Regulatory T Cells Play an Important Role in the Prevention of Murine Melanocytic Nevi and Melanomas

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 progresses to become malignant melanomas. The risk of melanoma varies based on type, size, number, and location of nevi. In approximately 4%–25% of nonfamilial 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 years (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 EMN revealed the presence of BRAF^V600E 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 immunologic 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 immunologic 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 because 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 7,12-dimethylbenz(a)anthracene (DMBA) followed by weekly application of 12-O-tetradecanoyl-phorbol-13-acetate (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 (LN; ref. 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 (Treg) were essential for immunosuppression and nevus development.

Male and female C3H/HeN mice ages 6–8 weeks, obtained from Charles River Laboratories, were housed in the University of Alabama at Birmingham (UAB, Birmingham, AL) 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 was 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 (Birmingham, AL) approved the animal protocol for the study (approval# 101009248). CD4- and CD8-knockout (KO) mice were backcrossed onto the C3H/HeN background and used as described in our earlier studies (11).

DMBA (≥95% purity), 1-fluoro-2,4-dinitrobenzene (DNFB), N6, 2′-O-dibutyryladenosine 3:5-cyclic monophosphate (dbcAMP), and sodium orthovanadate (Na₃VO₄) were purchased from Sigma-Aldrich Chemical Co. TPA was obtained from LC Laboratories. 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 eBioscience. FOXP3-v450 (AB_10611728), IFNγ-PE-Cy7 (AB_396766), CD8-Alexa-647 (AB_396792), and CD8-PE (AB_394571) were obtained from BD Pharmingen.

The DMBA/TPA nevogenesis protocol was applied as described previously (10). The shaved and naired backs of mice were painted with increasing DMBA doses [100–1,000 μg (∼400–4,000 nanomoles (nmoles))] of DMBA in 100 μL of acetone]. The animals were then treated biweekly with 12.5 μg TPA (20 nmol) in acetone. Lesional area was measured by 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.

LNs were digested in 200 μL of digestion buffer [collagenase D (Roche; 1 mg/mL) and DNase (20 μg/mL)] for 45 minutes. The cells were cultured in melanocyte growth media [OptiMEM with dbcAMP [0.1 millimolar (mmol)], Na₃VO₄ (1 μmol/L), horse serum (7%), and TPA (25 ng/mL)]. Medium was changed every 2 days.

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 preelicitation level was used to quantify the net ear thickness. Naïve 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 LN cells were obtained after 5 days. CD4⁺ T cells were isolated by Magnetic Bead Separation (Miltenyi Biotec) according to the manufacturer's instructions. Then, 10 × 10⁶ CD4⁺ T cells were adoptively transferred into wild-type (WT) mice. After 24 hours, mice were sensitized with DMBA as described. Elicitation and assessment of ear thickness were performed as described above.

Ear skin or LNs from DMBA/TPA-treated mice or age-matched untreated skin samples were collected as described previously (10). Ears from at least 3 mice were split in ventral and dorsal side, minced with scissors, and digested with collagenase D/DNase for 1 hour. Cells were passed through a 70-μm screen, counted, and used for staining with indicated antibodies. Single-cell suspensions were prepared individually by collagenase D/DNase I digestion. LN 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, and then stained with the appropriate primary antibody. After fixation/permeabilization, cells were further stained with the appropriate antibodies and fluorochromes. 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).

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 containing collagenase D (1 mg/mL, Roche Applied Sciences) and 20 μg/mL DNAse I (Sigma) for 45 minutes. After cells were passed through 100-μm mesh, they were counted and 2 × 10⁶ cells per mouse were stimulated with PMA (50 ng/mL) and ionomycin (250 ng/mL) for 5 hours in the presence of Brefeldin A (2 μmol/L) 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.

The total genomic DNA and RNA were extracted from samples using TRizol Reagent (Invitrogen) 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 260/280 nm > 1.8. cDNA was synthesized from 1 μg RNA using a Reverse Transcriptase Kit (Bio-Rad) according to the manufacturer's instructions. The expression of p16^INK4a and p19^ARF 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 1 minute 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 as per Parsons and colleagues (14), the WT allele–blocking primer was phosphorylated at the 3′ end to block primer extension.

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 < 0.05.

We sensitized mice with increasing DMBA doses [100 μL of 0.1% (400 nmol), 0.5% (2,000 nmol), and 1% (4,000 nmol)] and analyzed the allergic CHS responses to DMBA by challenging with 20 μL 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 (Fig. 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 (Fig. 1B). Next, we analyzed whether larger DMBA doses reduced the allergic CHS response to unrelated haptens as well. We painted mice with DMBA and 24 hours 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 (Fig. 1C). We next analyzed the effect of DMBA dose mutations in the skin. Mice were sensitized with DMBA on the abdomen and were treated 5 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 (Fig. 1D).

Because higher DMBA doses at immunization resulted in a reduced DMBA allergic CHS response, we next analyzed whether 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 (Fig. 2A and B). Furthermore, the average size of individual nevi also increased as the dose of DMBA increased (Fig. 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 with mice that received a lower DMBA dose (Fig. 2D). The nevus numbers and DMBA doses correlated significantly (P = 0.001; Fig. 3A). Furthermore, the percent loss of p16^INK4a expression in lesions correlated well with nevus numbers (P = 0.01; Fig. 3B). The percent LNs that produced an immortalized cell line also correlated significantly with average nevus numbers (P = 0.0068; Fig. 3C).

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 (Fig. 4A). However, there was a significant increase in frequency of CD25⁺Foxp3⁺CD4⁺ T cells as the DMBA dose increased (Fig. 4A). This was accompanied by a 10-fold increase in IL10 levels and a 50% decrease in IFNγ levels at highest dose compared with the lowest DMBA dose (Fig. 4B). Because 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 draining lymph nodes (dLNs), we observed an increase in CD4⁺ and decrease in CD8⁺ T-cell infiltration in the ear skin (Fig. 4C). To confirm that the regulatory CD4⁺ T cells mediate DMBA-induced immunosuppression, we applied increasing DMBA doses to CD4KO mice. We observed that CD4KO mice were resistant to DMBA-induced immunosuppression compared with WT mice (Fig. 4D). Consistent with our previous observations (11), we found an exaggerated DMBA CHS in CD4KO mice compared with WT mice (Fig. 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 with nonspecific and CD4⁺ T cells from low-dose DMBA-treated mice (Fig. 4E).

Because 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 with WT and CD8KO mice (Fig. 5A and B). Furthermore, the average size of pigmented lesions was also significantly reduced in CD4KO mice compared with 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 lesions vis-à-vis incidence (Fig. 5C). Categorizing the nevi in different size groups also revealed that smaller sized nevi were more prevalent in CD4KO mice compared with WT and CD8KO mice (Fig. 5D). The DMBA-induced CHS responses, as described earlier, were exaggerated in CD4KO and were significantly diminished in CD8KO compared with WT mice (Fig. 5E). The percent lesions that produced a cell line were also greater in CD8KO (35%) and WT (25%) compared with CD4KO (10%), suggesting a more benign behavior of lesions obtained from CD4KO mice.

Because 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 (Fig. 6A). The percent of CD25⁺Foxp3⁺ cells gated on CD4 was similar, but the overall percentage of Tregs 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 IL10 production from CD4 cells of CD8KO mice compared with the CD4⁺ cells of WT mice. Similarly, the CD8⁺ T cells from CD4KO mice produced increased IFNγ compared with CD8⁺ T cells from WT mice. We confirmed IL10 and IFNγ production using ELISA and observed IL10 levels were increased in CD8KO and WT mice and IFNγ was mainly produced by CD8⁺ T cells (Fig. 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⁺ cells were the major cell type that was present in the skin after DMBA application and CD8⁺ T cells were major IFNγ producers (Fig. 6C).

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 nonmelanoma 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, whether 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 Tregs might contribute to the suppressed DMBA CHS response and to the greater numbers and larger size of the melanocytic lesions.

Tregs suppress a variety of physiologic and pathologic 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 (DC) for antigen presentation. Furthermore, they are known to suppress DC polarization (20). Immunosuppression in the melanoma microenvironment is facilitated by Tregs and is a major mechanism of immune escape once melanomas are already present. Tregs accumulate in melanoma and the reduced ratio of CD8 to Tregs is used as a predictive marker for survival of patients with melanoma (21). Immunotherapies using neutralizing antibodies to cell surface markers of Tregs, such as CTLA-4, have been promising, especially in melanoma immunotherapy (6–9). Although there is an evidence of a Treg contribution to melanoma progression, little was known about their role in melanoma precursors.

We found, that employing our murine model for nevus initiation and progression 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 Tregs, 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 Treg component and hence, promote tumorigenesis. Others and our group has previously demonstrated that CD4⁺ T cells, presumably Tregs, promote squamous cell carcinoma tumor development in DMBA-, ultraviolet (UV)-, 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 IL10, 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 whether low amounts of IFNγ are important to maintain an antitumor response or whether 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 reduction in nevi numbers, indicating IFNγ may play an essential role in reducing early-oncogenic mutations, and, thus, nevus numbers. IFNγ is a pleiotropic molecule that is associated with antiproliferative, proapoptotic, and antitumor mechanisms. Although it is often considered as a major antitumor agent, and has been used in the treatment of many cancers due to its effective tumor immune surveillance. Type I IFNs play a role in DNA repair, and IFNγ early on treatment can induce cell-cycle arrest through Chk1 phosphorylation (25, 26).

However, many reports suggest that it may also play a protumorigenic role, because it can induce downregulation of MHC, and upregulation of indoleamine 2,3-dioxygenase and checkpoint inhibitors, such as programmed cell death ligand 1 (27, 28).

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

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, p16^INK4a and p19^ARF, the loss of which is critical for tumor development in many organ systems, including melanoma (31). Mutations in CDKN2A account for 35%–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 p16^INK4a expression (33, 34). Furthermore, it is well known that as nevi progress to melanomas, p16^INK4a expression gradually decreases. Thus, there is a negative association between p16^INK4a expression and melanoma progression (35).

The finding that deficiencies in immunologic 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 2-fold in metastatic LNs in comparison with both tumor-free LNs 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 Tregs (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, EMN frequently occur in the setting of immunosuppressive treatments (42). Treg 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.

T.H. Nasti reports a patent for exosome-containing preparations from post-irradiated mouse melanoma cells and delay melanoma growth in vivo by a natural killer cell-dependent mechanism pending and a patent for benzodiazepine mediated radiosensitization of metastatic melanoma pending. No disclosures were reported by the other authors.

T.H. Nasti: Conceptualization, resources, supervision, funding acquisition, methodology, project administration, writing-review and editing. N. Yusuf: Conceptualization, resources, supervision, funding acquisition, methodology, project administration, writing-review and editing. M.A. Sherwani: Formal analysis, investigation, visualization, methodology. M. Athar: Conceptualization, resources, supervision, funding acquisition, project administration. L. Timares: Conceptualization, resources, data curation, formal analysis, supervision, validation, methodology, writing-original draft, writing-review and editing. C.A. Elmets: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, validation, project administration, writing-review and editing.

This work was supported by NIH grant nos., P01CA210946, R01CA193885, P30 CA013148, N01 CN2012-00033, and 1R01AR071157- 01A1, and VA grant no., 101BX003395.

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.