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. Author manuscript; available in PMC: 2021 Aug 1.
Published in final edited form as: Cancer Prev Res (Phila). 2020 Nov 4;14(2):165–174. doi: 10.1158/1940-6207.CAPR-20-0360

Regulatory T cells play an important role in the prevention of murine melanocytic nevi and melanomas

Tahseen H Nasti 1, Nabiha Yusuf 1,2, Mohammad Asif Sherwani 1, Mohammad Athar 1, Laura Timares 1,3, Craig A Elmets 1,2,3
PMCID: PMC8093326  NIHMSID: NIHMS1643837  PMID: 33148679

Abstract

Melanocytic nevi are benign proliferations of pigment cells that can occasionally develop into melanomas. There is a significant correlation between increased nevus numbers and melanoma development. Our previous reports revealed that DMBA and TPA induced dysplastic nevi in C3H/HeN mice, with a potential to transform into melanomas. In order to understand the immune mechanisms behind this transformation, we applied increasing DMBA doses followed by TPA to the skin of C3H/HeN mice. We observed that increased doses of DMBA correlated well with increased numbers of nevi. The increased DMBA dose induced diminished immune responses and promoted the expansion of regulatory T cells that resulted in increased IL-10 and reduced IFNγ levels. Mice with increased nevus numbers had loss of p16 expression. These mice had increased migration of melanocytic cells to lymph nodes and a greater percent of lymph nodes produced immortalized melanocytic cell lines. DMBA-induced immunosuppression was lost in CD4KO mice. Lymphocytes in the CD4KO mice produced less IL-10 than CD8KO mice. Further, CD4KO mice had significantly reduced nevus numbers and size compared with wild type and CD8KO mice. These results suggest that regulatory T cells play a vital role in the incidence of nevi and their progression to melanoma.

Keywords: Regulatory T-cells, Melanoma, Melanocytic Nevi, Immunoprevention, DMBA

INTRODUCTION

Melanocytic nevi are abnormal clusters of pigment-producing cells that accumulate in the dermis and epidermis. Although they begin as benign lesions, and most remain so, a small proportion progress to become malignant melanomas. The risk of melanoma varies based on type, size, number, and location of nevi. In approximately 4–25% of non-familial cases, melanoma occurs in conjunction with a preexisting nevus (1). Patients with familial nevus syndrome have increased numbers of nevi that are dysplastic in nature. They have almost 100-times higher risk of developing melanoma than the general population, and approximately 50% will develop at least one melanoma by the age of 50 (2). Another situation in which individuals develop increased numbers of nevi is in eruptive melanocytic nevi (EMN). EMN are characterized by the quick appearance of large numbers (>100) of nevi, often in a grouped distribution. EMN are associated with systemic immunosuppression, such as that seen after organ transplantation (3,4). The paucity of EMN cases reported in the literature has made it difficult to assess a correlation between EMN and an increased risk for melanoma (3). However, sequencing of eruptive melanocytic nevi revealed the presence of BRAFV600E mutations in 85% of lesions (5). The role of mutated BRAF in the initiation and progression of melanoma is well known and suggests there may be a correlation between immunosuppression, EMN and melanoma development.

Basic research and clinical observations over the past several decades have clearly demonstrated that immunological mechanisms are capable of controlling melanoma growth. This line of investigation has led to the introduction of novel immunotherapeutic agents that prolong the survival of patients with advanced melanomas (6–9). Most melanomas begin as premalignant dysplastic nevi, which after months to years, may progress to become invasive melanomas. Thus, there is ample opportunity to prevent dysplastic nevi from evolving into melanomas. Identification of novel preventive agents that can avert melanoma development has proceeded slowly, at least in part because of the limited number of preclinical models that can be used to evaluate potential protective modalities. This is particularly true for immunological interventions that could prevent dysplastic nevi from becoming melanomas. Existing mouse melanoma models are largely restricted to transplantable syngeneic melanoma lines, such as B16, compatible only with C57BL/6 mice, or transgenic mice with enforced expression of mutant oncogenes. These animal models rapidly develop melanomas without first showing evidence of dysplastic nevi. Moreover, it is difficult to study immune-surveillance since the genes are expressed in all tissue specific cells (defined by promoter for transgene).

Our previous work (10) has shown that a single topical application of DMBA followed by weekly application of TPA induces melanocytic nevi in C3H/HeN mice. In this model, mice develop large numbers of pigmented nevi, some of which progress to become invasive melanomas and then metastasize to regional lymph nodes (10). Many of the clinical, genetic and biochemical features closely resemble those that occur in humans. This model gives us flexibility to study the role of immune mediators in melanoma, as well as to understand its initiating and promoting mechanisms.

In this study, we observed that increased DMBA doses correlated well with increased nevus numbers, and were also responsible for expansion of regulatory-T cells. Regulatory T cells were essential for immunosuppression and nevus development.

MATERIALS AND METHODS

Animals

Male and female C3H/HeN mice aged 6–8 weeks obtained from Charles River Laboratories (Wilmington, MA), were housed in the University of Alabama at Birmingham (UAB) pathogen-free animal facility. These mice were bred and female C3H/HeN mice so obtained were used for carcinogenesis experiments, beginning at 8–10 weeks of age. For other experiments a mix of male or female animals were used. Each group had the same number of age-matched male or female mice. All animals were fed a normal diet (standard chow) and were given water ad-libitum. The UAB Institutional Animal Care and Use Committee approved the animal protocol for the study (Approval# 101009248). CD4 and CD8 Knockout mice were backcrossed onto the C3H/HeN background and used as described in our earlier studies (11).

Chemicals and Antibodies

7,12-dimethylbenz(a)anthracene (DMBA) (≥95% purity); 1-fluoro-2,4-dinitrobenzene (DNFB); N6, 2’-O-dibutyryladenosine 3:5-cyclic monophosphate (dbcAMP); and sodium orthovandate (Na3VO4) were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO). 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was obtained from LC laboratories (Woburn, MA). CD4-PE (RRID:AB_465506), CD4-FITC (AB_464892), FOXP3-PE (AB_465936), IL-10-PE (AB_466176), CD45.2-Percp-Cy5.5 (AB_953590), and CD45.2-FITC (AB_465061) were obtained from eBiosciences. FOXP3-v450 (AB_10611728), IFNγ-PE-Cy7 (AB_396766), CD8-Alexa-647 (AB_396792), and CD8-PE (AB_394571) were obtained from BD-Pharmingen.

Nevus/Melanoma Induction Protocol

The DMBA/TPA nevogenesis protocol was applied as described previously (10). The shaved and naired backs of mice were painted with increasing DMBA doses [100–1000μg (~400–4000 nmoles)] of DMBA in 100μl of acetone]. The animals were then treated biweekly with 12.5μg TPA (20nmol) in acetone. Lesional area was measured using length and width of all nevi and using the following formula to calculate the area of an ellipse: [Area = π (d1/2)(d2/2)]. All mice were naired weekly to count and measure the nevi.

Development of melanocytic cell lines from lymph nodes (LNs)

Lymph nodes were digested in 200μl of digestion buffer [collagenase D (Roche) (1mg/ml) and DNase (20μg/ml)] for 45 minutes. The cells were cultured in melanocyte growth media [OptiMEM with dbcAMP (0.1mM), Na3VO4 (1μM), horse serum (7%), and TPA (25ng/ml)]. Medium was changed every 2 days.

Contact hypersensitivity response (CHS) and adoptive transfer

Mice were sensitized (initiation) on the abdomen with 100 μl of DMBA (0.1–1% w/v DMBA in acetone). Elicitation of specific responses was assessed 5 days after DMBA sensitization. DMBA (20 μl of 0.1–1% w/v DMBA in acetone) was painted on the ears for elicitation. Baseline ear thickness measurements were taken before elicitation and further measurements were taken after elicitation daily for the next 5 days using a dial thickness gauge spring-loaded micrometer (Mitutoyo 7301). The maximum increment in ear thickness compared with the baseline pre-elicitation level was used to quantify the net ear thickness. Naive mice, which were not sensitized but were challenged with DMBA, served as negative controls. To assess the extent of suppression by DMBA-specific regulatory-T cells of each group, mice were sensitized as described. Single cell suspensions of lymph node (LN) cells were obtained after 5 days. CD4+ T cells were isolated by magnetic bead separation (Miltenyi Biotech) according to the manufacturer’s instructions. Then, 10 × 106 CD4+ T cells were adoptively transferred into wild type (WT) mice. After 24h, mice were sensitized with DMBA as described. Elicitation and assessment of ear thickness was performed as described above.

Flow cytometry

Ear skin or LNs from DMBA/TPA treated mice or age matched untreated skin samples were collected as described (10). Ears from at least three mice were split in ventral and dorsal side, minced with scissors and digested with collagenase D/DNase for 1h. Cells were passed through a 70μm screen, counted and used for staining with indicated antibodies. Single cell suspensions were prepared individually by collagenase D/DNase I digestion. Lymph node cells were also prepared using collagenase digestion. Cells were collected, counted, and dispensed at 1×106 cells per sample and pretreated with Fc receptor block, then stained with the appropriate primary antibody. After fixation/permeablization, cells were further stained with the appropriate antibodies and flurochromes. Flow cytometric analysis was performed using a BD LSRII cytometer with BD FACS Diva software for acquisition and FlowJo 9.5.2 for data analysis (10).

T cell cultures and cytokine assay

For analysis of cytokines and T cell subsets during contact hypersensitivity (CHS), mice were sacrificed on day 3 after DMBA challenge. Ear draining LNs were removed and minced with scissors and digested in Hank’s Balanced salt solution (HBSS) containing collagenase D (1mg/ml, Roche Applied Sciences, Indianapolis, IN) and 20μg/ml DNAse I (Sigma, St. Louis, MO) for 45 minutes. After cells were passed through 100μm mesh, they were counted and 2×106 cells per mouse were stimulated with PMA (50ng/ml) and ionomycin (250ng/ml) for 5h in the presence of Brefeldin A (2 μM) for intracellular staining of cytokines. LNs from naïve mice were processed in parallel as a negative control. Staining profiles were obtained using LSRII flow cytometer (BD Biosciences) and FlowJo v9.5.2 for Mac or v10.0 for Windows computers.

RNA extraction and RT-PCR

The total genomic DNA and RNA was extracted from samples using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The concentration of total RNA was determined by measuring the absorbance at 260 nm using an Eppendorf biophotometer plus. Purity of isolated RNA was determined with the ratio of absorbance 260 nm/280 nm >1.8. cDNA was synthesized from 1μg RNA using a reverse transcriptase kit (Biorad) according to the manufacturer’s instructions. The expression of p16INK4a and p19ARF mRNA was detected using primers and conditions described elsewhere (12). For Gapdh primers were forward: 5’-CATGTTCCAGTATGACTCCACTC-3’ and reverse: 5’- GGCCTCACCCCATTTGATGT-3’ (13). The primers used for H-ras were WT forward 5’-CAGCAGGTCAAGAAGAGTATAGTGCCA-PO4–3; mutant forward 5’-CATCTTAGACACAGCAGGTCT-3’; and common reverse: 5’-GCGAGCAGCCAGGTCACAC-3’. The blocker allele and mutant allele-specific primers were used at a 4:1 ratio (WT:mutant) using RT-PCR protocols that were optimized for increased specificity and sensitivity in detecting the H-ras Q61L point mutation. Thermocycling conditions were 1minute at 95°C and 35 cycles of 20 seconds at 95°C, 30 seconds at 60°C and 20 seconds at 72°C. We followed the established allele-specific competitive blocker PCR (ACB-PCR) as per Parsons et al (14), the WT allele blocking primer was phosphorylated at the 3’ end to block primer extension.

Statistical Analysis.

Data were analyzed with GraphPad Prism 7.0 software. One-way ANOVA was performed to determine statistical significance. For correlations, equal distribution was assessed and linear regression was calculated and reported as R2. Statistical significance was set at a p value of < 0.05.

RESULTS

Larger doses of DMBA used for immunization decrease the DMBA allergic CHS response.

We sensitized mice with increasing DMBA doses (100 μl of 0.1% [400 nmol], 0.5% [2000 nmol] and 1% [4000 nmol]) and analyzed the allergic CHS responses to DMBA by challenging with 20μl of 0.1% (80 nmol) DMBA on the ears. We observed that there was a dose dependent decrease in ear swelling responses in the mice sensitized with larger DMBA doses (Figure 1A). Contrary to the reduction in CHS when higher amounts of DMBA were given for sensitization, increasing the dose at elicitation produced a dose dependent increase in the ear swelling response (Figure 1B). Next, we analyzed whether larger DMBA doses reduced the allergic CHS response to unrelated haptens as well. We painted mice with DMBA and 24h later applied DNFB at the same site. After challenging with DNFB on day 5 and measuring the ear swelling response, we observed that there was a dose dependent reduction in ear swelling as DMBA doses increased (Figure 1C). We next analyzed the effect of DMBA dose mutations in the skin. Mice were sensitized with DMBA on the abdomen and were treated five days later with 0.1% DMBA on the back. Five days after that, back skin was collected for mRNA analysis of mutant H-ras. We observed that mice immunized with larger DMBA doses showed greater numbers of H-ras mutations even when examined in the skin at a distant site (Figure 1D).

Figure 1. The magnitude of the DMBA-induced immune response is dependent on the dose at sensitization and not challenge.

Figure 1.

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(A) Mice were sensitized on the abdomen with increasing doses of DMBA (100μl of 0.1, 0.5 or 1%) and challenged 5 days later on the ears with 2μl of 0.1% DMBA. (B) Mice were sensitized on the abdomen with DMBA (100μl of 0.1%) and challenged 5 days later on the ears with increasing doses of DMBA (20μl of 0.1, 0.5 or 1%). (C) Mice were sensitized on the abdomen with increasing doses of DMBA (100μl of 0.1, 0.5 and 1%). 24h later mice were resensitized with 25μl of 0.3% (v/v) DNFB. 5 days later mice were challenged with 10μl of 0.2% (v/v) DNFB on the ear. There were 4–6 mice per group. (D) Relative mutant H-ras mRNA expression 5 days after DMBA application. Mice were sensitized with the indicated DMBA doses on the abdomen and challenged on the back 5 days later with 0.1% DMBA. 5 days later skin was collected for mRNA analysis of mutant H-ras as indicated in the figure. *p<0.05, **p<0.01,***p<0.001. There were 2–3 mice per group and the experiment was done twice.

Increased DMBA doses enhance nevus incidence and size

Since higher DMBA doses at immunization resulted in a reduced DMBA allergic CHS response, we next analyzed if increased dose of DMBA augmented the number and growth of nevi. We observed that the nevus size and numbers per mouse increased with a large DMBA dose (Figure 2A & 2B). Further, the average size of individual nevi also increased as the dose of DMBA increased (Figure 2C). Categorizing the nevi in different sizes also revealed that nevi that were greater in size were more prevalent in mice that received a large DMBA dose compared to mice that received a lower DMBA dose (Figure 2D). The nevus numbers and DMBA doses correlated significantly (p=0.001) (Figure 3A). Further, the percent loss of p16INK4a expression in lesions correlated well with nevus numbers (p=0.01) (Figure 3B). The percent LNs that produced an immortalized cell line also correlated significantly with average nevus numbers (p=0.0068) (Figure 3C).

Figure 2. A larger DMBA dose enhances the incidence and size of nevi.

Figure 2.

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Mice were painted on the backs with increasing doses of DMBA (100μl of 0.1, 0.5 and 1%) and promoted with 12.5 μg TPA beginning one week later. (A) Number of nevi per mouse (B) nevus area per mouse (C) average nevus size and (D) percent distribution of lesions, 15 weeks after initiation of the nevogenesis protocol. *p<0.05, **p<0.01,***p<0.001. There were at least 8–10 mice per group.

Figure 3.

Figure 3.

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The number of nevi correlates with DMBA dose and aggressiveness of melanoma progression. (A) Increasing DMBA doses correlates well with nevus incidence. (B) Loss of p16 in the lesions correlates well with the number of nevi present on the mice. (C) Increased nevus number correlates with increased ability to generate immortalized melanoma cells from lymph nodes. There were at 8–10 mice per group. Linear regression analysis was performed as described in materials and Methods and R2 and p values are shown in each figure.

Regulatory T cell expansion and function is increased by increasing the DMBA dose

In prior studies, we have shown that in C3H/HeN mice, CD8+ T-cells are the major effector cells for DMBA CHS; in contrast, CD4+ T-cells have a regulatory function (11). We next investigated the nature of T cells in draining LNs after DMBA induced CHS. We observed a minor increase in frequency of CD4+ T cells and a minor decrease in frequency of CD8+ T cells as the DMBA dose increased (Figure 4A). However, there was a significant increase in frequency of CD25+Foxp3+CD4+ T cells as the DMBA dose increased (Figure 4A). This was accompanied by a 10 fold increase in IL-10 levels and a 50% decrease in IFNγ levels at highest dose compared to the lowest DMBA dose (Figure 4B). Since immune responses at the site of exposure are also important, we next analyzed T cells in the ears. We observed a reduction in CD8+Ki67+ T cells in the LNs of the mice at the higher dose, indicating that the proliferation of CD8+ T cells is impaired or suppressed after a DMBA dose increase. Like the dLNs, we observed an increase in CD4+ and decrease in CD8+ T cell infiltration in the ear skin (Figure 4C). In order to confirm that the regulatory CD4+ T cells mediate DMBA-induced immunosuppression, we applied increasing DMBA doses to CD4 knockout (KO) mice. We observed that CD4KO mice were resistant to DMBA-induced immunosuppression compared to WT mice (Figure 4D). Consistent with our previous observations (11), we found an exaggerated DMBA CHS in CD4KO mice compared to WT mice (Figure 4D). Transfer of CD4+ T cells from mice that were sensitized with increasing doses of DMBA revealed that these cells showed an enhanced suppressive phenotype compared to non-specific and CD4+ T cells from low dose DMBA treated mice (Figure 4E).

Figure 4. Increased regulatory T cell expansion and function with increased DMBA dose in skin.

Figure 4.

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(A & B) Mice were sensitized on the abdomen with increasing doses of DMBA (100μl of 0.1, 0.5 and 1%) and challenged 5 days later on ears with 20μl of 0.1% DMBA. Ear draining LNs were isolated 3 days after DMBA challenge and stained with anti-CD4, CD8, CD25 and Foxp3 antibodies and analyzed by flow cytometry. One million cells from each group were stimulated with PMA/Ionomycin for 48h and supernatants were analyzed for IL-10 and IFNγ by ELISA. Histograms are 4 LNs pooled from two mice and experiments were done twice. (C) Mice were sensitized and challenged with DMBA as above. Three days after sensitization, ears were digested with collagenase D/DNase as described in the Materials and Methods. Cells were gated on CD45.2 as shown. Cells were stained for markers as shown in the figure. The ears from 3 mice were pooled and digested to release the cells. (D) WT and CD4KO mice were sensitized with increasing DMBA doses and challenged as described in A & B. (E) Mice were sensitized with increasing DMBA doses, 5 days later CD4 T cells were collected from each group and injected into WT mice. Twenty-four hours after the adoptive transfer, mice were sensitized with DMBA on the abdomen and were challenged on the ears 5 days later. Each group had 4–5 mice per group. *p<0.05, **p<0.01,***p<0.001, ****p<0.0001

Number and size of nevi is dependent on CD4 T cells

Since we observed an important role for regulatory-T cells in the magnitude of DMBA induced immune response, WT, CD4KO and CD8KO mice were subjected to our nevogenesis protocol to analyze the incidence and size of pigmented lesions. We observed that CD4KO mice were resistant to increases in the number and area of nevi compared to WT and CD8KO mice (Figure 5A & 5B). Further, the average size of pigmented lesions was also significantly reduced in CD4KO mice compared to WT and CD8KO mice. However, we also observed a slight increase in average size of lesions in CD8KO mice compared with WT mice, indicating that CD8 T cells control the growth of the lesion vis-à-vis incidence (Figure 5C). Categorizing the nevi in different size groups also revealed that smaller sized nevi were more prevalent in CD4KO mice compared to WT and CD8KO mice (Figure 5D). The DMBA-induced CHS responses, as described earlier, were exaggerated in CD4KO and were significantly diminished in CD8KO compared with WT mice (Figure 5E). The percent lesions that produced a cell line was also greater in CD8KO (35%) and WT (25%) compared to CD4KO (10%), suggesting a more benign behavior of lesions obtained from CD4KO mice.

Figure 5: Reduced number of pigmented lesions in CD4KO compared to WT and CD8KO mice.

Figure 5:

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DMBA (100μg/ mouse) was applied on the shaved and naired backs of mice and one week later TPA (12.5 μg/mouse) was applied twice weekly for 25 weeks. Nevi were counted and the area was measured weekly. (A) WT and CD8 KO had nearly equal lesion numbers and these numbers were significantly greater than CD4KO mice. (B) Reduced lesion area per mouse in CD4KO but not CD8KO or WT mice. Like the lesion numbers, the area was significantly reduced in CD4KO mice as compared to WT and CD8KO mice. The lesion area per mouse between CD8KO and WT groups was equal. (C) Area of each individual lesion at Week 25 for each group was measured. The mean lesion area is greatly reduced in CD4KO mice as compared to both WT and CD8KO. CD8KO mice have lesion whose size is slightly larger than WT mice. (D) Stacked bar graph for lesion area at week 25 from each group, shows that in CD8KO mice there is a higher percentage of lesions greater than 3 mm2 or 6 mm2 as compared to WT and CD4KO have larger percentage in the range below 3mm2. (E) Day 3 allergic contact hypersensitivity responses (CHS) in CD4KO is increased significantly compared to WT and CD8KO mice. Each group had 8–10 mice per group. *p<0.05, **p<0.01,***p<0.001

Immunosuppressive microenvironment created by CD4 cells

Since CD4 cells promoted nevus incidence and growth, we next investigated the cytokines being produced by the CD4KO mice. As CD4KO lack CD4 cells, the CD8+ cells compensated for the loss, and similarly CD4+ cells compensated for the loss in CD8KO (Figure 6A). The percent of CD25+Foxp3+ cells gated on CD4 were similar, but the overall percentage of regulatory T cells per LN was greater in CD8KO mice (11% vs 15%). The percentage of CD8+Foxp3+ cells was similar in CD4KO and WT mice. There was increased IL-10 production from CD4 cells of CD8KO compared to the CD4+ cells of WT mice. Similarly, the CD8+ T cells from CD4KO produced increased IFNγ compared to CD8+ T cells from WT mice. We confirmed IL-10 and IFNγ production using ELISA and observed IL-10 levels were increased in CD8KO and WT mice and IFNγ was mainly produced by CD8+ T cells (Figure 6B). Investigation of infiltrating cells revealed that a reduced number of CD45.2 cells infiltrated the skin in CD8KO mice compared with WT or CD4KO mice. The CD4+Foxp3 positive cells were the major cell type that was present in the skin after DMBA application and CD8+ T cells were major IFNγ producers (Figure 6C).

Figure 6. CD4 T cells are the major source of IL-10.

Figure 6.

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(A) Mice were sensitized on the abdomen with DMBA (100μl of 0.1%) and challenged 5 days later on ears with 20μl of 0.1% DMBA. Ear draining LNs were isolated 3 days after DMBA challenge and stained with anti-CD4, CD8, CD25 and Foxp3 antibodies and analyzed by flow cytometry. (B) One million cells from each group were stimulated with PMA/ionomycin for 6h and 48h for intracellular staining and ELISA, respectively, to detect IL-10 and IFNγ. Histograms are from four LNs pooled from two mice. The experiment was done twice. (C) Mice were sensitized and challenged as above. Three days after sensitization, ears were digested with collagenase D/DNAse as described in Materials and Methods. Cells were gated on CD45.2 as shown. Cells were stained for the markers as shown in the figure. The ears from 3 mice were pooled and digested to release the cells. **p<0.01

DISCUSSION

Melanocytic nevi can appear in both sun exposed and unexposed skin. Their etiology includes genetic factors, excessive exposure to sunlight and other environmental influences such as pollutants in the environment. An analysis of the scientific literature reveals many correlations between polyaromatic hydrocarbon exposure and the occurrence of malignant melanoma, particularly on cutaneous surfaces of skin that are protected from sunlight (15). The polyaromatic hydrocarbon DMBA is a model carcinogen that induces epidermal malignancies of skin, and a major focus of study has been its role in the initiation of non-melanoma skin cancers (16). Our previous work demonstrated that a single dose of 400 nmol of DMBA followed by weekly application of TPA resulted in melanocytic nevi in C3H/HeN mice that have the potential to progress to melanomas. This model gave us the opportunity to study the involvement of immune mediators on earlier stages of melanoma development, rather than on melanomas already present. This is an important issue if the immune system is to be manipulated to prevent melanomas in high-risk individuals. In this study, we report for the first time that CD4+ T cells promote initiation and growth of melanocytic nevi.

Our previous studies had shown that the generation of cytotoxic T-cells is in part responsible for limiting the development of the melanocytic nevi (17). During the course of these experiments, we observed that application of larger doses of DMBA produced greater numbers of nevi that grew to a larger size. While it might be expected that greater amounts of DMBA would produce more and larger-sized pigmented lesions, we found this occurred even at a distant skin site. Moreover, the larger doses of DMBA resulted in a smaller CHS response than lower doses. We therefore postulated that an increased number of regulatory T cells might contribute to the suppressed DMBA CHS response and to the greater numbers and larger size of the melanocytic lesions.

Regulatory T cells suppress a variety of physiological and pathological immune responses (18) and play a key role in progression of various cancers. They directly promote immunosuppression by regulating effector T cell functions (19). They can also directly eliminate effector T cells and compete for dendritic cells for antigen presentation. Further, they are known to suppress DC polarization (20). Immunosuppression in the melanoma microenvironment is facilitated by regulatory T cells and is a major mechanism of immune escape once melanomas are already present. Regulatory T cells accumulate in melanoma and the reduced ratio of CD8 to regulatory T cells is used as a predictive marker for survival of melanoma patients (21). Immunotherapies using neutralizing antibodies to cell surface markers of regulatory T cells such as CTLA-4 have been promising especially in melanoma immunotherapy (6–9). Although there is an evidence of a regulatory T cell contribution to melanoma progression, little was known about their role in melanoma precursors.

We found, employing our murine model for nevus initiation and progression, that by increasing the dose of DMBA, there was an expansion of functional CD4+ regulatory-T cells. These cells were essential for both suppression of DMBA CHS and the increase in nevi that develop. We also found that an increased percentage of LNs produced immortalized cell lines from mice that had fewer regulatory T-cells, indicating an association between immunosuppression and melanocyte immortalization in LNs.

Previous studies in mice have demonstrated that repeated DMBA doses over time have a greater carcinogenic affect than the same amount administered as a total single dose of DMBA (22). Repeated hapten exposure can induce a regulatory T cell component and hence promote tumorigenesis. Others and our group have previously demonstrated that CD4+ T cells, presumably regulatory T cells, promote SCC tumor development in DMBA, UVB and virus induced tumor models. In all cases, CD4+ T-cells enhanced the development of tumors (11,13, 23).

Previously, we have described the cytokine profile of CD8+ and CD4+ T cells after cutaneous application of DMBA on C3H/HeN mice (11). In this study, we confirmed our previous results that CD4+ T cells are the main cells that secrete IL-10, while CD8+ cells are the IFNγ producers. Specifically, infiltration of cells after DMBA challenge was impaired in CD8KO mice. Loss of CD8+ T cells did not affect the number of nevi, but, to an extent, affected their average size, indicating that CD8+ T cells play some role in controlling the growth of nevi. CD8+ T cells are generated against melanoma antigens and are able to mount an efficient anti-melanoma response (24). Although others have proposed that IFNγ levels secreted by macrophages promote melanoma development, it is unclear if low amounts of IFNγ are important to maintain an antitumor response or if IFNγ is an antitumor agent during early stages of nevogenesis. In this study, we observed that in CD4KO mice there was an increase in IFNγ production and reduced nevi numbers indicating IFNγ may play an essential role in reducing early oncogenic mutations and thus nevus numbers. IFN-γ is a pleiotropic molecule that is associated with anti-proliferative, pro-apoptotic and antitumor mechanisms. Although it is often considered as a major anti-tumor agent, and has been used in the treatment of many cancers due to its effective tumor immune surveillance. Type I interferons play a role in DNA repair, and IFNγ early on can induce cell cycle arrest through Chk1 phosphorylation (25,26).

However, many reports suggest that it may also play a pro-tumorigenic role, because it can induce down-regulation of major histocompatibility complex, up-regulation of indoleamine 2,3-dioxygenase and of checkpoint inhibitors, as programmed cell-death ligand 1 (27,28).

Previous studies suggest that the Langerhans cells (LCs) present low doses of hapten, while dermal dendritic cells (DCs) present high doses of the hapten (29). Dermal CD103− DCs constitutively produce retinoic acid and have the potential to induce Foxp3+ regulatory T cells (30). Our study did not investigate which antigen presenting cell (APC) subset is responsible for inducing regulatory T cells; however, we established that high DMBA doses induced expansion and the suppressive function of regulatory T cells.

The mice treated with the larger amount of DMBA and that had increased numbers of nevi were also observed to have loss of p16 and p19 in a higher percentage of lesions than in those that were given smaller amounts. The CDKN2A locus encodes the two proteins, p16INK4a and p19ARF, the loss of which is critical for tumor development in many organ systems, including melanoma (31). Mutations in CDKN2A, account for 35% to 40% of familial melanomas (32). Melanocytic nevi persist in a benign state for years and seldom progress to melanoma even when mutations are present (33). High activation of H-RAS in Spitz nevi or mutated BRAF in melanocytes is associated with elevated p16INK4a expression (33) (34). Further, it is well known that as nevi progress to melanomas, p16INK4a expression gradually decreases. Thus, there is a negative association between p16 INK4a expression and melanoma progression (35).

The finding that deficiencies in immunological function contribute to the development and growth of dysplastic nevi and potentially to their progression to melanoma is consistent with findings in humans. CD4+CD25+ T cells were increased two-fold in metastatic lymph nodes in comparison to both tumor-free lymph nodes in patients with melanoma. These cells expressed the Foxp3 transcription factor and displayed an activated phenotype. These CD4+CD25+Foxp3+ cells were found to inhibit in vitro the proliferation and cytokine production of infiltrating effector CD4+CD25- and CD8+ T cells through a cell-contact-dependent mechanism, thus behaving as T-regs (36). CD25+Foxp3+ Tregs were also prevalent both in junctional and compound atypical nevi and radial growth phase melanomas, suggesting that they can induce immunotolerance early during melanoma genesis, by positively regulating melanoma growth. The presence of these cells within a tumor site could be useful for prognosis and treatment of melanoma (37).

Organ transplant recipients on immunosuppressive therapy not only have a higher incidence of melanoma, but they also develop greater numbers of nevi as well (38–41). The nevi that develop in patients on immunosuppression have greater dermatoscopic changes than normal controls (41). Moreover, eruptive melanocytic nevi frequently occur in the setting of immunosuppressive treatments (42). Regulatory T cell therapies are in clinical trials to treat autoimmune diseases. If these results can be extrapolated to humans, then care should be taken when these immunosuppressive regimens are administered to individuals at increased risk for melanoma, and potentially other malignancies.

Acknowledgments

Funding sources:

NIH Grants P01CA210946, R01CA193885, P30 CA013148, N01 CN2012–00033, 1R01AR071157–01A1, and VA Grant 101BX003395

Footnotes

Conflict of interest:

The authors declare no potential conflicts of interest.

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