[CANCER RESEARCH 64, 1496–1501, February 15, 2004] Spontaneous Vitiligo in an Animal Model for Human Melanoma: Role of Tumor-specific CD8؉ T Cells Rene´e Lengagne,1 Fre´de´rique-Anne Le Gal,2 Maryle`ne Garcette,1 Laurence Fiette,3 Patrick Ave,3 Masashi Kato,4 Jean-Paul Briand,5 Christian Massot,6 Izumi Nakashima,4 Laurent Re´nia,1 Jean-Ge´rard Guillet,1 and Armelle Pre´vost-Blondel1 1De´partement d’Immunologie, Institut Cochin, INSERM U567, CNRS UMR 8104, Laboratoire membre de l’IFR 116, Universite´ R. Descartes, Paris, France; 2Laboratory of Tumor Immunology, Division of Oncology, Geneva, Switzerland; 3Unite´ de Recherche et d’Expertise en Histotechnologie et Pathologie, Institut Pasteur, Paris, France; 4Department of Immunology, Nagoya University School of Medicine, Nagoya, Japan; 5UPR 9021 CNRS, Strasbourg, France; and 6INSERM CRI/IFR69, Hopital P. Brousse, Villejuif, France ABSTRACT ated with positive clinical response. Indeed, it has been observed specifically in patients with metastatic melanoma who respond favor- Tumor antigen-reactive T cells can be detected in a large proportion of ably to interleukin (IL)-2 (4). Vitiligo also occurs in melanoma melanoma patients, but their efficacy on tumor control in vivo remains ϩ patients after infusion of tumor-specific CD8 T cells (5, 6). The unclear. On the other hand, vitiligo, a skin disorder characterized by patchy depigmented macules, may occur spontaneously or after antitumor prevalence of antibodies and T cells that are MDA specific in both therapies. Moreover, vitiligo is significantly associated with positive clin- melanoma and vitiligo patients highlights the similarities between ical response, but the mechanism is not understood. Therefore, the estab- autoimmunity observed in vitiligo and antitumor immunity observed lishment of a relevant animal model in which melanoma and vitiligo in melanoma immune surveillance (1). However, the exact role of the spontaneously develop stepwise may be useful for better understanding of immune response specific for MDA in vitiligo development and the parameters involved in the destruction of both benign and malignant melanoma control remains unclear. melanocytes. In a previous work, we established a mouse model for Progressive skin depigmentation occurs spontaneously in several melanoma in which MT/ret transgenic mice express the ret oncogene fused animal models of nonmelanoma-associated vitiligo, including swine, to the metallothionein promoter. Here we report that melanoma leads to Smyth chickens, and the C57BL6/Ler-vit/vit mouse strain (7). It has spontaneous vitiligo. We further investigate, for the first time in this been suggested that the vitiligo pathogenesis is similar in humans and model, the natural antitumor T-cell response and evaluate the role of cellular immunity in the development of the disease. Interestingly, the animals. Indeed, antibodies against the pigmented cell surface anti- occurrence of spontaneous tumor nodules in MT/ret mice with melanoma- gens have been found in dogs, cats, and horses with vitiligo (8), and associated vitiligo is significantly delayed when compared in melanoma T cells infiltrating feather tissue are abundant before visible signs of mice without vitiligo. Moreover, a significant proportion of mice with vitiligo and in tissues undergoing active loss of pigment in Smyth melanoma-associated vitiligo resisted a challenge with syngeneic mela- chickens (9). Frequent combination of melanoma and vitiligo has been noma cells in contrast to animals without vitiligo. Our results confirm that observed in Arabian horses and Sinclair swine (10), heralding regres- vitiligo is associated with clinical benefit and further demonstrate the sion or slow progression of the melanoma, but no relevant mouse ؉ crucial role of CD8 T cells for tumor control in melanoma-associated model is currently available. Therefore, the establishment of a suitable vitiligo. animal model in which melanoma and vitiligo spontaneously develop stepwise would contribute to a better understanding of parameters INTRODUCTION involved in the destruction of both benign and malignant melanocytes. By introducing the human ret oncogene fused to the murine metallo- A large proportion of melanoma patients spontaneously develop a thionein (MT) promoter-enhancer, Iwamoto et al. (11) produced MT/ strong response to melanocyte differentiation antigens (MDAs), pro- ret transgenic mouse lines with a mixed strain background (C57BL/ viding direct evidence for induction of antitumor immunity during 6 ϫ BALB/c). In these mice, skin melanosis and benign melanocytic cancer progression. However, the efficiency of tumor-reactive T cells tumors developed slowly. More recently, a new line was established in controlling tumor progression in vivo remains unclear. The onset of by back-crossing one MT/ret line with C57BL/6 mice. In this line, the vitiligo, a skin disorder characterized by patchy depigmented macules, process of tumor development and malignant transformation bears has been thought to be the result of immune-mediated destruction of multiple resemblances with that of the human giant congenital mela- melanocytes, although other mechanisms may lead to their destruction nocytic nevus that gives rise to cutaneous melanoma during aging (12). (1). Recent studies suggest the involvement of cellular immunity in In the present report, we show that a large proportion of MT/ret vitiligo development. Ogg et al. (2) were the first to identify, in animals spontaneously develop melanoma-associated vitiligo. We fur- patients with non-melanoma-associated vitiligo, skin-homing autore- ther used this model to evaluate the role of cellular immunity in the active circulating T cells that specifically recognize melanoma cells in disease development and to address the following questions: Can we vitro. Moreover, we recently studied T cells from vitiliginous skin ϩ detect melanoma-specific T cells in peripheral blood lymphocytes? margins, and our data clearly support the role of CD8 T cells specific What are their antigen specificities? Does T-cell frequency correlate for MDAs in vitiligo occurring naturally in melanoma patients (3). with disease severity? Does the occurrence of spontaneous vitiligo Antimelanoma therapies sometimes lead to the development of viti- correlate with a higher frequency of melanoma-specific circulating T ligo, and this permanent loss of skin pigment is significantly associ- cells? Does vitiligo correlate with regression of the spontaneous melanoma or protect mice against challenge with syngeneic mela- Received 9/8/03; revised 11/18/03; accepted 12/5/03. ϩ Grant support: French Ligue Nationale contre le Cancer, “axe immunologie des noma cells? Are CD8 T cells crucial for melanoma control in mice tumeurs”; and a fellowship from the Association Franc¸aise de Recherche contre le Cancer with melanoma-associated vitiligo? (to A. Pre´vost-Blondel). 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 MATERIALS AND METHODS 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Armelle Pre´vost-Blondel, Institut Cochin, De´partement d’Immunologie, 27 rue du Faubourg Saint Jacques, 75014 Paris, France. Phone: 33-1-40- Mice. MT/ret transgenic 304/B6 mice (12) were established by crossing b/d 51-65-24; Fax: 33-1-40-51-65-35; E-mail: [email protected]. line 304 MT/ret mice (originating from a BCF1 mouse, H-2 ) five times with 1496 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2004 American Association for Cancer Research. MOUSE MODEL FOR MELANOMA-ASSOCIATED VITILIGO C57BL/6J (H-2b) mice (Harlan, Gannat, France). The founder has been derived anti-mouse CD8 or purified rat IgG as described above. Mice were checked for from a cross between C57BL/6 and BALB/c ϫ (BALB/c ϫ C57BL/6). Thus, the presence of a palpable tumor, and tumor growth was measured twice a all of the mice used in the present report have been back-crossed six times with week. Animals were scored positive when the mean tumor diameter was Ͼ3 C57BL/6 mice and contain Ͻ1% of BALB/c genes. Mice were kept under mm, measured using a gauge caliper. standard conditions except that zinc (115 ␮g/l) was present in their drinking Statistical Analysis. Differences in the incidence of disease signs in ani- water. Clinical signs were assessed twice a month, and development of mals 6 months of age were evaluated using the ␹2 test. Associations between exophthalmus, facial or dorsal tumor nodules, and vitiligo was recorded. Mice disease sign and mortality were evaluated using Pearson correlation statistics. were used for experiments at different time points in the course of malignancy. The Mann-Whitney test was used to compare the frequency of IFN-␥-secreting Control mice were nontransgenic littermates. cells in response to Melan-ret cells in MT/ret mice with or without vitiligo. The Histology. Skin samples were taken from the periphery of vitiligo patches. ␹2 test and Fisher’s exact test were used to evaluate the significance between Skin tissues were fixed in a solution containing zinc acetate (0.5%), zinc responder and nonresponder mice groups. The difference in tumor develop- chloride (0.5%), and calcium acetate (0.05%) in Tris buffer at pH 7 for 72 h. ment in protection assays was compared using the log-rank test. Statistical They were then embedded in low-melting-point paraffin (37°C; polyethyleneg- analysis was performed using the