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Langerhans cells (LCs) are crucial regulators of anti-cancer immune responses. Cancer, however, can alter DCs functions leading to tolerance. The enzyme indoleamine 2,3-dioxygenase (IDO1) plays a crucial role in this process. In sentinel lymph nodes (SLNs) of patients with melanoma, LCs show phenotypical and functional alterations favoring tolerance. Herein we aimed to investigate IDO1 expression in SLN LCs from patients with melanoma. We showed by immunofluorescence analysis that a portion of Langerin+ LCs, located in the SLN T cell-rich area, displayed the typical dendritic morphology and expressed IDO1. There was no significant difference in the expression of IDO between SLN with or without metastases. Double IDO1/CD83 staining identified four LCs subsets: real mature IDO1−CD83+ LCs; real immature IDO1−CD83− LCs; tolerogenic mature IDO1+CD83+ LCs; tolerogenic immature IDO1+CD83− LCs. The latter subset was significantly increased in metastatic SLNs as compared to negative ones (p < 0.05), and in SLN LCs of patients with mitotic rate (MR) > 1 in primary melanoma, as compared to MR ≤ 1 (p < 0.05). Finally, immature SLN LCs, after in vitro stimulation by inflammatory cytokines, acquired a maturation profile by CD83 up-regulation. These results provide new input for immunotherapeutic approaches targeting in vivo LC of patients with melanoma.

Langerhans cells (LCs) from melanoma patients sentinel lymph nodes (SLN) are poor T cell activators mostly due to an immature immunophenotype. However Antigen Presenting Machinery (APM) role is unknown. We investigated HLA-class I APM components (Delta, LMP-7/10, TAP-1, Calnexin, Tapasin, β2-microglobulin and HLA-A,B,C) in LCs from healthy donors skin and melanoma patients SLN. APM component levels were low in immature epidermal LCs and significantly increased after maturation (p<0.05); their levels were significantly high in SLN LCs (p<0.01). APM component expression correlated with melanoma Breslow's thickness and SLN metastases: HLA-A,B,C level was significantly lower in SLN LCs from thick lesions patients compared with those from thin/intermediate lesions (p<0.05); β2-microglobulin level was significantly higher in positive SLN LCs compared to negative ones (p<0.05). Functionally, SLN LCs did not phagocytose exogenous antigens. These findings extend LCs knowledge indicating that they are not fully impaired by melanoma, contributing to design new LCs-based therapeutic approaches., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Electrochemotherapy (ECT) is a novel treatment for recurrent or in-transit unresectable melanoma metastases based on the administration of anti-neoplastic drugs followed by cancer cell electroporation. Whether ECT can also induce anti-tumour immunity is unclear. We addressed this issue investigating the presence of dendritic cells (DCs) in the inflammatory infiltrate of ECT-treated lesions. Biopsies from melanoma patients (n = 9) were taken before ECT (T0), at d7 and d14 after treatment and studied by immunofluorescence with DCs-related antibodies. Epidermal Langerin(+) Langerhans cells (LCs) were the most represented subset before treatment. ECT induced a significant reduction in epidermal LCs number at d7 (p < 0.001), while they were completely replaced at d14. Similarly, the few LCs observed intermingled with metastatic melanoma cells at T0 decreased after treatment (p < 0.001), suggesting an ECT-induced activation of LCs. Consistently, at d1 after ECT (n = 3 patients), LCs were found to express CCR7, which mediates LCs migration to regional lymph nodes, and CD83, the typical DCs maturation marker. In contrast, plasmacytoid DCs (pDCs) were not present at T0, but significantly increased after ECT both in melanoma metastasis (p < 0.001) and perilesionally (p < 0.05). Similarly, CD1c(+) dermal DCs (dDCs), observed in low number before ECT, strongly increased at d7 and even more at d14 (p < 0.05 and p < 0.001, respectively). Notably, some dDCs expressed CD83. These data suggest that ECT promotes LCs migration from the tumour to draining lymph nodes and pDCs and dDCs recruitment at the site of the lesion. These findings may help to design new strategies of in situ DCs vaccination in cancer patients.

On the basis of recent advances indicating a key role of microenvironment for tumor progression, we investigated the role of fibroblasts, macrophages and hypoxia, for primary melanoma aggressiveness. Our data indicate a key role of hypoxia in stromal reactivity, acting on both myofibroblasts and machrophages differentiation. Hypoxic myofibroblasts are more active than macrophages in inducing melanoma invasiveness and exploit their oxidative stress due to hypoxia to secrete soluble factors favouring melanoma invasion and chemotaxis. We underscore the key role of microenviroment on melanoma malignancy, highlighting reactive fibroblasts, intratumoral hypoxia and oxidative stress as promising targets for melanoma antimetastatic strategies., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Plasmacytoid dendritic cells (pDC) represent the main source of interferon-alpha, a cytokine with antitumor activity. However, in vitro studies point to pDC as a key subset for induction of tolerance. Herein, we investigated pDC in sentinel lymph nodes (SLN) of melanoma patients. We report that pDC were constantly found in SLN and represented, with Langerhans cells, the most frequent dendritic cell subset. Their frequency in positive (with metastasis) SLN was significantly higher than in negative (without metastasis) SLN. PDC were observed in the T cell-rich areas of lymph nodes, particularly around high endothelial venules and, in metastatic nodes, they accumulated in close vicinity with melanoma nests. Finally, pDC capability to produce interferon-alpha in situ was impaired. Consistently, pDC expressed CD86, but neither CD80 nor CD83, suggesting a not complete activation in melanoma-draining lymph nodes. These results are consistent with the hypothesis of a tolerogenic role played by pDC in tumor immunology.

Langerhans cells (LCs) are crucial regulators of anti-cancer immune responses. Cancer, however, can alter DCs functions leading to tolerance. The enzyme indoleamine 2,3-dioxygenase (IDO1) plays a crucial role in this process. In sentinel lymph nodes (SLNs) of patients with melanoma, LCs show phenotypical and functional alterations favoring tolerance. Herein we aimed to investigate IDO1 expression in SLN LCs from patients with melanoma. We showed by immunofluorescence analysis that a portion of Langerin+ LCs, located in the SLN T cell-rich area, displayed the typical dendritic morphology and expressed IDO1. There was no significant difference in the expression of IDO between SLN with or without metastases. Double IDO1/CD83 staining identified four LCs subsets: real mature IDO1−CD83+ LCs; real immature IDO1−CD83− LCs; tolerogenic mature IDO1+CD83+ LCs; tolerogenic immature IDO1+CD83− LCs. The latter subset was significantly increased in metastatic SLNs as compared to negative ones (p < 0.05), and in SLN LCs of patients with mitotic rate (MR) > 1 in primary melanoma, as compared to MR ≤ 1 (p < 0.05). Finally, immature SLN LCs, after in vitro stimulation by inflammatory cytokines, acquired a maturation profile by CD83 up-regulation. These results provide new input for immunotherapeutic approaches targeting in vivo LC of patients with melanoma. [ABSTRACT FROM AUTHOR]