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Background Chemokine ligand-20 (CCL20) expressed in the epidermis is a potent impetus for the recruitment of CC-chemokine receptor 6 (CCR6)-expressing subsets of DCs, B-cells and memory T-cells into the skin. CCL20 and CCR6+ immune cells have been detected in chronic inflammatory skin diseases and several malignancies, including melanoma. Yet, the functional contribution of the CCR6/CCL20 axis for melanoma progression remains controversial. Objective The functional contribution of CCR6-expressing immune cell subsets and local CCL20 in the tumor microenvironment for the immune control of melanoma was studied. Methods Homeostatic and inducible CCL20 secretion of murine (B16, Ret) and human (A375, C32) melanoma cells was analyzed by ELISA. To assess the functional relevance of CCR6/CCL20 interactions on local tumor progression, prestimulated or retrovirally transduced B16/F1 melanoma cells overexpressing CCL20 (B16-CCL20) were injected subcutaneously into C57BL/6 Wt mice and congenic CCR6-deficient (CCR6−/−) mice. Infiltrating leucocytes were examined by flow cytometry in tumors and draining lymph nodes (DLNs). Results Melanoma cell lines up-regulate CCL20 secretion upon stimulation with pro-inflammatory cytokines in vitro. While only moderate changes in phenotype and composition of leucocytes were detected in advanced tumors and DLNs, mice injected with CCR6+ B16-CCL20 cells developed smaller tumors compared to B16-Control injected littermates, with CCR6-/- mice displaying the most pronounced reduction in tumor growth and incidence. Conclusion Our results suggest that CCR6/CCL20 interactions and individual independent effects of CCL20 and CCR6 in the microenvironment may be essential for melanoma progression and suggest a decisive role of this chemokine axis for melanoma pathogenesis beyond chemoattraction.

Adenosine (Ado) produced by skin and skin migratory CD73

Dendritic cells (DCs) express the ecto-5′-nucleotidase CD73 that generates immunosuppressive adenosine (Ado) by dephosphorylation of extracellular Ado monophosphate and diphosphate. To investigate whether CD73-derived Ado has immune-suppressive activity, 2,4-dinitrothiocyanobenzene (DNTB) was applied to skin of wild-type (WT) or CD73-deficient (CD73–/–) mice, followed by sensitization and challenge with 2,4-dinitrofluorobenzene. In this model, we show the induction of tolerance by DNTB against 2,4-dinitrofluorobenzene only in WT but not in CD73–/– mice. Analysis of skin DCs showed increased expression of CD73 after application of DNTB in WT mice. That was accompanied by elevated concentrations of extracellular Ado in the lymph node. Moreover, T cells expressed markers for anergy, namely EGR2 and NDRG1 in DNTB-treated WT mice and they exhibited impaired proliferation upon ex vivo re-stimulation. Similarly, in vitro we observed that Ado-producing WT DCs, but not CD73–/– DCs, rendered transgenic T cells from OTII mice (OTII T cells) hyporeactive, decreased their T-cell costimulatory signaling, and induced up-regulation of EGR2 and NDRG1. Thus, these data show that expression of CD73 by DCs, which triggers elevated levels of extracellular Ado, is a crucial mechanism for the induction of anergic T cells and tolerance.

Dendritic cells (DCs) are important inducers and regulators of T-cell responses. They are able to activate and modulate the differentiation of CD4+ and CD8+ T cells. In the skin, there are at least five phenotypically distinct DC subpopulations that can be distinguished by differential expression of the cell surface markers CD207, CD103, and CD11b. Previous studies have suggested that dermal CD11b−CD207+ conventional type 1 DCs are indispensable for the priming of a skin homing cytotoxic T-lymphocyte response. However, conventional type 1 DCs are also the only skin DC subset capable of cross-presenting exogenous antigens on major histocompatibility complex class I. Thus, it remained unclear whether for antigens that do not require cross-presentation, such as viruses that infect DCs, other DC subtypes in the skin can contribute to cytotoxic T-lymphocyte priming. To address this question, we used a transgenic mouse model that allows inducible expression and presentation of a model antigen on selected subsets of dermal DCs. We show that for antigens presented via the conventional major histocompatibility complex class I presentation pathway, CD207– dermal DCs are fully competent to prime a skin homing cytotoxic T-lymphocyte response that is capable of protection against a local virus challenge and gives rise to skin resident memory CD8+ T cells.

Background Application of haptens to the skin induces release of immune stimulatory ATP into the extracellular space. This “danger” signal can be converted to immunosuppressive adenosine (ADO) by the action of the ectonucleotidases CD39 and CD73, expressed by skin and immune cells. Thus, the expression and regulation of CD73 by skin derived cells may have crucial influence on the outcome of contact hypersensitivity (CHS) reactions. Objective To investigate the role of CD73 expression during 2,4,6-trinitrochlorobenzene (TNCB) induced CHS reactions. Methods Wild type (wt) and CD73 deficient mice were subjected to TNCB induced CHS. In the different mouse strains the resulting ear swelling reaction was recorded along with a detailed phenotypic analysis of the skin migrating subsets of dendritic cells (DC). Results In CD73 deficient animals the motility of DC was higher as compared to wt animals and in particular after sensitization we found increased migration of Langerin + DC from skin to draining lymph nodes (LN). In the TNCB model this led to a stronger sensitization as indicated by increased frequency of interferon-γ producing T cells in the LN and an increased ear thickness after challenge. Conclusion CD73 derived ADO production slows down migration of Langerin + DC from skin to LN. This may be a crucial mechanism to avoid over boarding immune reactions against haptens.

Dendritic cells (DCs) express the ecto-5'-nucleotidase CD73 that generates immunosuppressive adenosine (Ado) by dephosphorylation of extracellular Ado monophosphate and diphosphate. To investigate whether CD73-derived Ado has immune-suppressive activity, 2,4-dinitrothiocyanobenzene (DNTB) was applied to skin of wild-type (WT) or CD73-deficient (CD73