Gianni Gerlini

Metastatic Melanoma Secreted IL-10 Down-Regulates CD1 Molecules on Dendritic Cells in Metastatic Tumor Lesions

CD1 molecules are expressed by antigen-presenting cells such as dendritic cells and mediate primary immune responses to lipids and glycolipids which have been shown to be expressed by various tumors. Glycolipids are expressed by melanoma cells but, despite their immunogenicity, no efficient spontaneous immune responses are elicited. As IL-10 has previously been shown to down-regulate CD1a on dendritic cells and is known to be expressed by various melanoma cell lines, we investigated if melanoma-derived IL-10 could down-regulate CD1 molecule expression on dendritic cells as a possible way to circumvent immune recognition. We found that CD1a, CD1b, CD1c, and CD1d were significantly down-regulated on dendritic cells in metastatic (n = 10) but not in primary melanoma lesions (n = 10). We further detected significantly higher IL-10 protein levels in metastatic than in primary melanomas. Moreover, supernatants from metastatic melanomas were significantly more effective in down-regulating CD1 molecules on dendritic cells than supernatants from primary melanoma cultures. This effect was blocked using a neutralizing IL-10 antibody in a dose dependent manner. Our findings suggest that metastatic but not primary melanomas can down-regulate CD1 molecules on infiltrating dendritic cells by secreting IL-10 which may represent a novel way to escape the immune response directed against the tumor.

HEDGEHOG‐GLI Signaling Drives Self‐Renewal and Tumorigenicity of Human Melanoma‐Initiating Cells

The question of whether cancer stem/tumor‐initiating cells (CSC/TIC) exist in human melanomas has arisen in the last few years. Here, we have used nonadherent spheres and the aldehyde dehydrogenase (ALDH) enzymatic activity to enrich for CSC/TIC in a collection of human melanomas obtained from a broad spectrum of sites and stages. We find that melanomaspheres display extensive in vitro self‐renewal ability and sustain tumor growth in vivo, generating human melanoma xenografts that recapitulate the phenotypic composition of the parental tumor. Melanomaspheres express high levels of Hedgehog (HH) pathway components and of embryonic pluripotent stem cell factors SOX2, NANOG, OCT4, and KLF4. We show that human melanomas contain a subset of cells expressing high ALDH activity (ALDHhigh), which is endowed with higher self‐renewal and tumorigenic abilities than the ALDHlow population. A good correlation between the number of ALDHhigh cells and sphere formation efficiency was observed. Notably, both pharmacological inhibition of HH signaling by the SMOOTHENED (SMO) antagonist cyclopamine and GLI antagonist GANT61 and stable expression of shRNA targeting either SMO or GLI1 result in a significant decrease in melanoma stem cell self‐renewal in vitro and a reduction in the number of ALDHhigh melanoma stem cells. Finally, we show that interference with the HH‐GLI pathway through lentiviral‐mediated silencing of SMO and GLI1 drastically diminishes tumor initiation of ALDHhigh melanoma stem cells. In conclusion, our data indicate an essential role of the HH‐GLI1 signaling in controlling self‐renewal and tumor initiation of melanoma CSC/TIC. Targeting HH‐GLI1 is thus predicted to reduce the melanoma stem cell compartment. Stem Cells2012;30:1808–1818

A Key Role for Poly(ADP-Ribose) Polymerase-1 Activity during Human Dendritic Cell Maturation

Poly(ADP-ribose) (PAR) polymerase (PARP)-1 is a nuclear enzyme regulating protein that functions by targeting PAR chains. Besides its classic role in DNA repair, PARP-1 is emerging as a key transcriptional regulator in different cell types including the immune ones. In this study, we investigated the role of PARP-1 in human dendritic cell (DC) function. We report that both PARP-1 mRNA and protein levels significantly increased during in vitro DC differentiation from monocytes. Of note, inhibitors of PARP-1 such as phenanthridinone and thieno[2,3-c]isoquinolin-5-one reduced expression of CD86 and CD83 in a concentration-dependent manner, having no effects on expression of CD80 and HLA-DR in mature DCs. In the same cultures, PARP-1 inhibitors also reduced production of IL-12 and IL-10. Addition of exogenous IL-12 to the culture medium partially restored CD86 expression in DCs exposed to PARP-1 inhibitors. In line with the role of PAR formation in NF-κB-dependent transactivation, we also report that phenanthridinone and thieno[2,3-c]isoquinolin-5-one impaired NF-κB and AP-1 subunit DNA binding activity in cellular extract of activated DCs. Finally, we show that PARP-1 inhibitors reduced the T cell allostimulatory activity of mature DCs, and that this reduction was prevented when DCs matured in the presence of PARP-1 inhibitors plus IL-12. Of note, nonproliferating T cells exposed to PARP-1 inhibitor-challenged DCs could undergo efficient proliferation when exposed to a subsequent activation stimulus such as anti-CD3 plus anti-CD-28. Together, data provide evidence for a key role of PARP-1 and poly ADP-ribosylation in DC immunocompetence and underscore the relevance of PARP-1 inhibitors to treatment of immune disorders.

Stromal fibroblasts synergize with hypoxic oxidative stress to enhance melanoma aggressiveness

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.

Induction of CD83+CD14+ Nondendritic Antigen-Presenting Cells by Exposure of Monocytes to IFN-α

IFN-α is a well-known agent for treatment of viral and malignant diseases. It has several modes of actions, including direct influence on the immune system. We investigated IFN-α effects on PBMC in terms of dendritic cell (DC) differentiation, as PBMC are exposed to high IFN-α levels during treatment of infections and cancers. We show that in vitro IFN-α exposure induced rapid and strong up-regulation of the DC-maturation markers CD80, CD86, and CD83 in bulk PBMC. Consistently, IFN-α induced up-regulation of these molecules on purified monocytes within 24 h. Up-regulation of CD80 and CD83 expression was IFN-α concentration-dependent. In contrast to GM-CSF + IL-4-generated DCs, most of the IFN-α-challenged CD83+ cells coexpressed the monocyte marker CD14. Despite a typical mature DC immunophenotype, IFN-α-treated monocytes conserved phagocytic activity and never acquired a dendritic morphology. In mixed lymphocyte reactions IFN-α-treated monocytes were less potent than GM-CSF + IL-4-generated DCs but significantly more potent than untreated monocytes to induce T cell proliferation in bulk PBMC. However, only GM-CSF + IL-4-generated DCs were able to induce a significant proliferation of naive CD4+ T cells. Notably, autologous memory CD4+ T cells proliferated when exposed to tetanus toxoid-pulsed IFN-α-treated monocytes. At variance with untreated or GM-CSF + IL-4-exposed monocytes, those challenged with IFN-α showed long-lasting STAT-1 phosphorylation. Remarkably, CD83+CD14+ cells were present in varicella skin lesions in close contact with IFN-α-producing cells. The present findings suggest that IFN-α alone promptly generates nondendritic APCs able to stimulate memory immune responses. This may represent an additional mode of action of IFN-α in vivo.

Characterization of circulating and monocyte-derived dendritic cells in obese and diabetic patients

Dendritic cells (DCs) are suspected to be involved in the development of atherogenesis, but their role is still unclear. The aim of this study was to characterize circulating DCs and monocyte-derived DCs (Mo-DCs) of obese and diabetic patients (T2D), and to study their interaction with human coronary smooth muscle cells (CASMCs). Obese post-menopausal women with or without insulin resistance were enrolled and were compared to age-matched healthy women. Myeloid circulating DCs significantly increased in obese T2D patients compared to healthy donors and a smaller increase was observed for plasmacytoid one. Mature Mo-DCs from obese T2D patients significantly decreased when compared to control, but they were significantly more capable of adhering to CASMCs compared to that from healthy controls and from not-T2D obese subjects. Altogether these data suggest that in conditions of insulin-resistance and obesity there is an up-regulation of myeloid DCs that might contribute to pathological vascular remodeling.

Enhancing anti-melanoma immunity by electrochemotherapy and in vivo dendritic-cell activation

Combining electrochemotherapy with dendritic cell-based immunotherapy is a promising strategy against human metastatic melanoma that deserves to be clinically assessed. While electrochemotherapy induces a rapid regression of metastases, immunotherapy generates systemic anticancer immunity, contributes to eradicate the tumor and maintains an immunological memory to control relapse.

Human Langerhans cells are immature in melanoma sentinel lymph nodes

To the editor: The specific function of Langerhans cells (LCs) in the skin immune system is controversial and a matter of intense debate.[1][1] As epidermal LCs are potential targets for cancer immunotherapy, the topic is of primary importance.[2][2] Recently, van de Ven et al showed that LCs are

Control of the differentiation state and function of human epidermal Langerhans cells by cytokines in vitro

Langerhans cells can originate in vitro from immature precursors stimulated with granulocyte macrophage‐colony‐stimulating factor (GM‐CSF), tumour necrosis factor (TNF)‐α and stem cell factor (SCF). We asked whether these cytokines also control the differentiation state of Langerhans cells within the epidermis and upon leaving this tissue. We harvested sheets of human epidermis by controlled dispase hydrolysis of keratomes, cultured them in RPMI and 10% fetal calf serum for 48 h and analysed the sheets and the cells migrated spontaneously into the medium, most of which were Langerhans cells containing Birbeck granules.1 By flow cytometry, the intensity of CD1a expression was reduced quite evenly among Langerhans cells migrated from sheets within 48 h. The cells in the sheets underwent loss of dendrites, with a significant reduction in the cell perimeter that was prevented by GM‐CSF and TNF‐α together. Either of these cytokines induced expression of CD18 by cells in the sheets and those in the medium. Moreover, TNF‐α induced expression of CD54 by cells in the medium, but not by those retained in the sheets, whereas human SCF induced, dose dependently, expression of CD54 by cells in the sheets, but not from those in the medium.2 The proliferation of allogeneic lymphocytes was much higher when stimulating Langerhans cells were harvested from cultures with any cytokine, rather than from cultures without cytokines. We conclude the following: (i) GM‐CSF and TNF‐α help to maintain full differentiation of Langerhans cells within the epidermis; (ii) cytokine influence on Langerhans cells adhesiveness is in part context dependent; and (iii) pretreatment with cytokines influences positively the number or accessory activity of Langerhans cells on lymphocytes during subsequent mixed leucocyte reaction.

Stem cell factor affects tumour progression markers in metastatic melanoma cells.

Stem cell factor (SCF), next to various relevant biological effects exerted on many cell types, is able to keep melanocyte homeostasis through its receptor c-kit. Only a minority of metastatic melanoma cells (MMC) express c-kit receptor, but c-kit positive MMC move more slowly towards tumour progression and have a more natural tendency to undergo apoptosis. In our study c-kit positive MMC from human melanoma metastases and a c-kit positive human melanoma cell line—SK-MEL-28—showed a clear-cut reduction of cytokines normally up-regulated along melanoma progression after SCF stimulation. SCF was also able to maintain all MMC and SK-MEL-28 cells in a well differentiated status with an increase in organellogenesis and in particular of melanosomes in various degree of differentiation, but it did not induce apoptosis as observed in other in vitro models. The increase of melanosomes matched an increase of tyrosinase production. SCF did not modify the expression of NOS while it enhanced the expression of HLA-DR molecules on MMC membranes. Taken altogether these data stress the biological activity of SCF as a cytokine which is able to maintain MMC in a well differentiated status, and suggest a more in depth evaluation of possible effects of SCF on melanoma cells.