- Email: [email protected]
ID: 76
Abstract / Cytokine 76 (2015) 66–112 numerous differences in gene expression (including interferon stimulated genes) between WT and STING / T cells before and after DMXAA stimulation were identified. The work was supported by the Russian Science Foundation (grant no. 115-1500100). http://dx.doi.org/10.1016/j.cyto.2015.08.103
ID: 74 Interleukin-6 globally influences stress-signaling by reducing the expression of the mTOR-Inhibitor REDD1 in a STAT3-dependent manner Anna Dittrich *, Pinno Jessica, Nicole Wundrack, Oliver Klepsch, Hannes Bongartz, Fred * Schaper, Otto-von-Guericke-University of Magdeburg, Germany Corresponding author. Mammalian target of rapamycin (mTOR) is a central mediator of cellular growth and survival which is potently activated by interleukin 6 (IL-6). However, the molecular mechanisms of IL-6-induced mTOR activation are still poorly understood. mTOR activity is negatively regulated by regulated in development and DNA damage response 1 (REDD1). Expression of REDD1 is induced by cellular stressors such as DNA damaging agents. Stress-induced REDD1 expression facilitates cellular damage repair by temporarily blocking mTOR-induced signaling. However, reduced REDD1 expression has been associated with proliferative diseases such as liver cancer. We show that REDD1 expression is reduced by IL-6. The reduction of REDD1 protein by IL-6 is independent of proteasomal or caspase-mediated degradation of REDD1 but occurs on the level of REDD1 mRNA. IL-6-induced reduction of REDD1 follows the same kinetics as mTOR activation, suggesting that it contributes to IL-6induced mTOR activation. The decrease in REDD1 expression is independent of phosphatidylinositide-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling but depends on the expression and activation of signal transducer and activator of transcription 3 (STAT3). Consequently, inhibition of STAT3 blocks IL-6induced mTOR activation. Of note, beside basal REDD1 expression also stress-induced REDD1 expression is reduced by IL-6 enabling IL-6 to globally interfere in stresssignaling. In summary, we present a novel STAT3-dependent mechanism of both IL-6induced activation of mTOR and IL-6-dependent reversion of stress-induced inhibition of mTOR (through repression of REDD1 expression) that might contribute to the development of proliferative diseases in inflammatory conditions. http://dx.doi.org/10.1016/j.cyto.2015.08.104
ID: 75 Unraveling the properties required for an effective therapeutic antibody targeting a chemokine Amanda Proudfoot *, Pauline Bonvin, Marie Novimmune, Switzerland * Corresponding author.
Kosco-Vilbois,
Nicolas
Fischer,
The control of cell migration by chemokines is mediated by interactions with seven transmembrane G protein-coupled receptors and cell surface proteoglycans. Monoclonal antibodies (mAbs) are a successful class of drugs especially when targeting cytokines. However, there has been limited success when targeting a chemokine. In order to further define what is required to achieve therapeutic efficacy when targeting a chemokine with a mAb, we have dissected the characteristics of two antimurine CXCL10 mAbs. Interestingly, despite having similar affinities and selectivity for CXCL10, they exhibit different key properties. Both mAbs are able to inhibit receptor mediated events such as chemotaxis in vitro, albeit with different potencies yet only one shows efficacy in several animal models of human disease. In addition, the PK of the two mAbs in mice was significantly different: the active mAb had a PK profile characteristic of IgGs with a T1/2 of 18 days whereas the other was rapidly cleared. In contrast to the belief that the active form of chemokine is that bound to cell surface GAGs, the mAb that showed activity in vivo did not recognize cell bound chemokine, whilst the mAb that was rapidly cleared did, leading to a proposal of a new paradigm in chemokine biology. Furthermore, in view of the unexpected abundance of chemokine protein, we have identified a pH dependent antibody which is could inhibit multiple target antigens, through release of the target in the endosomal compartment. Their head to head comparison in a CXCL10 dependent disease model will be presented. http://dx.doi.org/10.1016/j.cyto.2015.08.105
79
ID: 76 The MCMV protein M35 is a novel negative regulator of the type I IFN response Baca Chan 1, Vladimir Magalhães 1, Vanda Juranic´ Lisnic´ 2, Stipan Jonjic 2, Melanie M. Brinkmann 1,*, 1 Helmholtz Centre for Infection Research, Germany, 2 Faculty of Medicine, University of Rijeka, Croatia * Corresponding author. The early detection of herpesvirus infection by pattern recognition receptors (PRRs) triggers the production of type I interferons (IFN), which exert potent antiviral effects. Whilst MCMV is recognized to induce strong innate immune responses, UV inactivation of MCMV results in an enhanced type I IFN response, indicating that the virus actively downmodulates innate immune signaling. Using an unbiased luciferase-based assay to screen an MCMV cDNA library, we have identified the M35 protein as a negative regulator of PRR-dependent type I IFN signaling. Infection of primary bone marrow derived macrophages with an MCMV mutant lacking M35 (M35stop) results in increased secretion of type I IFN as compared to wild-type (WT) MCMV. To confirm the physiological role of M35, we infected BALB/c mice with MCMV M35stop to assess the subsequent type I IFN response. MCMV M35stop induces significantly higher type I IFN serum levels than WT MCMV 8 h post infection, which correlates well with expression of tegument M35 protein. MCMV M35stop replicates to significantly reduced titres compared to WT MCMV in the spleen at day 3 post infection. MCMV is then disseminated via secondary viremia to the salivary glands, a site of persistence and dissemination for MCMV. MCMV M35stop is undetectable from the salivary glands from as early as 7 days post infection. This exclusion of M35stop from the salivary glands indicates that M35 is a critical determinant of viral replication in vivo. Collectively, our data demonstrate the first characterisation of M35 as a novel antagonist of type I IFN signaling downstream of PRRs. http://dx.doi.org/10.1016/j.cyto.2015.08.106
ID: 77 Non-canonical TYK2 governs NK cell-mediated tumour surveillance Agnieszka Witalisz-Siepracka, Michaela Prchal-Murphy, Karoline Bednarik, Katrin Meissl, Eva Maria Putz, Dagmar Gotthardt, Veronika Sexl, Birgit Strobl, Mathias Müller *, University of Veterinary Medicine Vienna, Austria * Corresponding author. Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family. Loss of TYK2 reduces cytotoxicity of both natural killer (NK) and cytotoxic T cells (CTL) in tumour surveillance. In addition to the canonical cytokine receptor associated and enzymatic activities, JAKs function also non-canonical, i.e. kinase independent and/ or scaffolding. Recently, hyperactive TYK2 has been assigned to leukaemia forms in humans and therefore TYK2 inhibitors emerge in cancer therapy. In order to assess the non-canonical functions of TYK2 and potential harmful effects of its inhibition on tumour surveillance we have gene-targeted mouse strain expressing kinase-inactive TYK2 (TYK2K923E). TYK2K923E partially restored the impaired maturation of splenic NK cells from Tyk2 / mice. NK cell cytotoxicity against a range of target cells was significantly improved by TYK2K923E compared to loss of TYK2. IFNc production after IL-12 or NK cell receptor stimulation was fully dependent on enzymatically active TYK2. Most importantly, the control of NK cell targeted transplantable tumours was governed by TYK2K923E. In contrast to NK cells, Tyk2K923E CTLs show fully impaired cytotoxicity but, surprisingly, Tyk2K923E mice were capable to limit the growth of T cell targeted transplantable tumours. Reconstitution experiments in Rag2 / mice revealed that Tyk2K923E T cells induce NK cell tumour infiltration more efficiently than Tyk2 / T cells, which we propose to enable tumour eradication. We are currently investigating the mechanisms underlying this T cell-NK cell crosstalk and the dependence on non-canonical TYK2. VS, MM and BS are supported by the Austrian Science Fund (FWF SFB-F28, and DK-W1212). http://dx.doi.org/10.1016/j.cyto.2015.08.107
ID: 78 Recombinant MVA generating excess early double-stranded RNA transiently activates PKR and triggers enhanced innate immune responses Michael Wolferstätter 1, Marc Schweneker 1, Michaela Späth 1, Susanne Lukassen 1, Marieken Klingenberg 1, Kay Brinkmann 1, Ursula Wielert 1, Henning Lauterbach 1, Hubertus Hochrein 1, Paul Chaplin 1, Mark Suter 1,2, Jürgen Hausmann 1,*, 1 Bavarian Nordic GmbH, Germany, 2 University of Zurich, Zurich, Switzerland * Corresponding author.
ID: 74 Interleukin-6 globally influences stress-signaling by reducing the expression of the mTOR-Inhibitor REDD1 in a STAT3-dependent manner Anna Dittrich *, Pinno Jessica, Nicole Wundrack, Oliver Klepsch, Hannes Bongartz, Fred * Schaper, Otto-von-Guericke-University of Magdeburg, Germany Corresponding author. Mammalian target of rapamycin (mTOR) is a central mediator of cellular growth and survival which is potently activated by interleukin 6 (IL-6). However, the molecular mechanisms of IL-6-induced mTOR activation are still poorly understood. mTOR activity is negatively regulated by regulated in development and DNA damage response 1 (REDD1). Expression of REDD1 is induced by cellular stressors such as DNA damaging agents. Stress-induced REDD1 expression facilitates cellular damage repair by temporarily blocking mTOR-induced signaling. However, reduced REDD1 expression has been associated with proliferative diseases such as liver cancer. We show that REDD1 expression is reduced by IL-6. The reduction of REDD1 protein by IL-6 is independent of proteasomal or caspase-mediated degradation of REDD1 but occurs on the level of REDD1 mRNA. IL-6-induced reduction of REDD1 follows the same kinetics as mTOR activation, suggesting that it contributes to IL-6induced mTOR activation. The decrease in REDD1 expression is independent of phosphatidylinositide-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling but depends on the expression and activation of signal transducer and activator of transcription 3 (STAT3). Consequently, inhibition of STAT3 blocks IL-6induced mTOR activation. Of note, beside basal REDD1 expression also stress-induced REDD1 expression is reduced by IL-6 enabling IL-6 to globally interfere in stresssignaling. In summary, we present a novel STAT3-dependent mechanism of both IL-6induced activation of mTOR and IL-6-dependent reversion of stress-induced inhibition of mTOR (through repression of REDD1 expression) that might contribute to the development of proliferative diseases in inflammatory conditions. http://dx.doi.org/10.1016/j.cyto.2015.08.104
ID: 75 Unraveling the properties required for an effective therapeutic antibody targeting a chemokine Amanda Proudfoot *, Pauline Bonvin, Marie Novimmune, Switzerland * Corresponding author.
Kosco-Vilbois,
Nicolas
Fischer,
The control of cell migration by chemokines is mediated by interactions with seven transmembrane G protein-coupled receptors and cell surface proteoglycans. Monoclonal antibodies (mAbs) are a successful class of drugs especially when targeting cytokines. However, there has been limited success when targeting a chemokine. In order to further define what is required to achieve therapeutic efficacy when targeting a chemokine with a mAb, we have dissected the characteristics of two antimurine CXCL10 mAbs. Interestingly, despite having similar affinities and selectivity for CXCL10, they exhibit different key properties. Both mAbs are able to inhibit receptor mediated events such as chemotaxis in vitro, albeit with different potencies yet only one shows efficacy in several animal models of human disease. In addition, the PK of the two mAbs in mice was significantly different: the active mAb had a PK profile characteristic of IgGs with a T1/2 of 18 days whereas the other was rapidly cleared. In contrast to the belief that the active form of chemokine is that bound to cell surface GAGs, the mAb that showed activity in vivo did not recognize cell bound chemokine, whilst the mAb that was rapidly cleared did, leading to a proposal of a new paradigm in chemokine biology. Furthermore, in view of the unexpected abundance of chemokine protein, we have identified a pH dependent antibody which is could inhibit multiple target antigens, through release of the target in the endosomal compartment. Their head to head comparison in a CXCL10 dependent disease model will be presented. http://dx.doi.org/10.1016/j.cyto.2015.08.105
79
ID: 76 The MCMV protein M35 is a novel negative regulator of the type I IFN response Baca Chan 1, Vladimir Magalhães 1, Vanda Juranic´ Lisnic´ 2, Stipan Jonjic 2, Melanie M. Brinkmann 1,*, 1 Helmholtz Centre for Infection Research, Germany, 2 Faculty of Medicine, University of Rijeka, Croatia * Corresponding author. The early detection of herpesvirus infection by pattern recognition receptors (PRRs) triggers the production of type I interferons (IFN), which exert potent antiviral effects. Whilst MCMV is recognized to induce strong innate immune responses, UV inactivation of MCMV results in an enhanced type I IFN response, indicating that the virus actively downmodulates innate immune signaling. Using an unbiased luciferase-based assay to screen an MCMV cDNA library, we have identified the M35 protein as a negative regulator of PRR-dependent type I IFN signaling. Infection of primary bone marrow derived macrophages with an MCMV mutant lacking M35 (M35stop) results in increased secretion of type I IFN as compared to wild-type (WT) MCMV. To confirm the physiological role of M35, we infected BALB/c mice with MCMV M35stop to assess the subsequent type I IFN response. MCMV M35stop induces significantly higher type I IFN serum levels than WT MCMV 8 h post infection, which correlates well with expression of tegument M35 protein. MCMV M35stop replicates to significantly reduced titres compared to WT MCMV in the spleen at day 3 post infection. MCMV is then disseminated via secondary viremia to the salivary glands, a site of persistence and dissemination for MCMV. MCMV M35stop is undetectable from the salivary glands from as early as 7 days post infection. This exclusion of M35stop from the salivary glands indicates that M35 is a critical determinant of viral replication in vivo. Collectively, our data demonstrate the first characterisation of M35 as a novel antagonist of type I IFN signaling downstream of PRRs. http://dx.doi.org/10.1016/j.cyto.2015.08.106
ID: 77 Non-canonical TYK2 governs NK cell-mediated tumour surveillance Agnieszka Witalisz-Siepracka, Michaela Prchal-Murphy, Karoline Bednarik, Katrin Meissl, Eva Maria Putz, Dagmar Gotthardt, Veronika Sexl, Birgit Strobl, Mathias Müller *, University of Veterinary Medicine Vienna, Austria * Corresponding author. Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family. Loss of TYK2 reduces cytotoxicity of both natural killer (NK) and cytotoxic T cells (CTL) in tumour surveillance. In addition to the canonical cytokine receptor associated and enzymatic activities, JAKs function also non-canonical, i.e. kinase independent and/ or scaffolding. Recently, hyperactive TYK2 has been assigned to leukaemia forms in humans and therefore TYK2 inhibitors emerge in cancer therapy. In order to assess the non-canonical functions of TYK2 and potential harmful effects of its inhibition on tumour surveillance we have gene-targeted mouse strain expressing kinase-inactive TYK2 (TYK2K923E). TYK2K923E partially restored the impaired maturation of splenic NK cells from Tyk2 / mice. NK cell cytotoxicity against a range of target cells was significantly improved by TYK2K923E compared to loss of TYK2. IFNc production after IL-12 or NK cell receptor stimulation was fully dependent on enzymatically active TYK2. Most importantly, the control of NK cell targeted transplantable tumours was governed by TYK2K923E. In contrast to NK cells, Tyk2K923E CTLs show fully impaired cytotoxicity but, surprisingly, Tyk2K923E mice were capable to limit the growth of T cell targeted transplantable tumours. Reconstitution experiments in Rag2 / mice revealed that Tyk2K923E T cells induce NK cell tumour infiltration more efficiently than Tyk2 / T cells, which we propose to enable tumour eradication. We are currently investigating the mechanisms underlying this T cell-NK cell crosstalk and the dependence on non-canonical TYK2. VS, MM and BS are supported by the Austrian Science Fund (FWF SFB-F28, and DK-W1212). http://dx.doi.org/10.1016/j.cyto.2015.08.107
ID: 78 Recombinant MVA generating excess early double-stranded RNA transiently activates PKR and triggers enhanced innate immune responses Michael Wolferstätter 1, Marc Schweneker 1, Michaela Späth 1, Susanne Lukassen 1, Marieken Klingenberg 1, Kay Brinkmann 1, Ursula Wielert 1, Henning Lauterbach 1, Hubertus Hochrein 1, Paul Chaplin 1, Mark Suter 1,2, Jürgen Hausmann 1,*, 1 Bavarian Nordic GmbH, Germany, 2 University of Zurich, Zurich, Switzerland * Corresponding author.