- Email: [email protected]
ID: 99
Abstract / Cytokine 76 (2015) 66–112 ID: 95 Quantitative characterisation of STAT2 dimerisation Christin Pelzel *, David Jan Scott, Johnathan On Kay Ho, Uwe Vinkemeier, The University of Nottingham, United Kingdom * Corresponding author. Interferons (IFN) are released by cells as a response to microbial infection. They are best known for their antiviral and immunomodulatory functions. IFNs bind to cell surface receptors which then initiate a signalling cascade inside the cell, ultimately resulting in target gene transcription. Essential to this signalling cascade is the dimersation of signal transducer and activator of transcription proteins (STATs). The composition of these dimers determines the genes targeted. The STAT proteins, which are encoded by 7 different genes, can function as homodimers with the possible exception of STAT2. We will generate quanititative data on STAT2 homodimerisation to clarify this question. It is well known, in contrast, that STAT2 heterodimerises with STAT1. In fact, STAT1-STAT2 heterodimers are crucial for type I IFN signalling. STAT heterodimerisation is a commonly observed phenomenon that is nonetheless poorly understood. Our study will assess the interaction of STAT1 with STAT2 and thus for the first time generate quantitative data on the heterodimerisation of STAT proteins. We will utilise recombinant STAT1- and STAT2-proteins produced in insect cells, purified with affinity chromatography and gel filtration. Subsequently these proteins will be subject to sedimentation velocity and sedimentation equilibrium experiments using an analytical ultracentrifuge. These powerful methods allow quantitative characterisation of single- and multi-protein interactions, such as homodimer and heterodimer assembly respectively. These studies will further our understanding of STAT dimerisation and hence IFN signalling.
http://dx.doi.org/10.1016/j.cyto.2015.08.122
83
As a notorious pathogenic agent for humans, Influenza A virus (IAV) causes influenza diseases epidemically and pandemically, leading to the deaths of tens of millions of people during the last century. With new antigenic strains of influenza continuing to emerge, rapid generation of effective vaccines and antiviral medication remains a challenge. Therefore, novel strategies for the prevention and treatment of IAV infection are needed given the limitations of available antiviral strategies. By using in vitro transcription and replication system and also with the aid of Dicer, we generated a siRNA pool containing siRNAs targeting multiple conserved domain of IAV and we plan to evaluate the efficacy of specific siRNAs to prevent from the infection of different strains of IAV in human moDCs. The preliminary data showed that the replication of different strains of IAV in human moDCs was inhibited by the pre-transfection with these siRNAs 16 h before viral infection. And this inhibition was not solely due to the innate immune responses induced by pre-transfection with siRNAs. This study will address the potential use of novel synthesized siRNAs in limiting influenza viruses (including highly pathogenic avian influenza A/H5N1 viruses) replication in human monocyte-derived dendritic cells (moDCs) and also its adjuvant potential in vaccine development. These studies will provide more evidences for the feasibility of this novel strategy in the prevention and treatment of Influenza. http://dx.doi.org/10.1016/j.cyto.2015.08.124
ID: 98 H5N1 and H7N9 viruses induce impaired antiviral state in human immune cells by different mechanisms Veera Westenius 1, Sanna M. Mäkelä 1, Ilkka Julkunen 1,2, Pamela Österlund 1, National Institute for Health and Welfare (THL), Finland, 2 University of Turku, Department of Virology, Turku, Finland 1
ID: 96 Two mutations in the GTPase domain of interferon-induced Mx1 are responsible for high influenza susceptibility of wild-derived CAST/EiJ mice Cindy Nürnberger 1,2,*, Vanessa Zimmermann 1, Melanie Gerhardt 1, Katrin Friedrich 1, Staeheli Peter 1, 1 Institute of Virology, University Medical Center Freiburg, Freiburg, Germany, 2 Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Germany * Corresponding author at: Institute of Virology, University Medical Center Freiburg, Freiburg, Germany. Type I and III interferon-regulated Mx genes encode evolutionary conserved antiviral restriction factors which inhibit a broad range of RNA viruses in most vertebrate species. In mice, the Mx1 locus confers resistance to members of the Orthomyxoviridae, such as influenza A and Thogoto viruses. Mice of strain A2G carry a functional Mx1 gene, while most other laboratory inbred mouse strains carry truncated and therefore defective Mx1 alleles. A recent study (Ferris MT et al. PLOS pathogens 9(2) 2013) investigating how genetic factors might contribute to influenza susceptibility showed that CAST/EiJ mice, an inbred strain derived from wild Mus musculus castaneus, are susceptible to influenza virus challenge although they carry a full-length Mx1 gene. Here we characterized CAST-derived Mx1 in order to better understand the molecular basis of its altered antiviral properties. We were able to show that CASTderived Mx1 retains a low degree of anti-influenza activity but, surprisingly, is still able to confer full protection against Thogoto virus challenge. Sequencing revealed that CAST-derived Mx1 differs by two amino acids from A2G-derived Mx1 which are both localized in the GTPase (G) domain of the protein. These mutations lead to a reduction in GTPase activity. They further diminish Mx1 protein levels in organs of animals after interferon stimulation in vivo. Interestingly, these mutations have no effect on mRNA levels, suggesting that the reduced Mx1 protein levels might be attributed to differences in protein stability. In summary, our findings are able to explain the unexpected virus susceptibility of CAST/EiJ mice.
http://dx.doi.org/10.1016/j.cyto.2015.08.123
ID: 97 Novel RNA protection strategy against influenza A virus Miao Jiang 1,2,3,*, Pamela Österlund 1, Minna M. Poranen 2, Dennis H. Bamford 2,3, Ilkka Julkunen 4, 1 Virology Unit, Department of Infectious Disease Surveillance and Control, National Institute for Health and Welfare, Helsinki, Finland, 2 Department of Biosciences, University of Helsinki, Finland, 3 Institute of Biotechnology, University of Helsinki, Finland, 4 Department of Virology, University of Turku, Finland * Corresponding author at: Virology Unit, Department of Infectious Disease Surveillance and Control, National Institute for Health and Welfare, Helsinki, Finland.
The threatening H5N1 and H7N9 avian influenza viruses have infected humans repeatedly with high mortality (30–60%). Most H7N9 or H5N1 virus-infected patients suffer from acute respiratory distress syndrome (ARDS) and severe lower airway inflammation.This raises a question whether the H7N9 virus can induce a similar immune dysregulation as the H5N1 virus, which contributes to the devastating fatal outcome of the disease. We compared the antiviral-inducing mechanisms during the infection with seasonal H3N2 and avian-origin H5N1 and H7N9 influenza viruses in human immune cells. We have previously shown that the novel H7N9 virus, although replicating strongly in human DCs, did not efficiently induce antiviral IFN responses, and this deficiency was associated with a block in IRF3 activation. Interestingly, in H5N1 infection, in spite of the massive IFN induction the expression levels of IFN stimulated genes (ISGs) like MxA and IFITM3 remained relatively low. However, our new studies show that the H5N1 virus induces the robust IFN response by the incoming virus particles even if the virus replication is killed by UV treatment. By killing the H5N1 virus replication we were able to restore the expression of ISGs. Thus we further studied the role of the NS1 protein from different avian influenza viruses in antagonizing the IFN responses. We noticed that NS1 proteins from both H5N1 and H7N9 viruses were able to inhibit IFN-b promoter activation. Our data suggests that although both H7N9 and H5N1 avian-origin viruses cause severe pneumonia and ARDS in humans, the underlying mechanisms behind the pathology are distinct. http://dx.doi.org/10.1016/j.cyto.2015.08.125
ID: 99 Identification, function and clinical development of the heterodimeric IL-15 cytokine (hetIL-15) George N. Pavlakis 1, Cristina Bergamaschi 1, Sin-Man Ng 1, Stephanie Chen 1, Jenifer Bear 1, Candido Alicea 1, Bethany Nagy 1, Raymond Sowder 2, Elena Chertova 2, Barbara K. Felber 1, 1 National Cancer Institute at Frederick, USA, 2 AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, USA We have shown that the physiologically relevant Interleukin-15 molecule in vivo is a heterodimeric molecule consisting of the IL-15 polypeptide chain and the so-called IL-15 Receptor alpha chain (IL-15Ra), a membrane protein. There are two forms of IL-15 in the body: membrane bound heterodimer of IL-15 and IL-15Ra and soluble heterodimeric IL-15 produced by a specific proteolytic cleavage of the heterodimer at the extracellular domain of IL-15Ra. Our results indicate that IL-15Ra polypeptide is not a receptor but the other half of the heterodimeric IL-15 cytokine. We have characterized the soluble heterodimeric IL-15 (hetIL-15) and developed methods for its efficient production and purification. Injection of hetIL-15 resulted in a significant delay in tumor growth, in preclinical models of tumor-bearing mice. Intratumoral delivery of hetIL-15 induced regression
84
Abstract / Cytokine 76 (2015) 66–112
of established tumors. hetIL-15 has the unique property to increase intratumoral CD8 cells and to also increase dramatically the ratio of CD8 to Treg. We dissect the mechanisms leading to tumor control in hetIL-15-treated mice, revealing a novel interplay between IL-15 and the cognate cytokine interleukin-2 (IL-2). Repeated subcutaneous administration of purified hetIL-15 in macaques resulted in sustained plasma IL-15 levels and in dose-dependent expansion of NK and T cells in blood and tissues, demonstrating pharmacokinetics and in vivo bioactivity superior to monomeric IL-15. On the basis of these data, we have produced and characterized a cGMP preparation of hetIL-15 and we have initiated a Phase 1 dose escalation clinical trial in metastatic cancer to assess safety and activity of this cytokine. http://dx.doi.org/10.1016/j.cyto.2015.08.126
ID: 100 Regulation and B-cell help mediated by distinct subsets of IL10-producing T-cells Jens Geginat, INGM, Italy IL10 is a B-cell growth factor produced by CD4 + helper T-cells, but it also mediates suppression of T-cell responses by regulatory T-cells. We found that IL10-producing regulatory T-cells (Tr1) and IL10-dependent B-helper T-cells are distinct subsets in humans and mice. Tr1 cells that suppressed T cell proliferation and experimental colitis coexpressed CCR5 and PD1, but not CCR6 or IL-7R. They down-regulated IL-10 production in response to pro-inflammatory cytokines, and consequently produced only low levels of IL-10 in the intestine of IBD patients. Tr1 cells failed to up-regulate CD40L and did not promote B-cell responses, but conversely suppressed IgG production. However, Tr1 cells from lupus patients were unable to inhibit B cell responses. In marked contrast, IL10-producing IL-7R + CCR6 + helper T-cells expressed CD40L and induced B-cell antibody production via IL10. Notably, these IL10-dependent B-helper T-cells were distinct from conventional TFH and Th17-cells, were auto-reactive and consequently provided spontaneous B cell help in lupus patients. In summary, we defined a strategy to distinguish IL10 producing T-cell subsets with opposing functions in B-cell responses and in autoimmune diseases. http://dx.doi.org/10.1016/j.cyto.2015.08.127
ID: 101 Investigating the role of interferon k4 in hepatitis C virus infection Ewa Terczynska-Dyla 1,*, Stephanie Bibert 2, Francois H.T. Duong 3,4, Chris Lauber 5, Hans Henrik Gad 1, Lars Kaderali 5, Markus H. Heim 3,4, Pierre-Yves Bochud 2, Rune Hartmann 1, 1 Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark, 2 Infectious Diseases Service, Department of Medicine, University Hospital and University of Lausanne, Lausanne, Switzerland, 3 Department of Biomedicine, University of Basel, Basel, Switzerland, 4 Division of Gastroenterology and Hepatology, University Hospital Basel, Basel, Switzerland, 5 The Institute for Medical Informatics and Biometry, University of Technology Dresden, Dresden, Germany * Corresponding author. IFNL4, a recently discovered member of the interferon k family (IFNks or type III IFNs), is a pseudogene in a significant fraction of the human population. A single nucleotide polymorphism located in IFNL4, which determines the ability to express IFNk4, has been correlated with poor spontaneous and treatment-induced clearance of hepatitis C virus (HCV) infections. We show that IFNk4 is an active type III IFN that induces a typical subset of ISGs, signals through the classical type III IFN receptor complex and is antiviral against HCV and coronaviruses. However, its secretion is impaired and this impairment is caused by a yet unknown molecular determinant, but appears to be partially caused by a weak signal peptide and inefficient N-linked glycosylation. This glycosylation is not required for antiviral activity and secretion of IFNk4, but seems to improve its processing. The impaired secretion of IFNk4 appears to be a recently acquired feature of primates. A single amino acid substitution in IFNk4 changing a proline at position 70 to a serine (P70S) alters its activity. We demonstrate that the IFNk4-S70 variant has a significantly lower antiviral activity compared to IFNk4-P70. Our subsequent genetic study on a cohort of patients infected with HCV shows that individuals, who encode IFNk4-S70, display lower hepatic ISG expression, better treatment response rates and better spontaneous clearance rates than patients encoding IFNk4-P70. This study provides important evidence supporting a role for active IFNk4 as the driver of high hepatic ISG expression as well as the cause of poor HCV clearance.
ID: 102 Pretreatment prediction of individual rheumatoid arthritis patients’ response to anti-cytokine therapy using serum cytokine/chemokine/soluble receptor biomarkers Kazuko Uno 1,*, Kazuyuki Yoshizaki 2, Mitsuhiro Iwahashi 3, Miki Tanigawa 2, Katsumi Yagi 1, 1 Louis Pasteur Center for Medical Research, Japan, 2 The Institute of Scientific and Industrial Research, Osaka University, Japan, 3 Higashi Hiroshima Memorial Hospital, Japan * Corresponding author. The inability to match rheumatoid arthritis (RA) patients with the anti-cytokine agent most efficacious for them is a major hindrance to patients’ speedy recovery and to the clinical use of anti-cytokine therapy. Identifying predictive biomarkers that can assist in matching RA patients with more suitable anti-cytokine treatment was our aim in this research. The sample consisted of 138 RA patients (naïve and nonnaïve) who were administered tocilizumab or etanercept for a minimum of 16 weeks as a prescribed RA treatment. Pretreatment serum samples were obtained from patients and clinical measures of their disease activity were evaluated at baseline and 16 weeks after treatment commenced. Using patients’ pretreatment serum, we measured 31 cytokines/chemokines/soluble receptors and used multiple linear regression analysis to identify biomarkers that correlated with patients’ symptom levels (DAS28-CRP score) at week 16 and multiple logistic analyses for biomarkers that correlated with patients’ final outcome. The results revealed that sgp130, logIL-6, logIL-8, logEotaxin, logIP-10, logVEGF, logsTNFR-I and logsTNFR-II pretreatment serum levels were predictive of the week 16 DAS28-CRP score in naïve tocilizumab patients while sgp130, logGM-CSF and logIP-10 were predictive in non-naïve patients. Additionally, we found logIL-9, logVEGF and logTNF-a to be less reliable at predicting the week 16 DAS28-CRP score in naïve etanercept patients. The biomarkers for these two therapies differ suggesting that their efficacy will vary for individual patients. Biomarkers for tocilizumab, especially sgp130, are involved in RA pathogenesis and IL-6 signal transduction, which further suggests that they are highly reliable. http://dx.doi.org/10.1016/j.cyto.2015.08.129
ID: 103 Innate immune sensing of nucleic acids in airway epithelial cells compared to monocytic cells Davide Massa 1,*, Kora Przybyszewska 2, Jose A. Bengoechea 2, Andrew G. Bowie 1, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland, 2 Centre for Infection and Immunity, Queen’s University Belfast, Belfast, UK * Corresponding author. 1
In the last decade mechanisms for innate immune sensing of pathogen and self nucleic acids have been defined. For cytosolic sensing of DNA, the adaptor protein STING has a central role, operating downstream of DNA sensors such as cGAS and IFI16 to mediate transcription factor activation and cytokine and interferon induction. However most of the work in defining cytosolic DNA sensing pathways has been done in monocytic cells, while sensing mechanisms in other frontline sensing cells such as airway epithelial cells have been less well defined. Thus here we analysed the innate immune response of human airway epithelial cell line models to RNA and DNA, in comparison to a human monocytic cell line. Our results show that in both monocytic and epithelial cells the innate immune response to RNA virus and dsRNA was promptly activated. However compared to monocytic cells, A549 and BEAS-2B epithelial cells failed to produce inflammatory cytokines and interferons in response to DNA viruses and dsDNA. These cells lacked expression of IFI16 or STING, but did express cGAS. Ectopic expression of STING restored the DNA response in A549 cells, in a cGAS-dependent manner. In contrast to A549 and BEAS-2B cells, the hTERT-immortalised normal airway epithelial cell line NuLi-1 did express IFI16, cGAS and STING, and responded to DNA viruses and DNA in a STING-dependent manner. These results highlight the loss of DNA responses in epithelial cell lines lacking STING, and have relevance in considering different cell models for innate sensing studies in airway epithelial cells. http://dx.doi.org/10.1016/j.cyto.2015.08.130
ID: 104 Opposing roles of two dsRNA binding proteins PACT and TRBP on RIG-I induced interferon production Lauren S. Vaughn *, Rekha C. Patel, University of South Carolina, USA author.
*
Corresponding
http://dx.doi.org/10.1016/j.cyto.2015.08.128 An integral aspect of innate immunity is the ability to detect non-self molecules to initiate antiviral signaling via pattern recognition receptors (PRRs). One such
http://dx.doi.org/10.1016/j.cyto.2015.08.122
83
As a notorious pathogenic agent for humans, Influenza A virus (IAV) causes influenza diseases epidemically and pandemically, leading to the deaths of tens of millions of people during the last century. With new antigenic strains of influenza continuing to emerge, rapid generation of effective vaccines and antiviral medication remains a challenge. Therefore, novel strategies for the prevention and treatment of IAV infection are needed given the limitations of available antiviral strategies. By using in vitro transcription and replication system and also with the aid of Dicer, we generated a siRNA pool containing siRNAs targeting multiple conserved domain of IAV and we plan to evaluate the efficacy of specific siRNAs to prevent from the infection of different strains of IAV in human moDCs. The preliminary data showed that the replication of different strains of IAV in human moDCs was inhibited by the pre-transfection with these siRNAs 16 h before viral infection. And this inhibition was not solely due to the innate immune responses induced by pre-transfection with siRNAs. This study will address the potential use of novel synthesized siRNAs in limiting influenza viruses (including highly pathogenic avian influenza A/H5N1 viruses) replication in human monocyte-derived dendritic cells (moDCs) and also its adjuvant potential in vaccine development. These studies will provide more evidences for the feasibility of this novel strategy in the prevention and treatment of Influenza. http://dx.doi.org/10.1016/j.cyto.2015.08.124
ID: 98 H5N1 and H7N9 viruses induce impaired antiviral state in human immune cells by different mechanisms Veera Westenius 1, Sanna M. Mäkelä 1, Ilkka Julkunen 1,2, Pamela Österlund 1, National Institute for Health and Welfare (THL), Finland, 2 University of Turku, Department of Virology, Turku, Finland 1
ID: 96 Two mutations in the GTPase domain of interferon-induced Mx1 are responsible for high influenza susceptibility of wild-derived CAST/EiJ mice Cindy Nürnberger 1,2,*, Vanessa Zimmermann 1, Melanie Gerhardt 1, Katrin Friedrich 1, Staeheli Peter 1, 1 Institute of Virology, University Medical Center Freiburg, Freiburg, Germany, 2 Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Germany * Corresponding author at: Institute of Virology, University Medical Center Freiburg, Freiburg, Germany. Type I and III interferon-regulated Mx genes encode evolutionary conserved antiviral restriction factors which inhibit a broad range of RNA viruses in most vertebrate species. In mice, the Mx1 locus confers resistance to members of the Orthomyxoviridae, such as influenza A and Thogoto viruses. Mice of strain A2G carry a functional Mx1 gene, while most other laboratory inbred mouse strains carry truncated and therefore defective Mx1 alleles. A recent study (Ferris MT et al. PLOS pathogens 9(2) 2013) investigating how genetic factors might contribute to influenza susceptibility showed that CAST/EiJ mice, an inbred strain derived from wild Mus musculus castaneus, are susceptible to influenza virus challenge although they carry a full-length Mx1 gene. Here we characterized CAST-derived Mx1 in order to better understand the molecular basis of its altered antiviral properties. We were able to show that CASTderived Mx1 retains a low degree of anti-influenza activity but, surprisingly, is still able to confer full protection against Thogoto virus challenge. Sequencing revealed that CAST-derived Mx1 differs by two amino acids from A2G-derived Mx1 which are both localized in the GTPase (G) domain of the protein. These mutations lead to a reduction in GTPase activity. They further diminish Mx1 protein levels in organs of animals after interferon stimulation in vivo. Interestingly, these mutations have no effect on mRNA levels, suggesting that the reduced Mx1 protein levels might be attributed to differences in protein stability. In summary, our findings are able to explain the unexpected virus susceptibility of CAST/EiJ mice.
http://dx.doi.org/10.1016/j.cyto.2015.08.123
ID: 97 Novel RNA protection strategy against influenza A virus Miao Jiang 1,2,3,*, Pamela Österlund 1, Minna M. Poranen 2, Dennis H. Bamford 2,3, Ilkka Julkunen 4, 1 Virology Unit, Department of Infectious Disease Surveillance and Control, National Institute for Health and Welfare, Helsinki, Finland, 2 Department of Biosciences, University of Helsinki, Finland, 3 Institute of Biotechnology, University of Helsinki, Finland, 4 Department of Virology, University of Turku, Finland * Corresponding author at: Virology Unit, Department of Infectious Disease Surveillance and Control, National Institute for Health and Welfare, Helsinki, Finland.
The threatening H5N1 and H7N9 avian influenza viruses have infected humans repeatedly with high mortality (30–60%). Most H7N9 or H5N1 virus-infected patients suffer from acute respiratory distress syndrome (ARDS) and severe lower airway inflammation.This raises a question whether the H7N9 virus can induce a similar immune dysregulation as the H5N1 virus, which contributes to the devastating fatal outcome of the disease. We compared the antiviral-inducing mechanisms during the infection with seasonal H3N2 and avian-origin H5N1 and H7N9 influenza viruses in human immune cells. We have previously shown that the novel H7N9 virus, although replicating strongly in human DCs, did not efficiently induce antiviral IFN responses, and this deficiency was associated with a block in IRF3 activation. Interestingly, in H5N1 infection, in spite of the massive IFN induction the expression levels of IFN stimulated genes (ISGs) like MxA and IFITM3 remained relatively low. However, our new studies show that the H5N1 virus induces the robust IFN response by the incoming virus particles even if the virus replication is killed by UV treatment. By killing the H5N1 virus replication we were able to restore the expression of ISGs. Thus we further studied the role of the NS1 protein from different avian influenza viruses in antagonizing the IFN responses. We noticed that NS1 proteins from both H5N1 and H7N9 viruses were able to inhibit IFN-b promoter activation. Our data suggests that although both H7N9 and H5N1 avian-origin viruses cause severe pneumonia and ARDS in humans, the underlying mechanisms behind the pathology are distinct. http://dx.doi.org/10.1016/j.cyto.2015.08.125
ID: 99 Identification, function and clinical development of the heterodimeric IL-15 cytokine (hetIL-15) George N. Pavlakis 1, Cristina Bergamaschi 1, Sin-Man Ng 1, Stephanie Chen 1, Jenifer Bear 1, Candido Alicea 1, Bethany Nagy 1, Raymond Sowder 2, Elena Chertova 2, Barbara K. Felber 1, 1 National Cancer Institute at Frederick, USA, 2 AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, USA We have shown that the physiologically relevant Interleukin-15 molecule in vivo is a heterodimeric molecule consisting of the IL-15 polypeptide chain and the so-called IL-15 Receptor alpha chain (IL-15Ra), a membrane protein. There are two forms of IL-15 in the body: membrane bound heterodimer of IL-15 and IL-15Ra and soluble heterodimeric IL-15 produced by a specific proteolytic cleavage of the heterodimer at the extracellular domain of IL-15Ra. Our results indicate that IL-15Ra polypeptide is not a receptor but the other half of the heterodimeric IL-15 cytokine. We have characterized the soluble heterodimeric IL-15 (hetIL-15) and developed methods for its efficient production and purification. Injection of hetIL-15 resulted in a significant delay in tumor growth, in preclinical models of tumor-bearing mice. Intratumoral delivery of hetIL-15 induced regression
84
Abstract / Cytokine 76 (2015) 66–112
of established tumors. hetIL-15 has the unique property to increase intratumoral CD8 cells and to also increase dramatically the ratio of CD8 to Treg. We dissect the mechanisms leading to tumor control in hetIL-15-treated mice, revealing a novel interplay between IL-15 and the cognate cytokine interleukin-2 (IL-2). Repeated subcutaneous administration of purified hetIL-15 in macaques resulted in sustained plasma IL-15 levels and in dose-dependent expansion of NK and T cells in blood and tissues, demonstrating pharmacokinetics and in vivo bioactivity superior to monomeric IL-15. On the basis of these data, we have produced and characterized a cGMP preparation of hetIL-15 and we have initiated a Phase 1 dose escalation clinical trial in metastatic cancer to assess safety and activity of this cytokine. http://dx.doi.org/10.1016/j.cyto.2015.08.126
ID: 100 Regulation and B-cell help mediated by distinct subsets of IL10-producing T-cells Jens Geginat, INGM, Italy IL10 is a B-cell growth factor produced by CD4 + helper T-cells, but it also mediates suppression of T-cell responses by regulatory T-cells. We found that IL10-producing regulatory T-cells (Tr1) and IL10-dependent B-helper T-cells are distinct subsets in humans and mice. Tr1 cells that suppressed T cell proliferation and experimental colitis coexpressed CCR5 and PD1, but not CCR6 or IL-7R. They down-regulated IL-10 production in response to pro-inflammatory cytokines, and consequently produced only low levels of IL-10 in the intestine of IBD patients. Tr1 cells failed to up-regulate CD40L and did not promote B-cell responses, but conversely suppressed IgG production. However, Tr1 cells from lupus patients were unable to inhibit B cell responses. In marked contrast, IL10-producing IL-7R + CCR6 + helper T-cells expressed CD40L and induced B-cell antibody production via IL10. Notably, these IL10-dependent B-helper T-cells were distinct from conventional TFH and Th17-cells, were auto-reactive and consequently provided spontaneous B cell help in lupus patients. In summary, we defined a strategy to distinguish IL10 producing T-cell subsets with opposing functions in B-cell responses and in autoimmune diseases. http://dx.doi.org/10.1016/j.cyto.2015.08.127
ID: 101 Investigating the role of interferon k4 in hepatitis C virus infection Ewa Terczynska-Dyla 1,*, Stephanie Bibert 2, Francois H.T. Duong 3,4, Chris Lauber 5, Hans Henrik Gad 1, Lars Kaderali 5, Markus H. Heim 3,4, Pierre-Yves Bochud 2, Rune Hartmann 1, 1 Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark, 2 Infectious Diseases Service, Department of Medicine, University Hospital and University of Lausanne, Lausanne, Switzerland, 3 Department of Biomedicine, University of Basel, Basel, Switzerland, 4 Division of Gastroenterology and Hepatology, University Hospital Basel, Basel, Switzerland, 5 The Institute for Medical Informatics and Biometry, University of Technology Dresden, Dresden, Germany * Corresponding author. IFNL4, a recently discovered member of the interferon k family (IFNks or type III IFNs), is a pseudogene in a significant fraction of the human population. A single nucleotide polymorphism located in IFNL4, which determines the ability to express IFNk4, has been correlated with poor spontaneous and treatment-induced clearance of hepatitis C virus (HCV) infections. We show that IFNk4 is an active type III IFN that induces a typical subset of ISGs, signals through the classical type III IFN receptor complex and is antiviral against HCV and coronaviruses. However, its secretion is impaired and this impairment is caused by a yet unknown molecular determinant, but appears to be partially caused by a weak signal peptide and inefficient N-linked glycosylation. This glycosylation is not required for antiviral activity and secretion of IFNk4, but seems to improve its processing. The impaired secretion of IFNk4 appears to be a recently acquired feature of primates. A single amino acid substitution in IFNk4 changing a proline at position 70 to a serine (P70S) alters its activity. We demonstrate that the IFNk4-S70 variant has a significantly lower antiviral activity compared to IFNk4-P70. Our subsequent genetic study on a cohort of patients infected with HCV shows that individuals, who encode IFNk4-S70, display lower hepatic ISG expression, better treatment response rates and better spontaneous clearance rates than patients encoding IFNk4-P70. This study provides important evidence supporting a role for active IFNk4 as the driver of high hepatic ISG expression as well as the cause of poor HCV clearance.
ID: 102 Pretreatment prediction of individual rheumatoid arthritis patients’ response to anti-cytokine therapy using serum cytokine/chemokine/soluble receptor biomarkers Kazuko Uno 1,*, Kazuyuki Yoshizaki 2, Mitsuhiro Iwahashi 3, Miki Tanigawa 2, Katsumi Yagi 1, 1 Louis Pasteur Center for Medical Research, Japan, 2 The Institute of Scientific and Industrial Research, Osaka University, Japan, 3 Higashi Hiroshima Memorial Hospital, Japan * Corresponding author. The inability to match rheumatoid arthritis (RA) patients with the anti-cytokine agent most efficacious for them is a major hindrance to patients’ speedy recovery and to the clinical use of anti-cytokine therapy. Identifying predictive biomarkers that can assist in matching RA patients with more suitable anti-cytokine treatment was our aim in this research. The sample consisted of 138 RA patients (naïve and nonnaïve) who were administered tocilizumab or etanercept for a minimum of 16 weeks as a prescribed RA treatment. Pretreatment serum samples were obtained from patients and clinical measures of their disease activity were evaluated at baseline and 16 weeks after treatment commenced. Using patients’ pretreatment serum, we measured 31 cytokines/chemokines/soluble receptors and used multiple linear regression analysis to identify biomarkers that correlated with patients’ symptom levels (DAS28-CRP score) at week 16 and multiple logistic analyses for biomarkers that correlated with patients’ final outcome. The results revealed that sgp130, logIL-6, logIL-8, logEotaxin, logIP-10, logVEGF, logsTNFR-I and logsTNFR-II pretreatment serum levels were predictive of the week 16 DAS28-CRP score in naïve tocilizumab patients while sgp130, logGM-CSF and logIP-10 were predictive in non-naïve patients. Additionally, we found logIL-9, logVEGF and logTNF-a to be less reliable at predicting the week 16 DAS28-CRP score in naïve etanercept patients. The biomarkers for these two therapies differ suggesting that their efficacy will vary for individual patients. Biomarkers for tocilizumab, especially sgp130, are involved in RA pathogenesis and IL-6 signal transduction, which further suggests that they are highly reliable. http://dx.doi.org/10.1016/j.cyto.2015.08.129
ID: 103 Innate immune sensing of nucleic acids in airway epithelial cells compared to monocytic cells Davide Massa 1,*, Kora Przybyszewska 2, Jose A. Bengoechea 2, Andrew G. Bowie 1, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland, 2 Centre for Infection and Immunity, Queen’s University Belfast, Belfast, UK * Corresponding author. 1
In the last decade mechanisms for innate immune sensing of pathogen and self nucleic acids have been defined. For cytosolic sensing of DNA, the adaptor protein STING has a central role, operating downstream of DNA sensors such as cGAS and IFI16 to mediate transcription factor activation and cytokine and interferon induction. However most of the work in defining cytosolic DNA sensing pathways has been done in monocytic cells, while sensing mechanisms in other frontline sensing cells such as airway epithelial cells have been less well defined. Thus here we analysed the innate immune response of human airway epithelial cell line models to RNA and DNA, in comparison to a human monocytic cell line. Our results show that in both monocytic and epithelial cells the innate immune response to RNA virus and dsRNA was promptly activated. However compared to monocytic cells, A549 and BEAS-2B epithelial cells failed to produce inflammatory cytokines and interferons in response to DNA viruses and dsDNA. These cells lacked expression of IFI16 or STING, but did express cGAS. Ectopic expression of STING restored the DNA response in A549 cells, in a cGAS-dependent manner. In contrast to A549 and BEAS-2B cells, the hTERT-immortalised normal airway epithelial cell line NuLi-1 did express IFI16, cGAS and STING, and responded to DNA viruses and DNA in a STING-dependent manner. These results highlight the loss of DNA responses in epithelial cell lines lacking STING, and have relevance in considering different cell models for innate sensing studies in airway epithelial cells. http://dx.doi.org/10.1016/j.cyto.2015.08.130
ID: 104 Opposing roles of two dsRNA binding proteins PACT and TRBP on RIG-I induced interferon production Lauren S. Vaughn *, Rekha C. Patel, University of South Carolina, USA author.
*
Corresponding
http://dx.doi.org/10.1016/j.cyto.2015.08.128 An integral aspect of innate immunity is the ability to detect non-self molecules to initiate antiviral signaling via pattern recognition receptors (PRRs). One such