tumors was increased in RAG1–/– vs wild-type mice (Fig. 6C) whereas the absence of T, NK T, and B cells had no impact on the growth of the Mig-deficient MCA205-4 variants (Fig. 6D).


Figure 6
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  		FIGURE 6. In vivo growth of Mig-expressing, but not Mig-deficient, tumor cells is suppressed by NK and T cells. Wild-type syngeneic mice were treated with control rat IgG ({square}) or with anti-NK1.1 mAb ({blacksquare}) before Mig-expressing MCA205-10 (A) or Mig-deficient MCA205-4 (B) tumor cells were injected intradermally into groups of C57BL/6 mice. Mig-expressing MCA205-10 (C) or Mig-deficient MCA205-4 (D) tumor cells were injected intradermally into groups of syngeneic wild-type mice ({square}) or B6.RAG1–/– mice ({blacksquare}). Results indicate the mean tumor size on the indicated days in each recipient group of five mice. *, p < 0.05.

 
Expression of Mig in MCA205-4 cells reverses the high tumorigenicity

Finally, the role of Mig in MCA205 growth was tested by inducing constitutive Mig expression in the Mig-deficient MCA205-4 variants. MCA205-4 cells were transduced with either Mig-encoding or control viral vectors. Testing cell supernatants by ELISA confirmed that the Mig-transduced but not control-transduced MCA205-4 tumor cells constitutively produced high amounts of Mig during culture without IFN-{gamma} (data not shown). When transplanted with the MCA205-4 Mig-transductant, four of five recipients rejected the tumors by day 15 posttransplantation and the single remaining tumor grew very slowly. In contrast, recipients of the MCA205-4 control-transductants did not reject the tumors and the animals were sacrificed on day 25 posttransplant (Fig. 7A).


Figure 7
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  		FIGURE 7. Tumors constitutively producing Mig induce protective T cell-mediated immune responses. A, Mig-deficient MCA205-4 tumor cells were transduced with a control viral vector (•) or with a vector encoding Mig cDNA ({Upsilon}) and puromycin-selected transductants for each vector group were injected intradermally into groups of five syngeneic mice. The growth of solid tumors was monitored through day 25 posttransplantation. B, C57BL/6 mice were treated with control rat IgG (control), with anti-NK1.1 mAb (NK cell depleted), or with anti-CD4 plus anti-CD8 mAb (T cell depleted) and then received Mig-transduced MCA205-4 cells. The mean tumor size in each recipient group of five mice at day 14 posttransplantation is shown. *, p < 0.01. C, Mig-deficient tumor cells transduced with a control vector (control) or with a vector encoding Mig (Mig+) were injected intradermally into C57BL/6 mice. On day 8, CD4+ and CD8+ T cell-enriched cell suspensions were prepared from the draining lymph nodes of the tumor recipients or from naive mice using negative selection and tumor-specific IFN-{gamma}-producing cells were enumerated by ELISPOT. The mean number ± SEM of IFN-{gamma}-producing CD4+ ({square}) and CD8+ T cells ({blacksquare}) per 5 x 105 responder cells in triplicate cultures minus the number of spots from control cultures with syngeneic spleen cell stimulators for two individual mice is shown.

 
To begin to identify the cell population contributing to the suppressed growth of the Mig-transduced MCA205-4 tumors, mice were treated with mAb to deplete NK cells or T cells before transplantation with the Mig transductants. Tumors were either rejected or severely decreased in size on day 14 posttransplantation in control IgG-treated recipients (Fig. 7B). In NK cell-depleted recipients, the tumors grew slowly but were not rejected. In contrast, the Mig transductants grew rapidly in all recipients depleted of T cells. These results indicated that the restoration of Mig expression in Mig-deficient tumor cells reversed the high tumorigenicity and resulted in T cell-mediated suppression of in vivo growth. The development of tumor-specific T cells in the tumor-draining lymph nodes of mice bearing vector-transduced vs Mig-transduced tumors was tested by ELISPOT. A 3-fold increase in the numbers of tumor-specific CD4+ T cells producing IFN-{gamma} was observed in mice bearing Mig-transduced tumors as compared with mice bearing vector-transduced Mig-deficient tumors, whereas the numbers of IFN-{gamma}-producing CD8+ T cells were similar in both recipient groups (Fig. 7C).

Recipient mice that rejected the Mig-transduced tumors were rested for 3 wk and then rechallenged with the parental Mig-deficient tumor cells (MCA205-4). As previously observed, these tumor cells were highly tumorigenic, inducing rapidly growing intradermal tumors in naive wild-type mice (Fig. 8A, control recipients). In contrast, no tumors appeared by day 14 after inoculation of Mig-deficient tumor cells in recipient mice that had previously rejected the Mig-transduced tumors (Fig. 8A, tumor rechallenged recipients). Skin-draining lymph nodes were removed from these mice and tested by ELISPOT for the numbers of CD4+ T and CD8+ T cells producing IFN-{gamma} in response to the stimulator Mig-deficient tumor cells. Unlike naive mice transplanted with Mig-deficient tumor cells, mice that had previously rejected the Mig-transduced tumors and were then rechallenged with the Mig-deficient tumor cells had a high frequency of CD8+ T cells that produced IFN-{gamma} in response to in vitro stimulation with the tumor cells (Fig. 8B). Overall, this robust induction of tumor-specific T cells producing IFN-{gamma} in mice primed with Mig-transduced tumor cells correlated with the protection of these mice against subsequent challenge with the highly tumorigenic Mig-deficient tumor cells.


Figure 8
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  		FIGURE 8. Tumors constitutively producing Mig induce protective immune responses to Mig-deficient tumors. A, MCA205-4 cells transduced to constitutively express Mig cDNA were injected intradermally into syngeneic mice. Mice that rejected tumors were rested for 3 wk and then rechallenged with nontransduced, Mig-deficient MCA205-4 tumor cells (tumor rechallenged). A control group of naive mice also received the nontransduced, Mig-deficient MCA205-4 tumor cells (control). The results indicate the mean tumor size for each group of 5 mice 14 days after injection. ND, tumors were not detected. B, MCA205-4 cells transduced to constitutively express Mig cDNA were injected intradermally into syngeneic mice. Mice that rejected the tumors were rested for 3 wk and then rechallenged with nontransduced, Mig-deficient MCA205-4 tumor cells (tumor rechallenged). A control group of naive mice was transplanted with the same tumor cells (control). On day 8 after tumor challenge, lymph node CD4+ and CD8+ T cell-enriched cell suspensions were cultured with MCA205-4 tumor cells and the number of tumor-specific T cells producing IFN-{gamma} was tested by ELISPOT assay. The mean number ± SEM of IFN-{gamma}-producing CD4+ ({square}) and CD8+ T cells ({blacksquare}) per 5 x 105 responder cells in triplicate cultures minus the number of spots from control cultures with syngeneic spleen cell stimulators for two individual mice are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Clinical studies in humans and studies in murine models have suggested an important role for Mig and/or IP-10 in the suppression of malignant tumor growth (11, 12, 13, 14). The induction of Mig and IP-10 expression has been observed in renal cell carcinoma and melanoma patients that responded to IL-12 and IFN-{alpha}/IFN-beta immunotherapy (25, 26). In a murine model, treatment with recombinant IL-12 induced Mig and IP-10 expression in tumors and antitumor effects of the IL-12 treatment were dependent on these chemokines (15). Gene therapy to induce Mig or IP-10 expression either directly in tumors or in tumor sites has provided protective antitumor effects in mouse models (16, 17, 27, 28). In all of these studies, antitumor effects were associated with enhanced CD4+ and CD8+ T cell activity and with angiostatic effects presumably mediated by Mig or IP-10.

Although Mig and IP-10 can be induced in tumors during the course of immunotherapy, it remains unclear whether and how tumor cells produce these chemokines under physiologic growth conditions in vivo. In the current studies, we evaluated the significance of MCA205 fibrosarcoma production of Mig and IP-10 during tumorigenesis by isolating clones from the in vivo growing tumor. The majority of the tumor clones expressed low to undetectable levels of Mig mRNA following stimulation with IFN-{gamma} whereas IP-10 mRNA was detectable in all tested clones. Consistent with these results, production of Mig protein by tumor-derived Mig-deficient tumor cells was low to undetectable, while IP-10 production was clearly detected after stimulation with IFN-{gamma}. In contrast, both the tumor-derived Mig-expressing clone and parental MCA205 cells produced Mig following IFN-{gamma} stimulation. These results suggest that tumor cells deficient in Mig, but not in IP-10, expression are not present in high frequency in parental MCA205 cultures but are present in high frequency in MCA205 fibrosarcomas growing in vivo. Mig protein was clearly abundant during in vivo growth of the Mig-producing clone selected for study in this report. In contrast, Mig production by the Mig-deficient tumor cells was low in wild-type recipients and completely absent in Mig–/– recipients indicating that both tumor cells and tumor-infiltrating host cells are sources of Mig during tumor growth.

The results from these initial studies suggested that the loss of the ability of the tumor cells to produce Mig may be an important factor promoting tumor growth in vivo. In a direct test, the tumorigenicity of Mig-deficient tumor cells was significantly increased as these cells induced rapid growth of solid tumors when transplanted into syngeneic recipient mice, whereas Mig-expressing tumor cells grew slowly. The increased tumor growth of Mig-deficient tumor cells in vivo was not mediated by an enhanced ability of these cells to proliferate as their proliferation in vitro and in vivo in RAG1–/– hosts was similar to that of Mig-expressing tumor cells. It is also unlikely that the Mig-deficient tumor cells have impaired IFN-{gamma} signaling as class I MHC expression and IP-10 production was up-regulated at similar levels when the Mig-deficient and Mig-producing clones were stimulated with IFN-{gamma}. The most likely mechanism underlying the increased tumorigenicity of Mig-deficient tumor variants is that T cell and/or NK cell antitumor activity is impaired in the absence of tumor-derived Mig. Several findings in these studies support this mechanism. First, the absence of NK or T cells in the recipient mice did not change the in vivo growth of Mig-deficient tumor cells, while NK and T cells were clearly involved in the suppression of Mig-producing tumor cell growth. Consistent with other studies indicating NK and NK T cell regulation of MCA-induced fibrosarcoma growth (29, 30), the tumors expressing inducible Mig grew rapidly in NK cell-depleted wild-type or in RAG1–/– mice. In contrast, deficiency in NK/NK T or T cells did not promote Mig-deficient tumor growth indicating that, unlike the inducible Mig-expressing tumor cells, Mig-deficient tumor cells are not suppressed by NK or T cell immunity. Second, the induction of tumor-specific CD4+ T cells, was delayed in the immunocompetent recipients of Mig-deficient tumor cells when compared with recipients of Mig-producing tumor cells. This delay in CD4+ T cell activation suggests that tumor-derived Mig may participate in the activation of tumor-specific CD4+ T cells. This augmented T cell activation correlated with robust recruitment of CXCR3-expressing CD4+ and CD8+ T cells into Mig-producing tumors. Thus, Mig appears to enhance T cell immunity through both tumor-specific CD4+ T cell activation and recruitment of activated CD4+ and CD8+ T cells into the tumor. Apparently, these effects of tumor-derived Mig on T cell activation and recruitment are not substituted by IP-10, as the recipients bearing Mig-deficient tumors were able to produce IP-10 yet demonstrated significantly delayed induction of tumor-specific T cells and poor T cell infiltration into the tumors. This is reminiscent of previous studies suggesting nonredundant functions of Mig and IP-10 (8). Collectively, these results suggest that the increased tumorigenicity of Mig-deficient tumor cells is mediated by the inability of these cells to produce Mig, which impairs the early activation of tumor-specific CD4+ T cells and the subsequent recruitment of activated T cells into the tumor. Studies investigating the antitumor activities of Mig and IP-10 have focused on their angiostatic and chemoattraction functions whereas the distinct role of these chemokines in the activation of antitumor T cell-mediated responses, as suggested in the current studies, remains poorly investigated.

Constitutive expression of Mig not only reversed the high tumorigenicity of the Mig-deficient tumor cells but also induced a T cell response that rejected tumor implantation. Interestingly, the contribution of NK or NK T cells to the growth suppression of the transduced tumor cells constitutively producing Mig was marginal whereas NK or NK T cells suppressed the growth of the isolated clone that required stimulation with IFN-{gamma} to produce Mig. These results suggest that NK T and/or NK cells are the initial source of the IFN-{gamma} that stimulates the tumor cells to produce Mig in vivo. Without a need for this initial IFN-{gamma} stimulation to produce Mig, the transduced tumor cells constitutively produce Mig that may activate and/or attract effector T cells that eliminate the tumor cells.

Constitutive Mig production by tumor cells appears to induce robust protective antitumor responses. In this model, 50–80% of mice transplanted with MCA205 tumor cells genetically modified to constitutively produce Mig rejected the intradermal tumors induced by these cells and no tumor recurrence was observed when these recipients were monitored for up to two months (A. Gorbachev, unpublished observations). Furthermore, rechallenge of these mice with the highly tumorigenic Mig-deficient MCA205 tumor cell variant did not result in tumor growth. This resistance to tumor rechallenge correlated with robust development of IFN-{gamma}-producing T cells, primarily CD8+ T cells, in the skin-draining lymph nodes of recipient mice. Together with the results showing increased numbers of tumor-specific CD4+ T cells in mice bearing inducible Mig-producing and Mig- transduced tumors, these results support a critical role for tumor-derived Mig in promoting strong T cell-mediated antitumor responses. These findings are consistent with recent studies that demonstrated the important role of Mig as a costimulatory cytokine in the induction of alloreactive CD4+ and CD8+ T cells in a mouse heart allograft rejection model (31).

Another potential mechanism of Mig-dependent suppression of tumor growth is inhibition of tumor angiogenesis. In preliminary studies, however, we did not observe a difference in the development of a capillary network in either Mig-expressing or Mig-deficient tumors when accumulation of i.v. injected tetramethylrhodamine isothiocyanate-labeled dextran or immunostaining of tumors for expression of endothelial cell growth factor vascular endothelial growth factor was assessed (A. Gorbachev, unpublished observations). It is possible that the ability of Mig-deficient tumor cells to produce another potent angiostatic chemokine, IP-10, compensates for the absence of Mig in restricting vascularization of the tumor. Further studies are required to test the potential contribution of tumor-derived Mig to inhibit angiogenesis during tumor development.

Overall, these studies have indicated for the first time that tumor cells deficient in the production of Mig, but not in the production of IP-10, are present during tumor growth and that these Mig-deficient tumor cells are more tumorigenic than tumor cells expressing both Mig and IP-10. The deficiency in Mig production apparently allows these tumor cells to evade pressure from components of antitumor immunity including NKT, NK, and T cells. These findings suggest that a deficiency in the ability to produce Mig might provide a growth advantage to the tumor cells by attenuating the activation and/or strength of tumor-specific T cell responses. This mechanism may be commonly used by growing tumors to evade immune surveillance and might underlie the limited effect of many immunotherapeutic approaches aimed at enhancing T cell-mediated antitumor immunity. Mechanisms ablating tumor expression of the Mig gene and the selection pressures promoting growth of these variants from parental tumors clearly warrant further investigation.


   Acknowledgments
 
We thank the staff of the Cleveland Clinic Biological Resources Unit for excellent animal care.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 This work was supported by National Institutes of Health Grant RO1AI45888. Back

2 Address correspondence and reprint requests to Dr. Anton V. Gorbachev, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195-0001. E-mail address: gorbaca{at}ccf.org Back

3 A.V.G. and H.K. contributed equally to this work and share principal coauthorship. Back

4 Abbreviations used in this paper: Mig, monokine induced by IFN-{gamma}; IP-10, IFN-{gamma}-inducible protein 10; MCA, methylcholanthrene; RPA, RNase protection assay; RT, room temperature. Back

Received for publication May 22, 2006. Accepted for publication December 1, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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