hDectin-1 is involved in uptake and cross-presentation of cellular antigens.

Human Dectin-1 (hDectin-1) is a member of the C-type lectin-like receptor family that was shown to be the major receptor for fungal beta-glucans and to play an important role in the cellular responses mediated by these carbohydrates. In this study, we demonstrate that hDectin-1 is involved in the uptake and cross-presentation of cellular antigens. Furthermore, activation of monocyte-derived dendritic cells (MDCs) with toll-like receptor 3 (TLR3) ligand but not with TLR2 ligand or TLR7 ligand resulted in down-regulation of hDectin-1 expression and reduced phagocytosis of apoptotic tumor cells as well as presentation of pp65-derived T-cell epitopes upon engulfment of cytomegalovirus (CMV)-infected human foreskin fibroblasts.

Epithelial-specific transcription factor ESE-3 is involved in the development of monocyte-derived DCs.

Dendritic cells (DCs) are recognized as the most potent antigen-presenting cells of the immune system with the unique ability to initiate and maintain primary immune responses. In order to better characterize the functional and phenotypic features of DCs, a subtractive cDNA library to identify differentially expressed genes in monocyte-derived DCs (MDCs) was constructed. Using this approach, we found that the epithelial transcription factor ESE-3, which was previously shown to be exclusively expressed in cells of epithelial origin, is differentially expressed in MDCs. This was further confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analyses. The expression of ESE-3 is up-regulated upon maturation of MDCs and inhibited by treating the cells with IL-10 or IFN-gamma. Knockdown experiments using siRNA suggest that ESE-3 plays an important role during MDC development. Our results might help to improve the phenotypic characterization of DCs and lead to a better understanding of the cellular mechanisms involved in antigen presentation and T-cell stimulation.

Transfection of dendritic cells with in vitro-transcribed CMV RNA induces polyclonal CD8+- and CD4+-mediated CMV-specific T cell responses.

Transfection of dendritic cells (DCs) with RNA was shown to be effective in the generation of antigen-specific T cells, probably due to the induction of a polyclonal T cell response directed against multiple antigens. To verify this assumption we used DCs, generated from cytomegalovirus (CMV)-negative or -positive donors, that were electroporated with in vitro-transcribed RNA (in vitro transcript, IVT) coding for the CMV pp65 antigen. We found that transfection of DCs with pp65 IVT induces an expansion of polyclonal CD8(+) T lymphocytes that recognize peptide antigens presented on different HLA molecules. These T lymphocytes are able to lyse DCs pulsed with pp65-derived peptides or transfected with the cognate IVT. Furthermore, this approach allowed the identification of immunodominant epitopes presented upon IVT transfection. Interestingly, transfection of DCs with pp65 IVT resulted in the induction of CD4(+)-specific T cells. Cotransfection of DCs with IVTs coding for the CMV antigens pp65 and IE1 elicited polyclonal T lymphocytes specific for peptides derived from both antigens. More importantly, cytotoxic T cells could be generated in two of three CMV-negative donors. Finally, functional CMV-specific autologous cytotoxic T lymphocytes were successfully generated from immunosuppressed patients after allogeneic hematopoietic stem cell transplantation.

PPAR-gamma agonists inhibit toll-like receptor-mediated activation of dendritic cells via the MAP kinase and NF-kappaB pathways.

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.

Cotransfection of dendritic cells with RNA coding for HER-2/neu and 4-1BBL increases the induction of tumor antigen specific cytotoxic T lymphocytes.

Ribonucleic acid (RNA) transfection of dendritic cells (DCs) was shown to be highly efficient in eliciting CD8+ and CD4+ T-cell responses. We analyzed whether electroporation of DCs with RNA coding for a tumor-associated antigen (TAA) would elicit antigen-specific effector cytotoxic T lymphocyte (CTL) responses and whether these responses could be modulated by cotransfection with a second specific synthetic RNA. Therefore in vitro generated human monocyte-derived DCs were electroporated with in vitro transcribed RNA (in vitro transcript, IVT) encoding the TAA HER-2/neu. Additionally, these cells were cotransfected with IVT coding for human 4-1BBL. Transfection of DCs with 4-1BBL-IVT did not alter their typical phenotype. However, it increased the expression of the costimulatory molecules CD80 and CD40. Coadministration of HER-2/neu- and 4-1BBL-IVT resulted in an increased specific lysis of target cells by the in vitro induced CTL lines, indicating that 4-1BBL enhances their ability to elicit primary CTL responses. Interestingly, transfection of DCs with 4-1BBL-IVT did not augment their capacity to stimulate allogeneic lymphocyte responses. The here established approach of cotransfection of DCs with tumor-RNA and a second specific IVT could improve and optimize the in vitro manipulation of DCs for the induction of antigen-specific CTL responses.

Effects of imatinib on monocyte-derived dendritic cells are mediated by inhibition of nuclear factor-kappaB and Akt signaling pathways.

Dendritic cells are the most powerful antigen-presenting cells playing a decisive role for the initiation and maintenance of primary immune responses. However, signaling pathways involved in the differentiation of these cells have not been fully determined. Imatinib is a novel tyrosine kinase inhibitor effective against Abl kinases, c-Kit, and platelet-derived growth factor receptor. Using this compound, we show that human monocyte-derived dendritic cells generated in the presence of therapeutic concentrations of imatinib show a reduced expression of CD1a, MHC class I and II, and costimulatory molecules as well as decreased secretion of chemokines and cytokines resulting in an impaired capacity of dendritic cells to elicit primary T-cell responses. Using Western blot analyses, we found that these effects are mediated by inhibition of phosphatidylinositol 3-kinase/Akt pathways and a pronounced down-regulation of nuclear localized protein levels of nuclear factor-kappaB family members. Importantly, using blocking antibodies and tyrosine kinase inhibitors, we show that the inhibitory effects of imatinib on dendritic cell differentiation are not mediated via platelet-derived growth factor receptor and c-Kit. Taken together, our study reveals that imatinib inhibits dendritic cell differentiation and function via Akt and nuclear factor-kappaB signal transduction. Importantly, we show that imatinib can inhibit the function of normal, nonmalignant cells that may result in immunosuppression of these patients.

Processing and presentation of HLA class I and II epitopes by dendritic cells after transfection with in vitro-transcribed MUC1 RNA.

RNA transfection of dendritic cells (DCs) was shown to be highly efficient in eliciting CD8+ and CD4+ T-cell responses. However, antigen presentation pathways involved in generation of human leukocyte antigen (HLA) class I and class II peptides have remained elusive. To analyze this we incubated mucin 1 (MUC1) RNA-transfected DCs with compounds known to inhibit HLA class I presentation and used these cells in chromium 51 (51Cr)-release assays. As effectors, we used cytotoxic T lymphocyte (CTL) lines specific for the MUC1 peptides M1.1 and M1.2. We observed that the presentation of HLA-A*02 epitopes is inhibited by brefeldin A and lactacystin. To determine the requirement of a functional transporter associated with antigen processing (TAP), we cotransfected DCs with MUC1 and infected cell peptide 47 (ICP47) RNA. ICP47 could only inhibit the presentation of the M1.1 but not the M1.2 peptide, indicating that this epitope derived from the signal sequence is presented independently of TAP. Cocultivation of MUC1 RNA-transfected DCs with MUC1-specific CD4+ T lymphocytes revealed that the presentation of HLA class II peptides is sensitive to proteasomal inhibitors and brefeldin A. Furthermore, the presentation pathway requires lysosomal and endosomal processing and is mediated by autophagy. Our results demonstrate that the efficient presentation of cytosolic proteins on major histocompatibility complex (MHC) class II combines the proteolytic and lysosomal pathways.

Identification of C-met oncogene as a broadly expressed tumor-associated antigen recognized by cytotoxic T-lymphocytes.

PURPOSE: C-Met proto-oncogene is a receptor tyrosine kinase that mediates the oncogenic activities of the hepatocyte growth factor. Using a DNA chip analysis of tumor samples from patients with renal cell carcinoma and sequencing of peptides bound to the HLA-A*0201 molecules on tumor cells a peptide derived from the c-Met protein was identified recently. EXPERIMENTAL DESIGN: We used this novel HLA-A*0201 peptide for the induction of specific CTLs to analyze the presentation of this epitope by malignant cells. RESULTS: The induced CTL efficiently lysed target cells pulsed with the cognate peptide, as well as HLA-A*0201-matched tumor cell lines in an antigen-specific and HLA-restricted manner. Furthermore, the induced c-Met-specific CTLs recognized autologous dendritic cells (DCs) pulsed with the peptide or transfected with whole-tumor mRNA purified from c-Met-expressing cell lines. We next induced c-Met-specific CTLs using peripheral blood mononuclear cells and DC from an HLA-A*0201-positive patient with plasma cell leukemia to determine the recognition of primary autologous malignant cells. These CTLs lysed malignant plasma cells while sparing nonmalignant B- and T-lymphocytes, monocytes, and DCs. CONCLUSION: Our results demonstrate that c-Met oncogene is a novel tumor rejection antigen recognized by CTL and expressed on a broad variety of epithelial and hematopoietic malignant cells.

Induction of adipophilin-specific cytotoxic T lymphocytes using a novel HLA-A2-binding peptide that mediates tumor cell lysis.

Identification of tumor-associated antigens and advances in tumor immunology resulted in the development of vaccination strategies to treat patients with malignant diseases. Using a novel approach that combines DNA chip analysis of tumor samples with isolation of peptides on the surface of tumor cells, a HLA-A*0201-binding peptide derived from the adipophilin protein was identified. Adipophilin is involved in lipid storage and was thought to be expressed only in adipocytes, but it can be found in other cell types such as macrophages or tumor cells. In the present study, we analyzed the possible use of this peptide as a T-cell epitope presented by malignant cells. To accomplish this, we induced CTL responses using this HLA-A*0201-binding peptide. The in vitro-induced CTLs efficiently lysed cells pulsed with the adipophilin peptide and HLA-matched tumor cell lines in an antigen-specific and HLA-restricted manner. Finally, the induced CTLs recognized autologous dendritic cells (DCs) pulsed with the antigenic peptide or transfected with tumor RNA purified from an adipophilin-expressing tumor cell line. To further analyze the possible use of this peptide in immunotherapies of human malignancies, we induced adipophilin-specific CTLs using peripheral blood mononuclear cells and DCs from HLA-A*0201-positive patients with chronic lymphatic leukemia and plasma cell leukemia. The in vitro-generated CTLs recognized autologous chronic lymphatic leukemia cells and malignant plasma cells, whereas they spared nonmalignant resting or activated B and T lymphocytes, monocytes, or DCs. Our results demonstrate that this peptide might represent an interesting candidate for the development of cancer vaccines designed to target adipophilin-derived epitopes in a wide range of malignancies.

Imatinib mesylate affects the development and function of dendritic cells generated from CD34+ peripheral blood progenitor cells.

Imatinib mesylate (STI571) is a competitive Bcr-Abl tyrosine kinase inhibitor and has yielded encouraging results in treatment of chronic myelogenous leukemia (CML) and gastrointestinal stroma tumors (GISTs). Apart from inhibition of the Abl protein tyrosine kinases, it also shows activity against platelet-derived growth factor receptor (PDGF-R), c-Kit, Abl-related gene (ARG), and their fusion proteins while sparing other kinases. In vitro studies have revealed that imatinib mesylate can inhibit growth of cell lines and primitive malignant progenitor cells in CML expressing Bcr-Abl. However, little is known about the effects of imatinib mesylate on nonmalignant hematopoietic cells. In the current study we demonstrate that in vitro exposure of mobilized human CD34+ progenitors to therapeutic concentrations of imatinib mesylate (1-5 microM) inhibits their differentiation into dendritic cells (DCs). DCs obtained after 10 to 16 days of culture in the presence of imatinib mesylate showed concentration-dependent reduced expression levels of CD1a and costimulatory molecules such as CD80 and CD40. Furthermore, exposure to imatinib mesylate inhibited the induction of primary cytotoxic T-lymphocyte (CTL) responses. The inhibitory effects of imatinib mesylate were accompanied by down-regulation of nuclear localized RelB protein. Our results demonstrate that imatinib mesylate can act on normal hematopoietic cells and inhibits the differentiation and function of DCs, which is in part mediated via the nuclear factor kappaB signal transduction pathway.

Survivin is a shared tumor-associated antigen expressed in a broad variety of malignancies and recognized by specific cytotoxic T cells.

Survivin, a member of the inhibitor of apoptosis protein family, is expressed in almost all types of malignancies, making this protein a useful tool for the development of broadly applicable vaccination therapies. We used a recently identified HLA-A2 binding peptide and dendritic cells (DCs) from healthy donors to induce survivin-specific cytotoxic T lymphocytes (CTLs) in vitro. These T cells efficiently lysed target cells pulsed with the cognate peptide. Furthermore, survivin-specific CTLs recognized HLA-A2-matched tumor cell lines and primary malignant cells from patients with leukemia in an antigen-specific and HLA-restricted manner as demonstrated with the use of cold target inhibition assays and blocking antibodies. To validate the immunogenicity of survivin we performed the experiments in an autologous setting and used monocyte-derived DCs as targets. Interestingly, we found that DCs up-regulate survivin expression on stimulation with tumor necrosis factor alpha (TNF-alpha). However, these mature DCs were not recognized by survivin-specific CTLs, whereas they lysed autologous mature DCs pulsed with the antigenic peptide or transfected with whole tumor RNA purified from a survivin-expressing cell line. To further analyze the possible use of survivin-specific CTLs in cancer therapies, we induced survivin-specific CTLs using peripheral blood mononuclear cells (PBMNCs) and DCs from a patient with chronic lymphocytic leukemia (CLL). The in vitro-generated T cells efficiently recognized autologous malignant CLL cells, whereas they spared autologous-purified nonmalignant B cells or DCs. Our results demonstrate that survivin epitopes are presented on a broad variety of malignancies and can be applied in vaccination therapies.

Molecular and functional characterization of human Dectin-1.

OBJECTIVE: Dendritic cells (DC) have several unique features that differ from other antigen-presenting cells and that enable them to initiate primary immune responses. The aim of this study was to identify and characterize genes that are differentially expressed in DC generated from CD14+ peripheral blood monocytes in vitro. MATERIALS AND METHODS: Using a subtractive cDNA library, we identified the full-length cDNA sequence of human Dectin-1b by rapid amplification of cDNA ends. Expression profiles were performed by reverse transcriptase polymerase chain reaction and Western blotting. For functional analysis, HeLa cells were transfected with the Dectin-1b coding region and used as stimulators for purified peripheral blood T-cell populations. RESULTS: Our subtractive cloning strategy revealed the human Dectin-1b cDNA, which encodes for a transmembrane protein of the C-type lectin-like receptor family. It is selectively expressed in several purified DC subpopulations but not in monocytes and is up-regulated upon stimulation with lipopolysaccharide. The nucleotide sequence was submitted to GenBank (accession no. AY009090). Furthermore, we demonstrate that the Dectin-1b splice variant transfected in HeLa cells up-regulates the activation markers on human T lymphocytes, induces the production of interferon-gamma, and promotes proliferation of both CD4+ and CD8+ T lymphocytes. CONCLUSION: The Dectin-1 gene is expressed during the development of DC from peripheral blood monocytes and the transfection of the splice variant 1b into HeLa cells results in the stimulation of effector functions of human T lymphocytes.