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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.

RNA transfection of dendritic cells (DCs IntroductionDendritic cells (DCs) are recognized as the most powerful antigenpresenting cells that are able to induce and maintain primary immune responses in vitro and in vivo. [1][2][3] RNA transfection of DCs was demonstrated to be a highly efficient tool to elicit antigen-specific cytotoxic T lymphocytes (CTLs) capable of mediating tumor cell recognition and establishment of protective antitumor immunity. [4][5][6][7][8] Furthermore, several recent in vitro and in vivo studies indicate that RNA-transfected DCs can generate both CD8 ϩ -and CD4 ϩ -mediated immune responses, thus suggesting that DCs can process epitopes for human leukocyte antigen (HLA) class II-restricted presentation derived from newly synthesized cytosolic proteins. [9][10][11][12][13] Antigen processing for presentation on major histocompatibility complex (MHC) class II molecules involves a multitude of different compartments and proteases within the cell. 14 MHC class II molecules predominantly present peptides derived from exogenous proteins as well as epitopes from plasma membranes or endosomes. [15][16][17] However, biochemical and functional studies have shown that antigens localized within the cytoplasm can efficiently be processed for MHC class II-restricted presentation by human and murine antigen-presenting cells. [18][19][20][21] Several pathways involved in this process have been discussed and analyzed. Degradation of the proteins by the proteasome, translocation of epitopes from cytoplasm into membrane organelles, and autophagy were implicated in several experimental settings using defined antigens and delivery systems. 22,23 In our study, we addressed the pathways and requirements for processing and presentation of antigenic peptides for HLA class I and II presentation upon transfection of human monocyte-derived DCs with in vitro-transcribed RNA coding for a tumor-associated antigen. Our data demonstrate that the presentation of cytoplasmic proteins on HLA class II molecules requires the function of the proteasome, the involvement of lysosomal antigen degradation, processing in the lysosomal/endosomal vesicles, and autophagy. Materials and methods Reagents, 3-methyladenine (3-MA; 10 M), and wortmannin (10 to 30 nM) were all purchased from Sigma-Aldrich (Taufkirchen, Germany). Cathepsin B inhibitor II (10 M) was purchased from Calbiochem (Schwalbach, Germany). Cell isolation and generation of dendritic cells from adherent peripheral blood mononuclear cellsIn our study, we used buffy coats from healthy donors from the blood bank at the University of Tübingen, Germany. This was approved by our local ethics committee.Generation of DCs from peripheral blood monocytes was performed as described previously. 24 In brief, peripheral blood mononuclear cells The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 U.S.C. section 1734. For personal use only. o...

IntroductionImatinib mesylate, also known as STI571 or Glivec (Novartis, Basel, Switzerland), is a promising new treatment for chronic myelogenous leukemia (CML). Imatinib mesylate is a 2-phenylaminopyrimidine derivate that was designed as a selective competitive inhibitor of the Abl protein tyrosine kinases (v-Abl, Bcr-Abl, and c-Abl). [1][2][3][4][5] It also has strong activity against the platelet-derived growth-factor receptor (PDGF-R), c-Kit, ARG, and their fusion proteins Tel-Abl and Tel-PDGF-R, but does not affect other kinases. [6][7][8][9] Recent clinical trials of imatinib mesylate in the treatment of chronic-phase CML have demonstrated that the drug is well tolerated with only few adverse effects and can induce complete hematologic and cytogenetic responses in a significant proportion of patients. 2,3,[10][11][12] Moreover, activity of imatinib mesylate against more advanced, accelerated-phase blast crises and in patients with relapsed or refractory Philadelphia chromosomepositive (Ph ϩ ) acute lymphoid leukemias was reported. 13,14 Furthermore, in patients with gastrointestinal stroma tumors (GISTs), where activating mutations of c-Kit are likely responsible for the pathogenetic events, imatinib mesylate yielded encouraging results. 6,7,15,16 Because this tumor has so far been highly refractory to chemotherapy, imatinib mesylate is emerging as an important new therapeutic agent.However, the effects of imatinib mesylate on normal, nonmalignant hematopoietic cells have not been extensively evaluated so far. It is not clear whether some side effects like cytopenias that occur during treatment with imatinib mesylate may result from suppression of normal progenitor growth and differentiation. Recently, it was demonstrated that imatinib mesylate reduces the number of colony-forming cells in peripheral blood or bone marrow (BM) from patients with CML, with minimal effect on normal cells. 17 Dendritic cells (DCs) are recognized as the most powerful antigenpresenting cells (APCs) with the unique ability to initiate and maintain primary immune responses. They originate from BM-derived progenitor cells, spread via the bloodstream, and can be found in almost every organ as the sentinels of the immune system. In vitro, DCs can be generated from peripheral blood monocytes using granulocytemacrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The differentiation of DCs from CD34 ϩ progenitor cells can be mediated by different cytokines like GM-CSF, tumor necrosis factor ␣ (TNF-␣), IL-4, and FMS-like tyrosine kinase 3 (FLT3) ligand and stem cell factor (SCF). [18][19][20][21][22][23][24][25] Because SCF has been shown to play an important role in DC development, we here explored a potential effect of imatinib mesylate on the development of mobilized human CD34 ϩ peripheral blood progenitor cells (PBPCs) into DCs. We show that in vitro exposure of CD34 ϩ PBPCs to therapeutic concentrations of imatinib mesylate (1-5 M) affects the differentiation and functional properties of generated DCs via inhibit...

We analyzed herein whether members of the tetraspanin superfamily are involved in human immature dendritic cell (DC) functions such as foreign antigen internalization, phagocytosis, and cell migration. We show that CD63, CD9, CD81, CD82, and CD151 are present in immature DCs. Whereas CD9 and CD81 are mostly expressed at the cell surface, CD63 and CD82 are also located in intracellular organelles. Complexes of monoclonal antibody (Mab) FC-5.01-CD63 or Fab-5.01-CD63 were rapidly translocated "outside-in" and followed the endocytic pathway through early endosomes and lysosomes, reaching major histocompatibility complex (MHC) class IIenriched compartments (MIICs) in less than one hour. Internalization of CD63 was also observed during Saccharomyces cerevisiae phagocytosis. Moreover, an association of CD63 with the ␤-glycan receptor dectin-1 was observed. Mabs against CD9, CD63, CD81, and CD82 enhanced by 50% the migration induced by the chemokines macrophage inflammatory protein-5 (MIP- 5 IntroductionDendritic cells (DCs) comprise a family of professional antigenpresenting cells (APCs) that are sentinels of the immune system. 1 In this regard, DCs have been shown to efficiently stimulate both naive B and T cells and to elicit primary immune responses. 2,3 Their remarkable effectiveness is due to their ability to capture, process and present antigens along with costimulatory signals, and to migrate to secondary lymphoid tissues. 4 Different stages of maturation are responsible for the different functions of DCs. Immature DCs, widely present in peripheral tissues, efficiently uptake antigens but express moderate levels of major histocompatibility complex (MHC) class II and costimulatory molecules. In contrast, mature DCs poorly acquire antigens but express higher levels of MHC class II and costimulatory molecules, are able to migrate into lymph nodes, and become potent activators of resting T cells. [2][3][4][5] Since antigen internalization and processing, as well as cell migration, are essential properties of DCs, the study of the possible involvement of tetraspanins in these processes is a challenging question, as it could bring new insight into the physiologic role of these molecules in DCs and other cells. In recent years, considerable interest has arisen in the expanding tetraspanins family, which are integral membrane proteins with 2 extracellular domains (EC1 and EC2) that are variably glycosylated. 6 The most conspicuous members of this family are CD9, 7 CD63/lamp-3, 8 CD81/TAPA-1, 9 CD82/KAI1, 10 and CD151. 11 Tetraspanins form several specific complexes implicated in a variety of cellular processes such as migration, adhesion, proliferation, and signal transduction, initially leading to the idea that these proteins could play a "molecular facilitator" role. 6 There is growing evidence that points to a possible role of tetraspanins in antigen processing and presentation. In fact, it has been recently found that CD63 is modified after translation during maturation of DCs, and this event is accompanied by morph...