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The Journal of Immunology, 2007, 179: 365-371.
Copyright © 2007 by The American Association of Immunologists, Inc.
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IL-10 Controls Ultraviolet-Induced Carcinogenesis in Mice1

Karin Loser2,*, Jenny Apelt2,*, Maik Voskort*, Mariette Mohaupt{dagger}, Sandra Balkow*, Thomas Schwarz*,{ddagger}, Stephan Grabbe*,§ and Stefan Beissert3,*

* Department of Dermatology and Interdisciplinary Center of Clinical Research, Interdisziplinäres Zentrum für Klinische Forschung, University of Münster, Münster, Germany; {dagger} Max-Delbrück-Center for Molecular Medicine, Berlin, Germany; {ddagger} Department of Dermatology, University of Kiel, Kiel, Germany; and § Department of Dermatology, University of Mainz, Mainz, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
UV radiation-induced immunosuppression contributes significantly to the development of UV-induced skin cancer by inhibiting protective immune responses. IL-10 has been shown to be a key mediator of UV-induced immunosuppression. To investigate the role of IL-10 during photocarcinogenesis, groups of IL-10+/+, IL-10+/–, and IL-10–/– mice were chronically irradiated with UV. IL-10+/+ and IL-10+/– mice developed skin cancer to similar extents, whereas IL-10–/– mice were protected against the induction of skin malignancies by UV. Because UV is able to induce regulatory T cells, which play a role in the suppression of protective immunity, UV-induced regulatory T cell function was analyzed. Splenic regulatory T cells from UV-irradiated IL-10–/– mice were unable to confer immunosuppression upon transfer into naive recipients. UV-induced CD4+CD25+ T cells from IL-10–/– mice showed impaired suppressor function when cocultured with conventional CD4+CD25– T cells. CD4+CD25– T cells from IL-10–/– mice produced increased amounts of IFN-{gamma} and enhanced numbers of CD4+TIM-3+ T cells were detectable within UV-induced tumors in IL-10–/– mice, suggesting strong Th1-drived immunity. Mice treated with CD8+ T cells from UV-irradiated IL-10–/– mice rejected a UV tumor challenge significantly faster, and augmented numbers of granzyme A+ cells were detected within injected UV tumors in IL-10–/– animals, suggesting marked antitumoral CTL responses. Together, these findings indicate that IL-10 is critically involved in antitumoral immunity during photocarcinogenesis. Moreover, these results point out the crucial role of Th1 responses and UV-induced regulatory T cell function in the protection against UV-induced tumor development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The UV spectrum of sunlight, in particular the mid-wave range (290–320 nm, UVB) represents the most important risk factor for the development of nonmelanoma skin malignancies, including basal cell and squamous cell carcinomas (1, 2, 3). Both of these UV-induced skin cancers show a rapidly increasing incidence worldwide (4, 5, 6). During the development of skin tumors the immune system appears to play an important role. Evidence to support this hypothesis is provided from organ transplant patients who are therapeutically on long-term immunosuppressive medication (7, 8, 9). These patients present with up to 30-fold increased numbers of skin malignancies, primarily on sun-exposed areas such as the head, upper lip, and back of the hands. In murine experimental models of photocarcinogenesis, tumor development seems to be regulated by T cells (10). There is increasing evidence that UV-induced regulatory T cells play an important role during photocarcinogenesis, because they are able to inhibit antitumoral effector functions (11, 12). Although UV-induced regulatory T cells were detected as far back as two decades ago—in those days they were called UV-induced suppressor T cells—the exact phenotype and mode of action remained unclear for a long time. In the past few years several subtypes of UV-induced regulatory T cells have been described in a variety of experimental models; most of them are CD4+ and coexpress CD25 plus CTLA-4 (13, 14). Because CD4+ UV-induced regulatory T cells produce IL-10 upon stimulation, it was suggested that IL-10 mediates suppressor function (13, 15). Recent investigations show that CD4+ UV-induced regulatory T cells are involved in the inhibition of effector cells during the development of UV-induced immunotolerance or skin tumors (16, 17).

In addition to inducing regulatory T cells, UV radiation is able to inhibit the Ag-presenting function of dendritic cells such as epidermal Langerhans cells (18). Langerhans cells can present tumor Ags, thereby mediating both the induction and elicitation of protective immunity (19). UV radiation is able to inhibit the Ag-presenting function directly via UV-induced cytotoxicity as well as indirectly via the release of immunosuppressive cytokines such as IL-10 (20). From several reports it was concluded that UV-induced expression of IL-10 contributes to the development of photocarcinogenesis by suppressing protective cellular immune responses. This view was supported by findings showing that invasively growing basal cell carcinomas secreted IL-10 (21). The expression of IL-10 has also been reported in human melanoma cells, and IL-10 production of melanomas correlates with a poorer prognosis (22, 23, 24). However, the injection of IL-10-overexpressing tumor cells into mice did not result in enhanced tumor growth kinetics but rather in tumor rejection (25). Furthermore, transgenic mice that overexpressed viral IL-10 under the control of a skin-specific keratin-14 promoter, resulting in increased IL-10 serum concentrations, developed significantly reduced skin tumors upon chronic UV irradiation (26). These unexpected findings were explained by the fact that the activation of NK cell function may contribute to impaired photocarcinogenesis in viral IL-10 transgenic mice. According to these discrepancies, the exact role of UV-mediated IL-10 expression for the development of UV-induced skin tumors still remains to be determined.

To analyze the role of IL-10 during autochthonous tumor development after chronic UV irradiation, we used IL-10–/– mice and show that IL-10 deficiency results in protection from photocarcinogenesis.


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

IL-10–/– (knockout) and IL-10+/– (heterozygous) mice on a C57BL/6 background were generated as previously described (27). IL-10+/+ (wild type) C57BL/6 controls as well as nu/nu mice were purchased from Harlan-Winkelman. Mice were housed under specific pathogen-free-conditions and used according to institutional guidelines.

UV irradiation, tumor induction, and histology

For UV irradiation of mice, a bank of four Philips UV-B TL40W/12 sunlamps was used that has an emission spectrum ranging from 280 to 350 nm with a peak at 306 nm. The mice were placed on a shelf 20 cm below the light bulbs and the cage order was systematically rotated before each treatment to compensate for uneven lamp output (16, 28, 29). The location and growth of each tumor exceeding 2 mm in diameter was recorded. Sections from all tumor biopsies were stained with H&E and documented using an Olympus BX61 microscope with Visitron software (Visitron Systems).

Transfer of UV-induced skin tumors

Skin tumors that had reached a size of 5–7 mm in diameter were biopsied and tumor specimens were put in tissue culture flasks (BD Falcon) at 37°C and 5% CO2 containing RPMI 1640 supplemented with 10% heat inactivated FCS (PAA Laboratories), 100 U/ml penicillin (PAA Laboratories), 100 mg/ml streptomycin (PAA Laboratories), 0.1 mM essential and nonessential amino acids (PAA Laboratories), 2 mM L-glutamine (PAA Laboratories), 1 mM sodium pyruvate (PAA Laboratories), and 0.01 M HEPES buffer (PAA Laboratories). Tumor cells were allowed to grow to subconfluence. Subsequently, naive recipients (nu/nu or C57BL/6) were s.c. injected with 1 x 104 or 5 x 106 viable tumor cells, respectively, and tumor growth was documented over time.

Contact hypersensitivity (CHS)4

Mice were sensitized by painting 100 µl of 2,4,-dinitrofluorobenzene (DNFB; Sigma-Aldrich) solution (0.5% in acetone/olive oil, 4:1) on the shaved back as described (30). Each group consisted of at least five mice. Each experiment was performed at least three times.

UV irradiation

For induction of tolerance, mice were exposed to UV daily on four consecutive days (1 kJ/m2 per exposure) (13, 17). Twenty-four hours after the last UV exposure, DNFB was applied to the irradiated skin as described above or mice were sacrificed for cell preparation. For the induction of Th1 or CTL activity, mice were UV irradiated six times (5 kJ/m2 per exposure) at intervals of 48 h. Forty-eight hours after the last UV exposure, tumor cells were inoculated s.c. or mice were sacrificed for cell preparation.

Adoptive cell transfers

Donor mice were either UV irradiated or left untreated. Subsequently, regional lymph nodes were removed, single cell suspensions were prepared, and CD4+CD25–, CD4+CD25+, CD4+, or CD8+ T cells were purified by MACS (Miltenyi Biotec) and injected i.v. into recipient mice. After 24 h the recipients were challenged by painting 12 µl of 0.3% DNFB on both sides of the left ear and ear swelling was evaluated or they were inoculated with UV tumor cells and tumor growth was documented.

Cell preparations and flow cytometry

Single-cell suspensions of regional lymph nodes were prepared as described (31). The expression of cell surface and intracellular markers was analyzed by multicolor flow cytometry on a FACSCanto cytometer (BD Biosciences) and cells were stained in PBS containing 1% FCS using the following Abs from eBioscience/NatuTec: FITC-conjugated anti-Foxp3 (clone FJK-16s); PE-conjugated anti-TIM-3 (clone RMT3-23), anti-perforin (clone dG9), anti-CTLA-4 (clone UC10-4F10-11), and anti-IFN-{gamma} (clone XMG1.2); and allophycocyanin-conjugated anti-CD25 (clone PC61) and mouse polyclonal anti-T-bet. PerCP-conjugated anti-CD4 (clone RM4-5) and anti-CD8 (clone53-6.7) were obtained from BD Biosciences. Intracellular staining of CTLA-4, perforin, and IFN-{gamma} or intranuclear staining of Foxp3 and T-bet was performed using the Cytofix/Cytoperm kit (BD Biosciences) or the Foxp3 staining set (eBioscience/NatuTec).

Proliferation assays

CD4+CD25– and CD4+CD25+ cells from UV-irradiated or naive IL-10+/+ or IL-10–/– mice were sorted by MACS (Miltenyi Biotec). CD4+CD25+ and CD4+CD25– T cells (1 x 106/ml; alone or mixed at indicated ratios) were cultured in triplicate in 96-well-round-bottom plates and stimulated with 1 µg/ml anti-CD3 (clone 2c11) and 1 µg/ml anti-CD28 (clone 37.51; both eBioscience/NatuTec). Proliferation assays were cultured in a final volume of 200 µl, 1 µCi/well [3H]thymidine was added for the last 12 h of the experiment, and thymidine incorporation was measured by liquid scintillation counting.

Cytokine quantification

The cytokine activity in culture supernatants of CD4+CD25+ or CD4+CD25– T cells from IL-10+/+ and IL-10–/– mice was assayed using the mouse Th1/Th2 10plex kit (Bender MedSystems) according to the manufacturer’s instructions. T cells (2 x 106/ml) were incubated for 4 days with a combination of anti-CD3 and anti-CD28 Abs (1 µg/ml each Ab) at 37°C and 5% CO2 in 96-well round-bottom plates in a volume of 200 µl of RPMI 1640 containing 10% FCS. Supernatants were collected and subjected to cytokine quantification using mouse Th1/Th2 10plex kits.

Immunofluorescence stainings

Immunofluorescence stainings were performed on cryostat sections of tumors (3–4 µm) fixed in acetone according to standard methods. Slides were incubated in the appropriate dilutions of Abs (anti-CD4, anti-Foxp3, and anti-TIM-3; all purchased from eBioscience/NatuTec) or an isotype control (eBioscience/NatuTec). The anti-granzyme A (GrzA) Ab was kindly provided by Dr. M. M. Simon, Max-Planck-Institute for Immunobiology, Freiburg, Germany. Subsequently slides were incubated with Oregon Green- or Texas Red-labeled secondary Abs (Molecular Probes) and examined using a Leica confocal microscope.

Statistical analysis

The method of Kaplan and Meier was used to describe the probability of tumor development in the carcinogenesis study. Statistical differences for the development of tumors between the two strains of mice were determined using a log-rank test by Peto et al. (32). The differences in tumor latency periods were analyzed by using a Mann-Whitney U test. Tumor volumes were calculated as the product of the maximal tumor diameter in three perpendicular directions measured with a Vernier caliper (Mitutoyo). This method has previously been confirmed to correlate well with the tumor weight (19). Mice were sacrificed after the tumor volume exceeded 1,000 mm3. To evaluate statistical differences between the mean tumor volume in the various experimental groups, the "best-fit" slope of the tumor growth in each animal was determined (Cricket software, version 1.3.2; GraphPad Prism, version 5.0) on a MacIntosh G4 computer, and the significance of differences between the means of the slopes for the groups of interest were tested by the two-tailed Student t test for unpaired data. Data from the CHS experiments and proliferation assays were analyzed by Student’s t test and differences were considered significant at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Reduced development of UV-induced skin cancer in IL-10–/– mice

To determine the role of IL-10 for the generation of UV-induced cutaneous malignancies, groups of IL-10–/–, IL-10+/–, and IL-10+/+ mice (all H-2b) were chronically irradiated with UVB on their shaved backs and tumor development was documented over time. Upon irradiation, the vast majority of IL-10+/+ and IL-10+/– mice developed UV-induced skin tumors (Fig. 1). Tumor development occurred to a similar extent, latency period, and rate in both IL-10+/+ and IL-10+/– mice. Also, autochthonous tumor growth in vivo was similar both in IL-10+/+ and IL-10+/– mice (data not shown). In contrast, none of the IL-10–/– mice developed a skin tumor, not even after an observation period of 1 year. These findings strongly indicate that the expression of IL-10 is required for UV-induced skin tumor development in mice.


Figure 1
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  		FIGURE 1. Impaired development of UV-induced skin tumors in IL-10–/– mice. The rates of UV-induced skin cancer in wild-type (IL-10+/+; n = 20), heterozygous (IL-10+/–; n = 10), and knockout (IL-10–/–; n = 16) mice are shown. Mice were irradiated with 2.5 kJ/m2 UV for 4 wk, with 5 kJ/m2 UV for another 4 wk, and subsequently with 10 kJ/m2 UV for 6 mo and shaved weekly. This experiment was performed once. *, p < 0.00001 for IL-10+/+ vs IL-10–/–.

 
Analysis of skin tumors produced by UV irradiation

Tumor specimens were obtained and subjected to histopathological analysis. Most of the UV-induced skin tumors that developed in IL-10+/+ and IL-10+/– mice were located on the ears and backs (Table I). The majority of the primary skin tumors induced by UV irradiation in mice of both groups were poorly differentiated squamous cell carcinomas that grew rapidly in vivo (data not shown).


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  		Table I. Number, site, and histological type of skin tumor produced by chronic UV irradiation

 
In contrast, histopathological analysis of chronically UV-irradiated skin demonstrated that not even skin cancer in situ had developed in IL-10–/– mice. There were no signs of intraepidermal dysplastic keratinocytes or pathologic mitosis. Taken together, IL-10 deficiency resulted in no detectable UV-induced skin tumor development.

Previous studies have shown that the majority of murine skin cancers produced by UV irradiation are highly immunogenic and are therefore rejected upon injection into immunocompetent naive recipient mice (28, 33). Accordingly, the vast majority of cell lines derived from the skin tumors of both IL-10+/+ and IL-10+/– groups from the photocarcinogenesis experiment were rejected upon injection into naive immunocompetent recipients as indicated in Fig. 2A. To compare the growth kinetics of UV tumors from IL-10+/+ and IL-10+/– mice upon transplantation, tumor specimens were s.c. injected into immunodeficient nu/nu mice. The data depicted in Fig. 2B show that the tumor cell lines from the IL-10+/+ and IL-10+/– mice grew with similar kinetics in recipient animals, indicating a similar malignant phenotype of the UV-induced skin tumors from IL-10+/+ and IL-10+/– mice.


Figure 2
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  		FIGURE 2. Growth kinetics of UV-induced skin tumors from IL-10+/+ and IL-10+/– animals in nu/nu and C57BL/6 mice. A, Groups of naive C57BL/6 mice (n = 5) were injected s.c. with 5 x 106 tumor cells isolated from UV-tumors of IL-10+/+ or IL-10+/– mice. Afterward, tumor growth was documented over time by two independent investigators. B, UV-induced skin tumors were biopsied from IL-10+/+ and IL-10+/– mice and subsequently inoculated into nu/nu mice.

 
Impaired UV-induced immunotolerance in IL-10–/– mice

Because previous work has shown that the inhibitory effects of UV-induced regulatory T cells play a role in immunotolerance (13, 17), we were interested to analyze whether IL-10 is required for the in vivo function of UV-induced regulatory T cells (34, 35). Therefore, IL-10+/+ and IL-10–/– mice were DNFB-sensitized via UV-irradiated skin. Subsequently, splenic T cells were prepared from the different groups of animals and transferred into naive recipients. After cell transfer, all recipients were DNFB sensitized and ear challenged to DNFB to elicit CHS responses (Fig. 3). The transfer of splenic T cells from UV-tolerized IL-10+/+ donor mice suppressed CHS responses in recipients, indicating that UV-induced regulatory T cells had been induced. Interestingly, the transfer of splenic T cells from UV-tolerized IL-10–/– mice failed to transfer suppression, because recipient animals were still able to mount significant ear swelling responses (Fig. 3). Together, these results suggest that IL-10 is a critical mediator of UV-induced tolerance. The question arising is whether UV-induced regulatory T cells are not induced in IL-10–/– mice or whether IL-10 is important for their suppressor function.


Figure 3
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  		FIGURE 3. Transfer of T cells from UV-tolerized IL-10–/– mice failed to induce tolerance in naive recipients. Naive recipient mice were injected i.v. with 1 x 106 regulatory T cells obtained from donors that were tolerized against DNFB by an application of DNFB onto UV-exposed skin (4 x 1000 J/m2). Twenty-four hours after injection, mice (n = 8 mice per group) were sensitized against DNFB and 5 days later a challenge with DNFB was performed on the left ear. Positive (pos.) control mice were sensitized and challenged without transfer, negative (neg.) control mice were ear challenged only. Ear swelling was measured 24 h after challenge. Ear swelling response is expressed as the difference between the thickness (cm x 10–2) of the challenged left ear compared with the thickness of the vehicle-treated right ear. *, p < 0.0001 for positive vs negative control; positive vs recipients injected with UV-induced (UV-ind.) regulatory T cells from UV-tolerized IL-10+/+ mice.

 
Reduced suppressor function of UV-induced CD4+CD25+ T cells from IL-10–/– mice

There is accumulating evidence that UV-induced regulatory T cells belong to the CD4+CD25+ T cell subset (14, 16, 17). In mice, lineage development of naturally occurring CD4+CD25+ T cells is controlled by the transcription factor Foxp3 (36). Because our data indicate that the transfer of splenic T cells from UV-tolerized IL-10–/– mice failed to induce tolerance in IL-10+/+ recipient animals (Fig. 3), we were interested in determining the frequency and function of UV-induced CD4+CD25+Foxp3+ T cells. Naive IL-10–/– mice have slightly enhanced numbers of peripheral CD4+CD25+Foxp3+ T cells compared with IL-10+/+ controls (Fig. 4A). These numbers were increased upon UV irradiation, indicating the induction of UV-induced regulatory T cells. To test the function, UV-induced CD4+CD25+ T cells from IL-10+/+ as well as IL-10–/– mice were isolated and cocultured with CD4+CD25– effector T cells from IL-10+/+ mice. UV-induced CD4+CD25+ T cells from IL-10+/+ mice showed a significant suppressor function as evidenced by the decreased proliferative response of CD4+CD25– T cells (Fig. 4B). In contrast, UV-induced CD4+CD25+ T cells from IL-10–/– mice failed to inhibit CD4+CD25– T cell proliferation, suggesting a strongly impaired inhibitory effect of IL-10-deficient UV-induced CD4+CD25+ T cells. Hence, UV-induced CD4+CD25+Foxp3+ T cells are present in IL-10–/– mice; however, their suppressor function appears to be impaired. Because UV-induced CD4+CD25+ T cells have been identified as suppressing antitumoral immune responses, especially against incipient UV-induced skin cancer, these results suggest that IL-10-deficiency contributes to enhanced tumor immunity via the impaired inhibitory function of UV-induced CD4+CD25+ T cells.


Figure 4
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  		FIGURE 4. Reduced suppressor function of UV-induced CD4+CD25+ T cells from IL-10–/– mice. Groups of naive IL-10+/+ and IL-10–/– mice were irradiated with UV and CD4+CD25+ T cells were subsequently prepared from lymph nodes and spleen. A, Peripheral CD4+CD25+Foxp3+ T cells were quantitated before and after UV irradiation using multicolor flow cytometry (cells were gated for CD4). B, Equal numbers (2 x 105) of UV-induced CD4+CD25+ T cells from UV-irradiated IL-10+/+ or IL-10–/– mice were added to CD4+CD25– T cells from naive IL-10+/+ mice and stimulated with anti-CD3/anti-CD28. T cell proliferation was quantitated by [3H]thymidine incorporation. Mean values of [3H] uptake ± SD are shown from one of three independent experiments. *, p < 0.01 for CD4+CD25– vs CD4+CD25– plus UV-induced (UV-ind.) CD4+CD25+ T cells from IL-10+/+ mice.

 
Analysis of proliferation and cytokine production of CD4+CD25– or CD4+CD25+ T cells from IL-10–/– mice

Because T cell-derived mediators are critical for the stimulation of anti-tumoral immune responses, lymphocytes from IL-10+/+ and IL-10–/– mice have been phenotypically characterized. IL-10+/+ and IL-10–/– mice have similar numbers of peripheral CD4+ or CD8+ T cells (data not shown). Upon TCR stimulation with mitogenic Abs (anti-CD3/anti-CD28), splenic and lymph node CD4+CD25– T cells from IL-10–/– mice showed a reduced proliferative response compared with IL-10+/+ CD4+CD25– T cells (Fig. 5A). Subsequently, supernatants from stimulated CD4+CD25– and CD4+CD25+ lymphocytes were assayed for the presence of cytokines. The data shown in Fig. 5B indicate that stimulated CD4+CD25– T cells from IL-10–/– mice produced significantly more IFN-{gamma} and less IL-4 compared with stimulated CD4+CD25– T cells from IL-10+/+mice. Together, these findings support the concept that IL-10 plays an essential role in down-regulating Th1-driven immune responses. We speculate that this enhanced proliferative response plus cytokine expression contributes to the reduced photocarcinogenesis observed in IL-10–/– mice.


Figure 5
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  		FIGURE 5. Increased proliferation and IFN-{gamma} production by CD4+ CD25+ T cells from IL-10–/– mice. A, CD4+CD25– and CD4+CD25+ T cells from lymph nodes of IL-10+/+ and IL-10–/– mice were separated by MACS and proliferation assays were performed by stimulating 2 x 105 cells with anti-CD3 plus anti-CD28. T cell proliferation was quantitated by [3H]thymidine incorporation and mean values of [3H] uptake ± SD are shown from one of three independent experiments. *, p < 0.01 for CD4+CD25+ T cells from IL-10+/+vs IL-10–/– mice. B, For cytokine production, CD4+CD25– and CD4+CD25+ T cells (2 x 105) were stimulated with anti-CD3 and anti-CD28, and IL-4 as well as IFN-{gamma} levels were analyzed using the mouse Th1/Th2 10plex kit. Data show one of three different experiments.

 
Enhanced antitumoral immunity of CD4+ and CD8+ T cells from IL-10–/– mice

Because UV-induced CD4+CD25+ regulatory T cells can inhibit protective antitumoral immunity, we were interested to scrutinize whether UV-induced CD4+CD25+ T cells from IL-10–/– mice would regulate tumor rejection. To address this question, immunocompetent recipient mice were treated three times with CD4+CD25+ or CD4+CD25– T cells from IL-10+/+ or IL-10–/– mice and s.c. challenged with viable UV-induced regressor tumor cells. Mice that were injected with CD4+CD25– T cells from IL-10+/+ or IL-10–/– mice rejected the tumor challenge, similar to animals that received CD4+CD25+ T cells from IL-10–/– mice (Fig. 6A). However, treatment with CD4+CD25+ T cells from IL-10+/+ mice significantly inhibited tumor rejection, suggesting that IL-10 plays an important role in the suppressor function of CD4+CD25+ T cells during the down-regulation of antitumoral immune responses. This hypothesis is supported by the detection of reduced numbers of CD4+Foxp3+ T cells in tumor tissue of mice that had been treated with CD4+CD25+ T cells from IL-10–/– mice (Fig. 6B).


Figure 6
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  		FIGURE 6. CD4+CD25+ regulatory T cells from IL-10–/– mice are not immunosuppressive in vivo. A, Groups of wild type (wt) mice were injected i.v. with 2.5 x 106 CD4+CD25– or CD4+CD25+ T cells from IL-10+/+ or IL-10–/– mice at the time points indicated by the arrows. Twenty-four hours after the first injection all mice were inoculated s.c. with 5 x 106 IL-10+/– UV tumor cells and tumor growth was documented over time. Data are shown as mean tumor volume ± SD and are representative of six mice per group. *, p < 0.05 vs without injection of T cells. B, CD4+Foxp3+ regulatory T cells are absent in the tumors of mice injected with CD4+CD25+ T cells from IL-10–/– mice. Tumor biopsies of recipients that received CD4+CD25+ T cells from IL-10+/+ or IL-10–/– mice were stained with Abs to CD4 and Foxp3 and merged images are shown in yellow (original magnification, x400; scale bar, 20 µm).

 
To determine CTL responses and the role of IL-10-deficiency during UV irradiation, groups of mice were treated with two injections of CD8+ T cells from UV-irradiated IL-10–/– or IL-10+/+ mice. Subsequently, CD8+ T cells were phenotypically characterized in tumor-draining lymph nodes. Interestingly, mice that were treated with CD8+ T cells from UV-irradiated IL-10–/– mice rejected the tumor challenge significantly faster compared with controls, suggesting strong antitumoral CD8-mediated effector functions (Fig. 7A). Moreover, in the draining lymph nodes of IL-10–/– mice that had been challenged with UV-tumor cells and were UV irradiated, increased expression of T cell activation and cytotoxic markers was detected in CD8+ T cells (Fig. 7B). In addition, enhanced GrzA+ cells could be identified within the tumor tissue of IL-10–/– mice that were not further increased upon UV treatment, pointing to increased CTL activity against the injected UV tumor cells in IL-10–/– mice compared with the IL-10+/+ controls (Fig. 7C).


Figure 7
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  		FIGURE 7. UV-irradiated IL-10–/– mice contain CD8+ CTL reactive against syngeneic tumors and show enhanced Th1 activity. A, Groups of wild type (wt) mice were injected i.v. with 7 x 106 CD4+ or CD8+ T cells from UV-irradiated (6 x 5 kJ/m2 at intervals of 48 h) IL-10+/+ or IL-10–/– mice at the time points indicated by the arrows. Twenty-four hours after the first injection all mice were inoculated s.c. with 5 x 106 IL-10+/– UV-tumor cells and tumor growth was documented over time. Data are shown as mean tumor volume ± SD and are representative of five mice per group. *, p < 0.05 vs without the injection of T cells. B–D, Groups of IL-10+/+ and IL-10–/– mice were UV-irradiated (6 x 5 kJ/m2 at intervals of 48 h) and inoculated s.c. with 5 x 106 IL-10+/– UV-tumor cells. Seven days after the injection of tumor cells, tumor-draining lymph nodes and tumors were removed and analyzed for the presence of CTL-specific as well as Th1-specific markers by flow cytometry and immunofluorescence staining. B, Enhanced expression of cytotoxic markers in tumor draining lymph nodes of irradiated IL-10–/– mice. Cells were gated for CD8 and one representative of three independent experiments is shown. C, GrzA-expressing cytotoxic cells infiltrate the tumors of IL-10–/– mice. Tumor biopsies of UV-irradiated and nonirradiated IL-10+/+ and IL-10–/– mice were stained with anti-GrzA (original magnification, x400; scale bar, 20 µm). D, Increased infiltration of Th1 cells in tumors of IL-10–/– mice. Tumor biopsies of UV-irradiated and nonirradiated IL-10+/+ and IL-10–/– mice were stained with Abs to CD4 and TIM-3; merged images are shown in yellow (original magnification, x400; scale bar, 20 µm).

 
To investigate Th1-driven antitumoral immunity, IL-10+/+ and IL-10–/– mice were challenged with UV-tumors and the tumor tissue was subsequently analyzed for the presence of Th1 cells by staining for CD4 and TIM-3. TIM-3 is a specific molecular marker for Th1 cells. As shown in Fig. 7D, the tumor tissue of IL-10–/– mice contained more CD4+TIM-3+ T cells compared with controls, and this number was not increased by additional UV treatment. Together, these findings are suggestive of a marked Th1-mediated antitumoral immune response during IL-10 deficiency.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Investigating the development of UV-induced skin tumors is an ideal experimental model for studying carcinogenesis under control of the immune system. It is well established that UV-induced murine skin tumors are highly immunogenic and are therefore rejected upon transfer into immunocompetent recipients. Such tumors only grow in therapeutically immunosuppressed or UV-treated mice (28, 33, 37). It was suggested that tumor development and growth are controlled by UV-induced regulatory T cells and/or by skewing immunity toward Th2-type responses (12, 38, 39). This concept was supported by findings showing increased IL-10 as well as IL-4 production after UV irradiation.

The presented results indicate that IL-10–/– mice are resistant to photocarcinogenesis. Because other effects of UV on the skin such as epidermal hyperplasia, erythema, sunburn cell formation, and changes in Langerhans cell or dendritic epidermal T cell numbers were unaffected by IL-10 deficiency (data not shown), we surmise that the documented effects on UV-induced tumor development are due to the immunomodulation of IL-10. To this end, IL-10 has been shown to be a key mediator of UV-induced systemic immunosuppression (40, 41, 42). Irradiation of mice with UVB resulted in enhanced IL-10 serum concentrations and inhibition of the sensitization phase toward s.c. injected Ags even when the Ags were injected at a distant nonirradiated site (40, 43). This UV-induced systemic immunosuppression can be transferred to naive recipients by injecting either serum from UV-treated mice or supernatants from UV-exposed keratinocyte cultures. In line with these results, the treatment of UV-irradiated mice with a neutralizing anti-IL-10 Ab or the addition of anti-IL-10 Abs to the serum or cell supernatants abrogated the inhibitory effects of UV irradiation, suggesting that suppression was indeed mediated by IL-10 (43). Our present findings are in agreement with the importance of IL-10 for UV-induced immunosuppression.

UV-induced regulatory T cells appear to be critically involved in the control of UV-induced skin tumors (12). Although these experiments have been performed >20 years ago, the exact phenotype and the functional characterization have remained unclear for a long time. Recent investigations were aimed to determine the role of UV-induced CD4+CD25+ regulatory T cells during photocarcinogenesis. It was demonstrated that peripheral numbers of CD4+CD25+ T cells were increased after UV irradiation of mice. Interestingly, mice with strongly reduced peripheral numbers of CD4+CD25+ T cells such as CD80–/–CD86–/– double-deficient animals showed significantly reduced UV-induced tumor development (16). In addition, animals with an impaired suppressor function of UV-induced CD4+CD25+ T cells induced by treatment with anti-CTLA-4 Abs also demonstrated strongly reduced photocarcinogenesis. Our data indicate that IL-10 is dispensable for the development of UV-induced CD4+CD25+Foxp3+ T cells. Importantly, our results suggest that IL-10 is an essential factor for the suppressor function of UV-induced regulatory T cells because UV-induced CD4+CD25+ T cells from IL-10–/– mice show a significantly reduced ability to inhibit the proliferation of conventional CD4+CD25– T cells. This impaired suppressor activity detected in IL-10–/– mice allows for the enhanced stimulation of protective antitumoral immunity against incipient skin malignancies. Together, these results extend our understanding on the molecular mechanisms of how UV-induced regulatory T cells regulate cutaneous tumor growth and antitumoral immunity.

Other reports have argued that, in addition to CD4+CD25+ T cells, CD3+CD4+DX5+ NKT cells play a role in inhibiting antitumoral immunity against UV tumors. Upon UV irradiation of mice these CD3+CD4+DX5+ NKT cells were able to secrete significant concentrations of IL-4 (44). Treatment of mice with CD3+CD4+DX5+ NKT cells from UV-irradiated syngeneic donors resulted in the growth of a previously inoculated UV-induced regressor tumor that was otherwise rapidly rejected in control mice. Whether CD3+CD4+DX5+ NKT cells regulate immunity during ongoing tumor development after chronic UV exposure remains to be scrutinized. An analysis of NKT cells revealed increased numbers of splenic CD3+NK1.1+IFN-{gamma}+ NKT cells in IL-10–/– compared with IL-10+/+ mice (data not shown). Whether this enhanced number of activated NKT cells in IL-10–/– mice contributes to antitumoral immunity is currently under investigation.

The functional analysis of CD4+CD25– T cells from IL-10–/– mice demonstrated increased secretion of IFN-{gamma}. This is most likely due to the impaired Th1/Th2 balance in IL-10–/– mice in favor of Th1-mediated immunity, a hypothesis that is supported by the detection of increased numbers of CD4+Tim-3+ T cells within the UV tumor tissue of IL-10–/– mice. In line with this concept, previous photocarcinogenesis experiments have indicated that mice with impaired UV-induced Th2 responses were protected against UV-induced skin tumor generation (16, 45). Furthermore, IFN-{gamma} has been shown in other experimental tumor models to induce protective antitumoral immunity (46, 47). Our findings additionally indicate a strong CD8-mediated antitumoral CTL response induced by IL-10 deficiency (Fig. 7, A–C). Taken together, the reduced tumor development and the increased Th1 and CTL immunity in IL-10–/– mice indicate the importance of IL-10 for the inhibition of antitumoral immune responses during photocarcinogenesis and allow for the development of new strategies for the immunotherapy of skin cancer such as topical application of IL-10 inhibitors.


   Acknowledgments
 
We thank Thomas Blankenstein, Ph.D., Max-Delbrück-Center, Berlin, Germany, for critically reading the manuscript and offering helpful comments and Markus M. Simon, Ph.D., Max-Planck-Institute for Immunobiology, Freiburg, Germany, for providing the GrzA Ab. We also thank Meike Steinert and Birgit Geng for excellent technical assistance.


   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 German Research Association (Deutsche Forschungsgemeinschaft) Grants BE 1580/7-1 (to S.B.) and SCHW 1177/1-1 (to T.S.), Interdisciplinary Center of Clinical Research (Interdisziplinäres Zentrum für Klinische Forschung) Grant Lo2/017/07 (to K.L. and S.B.), and the fund "Innovative Medical Research" of the University of Münster Medical School Grant Lo11/06 03 (to K.L. and S.B.). Back

2 K.L. and J.A. share equal authorship. Back

3 Address correspondence and reprint requests to Dr. Stefan Beissert, Department of Dermatology, University of Münster, Von-Esmarch-Strasse 58, Münster, Germany. E-mail address: beisser{at}uni-muenster.de Back

4 Abbreviations used in this paper: CHS, contact hypersensitivity; DNFB, 2,4-dinitrofluorobenzene; GrzA, granzyme A. Back

Received for publication June 5, 2006. Accepted for publication April 13, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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