Carcinogen treatment in mouse selectively expressing activated N-Ras Q61K in melanocytes recapitulates metastatic cutaneous melanoma development.

The incidence of melanoma has significantly increased, and a better understanding of its pathogenesis and development of new therapeutic strategies are urgently needed. Here, we describe a murine model of metastatic cutaneous melanoma using C57BL/6 mice expressing a mutated human N-Ras gene under the control of a tyrosinase promoter (TyrRas). These mice were topically exposed to 7,12- dimethylbenzanthracene (DMBA) for brief exposure periods. Cutaneous melanoma developed at the site of exposure on average by 19 weeks of age and in 80% of mice. Importantly, as in humans, melanoma development was associated with subsequent metastasis to tumor-draining lymph nodes. Critically, such metastatic behavior is transplantable, as intradermal inoculation of melanoma cells from TyrRas-DMBA mice into non-transgenic mice led to the growth of melanoma and, again, metastasis to skin-draining lymph nodes. This metastatic melanoma model closely mimics human pathology and should be a useful tool for studying melanoma pathogenesis and developing new therapies.

Direct presentation of a melanocyte-associated antigen in peripheral lymph nodes induces cytotoxic CD8+ T cells.

Encounter of self-antigens in the periphery by mature T cells induces tolerance in the steady-state. Hence, it is not understood why the same peripheral antigens are also promiscuously expressed in the thymus to mediate central tolerance. Here, we analyzed CD8(+) T-cell tolerance to such an antigen constituted by ovalbumin under the control of the tyrosinase promoter. As expected, endogenous CD8(+) T-cell responses were altered in the periphery of transgenic mice, resulting from promiscuous expression of the self-antigen in mature medullary epithelial cells and deletion of high-affinity T cells in the thymus. In adoptive T-cell transfer experiments, we observed constitutive presentation of the self-antigen in peripheral lymph nodes. Notably, this self-antigen presentation induced persisting cytotoxic cells from high-affinity CD8(+) T-cell precursors. Lymph node resident melanoblasts expressing tyrosinase directly presented the self-antigen to CD8(+) T cells, independently of bone marrow-derived antigen-presenting cells. This peripheral priming was independent of the subcellular localization of the self-antigen, indicating that this mechanism may apply to other melanocyte-associated antigens. Hence, central tolerance by promiscuous expression of peripheral antigens is a mandatory, rather than a superfluous, mechanism to counteract the peripheral priming, at least for self-antigens that can be directly presented in lymph nodes. The peripheral priming by lymph node melanoblasts identified here may constitute an advantage for immunotherapies based on adoptive T-cell transfer.

T cell tolerance to the skin: a central role for central tolerance.

T cell tolerance to self-antigens is believed to be achieved in a two-step process. The first step, called central tolerance, takes place in the thymus. The second step takes place outside the thymus in secondary lymphoid organs. One may ask why two mechanisms are needed to insure T cell tolerance. These two mechanisms share redundant functions and dysfunctions, leading to T cell-mediated autoimmune syndromes. By reviewing the literature on relevant animal models for T cell tolerance and our own recent findings, we are providing evidences that only central tolerance is acting for the skin.

Extralymphatic tumors prepare draining lymph nodes to invasion via a T-cell cross-tolerance process.

Metastases often develop in lymphoid organs. However, the immunologic mechanism allowing such invasion is not known because these organs are considered to be hostile to tumor cells. Here, we analyzed the interactions between tumor cells and CD8(+) T cells in such lymphoid organs. Tumor cells implanted into lymph nodes were able to induce tumor-specific cytotoxic CD8(+) T-cell responses, conducting to tumor rejection, in contrast to primary extralymphatic tumors rapidly anergizing T cells in draining lymph nodes (DLN) via a cross-presentation process. This abortive CD8(+) T-cell response to extralymphatic tumor is independent of the subcellular localization of antigen in tumor cells and is consistent with a lack of CD4(+) T-cell help. Notably, this anergy was potent enough to allow successful DLN implantation of tumor cells. Such distant cross-tolerization of tumor-specific CD8(+) T cells may be a determinant permissive event leading to invasion of DLN. In this situation, metastatic tumor cells do not need to down-regulate their immunogenicity to spread.

Melanoma-infiltrating dendritic cells induce protective antitumor responses mediated by T cells.

Dendritic cells are the most potent antigen-presenting cells inducing innate and adaptive immune response. Dendritic cells infiltrate melanomas, but their ability to induce host antitumor immunity remains obscure. In a previous study, we have observed that melanoma-infiltrating dendritic cells have the capacity to process antigens and migrate to lymph nodes to prime T lymphocytes. Here, we observed that melanoma-infiltrating dendritic cells extracted from melanoma without any additional manipulations were able to protect naive mice against a lethal challenge with the tumor. Remarkably, this was achieved with reinjection of 10(5) melanoma-infiltrating dendritic cells, a number that did not exceed the total number of melanoma-infiltrating dendritic cells recovered from one single tumor. Three observations indicate that protection was due to the natural loading of melanoma-infiltrating dendritic cells with tumor antigens. First, the protective effect was not observed with equivalent numbers of bone marrow-derived dendritic cells. Second, the protection induced was specific for the tumor from which the tumor-infiltrating dendritic cells were isolated. Third, depletion experiments indicate that both CD4+ and CD8+ T lymphocytes were required during the effector phase of the antitumor response. Hence, designing strategies aimed at rendering melanoma-infiltrating dendritic cells visible to host T cells may boost spontaneous antitumor immunity.

Tumor-infiltrating dendritic cells are potent antigen-presenting cells able to activate T cells and mediate tumor rejection.

Dendritic cells (DC) are potent inducers of immune responses. DC have been shown to infiltrate tumors, but very little is known about the functional status of these naturally occurring tumor-infiltrating DC (TIDC). In this study, the status and function of TIDC from several types of mouse melanoma were investigated in detail. CD11c+/MHC II+ cells, consistent with a DC phenotype, were found in all of transplantable or spontaneous melanomas studied. These TIDC were predominantly myeloid (CD11c+/CD8alpha-/B220-) in nature with small numbers of plasmacytoid (CD11c+/B220+). TIDC had an intermediate maturation phenotype with some expression of costimulatory molecules and the capacity to take up particles. Upon culture overnight ex vivo, the TIDC markedly up-regulated the expression of costimulatory molecules and also increased IL-12 production. Importantly, such ex vivo-matured TIDC pulsed with OVA were able to migrate to lymph nodes, to activate naive OVA-specific CD4+ and CD8+ T cells, and to confer protection against a challenge with OVA-expressing tumor cells. In conclusion, melanomas are infiltrated by functional DC that can act as fully competent APC. These APC have the potential to be manipulated and may therefore represent a promising target for cancer immunotherapy.