Glucocorticoid-induced TNF-related protein (GITR) has been shown to stimulate T cell-mediated antitumor immunity in mice. However, the functional relevance of GITR and its ligand (GITRL) for non-T cells has yet to be fully explored. In addition, recent evidence suggests that GITR plays different roles in mice and humans. We studied the role of GITR-GITRL interaction in human tumor immunology and report for the first time that primary gastrointestinal cancers and tumor cell lines of different histological origin express substantial levels of GITRL. Signaling through GITRL down-regulated the expression of the immunostimulatory molecules CD40 and CD54 and the adhesion molecule EpCAM, and induced production of the immunosuppressive cytokine TGF-beta by tumor cells. On NK cells, GITR is constitutively expressed and up-regulated following activation. Blocking GITR-GITRL interaction in cocultures of tumor cells and NK cells substantially increased cytotoxicity and IFN-gamma production of NK cells demonstrating that constitutive expression of GITRL by tumor cells diminishes NK cell antitumor immunity. GITRL-Ig fusion protein or cell surface-expressed GITRL did not induce apoptosis in NK cells, but diminished nuclear localized c-Rel and RelB, indicating that GITR might negatively modulate NK cell NF-kappaB activity. Taken together, our data indicate that tumor-expressed GITRL mediates immunosubversion in humans.

Dendritic cells (DCs) play an important role in initiating and maintaining primary immune responses. However, mechanisms involved in the resolution of these responses are elusive. We analyzed the effects of 15d-PGJ2 and the synthetic peroxisome proliferator-activated receptor (PPAR)-gamma ligand troglitazone (TGZ) on the immunogenicity of human monocyte-derived DCs upon stimulation with toll-like receptor (TLR) ligands. Activation of PPAR-gamma resulted in a reduced stimulation of DCs via the TLR ligands 2, 3, 4, and 7, characterized by down-regulation of costimulatory and adhesion molecules and reduced secretion of cytokines and chemokines involved in T-lymphocyte activation and recruitment. MCP-1 (monocyte chemotactic protein-1) production was increased due to PPAR-gamma activation. Furthermore, TGZ-treated DCs showed a significantly reduced capacity to stimulate T-cell proliferation, emphasizing the inhibitory effect of PPAR-gamma activation on TLR-induced DC maturation. Western blot analyses revealed that these inhibitory effects on TLR-induced DC activation were mediated via inhibition of the NF-kappaB and mitogen-activated protein (MAP) kinase pathways while not affecting the PI3 kinase/Akt signaling. Our data demonstrate that inhibition of the MAP kinase and NF-kappaB pathways is critically involved in the regulation of TLR and PPAR-gamma-mediated signaling in DCs.

IntroductionDendritic cells (DCs) are recognized as the most efficient antigenpresenting cells that initiate antigen-specific immune responses. They reside in an immature state in peripheral tissues, where they sense their environment and take up antigens. Upon activation through inflammatory mediators or pathogen-derived products, they change their expression pattern of various cell-surface molecules and secreted mediators like cytokines and chemokines. These alterations enable DCs to migrate to lymphoid tissues, where they present antigens and induce differentiation of both naive CD4 ϩ and CD8 ϩ T lymphocytes. These functional changes are accompanied with a lower capacity to take up soluble and cellular antigens by phagocytosis or pinocytosis by mature DCs and an increased ability to stimulate T-cell responses. 1,2 Usually, antigens from exogenous sources are taken up by DCs, become processed, and are presented to CD4 ϩ T lymphocytes on MHC class II molecules while intracellular-derived peptides are loaded onto MHC class I molecules where they stimulate antigenspecific CD8 ϩ T lymphocytes. 3 However, DCs are able to present antigens taken up from their environment on MHC class I molecules in a process called cross-presentation. This mechanism enables these cells to raise immune responses against tumor cells or pathogens like viruses that do not infect themselves. [4][5][6][7][8] DCs employ various molecules to sense their environment for pathogen-associated molecular patterns (PAMPs), microbial elementary components that are not or only minimal subjected to host adaptation. The most prominent of these receptors resembles the Toll-like receptor (TLR) family. TLR family members recognize microbial components such as lipopolysaccharide (LPS), flagellin, lipopeptides, or nucleic acids, and therefore initiate specific signaling pathways that lead to distinct immune responses. 9 In our study, we analyzed the effects of TLR-mediated DC activation on uptake of cellular antigens and their crosspresentation on MHC class I molecules. We show that TLR3 or TLR4 but not TLR2 or TLR7/8 matured DCs are impaired in the phagocytosis of cellular material and subsequent cross-presentation of antigens on MHC class I molecules. Materials and methodsApproval was obtained from the ethics committee of the University of Tübingen for these studies. In our study we used buffy coats provided by the local blood bank. Informed consent was provided according to the Declaration of Helsinki. CellsHEK-293 cells (embryonal kidney; DSMZ, Braunschweig, Germany) were cultured in RP10 medium (RPMI 1640 with glutamax I, supplemented with 10% inactivated fetal calf serum [FCS] and antibiotics; Invitrogen, Karlsruhe, Germany). Generation of DCsDCs were generated from peripheral blood monocytes by magnetic cell sorting as described previously. 10 In brief, peripheral blood mononuclear cells were isolated by Ficoll/Paque (Biochrom, Berlin, Germany) density gradient centrifugation of blood obtained from buffy coats of healthy volunteers from the blood ba...

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