Recurrent genetic and chromosomal aberrations drive multiple myeloma (MM) pathogenesis. Among these, the t(4;14) translocation leads to overexpression of fibroblast growth factor receptor 3 (FGFR3) and is associated with poor prognosis. However, therapeutic approaches directly targeting FGFR3-driven myeloma progression remain limited. Here, we investigated the single-agent activity of ponatinib, a multikinase inhibitor, in MM. KMS18 and U266 myeloma cell lines were treated with ponatinib, and apoptosis induction, as well as VEGF and IL-6 secretion, was assessed. RNA sequencing of MM cells revealed pathway alterations induced by ponatinib treatment, which were subsequently validated by Western blot analysis. In vivo, mice inoculated with 5T33 myeloma cells received ponatinib, and survival was monitored. Notably, ponatinib exerted potent single-agent antimyeloma activity in an FGFR3-dependent manner by inducing apoptosis and suppressing VEGF and IL-6 secretion through inhibition of JAK/STAT, PI3K/AKT, and MAPK signaling. In vivo administration prolonged survival in myeloma-bearing mice. Collectively, our findings demonstrate the therapeutic efficacy of ponatinib in FGFR3-expressing MM beyond selective FGFR3 inhibition, suggesting that concurrent suppression of multiple signaling pathways is a critical mechanism of action. These results highlight the therapeutic potential of combined FGFR3-targeted strategies in multiple myeloma and provide a rationale for further clinical investigation.

Systemic candidiasis is a serious complication in immunocompromised patients, with Candida albicans emerging as the most common opportunistic pathogen. In various therapeutic treatment regimens the immunosuppressive agent Dexamethasone is used. Dexamethasone itself impairs the function of dendritic cells and reduces thereby their capacity for T-cell proliferation through the activation of Dectin-1 by β-glucans. In the present study, we reveal that these tolerogenic dendritic cells (Dex-DCs) have an increased secretion of IL-1ß and IL-18 when stimulated with β-glucans. We show an increased formation of ASC specks, which are crucial for recruiting pro-caspase-1, indicating an elevated inflammasomal activity. In line with this, we were able to show that treatment of tolerogenic dendritic cells with a NLRP3 inhibitor prior to Dectin-1 stimulation normalized the secretion of IL-1ß and IL-18. Furthermore, the addition of Caspase- and Syk-inhibitors led to diminished inflammasome activation as well as to less pyroptosis and apoptosis in response to β -glucan stimulation. Finally, we identified elevated production of reactive oxygen species (ROS) upon β-glucan stimulation in DexDCs as a possible mechanism for apoptosis induction as it can be reversed by the treatment with a specific anti-Dectin-1 antibody. Moreover, the underlying mechanism of the NLRP3 activation seems to be mediated through mitochondrial DNA release induced by mitochondrial ROS. Taken together, the present study demonstrates that Dectin-1 stimulation of tolerogenic DCs can result in severe pro-inflammatory responses due to cytokine release and subsequent NLRP3 inflammasome activation. In conclusion, the application of NLRP3 inflammasome inhibitors to patients treated with corticosteroids like Dexamethasone may significantly improve their outcome as they might be well-protected against local or severe systemic fungal infections.

Supplementary Figures S1-S5 Supplementary Tables S1-S3 revised

Blastic plasmacytoid dendritic-cell neoplasm (BPDCN) is an extremely rare disease that originates from dendritic cells and is associated with a poor overall survival (OS). Diagnostic and therapeutic standards are less well-established in comparison to other leukemic conditions and standards of care are lacking. Morphologic and molecular similarities to acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) are hard to distinguish. We here report a BPDCN patient with a long, challenging diagnostic period. While bone marrow biopsies initially failed to prove the correct diagnosis, a cutaneous biopsy finally identified a CD45+/CD56+/CD4+/CD123+/CD33+/MPO- population suggestive of BPDCN which was confirmed by flow cytometry. Molecular analysis revealed an ASXL-1, TET2 and SRSF2-mutation, cytogenetic analysis showed a normal karyotype. Treatment with the recently approved CD123-cytotoxin Tagraxofusp showed initially a very good response. This case reflects diagnostic and therapeutic difficulties in BPDCN as very rare, easily misdiagnosed neoplasia and the need for precise diagnostic care.

Management of patients with metastatic squamous cell cancer of the head and neck, refractory to initial therapy with standard chemo- and radiation protocols, remains difficult with poor overall prognosis and limited therapeutic options. Nivolumab (a programmed death receptor-1 ,PD-1 blocking antibody) has been approved in Germany since June 2017 for the treatment of squamous cell carcinoma of the head and neck in adults. Nivolumab in combination with Ipilimumab (an anti-CTLA 4 antibody) is licenced in the EU for the treatment of advanced (unresectable or metastatic) melanoma in adults. In April 2018, the Food and Drug Administration granted approvals to nivolumab and Ipilimumab in combination for the treatment of intermediate or poor risk, previously untreated advanced renal cell carcinoma. So far, there are no clinical data on the therapeutic efficacy of nivolumab in combination with ipilimumab in squamous cell cancer of the head and neck. This report is about a patient with metastastic squamous cell cancer of the head and neck refractory to nivolumab monotherapy, who showed a remarkable response to combination therapy with nivolumab and ipilimumab over a period of 2 years, but developed serious renal failure and a Takotsubo cardiomyopathy.

Treatment options for patients with platinum-refractory, recurrent, metastatic head and neck squamous cell carcinoma (HNSCC) are limited, and prognosis is poor. Nivolumab (Opdivo) has been approved by the US Food and Drug Administration (FDA) for the treatment of patients with recurrent or metastatic HNSCC who have disease progression on or after platinum-based therapy. Recently, in patients with metastatic malignant melanoma a significant improvement of outcome and response was achieved with the combination of ipilimumab (CTLA4 antibody) and the programmed death (PD)-1 inhibitor nivolumab compared with monotherapy. Based on these results, the combination of nivolumab and ipilimumab has been approved by the FDA for the treatment of patients with unresectable or metastatic melanoma. So far, there have been no data concerning the combination of nivolumab and ipilimumab in squamous cell head and neck cancer. We here present the case of a 46-year-old male with refractory squamous cell head and neck cancer, who was successfully treated with the PD-1 inhibitor nivolumab in combination with the anti-CTLA4 antibody ipilimumab.

The endocannabinoid system (ECS) with its binding receptors CB1 and CB2 impacts multiple pathophysiologies not only limited to neuronal psychoactivity. CB1 is assigned to cerebral neuron action, whereas CB2 is mainly expressed in different non-neuronal tissues and associated with immunosuppressive effects. Based on these tissue-selective CB receptor roles, it was the aim of this study to analyze potential expression in periodontal tissues under physiological conditions and inflammatory states. In vivo, CB receptor expression was investigated on human periodontal biopsies with or without bacterial inflammation and on rat maxillae with or without sterile inflammation. In vitro analyses were performed on human periodontal ligament (PDL) cells at rest or under mechanical strain via qRT-PCR, Western blot, and immunocytochemistry. P < 0.05 was set statistical significant. In vivo, CB1 expression was significantly higher in healthy PDL structures compared to CB2 (13.5% ± 1.3 of PDL tissues positively stained; 7.1% ± 0.9). Bacterial inflammation effected decrease in CB1 (9.7% ± 2.4), but increase in CB2 (14.7% ± 2.5). In contrast, sterile inflammation caused extensive CB1 (40% ± 1.9) and CB2 (41.7% ± 2.2) accumulations evenly distributed in the tooth surrounding PDL. In vitro, CB2 was ubiquitously expressed on gene and protein level. CB1 was constitutively expressed on transcriptional level (0.41% ± 0.09), even higher than CB2 (0.29% ± 0.06), but undetectable on protein level. Analyses further revealed expression changes of both receptors in mechanically loaded PDL cells. CB1 and CB2 are varyingly expressed in periodontal tissues, both adjusted by different entities of periodontal inflammation and by mechanical stress. This indicates potential ECS function as regulatory tool in controlling of periodontal pathophysiology.

The recently approved Jak-Inhibitor ruxolitinib improves clinical symptoms in patients with myelofibrosis and autoimmune diseases such as rheumatoid arthritis. It has been shown in myelofibrosis that the clinical benefit occurs independent of the Jak mutational status, indicating that not only diseased, but also healthy cells like immune cells might be affected by the treatment. In a previous study we investigated the effects of ruxolitinib on dendritic cells (DCs) as the most professional antigen presenting immune cells. We were able to show that ruxolitinib markedly impairs human and murine DC phenotype, DC activation and migration and the induction of allogeneic and antigen-specific T cell responses in vitro and in vivo. In regard to good clinical responses in myelofibrosis patients lacking the Jak-2-mutation, it seems rather unlikely that Jak-inhibition is the only responsible for the observed in vivo and in vitro effects. In the present study, we therefore aimed to investigate whether other signalling mechanisms might be additionally involved. To exclude significant effects on Jaks other than Jak-2, we first analyzed the more selective Jak-2 inhibitor TG101348 in our in vitro models. Here, we were able to show that both inhibitors impair DC phenotype and function to a similar extend. We next investigated the role of the MAP-kinase and the NF-kB signalling pathways. Therefore, human monocyte-derived immature and mature DCs were treated with different concentrations of ruxolitinib for distinct durations, followed by preparation of whole cell lysates and nuclear extracts to perform Western blot analyses. We found that ruxolitinib treatment resulted in a reduced phosphorylation and expression of the MAP-kinases ERK and p38, while the housekeeping gene GAPDH was not affected. This effect was independent of apoptosis induction as the expression of caspase 3 was constant. Moreover, the expression of the NF-kB family members RelA and cRel were markedly reduced.

Innate immunity is crucial for an effective host defense against pathogenic microorganisms in periodontal tissues. As periodontal ligament (PDL) cells synthesize immunomodulatory cytokines, the aim of this in vitro study was to investigate whether these cells can interact with innate immune cells. Resting and inflammatory primed (IL-1β, TNF-α, HMGB1) human PDL cells were co-cultured with human monocyte-derived dendritic cells or macrophages. Migration, phenotypic maturation and modulation of phagocytosis of Porphyromonas gingivalis by immune cells were investigated upon co-culture with PDL cells and/or their released soluble factors. PDL cells interacted with immune cells under both non-inflammatory and inflammatory conditions. Immune cell migration was significantly enhanced by co-culture with PDL cells, which also affected their phenotypic maturation both through cell-cell contact and through released soluble mediators. The dendritic cell maturation markers CD83 and CD86 were upregulated as much as both ‘alternatively activated’ M2 macrophage maturation markers CD23 and CD163. In contrast, the ‘classically activated’ M1 macrophage maturation marker CD64 was downregulated. Finally, PDL cells significantly enhanced the phagocytosis of Porphyromonas gingivalis by immune cells. Our experiments revealed that PDL cells are not only structural elements of the periodontium, but actively influence immune responses by interaction with innate immune cells.

Acute and chronic graft-versus-host disease (GVHD) are potentially lethal complications after stem cell transplantation (SCT). Steroids are the appropriate first-line treatment for both. However, if patients do not adequately benefit from steroid therapy, mortality is high and standardized treatment algorithms are lacking. This is mainly because of limited data from prospective, randomized clinical trials. In addition, most of the available treatment options only induce clinical benefits in a limited proportion of patients. Thus, there is an urgent clinical need to develop more potent immunosuppressive treatment strategies for patients suffering from acute or chronic steroid-refractory GVHD while maintaining the graft versus tumor effect to avoid a potential rise in relapse-related mortality. The increasing knowledge about host- as well as donor-derived variables favoring GVHD development and the increasing armamentarium of immune-modulatory agents entering preclinical and clinical research will probably allow more effective treatment of GVHD in the future. This review describes novel developments in the treatment of steroid-refractory GVHD, with a special focus on the rationale behind promising pharmacologic compounds or up-coming cellular therapies.

Abstract 2748 The tyrosine kinase inhibitors (TKIs) Imatinib mesylate (IM, Gleevec, Glivec) and nilotinib (NI, Tasigna, AMN) are currently used in treatment of chronic myeloid leukaemia (CML). IM has been described to influence the function and differentiation of antigen presenting cells, to inhibit the effector function of T lymphocytes and to decrease the immunogenicity of CML cells by downregulation of tumor associated antigens. In the present study, we analyzed the effect of IM and NI on proteasomal activity in IM-sensitive or IM/NI- resistant CML cells as well as in patient samples using a biotinylated active site-directed probe, which, covalently binds and labels proteasomal subunits beta-1, beta-2 and beta-5 and their immunosubunit counterparts beta-1i, beta-2i and beta-5i, in an activity-dependent fashion. Incubation of CML cell lines and primary CML cells with IM or NI resulted in a concentration dependent inhibition of proteasomal activity that was independent of BCR-ABL, as these effects were observed in TKI-resistant and BCR-ABL negative cells. In addition, these effects were not due to a downregulation of the expression of proteasomal subunits as analyzed by Western blot and independent of apoptosis induction. To further analyze and confirm the direct effects of TKIs on proteasomal function, isolated h20S proteasome assays were performed. In line with the results of activity site labeling, IM or NI treated isolated h20S, showed a reduced concentration-dependent activity, as measured by fluorometric cleavage of the substrate suc-leu-leu-val-tyr-AMC. Furthermore, incubation of purified h20S proteasomes with serine or tyrosine specific phosphatases reduced the proteasomal activity as observed with the TKIs, indicating that serine and tyrosine phosphorylation plays an important role in the regulation of the proteasome function. In the next set of experiments we analyzed the effects of IM and NI on the proteasomal generation of antigenic peptides derived from BCR-ABL. In in vitro digestion experiments using purified proteasomes in the presence of NI or IM, we found that the treatment of immunoproteasome i20S with the TKIs almost completely abolished the generation of the long precursor peptides for the HLA-A3/A11 (KQSSKALQR) and the HLA-B8 (GFKQSSKAL) epitopes, while the cleavage of the short peptides significantly increased. Both epitopes have been shown to be naturally processed and presented in CML cells. However, we show that both epitopes can be generated as N-terminal elongated precursors in vitro by both constitutive and immuno-20S proteasomes. Interestingly, in all performed experiments NI was more effective as compared to IM, while other TKIs had no effect. Our results demonstrate that treatment with IM and NI can affect the immunogenicity of malignant cells by affecting proteasomal degradation of cytosolic antigens, thereby modulating the repertoire of presented antigens. These strong effects of the TKIs IM and NI on proteasomal activity might be a result of changes in the phosphorylation of proteasomal subunits akin to the recently showed endogenous phosphorylation sites of the mammalian 20S proteasome. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3393 Introduction: Chronic myeloid leukemia (CML) is a myeloproliferative disease which resolves in the constitutive activity of the BCR-ABL protein. Since the introduction of imatinib as first line therapy for CML, the overall survival of patients could be significantly extended. However, mutations of the BCR-ABL protein and environmental effects mediated by adhesion molecules, cytokines and growth factors were shown to induce resistance to TKIs. For the development of immunotherapeutic approaches that combine TKIs with vaccination strategies or donor lymphocyte infusions it is important to understand the mechanisms of possible interactions and inhibition. Therefore, we thought to determine the role of activated T cells and to analyze a possible antiapoptotic effect of released IFNγ on the CML cells. Methods: CML cell lines and primary cells from patients with CML were treated with imatinib or nilotinib and incubated with activated (PMA/ionomycin) or resting T cells in transwell experiments. Induction of apoptosis was analyzed by flow cytometry. In addition, IFNγ was used together with TKIs and apoptotic cell death and proliferation was determined. Immunoblotting was performed to analyze the involved pathways. Result: Incubation of CML cells in transwell assays with activated but not resting T lymphocytes resulted in significant inhibition of the apoptosis induction by imatinib and nilotinib. To further determine the mechanisms mediating these effects, CML cells were treated with IFNγ in addition to the TKIs. In line with the results from previous experiments, IFNγ reduced the rate of cell death and proliferation inhibition mediated by imatinib and nilotinib. Interestingly, in contrast to IFNα, IFNγ inhibited the TKIs induced downregulation of MHC-class I molecules on CML cells. In line with these results, Western blot analysis of K-562 cells incubated with imatinib or nilotinib showed an increased caspase-3 activation and PARP cleavage as well as reduced down regulation of anti-apoptotic molecules such as xIAP as compared to IFNγ treated cells. Furthermore, we observed a diminished TKI induced downregulation of Jak-2 and STAT-5 phosphorylation as well as increased nuclear expression of RUNX-1 in cells treated with IFNγ. Conclusion: Our results demonstrate that IFNγ released by activated T-cells can interfere with the action of TKIs in CML patients and might have important implications in the development of immunotherapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

Abstract 1645 Poster Board I-671 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 pathogens. Activation of Dectin-1 via extracts such as zymosan or more specifically with curdlan can lead to DC activation characterized by upregulation of surface molecules or secretion of cytokines. Futhermore, Dectin-1 is important for recruitment of leukocytes and production of inflammatory mediators at the site of infection.. Peroxisome proliferator-activated receptor (PPAR) and its ligands, cyclopentenone prostaglandins or thiazolidinediones, have been shown to have profound modulatory effects on B and T lymphocytes as well as DCs. Cyclopentenone prostaglandins are produced during the late phase of inflammation due to up-regulation of cyclooxygenase 2 (COX2), a key enzyme for the synthesis of cyclopentenone prostaglandins, that mediate their effects by activation of PPAR-gamma dependent and independent pathways. In our study we analyzed the effects of troglitazone (TGZ), a high affinity synthetic ligand of PPAR-gamma, on the Dectin-1 mediated activation of antigen presenting cells (APC). TGZ was added to the cell culture medium during the differention of monocyte derived DC. On day 5-6 of culture the cells were additionally treated with curdlan or zymosan and phenotypical and functional analyses were performed. Activation of PPAR-gamma resulted in a reduced stimulation of APC via Dectin-1 characterized by down-regulation of CD1a, CD83 and costimulatory molecules on the cell surface and reduced secretion of cytokines and chemokines involved in T-lymphocyte activation and recruitment including IL-1beta, TNF-a, IL-6, IL-12, MIP-1a and Rantes. Western blot analyses revealed that these inhibitory effects were mediated by the inhibition of the mitogen-activated protein (MAP) kinase pathways characterized by a reduced phosphorylation of ERK1/2 and p38. Furthermore, we found that Dectn-1 induced c-Jun phosphorylation was reduced by pretreatment with TGZ. In addition, Dectin-1 induced nuclear translocation of NF-kappaB family members RelB and cRel was dramatically reduced in APC incubated with TGZ. The observed inhibition of signaling pathways was not due to a reduced expression of Dectin-1, phosphorylation of Syk or increased secretion of IL-10 and TGF-beta. TGZ had no effect on the Dectin-1 induced phosphorylation and nuclear expression of NFAT. Our data demonstrate that PPAR-gamma ligands inhibit Dectin-1 mediated activation by interfering with the MAP kinase, c-Jun and NF-kappaB signaling pathways, thus confirming their important role as a negative feedback mechanisms in the regulation of potentially harmful inflammation signaling pathways. Disclosures No relevant conflicts of interest to declare.

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.

Imatinib mesylate (IM; Gleevec) is currently used in the treatment of CML and GIST. IM has been described to influence the function and differentiation of antigen presenting cells, to inhibit the effector function of T lymphocytes and to decrease the immunogenicity of CML cells by downregulation of tumor associated antigens. In the present study, we analyzed the possible effects of IM on antigen processing and presentation. To accomplish this we employed active site labelling of proteasomal subunits to measure proteasomal activities in cells treated with IM and other tyrosine kinase inhibitors (TKIs). Proteasomal activity was determined in cell lysates from established CML cell lines and primary cells from CML patients using a biotinylated active site-directed probe, which, covalently binds and labels proteasomal subunits beta-1, beta-2 and beta-5 depending on their activity. In addition, in order to analyze a possible direct effect of IM, isolated 20S and 26S proteasomes were examined. We found that treatment with IM led to a concentration-dependent decrease of proteasomal activity in BCR-ABL positive cells. In line with these results IM reduced the expression of HLA-class I molecules in HLA-A2 transfected K-562 cells. This inhibitory effect was independent of protein expression of proteasome subunits as analyzed by western blotting and it was not due to the induction of apoptosis as only samples with nuclei fragmentation below 40% were utilized. Furthermore, these effects were not inhibited by addition of zVAD, a pan-caspase inhibitor of apoptosis. The inhibition of proteasomal activity was independent of BCR-ABL as analyzed using IM resistant CML cell lines including BA/F-T315I cell line which is transfected with the multiresistent T315I mutation. Application of BCR-ABL specific siRNA to knock down its expression had no significant influence on proteasomal activity. Accordingly, incubation of isolated 20S and 26S proteasomes with IM resulted in a profound inhibition of their activity as determined by reduced proteolytic cleavage of fluorogenic substrates. Interestingly, this inhibition of the proteasomal activity was not IM specific as nilotinib, a TKI with a higher BCR-ABL affinity had similar effects. Furthermore, the multikinase inhibitor sorafenib but not sunitinib reduced the proteasomal activity in cell lysates and isolated proteasomes. Incubation of cells with the mTOR inhibitor rapamycin inhibited the activity of proteasomal subunits while the PI3 kinase inhibitor LY-294002 that acts upstream of rapamycin had no effect. Our results demonstrate that IM and several other TKIs currently applied in the treatment of patients can affect the immunogenicity of malignant cells by a direct inhibition of the proteasomal activity.

Dendritic cells (DCs) play a significant role in the initiation and maintenance of primary immune responses. The identification of new genes that are involved in DC biology is crucial for a better understanding of the unique DC functions. We approached this issue by generating a substractive cDNA library based on suppression hybridization between monocyte derived DCs (mDCs) cDNA and the reference monocyte cDNA. Among various differentially in mDC expressed genes, we identified the gene p306 with unknown functions. In the following, we characterized the gene p306 and the translated protein splice variants. We confirmed the p306 expression in mDCs on mRNA level by RACE and RT-PCR with subsequent sequencing. Besides the 2 known splice variants, we found a third novel form. Two splice variants were expressed in activated myeloid and plasmacytoid DCs sorted from blood. To characterize the p306 protein forms, we analyzed the hypothetical proteins by various prediction programs. We found that the N-terminus of the protein isoforms is homologue to a DNA-binding protein. Structural features are an oligonucleotide-binding fold with an integrated zinc ribbon. To detect p306 protein expression, we generated a polyclonal antibody and used it in Western blotting experiments. We were able to detect 3 protein bands with sizes of 36 kDa, 84 kDa and about 200 kDa, which differed from the expected sizes of 20 kDa and 111 kDa. We found that the unexpected sizes are in part due to N-glycosylation using the inhibitor tunicamycin. When analyzing cell lysates and nuclear extracts, we found that the 36 kDa protein form is expressed exclusively in the nucleus, while other forms could be detected in nucleus and cytosol. Interestingly, activation of mDCs with Toll like receptor ligands Pam3Cys, Poly I:C, LPS and R848 selectively upregulated the expression of the 84 kDa form in the nucleus. DC treatment with IL-10 or Imatinib, compounds that inhibit DC differentiation and function, abolished the expression of all 3 protein forms. In order to analyze the possible effect of proteases on the post-transcriptional regulation of the protein expression and generation of the identified splice variants, mDCs were treated with several protease inhibitors. DC treatment with cathepsin/subtilisin inhibitor led to the downregulation of all 3 protein variants, indicating that p306 might be involved in the regulation of apoptosis induction. The pan-caspase inhibitor zVAD caused the upregulation of all protein forms. In conclusion, we identified a novel gene p306 that is differentially expressed in mDC, which encodes for at least 3 different splice variants and protein forms with diverse cellular localization. Although the functions of the p306 proteins still have to be determined, the gene p306 is probably involved in binding nucleic acids and in regulating apoptosis induction.

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.