Daniela Werth

Sorafenib, but not sunitinib, affects function of dendritic cells and induction of primary immune responses

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.

Histone Deacetylase Inhibitors Affect Dendritic Cell Differentiation and Immunogenicity

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.

BCR-ABL Activity Is Critical for the Immunogenicity of Chronic Myelogenous Leukemia Cells

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.

Proliferation of human primary vascular smooth muscle cells depends on serum response factor.

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 alpha-actin (ACTA2) or SM22alpha (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 p27(kip1) (CDKN1B) protein levels. SRF-depleted cells expressed senescence-associated beta-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.

Zoledronic acid inhibits the function of Toll-like receptor 4 ligand activated monocyte-derived dendritic 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.