Abstract Abstract 1032 Myeloid derived suppressor cells (MDSC) play an important role in the regulation of immune responses by suppressing the function of antigen presenting cells and T cells. In humans several populations of MDSC with different phenotypes were characterized due to their distinct immunosuppressive function. However, the origin and development of these cells is not well understood. We observed that incubation of peripheral blood monocytes with IL-10 during their differentiation to dendritic cells (DC) in the presence of GM-CSF and IL-4 results in the generation of an APC population with dramatically reduced stimulatory capacity and a CD14+ and HLA-DR low phenotype (IL-10-APC) similar to the recently described human MDSC subpopulation. In coincubation experiments we found that the addition of these cells to immature or LPS activated DC generated from peripheral blood monocytes resulted in a cell number dependent inhibition of their stimulatory capacity in the mixed lymphocyte reaction assay. Furthermore, these IL-10-APCs reduced the expression of CD1a and costimulatory molecules on DC as well as their activation by LPS characterized by diminished expression of maturation markers including CD83, CD80, CD86 or CD40. IL-10-APC almost completely abolished the secretion of cytokines and chemokines by mature and immature DC involved in T cell stimulation and migration such as TNF-a, IL-6, MIP-1a or Rantes. These effects were not due to induction of IL-10 production and were not observed when purified CD14+ monocytes were used as a control in the experiments. In order to analyze the possible mechanisms and molecules involved in these inhibitory effects we found that IL-10-APC expressed higher levels of PD-1, GITR, GITRL and osteoactivin, an immunosuppressive molecule that was shown to inhibit the function of T cells. The effects mediated by these molecules were further confirmed by utilizing blocking antibodies. Interestingly, addition of IL-10-APC to mature or immature DC induced an increased expression of osteoactivin and its corresponding receptor syndecan 4 on DC thus demonstrating that osteoactivin mediates its effects by upregulating its own receptor. In the next set of experiments we isolated the CD14+ HLA-DR low cell population from buffy coats of healthy donors. We found, that these cells similar to the IL-10-APCs express high levels of osteoactivin and syndecan-4 that can be further upregulated by the addition of IL-10. Our results demonstrate that osteoactivin mediates its inhibitory effects by induction of its cognate receptor syndecan 4. In addition, cells with the phenotype and function of MDSC can differentiate from human peripheral blood monocytes in the presence of GM-CSF, IL-4 and IL-10. This immunosuppressive cell population can easily be generated in vitro and might be applied for the treatment of patients with GVHD or autoimmune disorders. Disclosures: No relevant conflicts of interest to declare.

Much progress and significant therapeutic changes have been made in the field of tumor therapy in the past decades. Besides chemotherapy and radiotherapy, a special focus was laid on targeted therapies such as small molecule tyrosine kinase inhibitors (TKIs) and other immunomodulatory drugs, which have become standard therapies and important combination partners in a variety of malignancies. In contrast to the widely established use of these often anti-angiogenic drugs, many functional molecular mechanisms are yet not completely understood. Recent analyses focused not only on their direct anti-tumor responses, but also on their influence on tumor microenvironment, as well as on their effects on malignant and healthy cells. Different anti-angiogenic compounds targeting the vascular endothelial growth factor (VEGF) or platelet-derived growth factor pathways seem to be capable of modulating immune responses, in a positive, as well as apparently harmful manner. For an optimal clinical anti-cancer treatment, a better understanding of these immunomodulatory effects is necessary. Here we summarize recent reports on the immunomodulatory function of lately introduced clinically applied anti-angiogenic compounds, such as the humanized monoclonal antibody against VEGF bevacizumab, the small molecule TKIs sunitinib, sorafenib, imatinib, dasatinib, nilotinib and the proteasome inhibitor bortezomib.

Abstract 4071 Introduction: Multiple myeloma (MM) is a malignant plasma cell disorder characterized by clonal expansion of single plasma cells in the bone marrow. Despite high-dose melphalan therapy with autologous stem cell transplantation (ASCT) and the introduction of active drugs like bortezomib or lenalidomide that have been associated with improved survival MM is still incurable. Thus, the identification of novel molecular targets and additional treatment options are warranted. In B-cell malignancies such as chronic lymphocytic leukaemia (CLL) or diffuse large B cell lymphoma, the inhibition of the tyrosine kinase Syk gave promising preclinical and first clinical results.In our study, we analyzed the potential of Syk as a target in MM. Methods: The MM cell lines AMO-1, U266, MMS-1 and RPMI8226 and primary MM cells were treated with the Syk-inhibitors BAY61-3606 or piceatannol and proliferation, migration and apoptosis induction were analyzed. Effects on involved intracellular signaling cascades were determined by Western blotting. Results: Incubation of MM cell lines with Syk-inhibitors resulted in a reduced proliferation and and SDF-1 induced migration, that was accompanied by a concentration dependent inhibition of the MAP kinase signaling characterized by reduced phosphorylation of ERK an p38 molecules. Furthermore, the nuclear localized expression of the NF-kB members RelA and RelB was inhibited in treated cells. In addition, Syk inhibition induced apoptosis in MM cell lines and primary MM cells in a dose-dependent manner as demonstrated by flow cytometry, caspase-3 activity and PARP-1 cleavage. While there was no effect on the expression of xIAP, survivin or MCL-1, Syk inhibition reduced the expression of pro-apoptotic Bcl-2 and Bcl-xl molecules and increased the release of cytochrome c, indicating that the apoptotic cell death is mediated via the internal mitochondrial pathway. Combination of piceatannol with lenalidomide and orally bioavailable dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235but not with bortezomib or dexamethasone enhanced the cytotoxic effects of the compound. In line with the results from previous experiments the addition of MAPK inhibitors PD98059, SP600125, U0126, SB202190 and SB203580 to piceatannol further increased the efficacy of Syk inhibition. Conclusions: Our results show that Syk inhibition might represent a promissing new treatment option in MM with an increased efficacy when combined with lenalidomide or MAP kinase inhibitors. Disclosures: No relevant conflicts of interest to declare.

In order to analyze the effects of peroxisome proliferator-activated receptor-γ (PPARγ) activation on renal cell carcinomas we utilized several cell lines that were treated with the high affinity PPARγ agonist, troglitazone. Incubation of RCC cells with troglitazone resulted in reduced secretion of growth factors that was due to the inhibition of MAP kinase signaling and reduced nuclear localized expression of relB and HIF1alpha. Interestingly, the cell lines used showed a different sensitivity towards apoptosis induction that did not correlate with the inhibition of growth factors or expression of pro- and antiapoptotic molecules. To overcome this resistance the cells were treated with a combination of troglitazone and the proteasome inhibitor, bortezomib. The combination of both compounds induced apoptosis even in cells resistant to both agents alone, due to increased induction of ER-stress and caspase-3 mediated cell death.

Using dendritic cells (DCs) electroporated with whole RNA isolated from blasts of a patient with acute myeloid leukemia (AML), we were able to generate leukemia-specific cytotoxic T lymphocytes (CTLs) capable of recognizing the leucemic cells. To identify T-cell epitopes mediating lysis of malignant cells, peptides were eluted from the patient's blasts and analyzed by mass spectrometry (LC/MS)-based peptide sequencing. Using this approach, an HLA-A24-binding peptide derived from Bax inhibitor-1 (BI-1), a regulator of apoptosis pathways, was identified as an epitope recognized by the generated CTLs. To further characterize this novel antigenic peptide, CTLs were induced using DCs electroporated with RNA coding for BI-1 or pulsed with the cognate peptide. These CTLs generated from healthy donors in vitro efficiently lysed the patient's blasts as well as other HLA-matched leukemic cells. In conclusion, we identified a BI-1 peptide as a novel immunogenic tumor-associated antigen (TAA) in AML. In vitro induction of BI-1-specific CTLs by RNA transfection or pulsing of DCs with the synthetically generated peptide was a feasible and highly effective method to generate leukemia-specific CTLs. As BI-1 is (over-) expressed in a broad variety of malignancies, it may represent an interesting novel TAA in the context of cancer vaccines.

Toll-like receptors (TLRs) act to sense the environment for microbial products and submit danger signals to antigen-presenting cells (APCs) resulting in activation of complex immune responses. In this study, we analyzed the function of human monocyte-derived APCs generated in vitro in the presence of interleukin (IL)-10 upon activation by TLR ligands. Exposure of these APCs to IL-10 resulted in a skewed phenotypic maturation in response to stimuli provided by the TLR ligands, a reduced cytokine production, such as IL-12, IL-6 or tumor necrosis factor-α, and impaired capacity to stimulate T-cell activation. Furthermore, CCR7 upregulation in APCs exposed to TLR stimulation as well as migration towards CCL19/MIP-3β were strongly reduced. IL-10 was found to downregulate MyD88, IRAK1 (IL-1 receptor-associated kinase) and tumor necrosis factor receptor-associated factor 6, essential adaptor molecules for TLR signaling, and to decrease TLR-induced nuclear expression of the nuclear factor-κB transcription factors c-Rel and Rel-B as well as interferon regulatory factor (IRF)-3 and IRF-8. This was not due to the inhibition of the mitogen-activated protein kinase pathway, but was rather mediated by the blockage of the PI3K signaling cascade. Interestingly, the inhibition of proteins involved in TLR signaling, such as MyD88, IRAK1 and mammalian target of rapamycin, was due to a selective post-transcriptional regulation.

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.