Smooth muscle cells (SMCs) can switch between a differentiated/contractile and an alternative proliferative phenotype. The transcription factor serum response factor (SRF) has been implicated in the regulation of gene expression profiles determining both phenotypes. Whereas strong evidence exists for a role of SRF in SMC differentiation, the contribution of SRF to SMC proliferation is less well defined. For primary human vascular SMCs in particular, existing data are non-conclusive. To study SRF functions in primary human vascular SMCs, we used an siRNA approach. siRNA-mediated SRF suppression affected the expression of established SRF target genes such as smooth muscle α-actin (ACTA2) or SM22α (TAGLN) and decreased both F-actin formation and cell migration. Furthermore, SRF knockdown caused a cell-cycle arrest in G1 associated with reduced hyperphosphorylated pRB, cyclin A and SKP2 levels, and increased p27kip1 (CDKN1B) protein levels. SRF-depleted cells expressed senescence-associated β-galactosidase indicating an irreversible G1 arrest. siRNA-mediated suppression of SKP2 triggered senescence to a similar extent as SRF depletion, indicating that SRF knockdown-induced senescence may be dependent on a decrease in SKP2. Thus, SRF is an essential regulator of primary human vascular SMC proliferation and senescence. Interfering with SRF function may therefore be a promising strategy for the treatment of hyperproliferative SMC disorders such as atherosclerosis and in-stent restenosis.

Interferon regulatory factor 8 (IRF-8) is a member of the IRF family of transcription factors, which are stimulated through interferon mediated pathways. In mice, IRF-8 seems to play an essential role in the development and maturation of dendritic cells (DCs). However, very limited knowledge is available about the potential role of IRF-8 in the human system. To bridge this gap we analyzed function and activation of human monocyte-derived dendritic cells (mDCs) lacking IRF-8 expression. To knockdown IRF-8 protein levels, we electroporated mDCs with different siRNAs against IRF-8. Additionally, we stimulated the electroporated mDCs with the Toll like receptor (TLR) 2 ligand Pam3Cys or the TLR 7/8 ligand R848. IRF-8 knockdown in mDCs was verified constantly by Western Blot analysis using an anti-IRF-8 antibody. We found that IRF-8 knockdown clearly diminished the expression of the human lymphocyte antigen molecules HLA-ABC and HLA-DR in Pam3Cys and R848 stimulated mDCs. To gain functional data, we performed ELISAs to determine cytokine and chemokine secretion. The electroporation of mDCs with IRF-8 specific siRNA resulted in profound inhibition of secretion of the cytokines IL-6, IL-12 and TNF-a as well as the chemokines MIP-1a (CCL3), MCP-1 (CCL2) and RANTES (CCL5). To get additional information on IRF-8 function in human mDCs, the regulation of signal transduction pathways was determined by Western Blot analysis. The suppression of IRF-8 diminished the nuclear translocation of the NF-kB family member's c-Rel and RelB as well as PU.1 and IRF-3 in activated mDCs. In addition, we showed that the suppression of IRF-8 caused a reduced phosphorylation of ERK and JNK, but had no effect on the expression of STAT3. In summary, the knockdown of IRF-8 reduced the capability of mDCs to develop appropriate phenotype and functions in response to activating stimuli. Our results indicate that these effects are mediated via the ERK, NF-kB and PU.1 signalling pathways. IRF-8 plays an important role in the activation and function of human mDCs.

The tyrosine kinase inhibitors sorafenib and sunitinib are approved for the treatment of patients with malignant diseases. To analyze the possible use of these compounds in combination with immunotherapeutic approaches, we analyzed the effects of both inhibitors on the immunostimulatory capacity of human dendritic cells (DCs) and the induction of primary immune responses in vivo. Sorafenib, but not sunitinib, inhibits function of DCs, characterized by reduced secretion of cytokines and expression of CD1a, major histocompatibility complex, and costimulatory molecules in response to TLR ligands as well as by their impaired ability to migrate and stimulate T-cell responses. These inhibitory effects are mediated by inhibition of PI3 and MAP kinases and NFkappaB signaling. In contrast, sorafenib had no influence on the phenotype and proliferation of T cells. To analyze the effects of both TKIs on cytotoxic T-cell induction in vivo, C57BL/6 mice were pretreated with sorafenib or sunitinib and immunized with OVA(257-264) peptide. Sorafenib, but not sunitinib, application significantly reduced the induction of antigen-specific T cells. Numbers of regulatory T cells were reduced in peripheral blood mononuclear cells from mice treated with sunitinib. These results indicate that sunitinib, but not sorafenib, is suitable for combination with immunotherapeutic approaches for treatment of cancer patients.

Dendritic cells (DCs) have the unique ability to efficiently present T-cell epitopes from exogenous antigens on MHC class I molecules, a process called cross-presentation. In our study we demonstrate that stimulation of monocyte-derived DCs with Toll-like receptor (TLR) ligands differentially affects the uptake and cross-presentation of cellular antigens. Activation of DCs with TLR3 or TLR4 but not with TLR2 or TLR7/8 ligands inhibited phagocytosis of apoptotic tumor cells and resulted in a reduced cross-presentation of pp65-derived T-cell epitopes on MHC class I molecules upon engulfment of cytomegalovirus (CMV)-infected fibroblasts. These results have an important impact on the understanding of the interactions between the immune system and pathogens and the development of vaccination strategies to treat malignant diseases.

Human (h)Dectin-1 is a member of the C-type-lectin-like receptor family that was shown to be the major receptor for fungal β-glucans and to play an important role in cellular responses mediated by these carbohydrates. It is mainly expressed on human DCs and macrophages. In our study, we observed that activation of monocyte-derived dendritic cells (MDCs) with TLR3 ligand (poly I:C) but not with TLR ligand 7/8 (R848) resulted in down-regulation of hDectin-1 expression and this down-regulation correlated with a reduced uptake of apoptotic cells in phagocytosis assays. In order to analyze the possible cross-presentation of engulfed antigens we used CMV infected human fibroblasts (HFF). We found that hDectin-1 is involved in the uptake of CMV-infected HFF leading to cross-presentation of CMV-derived peptides on MHC class I molecules and activation of CMV pp65-specific CD8+ T-lymphocytes. To further delineate the pathway leading to presentation, we pretreated MDCs with compounds that inhibit processing of antigens at defined steps during presentation. Cytosolic protein degradation is performed by the proteasome, a large multicatalytic protease complex. Lactacystin specifically inhibits the 20S and 26S proteasome activity by targeting the catalytic subunit. In standard 51Cr-release assays, addition of lactacystin completely inhibited the presentation of pp65-derived peptides indicating the involvement of the proteasome in these process. The fungal product brefeldin A blocks the MHC class I processing pathway by specifically inhibiting the vesicular egress from the ER and the Golgi complex. In line with previous findings, incubation with brefeldin A almost completely abolished the lysis of MDCs incubated with CMV+ HFF. To further analyze whether the cross-presentation of CMV-derived peptides on HLA class I molecules was dependent on lysosomal proteases, MDCs that were co-incubated with HFF as above were treated with the lysosomotropic agent chloroquine that prevents acidification of the lysosomal compartment involved in the exogenous pathway of antigen presentation. The addition of chloroquine had no effect on the cross-presentation of CMV-derived epitopes on HLA class I-molecules. Summarized, the data reported here show that hDectin-1 functions not only as a pattern recognition receptor in innate immunity but is also involved in the clearing of apoptotic cells and cross-presentation of cellular antigens on MHC class I molecules to specific CTLs.

Purpose: Histone deacetylases (HDAC) modulate gene transcription and chromatin assembly by modifying histones at the posttranscriptional level. HDAC inhibitors have promising antitumor activity and are presently explored in clinical studies. Cumulating evidence in animal models of immune disorders also suggests immunosuppressive properties for these small molecules, although the underlying mechanisms remain at present poorly understood. Here, we have evaluated the effects of two HDAC inhibitors currently in clinical use, sodium valproate and MS-275, on human monocyte-derived DCs. Experimental Design: DCs were generated from monocytes through incubation with granulocyte macrophage colony-stimulating factor and interleukin-4. DC maturation was induced by addition of polyinosinic-polycytidylic acid. DC phenotype, immunostimulatory capacity, cytokine secretion, and migratory capacity were determined by flow cytometry, mixed leukocyte reaction, ELISA, and Transwell migration assay, respectively. Nuclear translocation of RelB, IFN regulatory factor (IRF)-3, and IRF-8 were determined by immunoblotting. Results: HDAC inhibition skews DC differentiation by preventing the acquisition of the DC hallmark CD1a and by affecting the expression of costimulation and adhesion molecules. In addition, macrophage inflammatory protein-3β/chemokine, motif CC, ligand 19–induced migration, immunostimulatory capacity, and cytokine secretion by DCs are also profoundly impaired. The observed defects in DC function on exposure to HDAC inhibitors seem to reflect the obstruction of signaling through nuclear factor-κB, IRF-3, and IRF-8. Conclusions: HDAC inhibitors exhibit strong immunomodulatory properties in human DCs. Our results support the evaluation of HDAC inhibitors in inflammatory and autoimmune disorders.

3504 Background: The tyrosine kinase inhibitors (TKIs) sorafenib and sunitinib are approved for the treatment of patients with metastatic renal cell cancer. To analyze the possible use of these compounds in combination with immunotherapeutic approaches we investigated the effects of both TKIs on function of human dendritic cells (DCs) and induction of primary immune responses in vitro and in vivo. Methods: Human monocytes-derived DCs were treated with DMSO, sorafenib or sunitinib. Functional and phenotypic analyses as well as the possible impact on signal transduction pathways were performed. Furthermore, induction of immune responses in vivo was analyzed in animals treated with both compounds. Results: Sorafenib but not sunitinib inhibits function of DCs. Exposure of DCs to sorafenib reduces expression of CD1a, MHC and costimulatory molecules in response to stimuli via TLR ligands. Sorafenib reduces cytokine production by DCs as well as their ability to migrate and stimulate T cell (TC) responses. We found that these inhibitory effects of sorafenib are mediated via inhibition of PI3K, MAP kinases and NFκB signaling. The TKIs have no influence on phenotype and proliferation of TCs. To analyze the effects of TKIs on the generation of immune responses in vivo, induction of TC responses was assessed by peptide vaccination with the model antigen OVA-001 in C57BL/6 mice. When mice were pretreated with both TKIs which were also given during vaccination, it was observed that sorafenib, but not sunitinib significantly reduces the generation of vaccine-specific CD8+ TCs. Numbers of CD4+ CD25+ regulatory TCs are reduced in sunitinib-treated mice, but not in sorafenib-treated animals. All effects of the TKIs are reversible, and the immune responses go back to normal levels if mice are immunized after discontinuation of treatment. Conclusions: In summary, sunitinib represents an interesting compound to be used in combination with immunotherapeutic approaches to treat cancer patients. No significant financial relationships to disclose.

Imatinib mesylate (Gleevec) is a specific tyrosine kinase inhibitor, which inhibits phosphorylation of downstream proteins involved in BCR-ABL signal transduction. In the treatment of CML it has become indispensable and shows few side effects. Nevertheless, imatinib has been described to influence the function and differentiation of APCs, inhibit the effector function of T lymphocytes and decrease the immunogenicity of CML cells. In the present study, we analyzed the possible effects of imatinib on proteasomal activity in CML cells. Proteasomal activity was determined using a biotinylated active site-directed probe which, depending on their activity, covalently binds and labels proteasomal subunits beta-1, beta-2 and beta-5. We show that Imatinib treatment leads to a decreased activity of the proteasome in BCR-ABL positive cells. Imatinib sensitive K-562 cells as well as HLA-A2 transfected K-562 cells displayed a profound downregulation of proteasome activity at sub-therapeutic concentrations of imatinib. This effect was not due to downregulation of the protein expression of proteasome subunits or the induction of apoptosis. In imatinib resistant K-562R cells a higher concentration of imatinib was required for a detectable inhibition of proteasomal activity corresponding to a higher expression of BCR-ABL in these cells. Interestingly, the use of specific siRNA against the BCR-ABL fusion site b3a2 to downregulate the expression of the protein in K-562 and K-562R cells did not influence the activity of the proteasome indicating that this effect is independent of the expression and activity of BCR-ABL. In line with these results transfection of BaF/3 cells with BCR-ABL had no effect on proteasomal activity. To further analyze the mechanisms involved in proteasome inhibition by imatinib we performed activity assays with isolated 20S and 26S proteasomes. Incubation of both proteasome subtypes with imatinib resulted in a reduced acitivity in a concentration dependent manner as shown by reduced proteolytic cleavage of fluorogenic substrates. As the reduction in activity was seen in the ATP-independent 20S proteasome as well as the ATP-dependent 26S proteasome, a competition of imatinib with ATP similar to the mechanism of BCR-ABL inhibition is not responsible for this effect. In order to expand these studies, we additionally examined HLA class I expression on the cell surface to determine the influence of imatinib and subsequent proteasome inhibition on antigen presentation. Correlating with the inhibition of proteasome activity, HLA class I expression was strongly reduced on HLA-A2 transfected K-562 cells after imatinib treatment. Taken together these findings show that imatinib directly affects proteasomal activity in vitro. This effect is more pronounced in BCR-ABL positive cells, which might be due to BCR-ABL mediated deregulation of cellular pathways, but seems to be, however, independent of BCR-ABL.

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder caused by excessive granulopoiesis due to the formation of the constitutively active tyrosine kinase BCR-ABL. An effective drug against CML is imatinib mesylate, a tyrosine kinase inhibitor acting on Abl kinases, c-KIT, and platelet-derived growth factor receptor. Recently, a study revealed that patients treated with imatinib showed impaired CTL responses compared with patients treated with IFN-alpha, which might be due to a treatment-induced reduction in immunogenicity of CML cells or immunosuppressive effects. In our study, we found that inhibition of BCR-ABL leads to a down-regulation of immunogenic antigens on the CML cells in response to imatinib treatment, which results in the inhibition of CML-directed immune responses. By treating CML cells with imatinib, we could show that the resulting inhibition of BCR-ABL leads to a decreased expression of tumor antigens, including survivin, adipophilin, hTERT, WT-1, Bcl-x(L), and Bcl-2 in correlation to a decreased development of CML-specific CTLs. In contrast, this reduction in immunogenicity was not observed when a CML cell line resistant to the inhibitory effects of imatinib was used, but could be confirmed by transfection with specific small interfering RNA against BCR-ABL or imatinib treatment of primary CML cells.

Zoledronic acid (ZA) is a nitrogen-containing bisphosphonate with antitumor activity used to treat patients with malignant diseases. ZA treatment induces, as a side effect, inflammatory responses, which are accompanied by expansion of γδ T cells. In our study, we analyzed the function and differentiation of monocyte-derived immature and lipopolysaccharide (LPS)-stimulated dendritic cells (moDCs) treated with different ZA concentrations, which are achieved in patients. We found that moDC activation with TLR4 ligand LPS is modulated by ZA. The expression of maturation markers was diminished with increasing ZA levels upon LPS activation. The migratory capacity, interleukin-12 secretion and generation of cytotoxic- T-cell responses were reduced at higher ZA levels. Increasing ZA concentrations downregulated nuclear factor-κB family members and interferon-regulatory factor (IRF)-3. Surprisingly, in immature moDCs, low ZA concentrations caused upregulation of RelB, c-Rel, IRF-3 and IRF-8. We conclude that ZA concentrations used to treat patients have inhibitory effects on DC activation. This might lead to immunosuppression or result in infectious complications.

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.