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Abstract / Cytokine 70 (2014) 28–79
Hepatitis C virus (HCV) infections are the major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma worldwide. Both spontaneous and treatment induced clearance of HCV depend upon genetic variation within the interferon lambda locus, but until now no clear causal relationship has been established. We demonstrate that an amino acid substitution in the IFNk4 protein changing a proline at position 70 to a serine (P70S) substantially alters its antiviral activity. Paradoxically the lower antiviral activity of the S70 version of IFNk4 leads to an improved prognosis for HCV infected patients. Patients harboring the impaired IFNk4-S70 variant display lower interferon-stimulated gene (ISG) expression levels, better treatment response rates and better spontaneous clearance rates, compared to patients coding for the fully active IFNk4-P70 variant. High expression of Interferon Stimulated Genes (ISGs) in the liver of HCV infected patients are known to predict a poor response to interferon based treatments, however, the interferon driving this high ISG expression has not been identified. Our data provide compelling evidence that the active IFNk4 protein is the driver of high hepatic ISG expression in HCV infected individuals as well as being the cause of poor HCV clearance.
infection with recombinant IFN-a (rIFN-aÞ. Interestingly, under such conditions MyTrCa/ mice mounted normal adaptive immune responses and approximately 76% of the mice survived. On the contrary, initiation of the rIFN-a treatment scheme 4 h before VSV infection significantly prolonged survival, whereas reduced VSV-specific cytotoxic T-lymphocytes and basically no VSV neutralizing antibody responses were induced and 100% of the mice finally died. Long-term rIFN-a treatment for 9 days initiated 4 h after VSV infection promoted 100% survival of MyTrCa/ mice and protective immunity was induced normally as verified by 100% of the animals surviving even a VSV re-challenge 3 weeks after discontinuation of the rIFN-a treatment. Initiation of the 9 days rIFN-a regimen 4 h prior to infection also rescued 100% of the mice, however, no adaptive immunity developed and 100% of the animals succumbed to re-challenge. In conclusion, long-term rIFN-a treatment initiated before virus infection prevented induction of protective adaptive immunity, whereas initiation of rIFN-a treatment hours after infection supported development of normal adaptive immunity and protective memory response. These observations have implications for immunotherapies and vaccination strategies, in particular for patients under long-term IFN-I therapy who are treated with live attenuated vaccines.
http://dx.doi.org/10.1016/j.cyto.2014.07.082 http://dx.doi.org/10.1016/j.cyto.2014.07.084
76 Regulation of activins and the activin-binding protein, follistatin, by Toll-like receptor ligands in the adult mouse Mark P. Hedger 1, David J. Phillips 2, Ashley Mansell 1, Kim Sebire 1, Kathy Wilson 1, Helen Ludlow 3, David M. de Kretser 1, 1 MIMR-PHI Institute of Medical Research, Clayton, VIC, Australia, 2 Epworth Research Institute, Richmond, VIC, Australia, 3 Oxford Brookes University, Oxford, United Kingdom The activins, A and B, are members of the transforming growth factor-b cytokine family, and are regulators of inflammation, immunity and fibrosis. Production of activin A and the activin-binding protein, follistatin, increases during inflammation, although aspects of this regulation require clarification and activin B has not been studied in inflammation, previously. Adult male mice were injected with Toll-like receptor (TLR) ligands, acting though MyD88-dependent (TLR2, 4, 7 and 9) and MyD88-independent (TLR3, 4) signalling pathways. Activation of TLR4 by lipopolysaccharide (LPS) was also examined in MyD88-deficient mice, and in mice pre-treated with cycloheximide and actinomycin D. Activin A, B and follistatin were measured in serum by specific immunoasssays, and mRNA expression was measured in the liver by qRT-PCR. Activin A secretion, but not mRNA expression, was stimulated via activation of TLR2, 3, 4 and 7, reaching a peak in the serum around 2–3 h. By contrast, all ligands stimulated activin B at both protein and mRNA level. Compared with activin A, activin B in serum increased more slowly (3–5 h), but reached much higher maximum concentrations. Follistatin was stimulated at the mRNA level by TLR2, 3, 4 and 7. In the absence of MyD88, induction of activin A and follistatin by LPS was prevented, but activin B production was unaffected. While production of activin A was inhibited by cycloheximide, but not by actinomycin D, activin B was inhibited to a similar degree by both compounds. These data indicate that viral and bacterial ligands, acting through both MyD88-dependent and MyD88-independent signaling pathways, stimulate production of activin A, B and follistatin. These data further indicate that activin B, like follistatin, but unlike activin A, is primarily regulated at the transcriptional level during inflammation. These studies suggest that delayed production of activin B may complement the more rapid release of activin A during inflammatory responses. http://dx.doi.org/10.1016/j.cyto.2014.07.083
78 Insights into the function of an anti-leukaemia antibody: structural studies of CSL362 bound to soluble CD123 Tim R. Hercus 1, Sophie E. Broughton 2, Matthew P. Hardy 3, Tracy L. Nero 2, Barbara J. McClure 1, Mara Dottore 1, Urmi Dhagat 2, Emma F. Barry 1, Winnie L. Kan 1, Samantha J. Busfield 3, Andrew D. Nash 3, Nicholas J. Wilson 3, Michael W. Parker 2, Angel F. Lopez 1, 1 Centre for Cancer Biology, SA Pathology and University of South Australia, Adelaide, SA, Australia, 2 ACRF Rational Drug Discovery Centre, St. Vincent’s Institute of Medical Research, Melbourne, Victoria, Australia, 3 CSL Limited, Melbourne, Victoria, Australia The ßc family of cytokines (GM-CSF, IL-3 and IL-5) are largely products of activated T cells and regulate the survival, proliferation, differentiation and functional activation of hematopoietic cells following infection or bleeding. They are variously able to target myeloid hematopoietic stem and progenitor cells as well as mature neutrophils, eosinophils, macrophages and mast cells. Recent evidence suggests that this family of cytokines plays a role in other biological systems and importantly also in cancer, through the development and metastasis of solid tumours. Intriguingly, CD123, the IL-3 receptor alpha subunit (IL3RaÞ is overexpressed by stem cells and primary blast cells of patients with acute myelogenous leukaemia (AML) and this correlates with poor prognosis leading to the development of multiple strategies that target CD123. We have now solved the structure of soluble IL3Ra, in complex with a Fab fragment of CSL362, a humanised monoclonal antibody that binds IL3Ra, blocks IL-3 function and is optimised for NK cell-mediated killing of leukaemic cells [1]. CSL362 is currently in a Phase 1 clinical trial for the treatment of patients with AML (Clinical Trials Gov. Identifier: NCT01632852). The three domain IL3Ra structure unexpectedly revealed two alternative conformations, an open and a closed form based on the orientation of the N-terminal domain (NTD). The open conformation has not previously been reported for other Type I cytokine receptors with a similar domain structure, IL5Ra, IL13Ra1 and IL13Ra2. The IL3Ra structure will be presented together with data that supports a mechanism of IL-3 recognition and receptor signalling that may be applicable to other members of the Type I cytokine receptor superfamily. An unexpected dual mechanism of IL-3 antagonism utilised by CSL362 that involves direct antagonism of IL-3 binding as well as blockade of IL-3 receptor assembly, will also be presented.
Reference 77 Kinetics of Type I interferon responses determine either establishment of antiviral memory or no induction of adaptive immunity Julia Heinrich 1, Peter Staeheli 2, Ulrich Kalinke 1, 1 Institute for Experimental Infection Research, TWINCORE, Hanover, Germany, 2 Department of Virology, Institute for Medical Microbiology and Hygiene, Albert-Ludwigs-Universität, Freiburg, Germany Many pathogens trigger type I interferon (IFN-I) responses early after infection that confer protection until adaptive immunity is induced. Upon infection with vesicular stomatitis virus (VSV) Toll-like receptor (TLR) and RIG-I-like helicase (RLH) signaling platforms are indispensable for the induction of protective IFN-I. Hence, TLR- and RLH-ablated MyTrCa/ mice challenged with VSV do not mount IFN-I responses and are inevitably susceptible to lethal VSV infection. To study the impact of the kinetics of IFN-I responses on the induction of adaptive immunity, we infected MyTrCa/ mice with VSV and treated them 4, 8, 16, and 24 h after
[1] Broughton, et al. Cell reports; 2014 [in press]. http://dx.doi.org/10.1016/j.cyto.2014.07.085
79 S100 proteins regulate LPS-induced inflammation in murine lung Yuka Hiroshima, Kenneth Hsu, Nicodemus Tedla, Sharron Chow, Naomi Kawaguchi, Carolyn L. Geczy, University New South Wales, Sydney, NSW, Australia Aims: S100A8 and S100A9 are calcium binding proteins that comprise 40% of the neutrophil cytosol and induced in several cell types by inflammatory mediators or by
Abstract / Cytokine 70 (2014) 28–79
Hepatitis C virus (HCV) infections are the major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma worldwide. Both spontaneous and treatment induced clearance of HCV depend upon genetic variation within the interferon lambda locus, but until now no clear causal relationship has been established. We demonstrate that an amino acid substitution in the IFNk4 protein changing a proline at position 70 to a serine (P70S) substantially alters its antiviral activity. Paradoxically the lower antiviral activity of the S70 version of IFNk4 leads to an improved prognosis for HCV infected patients. Patients harboring the impaired IFNk4-S70 variant display lower interferon-stimulated gene (ISG) expression levels, better treatment response rates and better spontaneous clearance rates, compared to patients coding for the fully active IFNk4-P70 variant. High expression of Interferon Stimulated Genes (ISGs) in the liver of HCV infected patients are known to predict a poor response to interferon based treatments, however, the interferon driving this high ISG expression has not been identified. Our data provide compelling evidence that the active IFNk4 protein is the driver of high hepatic ISG expression in HCV infected individuals as well as being the cause of poor HCV clearance.
infection with recombinant IFN-a (rIFN-aÞ. Interestingly, under such conditions MyTrCa/ mice mounted normal adaptive immune responses and approximately 76% of the mice survived. On the contrary, initiation of the rIFN-a treatment scheme 4 h before VSV infection significantly prolonged survival, whereas reduced VSV-specific cytotoxic T-lymphocytes and basically no VSV neutralizing antibody responses were induced and 100% of the mice finally died. Long-term rIFN-a treatment for 9 days initiated 4 h after VSV infection promoted 100% survival of MyTrCa/ mice and protective immunity was induced normally as verified by 100% of the animals surviving even a VSV re-challenge 3 weeks after discontinuation of the rIFN-a treatment. Initiation of the 9 days rIFN-a regimen 4 h prior to infection also rescued 100% of the mice, however, no adaptive immunity developed and 100% of the animals succumbed to re-challenge. In conclusion, long-term rIFN-a treatment initiated before virus infection prevented induction of protective adaptive immunity, whereas initiation of rIFN-a treatment hours after infection supported development of normal adaptive immunity and protective memory response. These observations have implications for immunotherapies and vaccination strategies, in particular for patients under long-term IFN-I therapy who are treated with live attenuated vaccines.
http://dx.doi.org/10.1016/j.cyto.2014.07.082 http://dx.doi.org/10.1016/j.cyto.2014.07.084
76 Regulation of activins and the activin-binding protein, follistatin, by Toll-like receptor ligands in the adult mouse Mark P. Hedger 1, David J. Phillips 2, Ashley Mansell 1, Kim Sebire 1, Kathy Wilson 1, Helen Ludlow 3, David M. de Kretser 1, 1 MIMR-PHI Institute of Medical Research, Clayton, VIC, Australia, 2 Epworth Research Institute, Richmond, VIC, Australia, 3 Oxford Brookes University, Oxford, United Kingdom The activins, A and B, are members of the transforming growth factor-b cytokine family, and are regulators of inflammation, immunity and fibrosis. Production of activin A and the activin-binding protein, follistatin, increases during inflammation, although aspects of this regulation require clarification and activin B has not been studied in inflammation, previously. Adult male mice were injected with Toll-like receptor (TLR) ligands, acting though MyD88-dependent (TLR2, 4, 7 and 9) and MyD88-independent (TLR3, 4) signalling pathways. Activation of TLR4 by lipopolysaccharide (LPS) was also examined in MyD88-deficient mice, and in mice pre-treated with cycloheximide and actinomycin D. Activin A, B and follistatin were measured in serum by specific immunoasssays, and mRNA expression was measured in the liver by qRT-PCR. Activin A secretion, but not mRNA expression, was stimulated via activation of TLR2, 3, 4 and 7, reaching a peak in the serum around 2–3 h. By contrast, all ligands stimulated activin B at both protein and mRNA level. Compared with activin A, activin B in serum increased more slowly (3–5 h), but reached much higher maximum concentrations. Follistatin was stimulated at the mRNA level by TLR2, 3, 4 and 7. In the absence of MyD88, induction of activin A and follistatin by LPS was prevented, but activin B production was unaffected. While production of activin A was inhibited by cycloheximide, but not by actinomycin D, activin B was inhibited to a similar degree by both compounds. These data indicate that viral and bacterial ligands, acting through both MyD88-dependent and MyD88-independent signaling pathways, stimulate production of activin A, B and follistatin. These data further indicate that activin B, like follistatin, but unlike activin A, is primarily regulated at the transcriptional level during inflammation. These studies suggest that delayed production of activin B may complement the more rapid release of activin A during inflammatory responses. http://dx.doi.org/10.1016/j.cyto.2014.07.083
78 Insights into the function of an anti-leukaemia antibody: structural studies of CSL362 bound to soluble CD123 Tim R. Hercus 1, Sophie E. Broughton 2, Matthew P. Hardy 3, Tracy L. Nero 2, Barbara J. McClure 1, Mara Dottore 1, Urmi Dhagat 2, Emma F. Barry 1, Winnie L. Kan 1, Samantha J. Busfield 3, Andrew D. Nash 3, Nicholas J. Wilson 3, Michael W. Parker 2, Angel F. Lopez 1, 1 Centre for Cancer Biology, SA Pathology and University of South Australia, Adelaide, SA, Australia, 2 ACRF Rational Drug Discovery Centre, St. Vincent’s Institute of Medical Research, Melbourne, Victoria, Australia, 3 CSL Limited, Melbourne, Victoria, Australia The ßc family of cytokines (GM-CSF, IL-3 and IL-5) are largely products of activated T cells and regulate the survival, proliferation, differentiation and functional activation of hematopoietic cells following infection or bleeding. They are variously able to target myeloid hematopoietic stem and progenitor cells as well as mature neutrophils, eosinophils, macrophages and mast cells. Recent evidence suggests that this family of cytokines plays a role in other biological systems and importantly also in cancer, through the development and metastasis of solid tumours. Intriguingly, CD123, the IL-3 receptor alpha subunit (IL3RaÞ is overexpressed by stem cells and primary blast cells of patients with acute myelogenous leukaemia (AML) and this correlates with poor prognosis leading to the development of multiple strategies that target CD123. We have now solved the structure of soluble IL3Ra, in complex with a Fab fragment of CSL362, a humanised monoclonal antibody that binds IL3Ra, blocks IL-3 function and is optimised for NK cell-mediated killing of leukaemic cells [1]. CSL362 is currently in a Phase 1 clinical trial for the treatment of patients with AML (Clinical Trials Gov. Identifier: NCT01632852). The three domain IL3Ra structure unexpectedly revealed two alternative conformations, an open and a closed form based on the orientation of the N-terminal domain (NTD). The open conformation has not previously been reported for other Type I cytokine receptors with a similar domain structure, IL5Ra, IL13Ra1 and IL13Ra2. The IL3Ra structure will be presented together with data that supports a mechanism of IL-3 recognition and receptor signalling that may be applicable to other members of the Type I cytokine receptor superfamily. An unexpected dual mechanism of IL-3 antagonism utilised by CSL362 that involves direct antagonism of IL-3 binding as well as blockade of IL-3 receptor assembly, will also be presented.
Reference 77 Kinetics of Type I interferon responses determine either establishment of antiviral memory or no induction of adaptive immunity Julia Heinrich 1, Peter Staeheli 2, Ulrich Kalinke 1, 1 Institute for Experimental Infection Research, TWINCORE, Hanover, Germany, 2 Department of Virology, Institute for Medical Microbiology and Hygiene, Albert-Ludwigs-Universität, Freiburg, Germany Many pathogens trigger type I interferon (IFN-I) responses early after infection that confer protection until adaptive immunity is induced. Upon infection with vesicular stomatitis virus (VSV) Toll-like receptor (TLR) and RIG-I-like helicase (RLH) signaling platforms are indispensable for the induction of protective IFN-I. Hence, TLR- and RLH-ablated MyTrCa/ mice challenged with VSV do not mount IFN-I responses and are inevitably susceptible to lethal VSV infection. To study the impact of the kinetics of IFN-I responses on the induction of adaptive immunity, we infected MyTrCa/ mice with VSV and treated them 4, 8, 16, and 24 h after
[1] Broughton, et al. Cell reports; 2014 [in press]. http://dx.doi.org/10.1016/j.cyto.2014.07.085
79 S100 proteins regulate LPS-induced inflammation in murine lung Yuka Hiroshima, Kenneth Hsu, Nicodemus Tedla, Sharron Chow, Naomi Kawaguchi, Carolyn L. Geczy, University New South Wales, Sydney, NSW, Australia Aims: S100A8 and S100A9 are calcium binding proteins that comprise 40% of the neutrophil cytosol and induced in several cell types by inflammatory mediators or by