ALBERT

Description: 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 chronic myelogenous leukemia (CML) it has become indispensable and shows few side effects. Recently, it was shown that patients treated with imatinib showed impaired CTL responses in comparison to patients treated with IFN-α, which might be due to a reduced immunogenicity of CML cells or result from an inhibitory effect of imatinib on the function of antigen presenting cells and T lymphocytes. In the present study, we show that imatinib treatment leads to a downregulation of immunogenic antigens on the CML cells, which in turn inhibits the development of CML-specific cytotoxic T lymphocytes (CTLs). To achieve this, we treated the CML cell line K562 and an imatinib-resistant K562 variant, K562R, with imatinib or DMSO, isolated the total RNA and used it to electroporate monocyte-derived dendritic cells (DCs). These cells were then used as antigen presenting cells (APCs) for the induction of polyclonal CTL responses. The cytolytic activity of the CTLs was assayed in standard 51Cr-release assays and their fine specificity in IFNγ-Elispot assays. CTLs generated using RNA from imatinib-treated K562 cells were completely incapable of specific killing and did not react in Elispot assays, whereas those CTLs induced using RNA from K562 cells subjected to DMSO treatment as well as RNA from imatinib-treated K562R cells showed specific cytolytic activity against targets electroporated with RNA from CML cells and were able to recognize several CML-associated antigens, like survivin, PRAME, WT-1 and PR3 in Elispot assays. To confirm that this effect is mediated by BCR-ABL inhibition, we used specific siRNA against the bcr-abl fusion site b3a2 to downregulate the protein expression and found essentially the same results. Even in K562R cells, that constitutively overexpress BCR-ABL, targeting the expression of the protein directly by specific siRNA leads to an impairment of CTL induction. In order to confirm and expand these studies, we additionally analyzed the expression of antigens connected to immune responses to CML in Western Blot and Real-time PCR experiments. We found, that imatinib-mediated inhibition of BCR-ABL in K562 cells leads to a decreased expression of tumor antigens and cellular proteins including survivin, adipophilin, hTERT, WT-1, Bcl-xL and Bcl-2 in correlation to the decreased development of specific CTLs. Matching the results of the 51Cr-release assays, these effects were not observed in K562R cells. In primary CML cells subjected to imatinib a downregulation of hTERT and survivin could be detected, which corresponded to a decreased lysis of DCs electroporated with RNA from these cells in standard 51Cr-release assays. Our results demonstrate, that BCR-ABL directly influences the expression of immunogenic tumor associated antigens by its uncontrolled tyrosine kinase activity and therefore substantially contributes to the immunogenicity of CML cells.

Description: Identification of tumor-associated antigens (TAA) has resulted in the development of therapeutic vaccines for the treatment of cancer. We applied an integrated functional genomics approach to identify TAA in malignant tissues of patients with renal cell carcinoma (RCC). A comparative DNA chip analysis of tumor and the corresponding non-malignant tissue from patients with RCC followed by sequencing of peptides bound to the HLA-class I molecules by mass spectrometry was applied to identify novel TAA in RCC. To confirm the immunogenicity of identified epitopes cytotoxic T lymphocytes (CTL) were generated using dendritic cells (DC). Utilizing this approach, two peptides derived from RGS5 binding to either HLA-A*02 or 03 were identified. The key function of regulators of G protein-signalling (RGS) is to bind to G protein α subunits and to stimulate their intrinsic GTPase activity. The hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) thereby is accelerated and the inactive heterotrimer more rapidly restored. Thus, RGS proteins inhibit the biological activity of G proteins. Interestingly, it was recently shown that RGS5 is overexpressed in pericytes of newly developing tumor vessels, indicating that RGS5 plays an important role during tumor angiogenesis. Using RT-PCR analysis we found that RGS5 is expressed on a broad variety of tumor cells including RCC, colorectal, breast and ovarian cancer, malignant melanoma and multiple myeloma as well as in acute and chronic leukemias making this protein an interesting candidate for the development of vaccination strategies to target the tumor cells and the tumor vessels. CTL that were induced using the RGS5 peptides lysed autologous DC pulsed with the cognate peptide or transfected with in vitro transcribed RGS5 RNA as well as HLA-matched tumor cell lines. The specificity and HLA restriction was confirmed using blocking monoclonal antibodies and in cold-target inhibition assays. We next utilized DC transfected with RGS5 RNA to generate specific CTL. Using this approach we confirmed the processing and presentation of the identified peptides by malignant cells. These CTL lysed tumor cells in an antigen specific manner while sparing non-malignant cells. To analyze the induction of RGS5 specific CTL in an autologous setting in patients with malignant diseases, we used blood samples from a patient with acute myeloid leukemia (AML) in complete remission after chemotherapy and were able to generate specific CTL capable of recognizing autologous AML blasts while sparing non-malignant cells. Our results demonstrate that RGS5 is a novel tumor rejection antigen expressed in a wide range of malignancies that can be applied to target malignant cells and tumor angiogenesis.

Description: 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 NFκB 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 OVA257-264 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.

Description: Evidence from the animal model suggests that proteasome inhibitors may have immunosuppressive properties; however, their effects on the human immune system remain poorly investigated. Here, we show that bortezomib, a proteasome inhibitor with anticancer activity, impairs several immune properties of human monocyte-derived dendritic cells (DCs). Namely, exposure of DCs to bortezomib reduces their phagocytic capacity, as shown by FITC-labeled dextran internalization and mannose-receptor CD206 down-regulation. DCs treated with bortezomib show skewed phenotypic maturation in response to stimuli of bacterial (lipopolysaccharide [LPS]) and endogenous sources (including TNF-α and CD40L), as well as reduced cytokine production and immunostimulatory capacity. LPS-induced CCL-2/MCP-1 and CCL5/RANTES secretions by DCs were prevented by DC treatment with bortezomib. Finally, CCR7 up-regulation in DCs exposed to LPS as well as migration toward CCL19/MIP-3β were strongly impaired. As a suitable mechanism for these effects, bortezomib was found to down-regulate MyD88, an essential adaptor for TLR signaling, and to relieve LPS-induced activation of NF-κB, IRF-3, and IRF-8 and of the MAP kinase pathway. In summary, inhibition of DC function may represent a novel mechanism by which proteasome inhibitors exert immunomodulatory effects. These compounds could prove useful for tuning TLR signaling and for the treatment of inflammatory and immune-mediated disorders.

Description: Zoledronic acid (ZA; Zometa®) is a cyclic nitrogen-containing bisphosphonate with anti-tumor activity used to treat patients with malignant diseases. As a side effect, ZA induces inflammatory responses which are accompanied by expansion of gd T cells that probably mediate its anti-tumor activity. In our study we analyzed the effects of ZA on the function and differentiation of monocyte-derived dendritic cells (DC). We investigated the differentiation of immature and LPS-stimulated DCs incubated with different ZA concentrations which are usually achieved in treated patients. Expression of DC-specific marker molecules was assessed by FACS. Furthermore, the activation state and involvement of different signal transduction pathways were evaluated by Western blot. To monitor DC function we used a migration assay and analyzed the cytokine secretion. We found that DC activation with TLR4 ligand LPS is modulated by ZA. We show that the expression of the surface maturation markers CD80, CD86, CD40, CD54, CD83 and DC-SIGN is diminished with increasing ZA levels upon LPS activation. The migratory capacity of LPS stimulated DCs towards the chemokine CCL19/MIP-3beta and the secretion of the activatory cytokine IL-12 are reduced at higher ZA levels. Increasing ZA concentrations lead to the down-regulation MyD88 and diminish the nuclear localization of NF-kB family members RelB and c-Rel as well as interferon-regulatory factors IRF-3 and IRF-8 in LPS activated DCs. Hence, ZA concentrations used to treat patients have inhibitory effects on DC differentiation and activation. This might lead to immuno-suppression and negatively affect tumor rejection or result in infectious complications.

Description: 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.

Description: Danger signals provided by pathogens are sensed by specialized receptors, Toll-like receptors (TLR), on the cell surface of antigen presenting cells (APC) leading to activation of dendritic cells (DC) accompanied by their phenotypic and functional changes. Activation of DC via the TLR is believed to be a critical event regulating the strength and outcome of an immunological response. IL-10 is an immunosuppressive cytokine shown to have inhibitory effects on function and differentiation of APC by interfering with signaling pathways involved in DC activation like the PI3-kinase or NFκB pathway. In our study we analyzed the function of human dendritic cells generated in the presence of IL-10 upon activation by Toll-like receptor ligands (TLR2, TLR3, TLR4, TLR7/8). Exposure of DCs to IL-10 resulted in a skewed phenotypic maturation in response to stimuli provided by TLR ligands, a reduced production of cytokines like IL-12, IL-6 or TNF-α and an impaired capacity to stimulate T cell activation. Furthermore, CCR7 upregulation in DC exposed to TLR stimulation as well as migration toward CCL19/MIP-3β were strongly reduced. As a suitable mechanism for these effects, IL-10 was found to downregulate MyD88, an essential adaptor molecule for TLR signaling, and to decrease TLR-induced nuclear expression of the NFκB transcription factor members c-Rel and Rel-B as well as IRF-3 and IRF-8. This was not due to the inhibition of the MAP kinase pathway as phosphorylation of p38 and ERK was not affected but was rather mediated by inhibitory effects on the PI3-kinase pathway. IL-10 treatment of APC resulted in a reduced TLR induced mTOR phosphorylation. In summary, our results demonstrate that IL-10 can inhibit TLR-mediated activation of APC via a MyD88-dependent and independent pathway.