Melanomas resist T-cell therapy through inflammation-induced reversible dedifferentiation.

Adoptive cell transfer therapies (ACTs) with cytotoxic T cells that target melanocytic antigens can achieve remissions in patients with metastatic melanomas, but tumours frequently relapse. Hypotheses explaining the acquired resistance to ACTs include the selection of antigen-deficient tumour cell variants and the induction of T-cell tolerance. However, the lack of appropriate experimental melanoma models has so far impeded clear insights into the underlying mechanisms. Here we establish an effective ACT protocol in a genetically engineered mouse melanoma model that recapitulates tumour regression, remission and relapse as seen in patients. We report the unexpected observation that melanomas acquire ACT resistance through an inflammation-induced reversible loss of melanocytic antigens. In serial transplantation experiments, melanoma cells switch between a differentiated and a dedifferentiated phenotype in response to T-cell-driven inflammatory stimuli. We identified the proinflammatory cytokine tumour necrosis factor (TNF)-α as a crucial factor that directly caused reversible dedifferentiation of mouse and human melanoma cells. Tumour cells exposed to TNF-α were poorly recognized by T cells specific for melanocytic antigens, whereas recognition by T cells specific for non-melanocytic antigens was unaffected or even increased. Our results demonstrate that the phenotypic plasticity of melanoma cells in an inflammatory microenvironment contributes to tumour relapse after initially successful T-cell immunotherapy. On the basis of our work, we propose that future ACT protocols should simultaneously target melanocytic and non-melanocytic antigens to ensure broad recognition of both differentiated and dedifferentiated melanoma cells, and include strategies to sustain T-cell effector functions by blocking immune-inhibitory mechanisms in the tumour microenvironment.

Cannabinoid 1 receptors in keratinocytes modulate proinflammatory chemokine secretion and attenuate contact allergic inflammation.

Epidermal keratinocytes (KCs) and cannabinoid (CB) receptors both participate in the regulation of inflammatory responses in a mouse model for allergic contact dermatitis, the contact hypersensitivity (CHS) response to the obligate sensitizer 2,4-dinitrofluorobenzene. In this study, we investigated the cellular and molecular mechanisms how CB1 receptors attenuate CHS responses to 2,4-dinitrofluorobenzene. We used a conditional gene-targeting approach to identify the relative contribution of CB1 receptors on epidermal KCs for the control of CHS responses. To determine the underlying cellular and molecular mechanisms that regulate inflammatory responses in the effector phase of CHS, we performed further investigations on inflamed ear tissue and primary KC cultures using morphologic, molecular, and immunologic methods. Mice with a KC-specific deletion of CB1 receptors developed increased and prolonged CHS responses. These were associated with enhanced reactive epidermal acanthosis and inflammatory KC hyperproliferation in the effector phase of CHS. In vitro, primary cultures of CB1 receptor-deficient KC released increased amounts of CXCL10 and CCL8 after stimulation with IFN-γ compared with controls. In vivo, contact allergic ear tissue of CB1 receptor-deficient KCs showed enhanced expression of CXCL10 and CCL8 compared with controls. Further investigations established CCL8 as a proinflammatory chemokine regulated by CB1 receptors that promotes immune cell recruitment to allergen-challenged skin. Taken together, these results demonstrate that CB1 receptors are functionally expressed by KCs in vivo and help to limit the secretion of proinflammatory chemokines that regulate T cell-dependent inflammation in the effector phase of CHS.

Autochthonous primary and metastatic melanomas in Hgf-Cdk4 R24C mice evade T-cell-mediated immune surveillance.

Genetically engineered mouse models offer new opportunities to investigate the role of cell-mediated immunity in the natural progression of melanoma in an immunocompetent host. Here we report that Hgf-Cdk4(R24C) mice spontaneously develop a spectrum of primary melanomas with high penetrance during their first year of life. Malignant transformation proceeds in a stepwise manner from multiple melanocytic nevi to single nodular melanomas and disseminated metastases in most mice. Migrating melanoma cells invade the draining lymph nodes without activating the immune system. Autochthonous primary tumors are destroyed following experimental introduction of immune surveillance using an adoptive lymphocyte transfer approach. However, some tumor cells are able to survive, evade immune cell control, and recur both locally and systemically. Immune tolerance in recurring tumors may be supported by immunosuppressive Gr1(+) myeloid cells. Taken together, our results demonstrate that primary and metastatic melanomas developing spontaneously in Hgf-Cdk4(R24C) mice effectively evade cellular immune surveillance.

Complete regression of advanced primary and metastatic mouse melanomas following combination chemoimmunotherapy.

The development of therapeutic strategies which induce effective cellular antitumor immunity represents an important goal in cancer immunology. Here, we used the unique features of the genetically engineered Hgf-Cdk4(R24C) mouse model to identify a combination chemoimmunotherapy for melanoma. These mice develop primary cutaneous melanomas which grow progressively and metastasize in the absence of immunogenic foreign proteins such as oncogenes or antigens. Primary and metastatic tumors evade innate and adaptive immune defenses, although they naturally express melanocytic antigens which can be recognized by antigen-specific T cells. We found that primary melanomas continued to grow despite infiltration with adoptively transferred, in vivo-activated, tumor-specific CD8(+) T cells. To promote tumor immune defense, we developed a treatment protocol consisting of four complementary components: (a) chemotherapeutic preconditioning prior to (b) adoptive lymphocyte transfer and (c) viral vaccination followed by (d) adjuvant peritumoral injections of immunostimulatory nucleic acids. Lymphocyte ablation and innate antiviral immune stimulation cooperatively enhanced the expansion and the effector cell differentiation of adoptively transferred lymphocytes. The efficacy of the different treatment approaches converged in the tumor microenvironment and induced a strong cytotoxic inflammatory response enabling preferential recognition and destruction of melanoma cells. This combination chemoimmunotherapy caused complete regression of advanced primary melanomas in the skin and metastases in the lung with minimal autoimmune side effects. Our results in a clinically highly relevant experimental model provide a scientific rationale to evaluate similar strategies which unleash the power of innate and adaptive immune defense in future clinical trials.