Notably, the slight inhibitory activity elicited by normal fibroblasts may not reflect their actual function in vivo, since, in culture, they may undergo artifactual activation (as also suggested by their faint but detectable, FAP expression;Table 1). fibroblasts was far stronger and directly correlated with their ability to produce PGE2either constitutively or upon induction by NK cells. Keywords:activating receptors, cytotoxicity, tumor microenvironment Over the past two decades, major advances have been made in the definition of NK cell function including their cytolytic activity against virus infected or tumor cells (13). The molecular mechanisms regulating AP521 NK cell functions have been defined thanks to the discovery of various activating and inhibitory receptors. These receptors primarily include inhibitory receptors specific for HLA class I molecules, such as killer-Ig-like receptors (KIRs) and NKG2A and activating receptors, such as NKp30, NKp46, NKp44 (collectively termed natural cytotoxicity receptors; NCRs), NKG2D, DNAM-1, NKp80, 2B4, NTBA, CRTAM. The activating receptors recognize various ligands expressed by transformed cells and/or by certain stressed or activated cells (48). Certain activating receptors including NKp44 and CD69 are induced (9,10), while others are up-regulated (NKp30, NKG2D, and, partially, NKp46) at the NK cell surface by cytokines, such as IL-2, known to potentiate NK cell activity (11). Upon recognition of specific ligands, NK-receptors can focalize NK cell responses against potentially harmful target cells avoiding reactivity with autologous HLA-class I+normal cells (4). Initially, this information could be achieved by the in vitro analysis of purified peripheral blood NK cell populations and clones expanded in the presence of IL-2 (10,12). Those studies however, did not explain how NK cell functions could actually be modulated in tissues in vivo. Recently, several studies attempted to clarify this point. For example, it has been shown that NK cell function can be modified sharply by several cytokines or AP521 enzymes including IL-15, IL-18, IL-21, IL-23, TGF-, or indoleamine 2,3-dioxigenase (IDO) (11,1318). In addition, different cell types including dendritic cells (DCs), plasmacytoid DC (pDC), monocytes, and mesenchymal stem cells (MSCs) have been demonstrated to interact with NK cells giving rise to different functional outcomes (1922). Finally, in pathologic conditions including allergy, NK-type lymphoproliferative disease of granular lymphocytes, non-small cell lung cancer, and AML, NK cells have AP521 been shown to display unique functional and/or phenotypic alterations (2326). These findings imply that effective NK cell responses established in vitro against transformed cells and/or pathogens may substantially differ in vivo as a consequence of NK cell interaction with different cell types in different inflammatory or tumor sites. In this context, it became increasingly evident that tumors can influence the nature and the efficacy of the immune responses of the host (27). For example, induction of regulatory T cells (Tregs) or myeloid suppressor cells and/or production of several immunosuppressive factors [including arginase-1, NOS-2, IDO, or TGF-] by tumor cells or components of tumor microenvironment may result in functional subversion of tumor-infiltrating immunocompetent cells (27). In this context, tumor-associated fibroblasts are a relevant component of the tumor microenvironment (28). However, very limited information is available on their ability to suppress immune responses. In this study, we show that fibroblasts derived from melanomas profoundly affect NK cell functions. Thus, NK cells reaching the tumor site may be greatly impaired in their anti-tumor activity by the tumor stroma itself. == Results == == Comparative Analysis of Fibroblasts Derived from Melanomas or Normal Skin. == Four primary fibroblast cell lines were established either from two melanoma metastases (lines TF1 and TF2) or from skin of two healthy individuals (lines HF1 and HF2). Their surface phenotype was analyzed by immunofluorescence and cytofluorimetric analysis. As shown inTable 1, all expressed prolyl-4-hydroxylase (P4H; an enzyme involved in the biosynthesis of collagen), but not the CD106 adhesion molecule (that is expressed by MSCs) (22,29) nor the tumor marker GD2 ganglioside. Notably, fibroblasts derived from tumor or normal tissue displayed some phenotypic variations: TF1 and TF2 were CD56-bad and indicated high levels of fibroblast activation protein (FAP); while HF1 and HF2 were CD56+and indicated lower levels of FAP. The manifestation of FAP has been reported as a distinctive feature of triggered fibroblasts (30). Therefore, the phenotypic profile suggests a different activation level on tumor-derived or normal fibroblasts, respectively. Concerning the known ligands for activating NK receptors, both tumor and IGF2R normal fibroblasts indicated the DNAM-1 receptor ligands PVR and Nectin-2, whereas NKG2D ligands were not indicated at significant levels.