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Gideon Schreiber - One of the best experts on this subject based on the ideXlab platform.
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Dose-Dependent Differences in HIV Inhibition by Different Interferon Alpha Subtypes While Having Overall Similar Biologic Effects
American Society for Microbiology, 2019Co-Authors: Erika Schlaepfer, Gideon Schreiber, Audrey Fahrny, Maarja Gruenbach, Stefan P. Kuster, Viviana Simon, Roberto F. SpeckAbstract:Elucidating the functional role of the IFN-α subtypes is of particular importance for the development of efficacious therapies using exogenous IFN-α. Specifically, this will help define whether IFN therapy should be based on the use of pathogen-dependent IFN subtypes or, rather, IFN mutants with optimized IFNAR binding properties.Type I interferons (IFNs) are key players in the antiviral immune response. Interferon alpha (IFN-α) belongs to this class of IFNs and comprises 12 subtypes that differ from each other in their binding affinities for a common receptor and, thus, in their signaling potencies. Recent data suggest that IFN-α6 and -α14 are the most potent IFN-α subtypes in restricting HIV replication when applied exogenously. However, in the context of antiviral therapy, IFNs are administered at high doses, which may compensate for differences in potency seen between IFN-α subtypes. In this study, we reexamined whether IFN-α subtypes induce different biological activities, with a focus on how IFN-α treatment dose affects cellular responses to HIV in primary CD4+ T cells, peripheral blood mononuclear cells (PBMCs), and macrophages. We found that the subtypes’ antiviral activities were dose dependent, with >90% inhibition of HIV replication at a high dose of all IFN-αs except the weak IFN-α/β receptor (IFNAR) binder, IFN-α1. The quality of the responses engendered by IFN-α1, -α2, -α6, and -α14 was highly comparable, with essentially the same set of genes induced by all four subtypes. Hierarchal cluster analysis revealed that the individual donors were stronger determinants for the IFN-stimulated-gene (ISG) responses than the specific IFN-α subtype used for stimulation. Notably, IFN-α2-derived mutants with substantially reduced IFNAR2 binding still inhibited HIV replication efficiently, whereas mutants with increased IFNAR1 binding potentiated antiviral activity. Overall, our results support the idea that IFN-α subtypes do not induce different biological responses, given that each subtype is exogenously applied at bioequivalent doses
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multifaceted activities of type i interferon are revealed by a receptor antagonist
Science Signaling, 2014Co-Authors: Doron Levin, Hansheinrich Hoffmann, Nanaocha Sharma, William M Schneider, Ganit Yarden, Alberto Giovanni Busetto, Ohad Manor, Charles M Rice, Gideon SchreiberAbstract:Type I interferons (IFNs), including various IFN-α isoforms and IFN-β, are a family of homologous, multifunctional cytokines. IFNs activate different cellular responses by binding to a common receptor that consists of two subunits, IFNAR1 and IFNAR2. In addition to stimulating antiviral responses, they also inhibit cell proliferation and modulate other immune responses. We characterized various IFNs, including a mutant IFN-α2 (IFN-1ant) that bound tightly to IFNAR2 but had markedly reduced binding to IFNAR1. Whereas IFN-1ant stimulated antiviral activity in a range of cell lines, it failed to elicit immunomodulatory and antiproliferative activities. The antiviral activities of the various IFNs tested depended on a set of IFN-sensitive genes (the "robust" genes) that were controlled by canonical IFN response elements and responded at low concentrations of IFNs. Conversely, these elements were not found in the promoters of genes required for the antiproliferative responses of IFNs (the "tunable" genes). The extent of expression of tunable genes was cell type-specific and correlated with the magnitude of the antiproliferative effects of the various IFNs. Although IFN-1ant induced the expression of robust genes similarly in five different cell lines, its antiviral activity was virus- and cell type-specific. Our findings suggest that IFN-1ant may be a therapeutic candidate for the treatment of specific viral infections without inducing the immunomodulatory and antiproliferative functions of wild-type IFN.
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Bridging the Species Divide: Transgenic Mice Humanized for Type-I Interferon Response
2014Co-Authors: Daniel Harari, Renne Abramovich, Alla Zozulya, Paul Smith, Rine Pouly, Gideon SchreiberAbstract:We have generated transgenic mice that harbor humanized type I interferon receptors (IFNARs) enabling the study of type I human interferons (Hu-IFN-Is) in mice. These ‘‘HyBNAR’ ’ (Hybrid IFNAR) mice encode transgenic variants of IFNAR1 and IFNAR2 with the human extracellular domains being fused to transmembrane and cytoplasmic segments of mouse sequence. B16F1 mouse melanoma cells harboring the HyBNAR construct specifically bound Hu-IFN-Is and were rendered sensitive to Hu-IFN-I stimulated anti-proliferation, STAT1 activation and activation of a prototypical IFN-I response gene (MX2). HyBNAR mice were crossed with a transgenic strain expressing the luciferase reporter gene under the control of the IFN-responsive MX2 promoter (MX2-Luciferase). Both the HyBNAR and HyBNAR/MX2-Luciferase mice were responsive to all Hu-IFN-Is tested, inclusive of IFNa2A, IFNb, and a human superagonist termed YNSa8. The mice displayed dose-dependent pharmacodynamic responses to Hu-IFN-I injection, as assessed by measuring the expression of IFN-responsive genes. Our studies also demonstrated a weak activation of endogenous mouse interferon response, especially after high dose administration of Hu-IFNs. In sharp contrast to data published for humans, our pharmacodynamic readouts demonstrate a very short-lived IFN-I response in mice, which is not enhanced by sub-cutaneous (SC) injections in comparison to other administration routes. With algometric differences between humans and mice taken into account, the HyBNAR mic
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stochastic receptor expression determines cell fate upon interferon treatment
Molecular and Cellular Biology, 2011Co-Authors: Doron Levin, Daniel Harari, Gideon SchreiberAbstract:Type I interferons trigger diverse biological effects by binding a common receptor, composed of IFNAR1 and IFNAR2. Intriguingly, while the activation of an antiviral state is common to all cells, antiproliferative activity and apoptosis affect only part of the population, even when cells are stimulated with saturating interferon concentrations. Manipulating receptor expression by different small interfering RNA (siRNA) concentrations reduced the fraction of responsive cells independent of the interferon used, including a newly generated, extremely tight-binding variant. Reduced receptor numbers increased 50% effective concentrations (EC50s) for alpha interferon 2 (IFN-α2) but not for the tight-binding variant. A correlation between receptor numbers, STAT activation, and gene induction is observed. Our data suggest that for a given cell, the response is binary (+/−) and dependent on the stochastic expression levels of the receptors on an individual cell. A low number of receptors suffices for antiviral response and is thus a robust feature common to all cells. Conversely, a high number of receptors is required for antiproliferative activity, which allows for fine-tuning on a single-cell level.
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the stability of the ternary interferon receptor complex rather than the affinity to the individual subunits dictates differential biological activities
Journal of Biological Chemistry, 2008Co-Authors: Eyal Kalie, Jacob Piehler, Diego Jaitin, Yulia Podoplelova, Gideon SchreiberAbstract:Type I interferons (IFNs) signal for their diverse biological effects by binding a common receptor on target cells, composed of the two transmembrane IFNAR1 and IFNAR2 proteins. We have previously differentially enhanced the antiproliferative activity of IFN by increasing the weak binding affinity of IFN to IFNAR1. In this study, we further explored the affinity interdependencies between the two receptor subunits and the role of IFNAR1 in differential IFN activity. For this purpose, we generated a panel of mutations targeting the IFNAR2 binding site on the background of the IFNalpha2 YNS mutant, which increases the affinity to IFNAR1 by 60-fold, resulting in IFNAR2-to-IFNAR1 binding affinity ratios ranging from 1000:1 to 1:1000. Both the antiproliferative and antiviral potencies of the interferon mutants clearly correlated to the in situ binding IC(50) values, independently of the relative contributions of the individual receptors, thus relating to the integral lifetime of the complex. However, the antiproliferative potency correlated throughout the entire range of affinities, as well as with prolonged IFNAR1 receptor down-regulation, whereas the antiviral potency reached a maximum at binding affinities equivalent to that of wild-type IFNalpha2. Our data suggest that (i) the specific activity of interferon is related to the ternary complex binding affinity and not to affinity toward individual receptor components and (ii) although the antiviral pathway is strongly dependent on pSTAT1 activity, the cytostatic effect requires additional mechanisms that may involve IFNAR1 down-regulation. This differential interferon response is ultimately mediated through distinct gene expression profiling.
John J. Krolewski - One of the best experts on this subject based on the ideXlab platform.
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Sequential Proteolytic Processing of an Interferon-Alpha Receptor Subunit by TNF-Alpha Converting Enzyme and Presenilins
Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research, 2012Co-Authors: Peter D. Pioli, Ashraf El Fiky, Kent L. Nastiuk, Abu Z. M. Saleh, John J. KrolewskiAbstract:It is well established that interferons trigger tyrosine-kinase-dependent signaling via JAK kinases and STAT transcription factors. However, we have observed both IFNAR2 receptor cleavage and functional activity of the liberated intracellular domain (ICD), suggesting that interferon-alpha (IFN-alpha) can also signal via regulated intramembrane proteolysis (RIP), an evolutionarily conserved mechanism of receptor-mediated signaling. Sequential cleavage of the receptor ectodomain and transmembrane domain is a hallmark of the most common class of RIP. To investigate the mechanisms of IFNAR2 RIP signaling, we examined IFNAR2 cleavage by TNF-alpha converting enzyme (TACE) and presenilin proteases. We tracked the fate of epitope-tagged and fusion variants of IFNAR2 in cells expressing wild-type, mutant, or null versions of TACE and presenilins 1 and 2. Cleavage and subcellular location were determined by immunoblot, fluoresence microscopy, and reporter assays. We found that both TACE and presenilin 1/2 cleave IF...
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Nuclear transit of the intracellular domain of the interferon receptor subunit IFNAR2 requires Stat2 and Irf9.
Cellular signalling, 2008Co-Authors: Ashraf El Fiky, Peter D. Pioli, Arif Azam, Kiwon Yoo, Kent L. Nastiuk, John J. KrolewskiAbstract:Regulated intramembrane proteolysis (RIP) is the primary signaling mechanism for some receptors, such as Notch and the amyloid precursor protein. In addition, some receptor type tyrosine kinases, such as HER4, are able to signal via both kinase activation and regulated receptor proteolysis. Previously, we showed that the IFNAR2 subunit of the type I interferon receptor can be cleaved in a two step process that resembles RIP and that the IFNAR2 intracellular domain (IFNAR2-ICD) can mediate gene transcription in a Stat2 dependent manner. Here, we demonstrate that IFNAR2-ICD, Stat2 and Irf9 form a ternary complex. Furthermore, Stat2 and Irf9 are required for the nuclear transit of a GFP-linked IFNAR2-ICD construct (GFP-ICD). Additional experiments monitoring the nuclear localization of GFP-ICD demonstrate that Stat2 serves an adaptor role, mediating the interaction between the IFNAR2-ICD and Irf9, while the bipartite nuclear localization signal within Irf9 is the primary determinant driving nuclear transit of the ICD containing complex. Overall, the data suggest that liberation of the IFNAR2-ICD by regulated proteolysis could trigger a novel mechanism for moving the transcription factor Stat2 to the nucleus.
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Stat2 Binding to the Interferon-α Receptor 2 Subunit Is Not Required for Interferon-α Signaling
The Journal of biological chemistry, 2002Co-Authors: Vinh-phuc Nguyen, Hai Yan, Abu Z. M. Saleh, Allison E. Arch, Flavia Piazza, John S. Kim, John J. KrolewskiAbstract:Abstract The interferon-α (IFNα) receptor consists of two subunits, the IFNα receptor 1 (IFNaR1) and 2 (IFNAR2) chains. Following ligand binding, IFNaR1 is phosphorylated on tyrosine 466, and this site recruits Stat2 via its SH2 domain. In contrast, IFNAR2 binds Stat2 constitutively. In this study we have characterized the Stat2-IFNAR2 interaction and examined its role in IFNα signaling. Stat2 binds the major IFNAR2 protein but not a variant containing a shorter cytoplasmic domain. The interaction does not require a STAT SH2 domain. Both tyrosine-phosphorylated and non-phosphorylated Stat2 bind IFNAR2 in vitro; however, relatively little phosphorylated Stat2 associates with IFNAR2 in vivo. In vitro binding assays defined IFNAR2 residues 418–444 as the minimal interaction domain and site-specific mutation of conserved acidic residues within this domain disrupted in vitro and in vivobinding. An IFNAR2 construct carrying these mutations was either (i) overexpressed in 293T cells or (ii) used to complement IFNAR2-deficient U5A cells. Unexpectedly, the activity of an IFNα-dependent reporter gene was not reduced but, instead, was enhanced up to 2-fold. This suggests that this particular IFNAR2-Stat2 interaction is not required for IFNα signaling, but might act to negatively inhibit signaling. Finally, a doubly truncated recombinant fragment of Stat2, spanning residues 136–702, associated with IFNAR2 in vitro, indicating that the interaction with IFNAR2 is direct and occurs in a central region of Stat2 marked by a hydrophobic core.
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definition of the interferon α receptor binding domain on the tyk2 kinase
Journal of Biological Chemistry, 1998Co-Authors: Hai Yan, Kartik Krishnan, Flavia Piazza, Richard Pine, John J. KrolewskiAbstract:Abstract Interferons and cytokines modulate gene expression via a simple, direct signaling pathway containing receptors, JAK tyrosine kinases, and STAT transcription factors. The interferon-α pathway is a model for these cascades. Two receptors, IFNaR1 and IFNAR2, associate exclusively in a constitutive manner with two JAK proteins, TYK2 and JAK1, respectively. Defining the molecular interface between JAK proteins and their receptors is critical to understanding the signaling pathway and may contribute to the development of novel therapeutics. This report defines the IFNaR1 interaction domain on TYK2. In vitro binding studies demonstrate that the amino-terminal half of TYK2, which is ∼600 amino acids long and contains JAK homology (JH) domains 3–7, comprises the maximal binding domain for IFNaR1. A fragment containing amino acids 171–601 (JH3–6) also binds IFNaR1, but with reduced affinity. Glutathione S-transferase-TYK2 fusion proteins approximating either the JH6 or JH3 domain affinity-precipitate IFNaR1, suggesting that these are major sites of interaction within the larger binding domain. TYK2 amino acids 1–601 act in a dominant manner to inhibit the transcription of an interferon-α-dependent reporter gene, presumably by displacing endogenous TYK2 from the receptor. This same fragment inhibits interferon-α-dependent tyrosine phosphorylation of TYK2, STAT1, and STAT2.
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dimerization of a chimeric cd4 interferon alpha receptor reconstitutes the signaling events preceding stat phosphorylation
Oncogene, 1996Co-Authors: Kartik Krishnan, Hai Yan, J. T. E. Lim, John J. KrolewskiAbstract:Interferon-alpha induces the rapid tyrosine phosphorylation of a number of molecules, including the cognate receptors, JAK-family kinases (Jak1 and tyk2), and latent transcription factors (STATs 1 and 2). Here, we describe the use of chimeric molecules composed of the extracellular domain of CD4 fused to the intracellular domain of the interferon-alpha receptor subunit 1 (IFNaR1). Antibody mediated crosslinking dimerizes the transfected chimeras, activates tyk2 and induces a tyk2-dependent tyrosine phosphorylation of the intracellular domain of the chimera. We further define the major site of IFNaR1 phosphorylation, and show that phosphorylation of this site is required for association with STAT2. Finally, we show that homodimerization of IFNaR1 is not sufficient to activate the STATs, suggesting a role for the IFNAR2 subunit and Jak1 in the transduction of the interferon-alpha signal.
Nicole A De Weerd - One of the best experts on this subject based on the ideXlab platform.
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a hot spot on interferon α β receptor subunit 1 ifnar1 underpins its interaction with interferon β and dictates signaling
Journal of Biological Chemistry, 2017Co-Authors: Antony Yaron Matthews, Phillip R. Pattie, Nollaig M. Bourke, San Sui Lim, Nicole A De Weerd, Julian P VivianAbstract:The interaction of IFN-β with its receptor IFNAR1 (interferon α/β receptor subunit 1) is vital for host-protective anti-viral and anti-proliferative responses, but signaling via this interaction can be detrimental if dysregulated. Whereas it is established that IFNAR1 is an essential component of the IFNAR signaling complex, the key residues underpinning the IFN-β-IFNAR1 interaction are unknown. Guided by the crystal structure of the IFN-β-IFNAR1 complex, we used truncation variants and site-directed mutagenesis to investigate domains and residues enabling complexation of IFN-β to IFNAR1. We have identified an interface on IFNAR1-subdomain-3 that is differentially utilized by IFN-β and IFN-α for signal transduction. We used surface plasmon resonance and cell-based assays to investigate this important IFN-β binding interface that is centered on IFNAR1 residues Tyr240 and Tyr274 binding the C and N termini of the B and C helices of IFN-β, respectively. Using IFNAR1 and IFN-β variants, we show that this interface contributes significantly to the affinity of IFN-β for IFNAR1, its ability to activate STAT1, the expression of interferon stimulated genes, and ultimately to the anti-viral and anti-proliferative properties of IFN-β. These results identify a key interface created by IFNAR1 residues Tyr240 and Tyr274 interacting with IFN-β residues Phe63, Leu64, Glu77, Thr78, Val81, and Arg82 that underlie IFN-β-IFNAR1-mediated signaling and biological processes.
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structural basis of a unique interferon beta signaling axis mediated via the receptor ifnar1
Nature Immunology, 2013Co-Authors: Julian P Vivian, Thao Kim Thi Nguyen, Niamh E Mangan, Nicole A De Weerd, Jodee A Gould, Susie-jane BraniffAbstract:Type I interferons regulate immune responses by signaling via heterodimeric IFNAR1-IFNAR2 complexes. Hertzog and colleagues reveal a unique IFN-β–IFNAR1 signaling complex that is IFNAR2-independent and modulates expression of a distinct set of interferon-inducible genes.
Thao Kim Thi Nguyen - One of the best experts on this subject based on the ideXlab platform.
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structural basis of a unique interferon beta signaling axis mediated via the receptor ifnar1
Nature Immunology, 2013Co-Authors: Julian P Vivian, Thao Kim Thi Nguyen, Niamh E Mangan, Nicole A De Weerd, Jodee A Gould, Susie-jane BraniffAbstract:Type I interferons regulate immune responses by signaling via heterodimeric IFNAR1-IFNAR2 complexes. Hertzog and colleagues reveal a unique IFN-β–IFNAR1 signaling complex that is IFNAR2-independent and modulates expression of a distinct set of interferon-inducible genes.
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structural basis of a unique interferon beta signaling axis mediated via the receptor ifnar1
Nature Immunology, 2013Co-Authors: Nicole Anne De Weerd, Jodee Gould, Julian P Vivian, Thao Kim Thi Nguyen, Niamh E Mangan, Susie-jane Braniff, Leyla ZakertabriziAbstract:Type I interferons are important in regulating immune responses to pathogens and tumors. All interferons are considered to signal via the heterodimeric IFNAR1-IFNAR2 complex, yet some subtypes such as interferon-β (IFN-β) can exhibit distinct functional properties, although the molecular basis of this is unclear. Here we demonstrate IFN-β can uniquely and specifically ligate to IFNAR1 in an IFNAR2-independent manner, and we provide the structural basis of the IFNAR1-IFN-β interaction. The IFNAR1-IFN-β complex transduced signals that modulated expression of a distinct set of genes independently of Jak-STAT pathways. Lipopolysaccharide-induced sepsis was ameliorated in Ifnar1(-/-) mice but not IFNAR2(-/-) mice, suggesting that IFNAR1-IFN-β signaling is pathologically relevant. Thus, we provide a molecular basis for understanding specific functions of IFN-β.
Julian P Vivian - One of the best experts on this subject based on the ideXlab platform.
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a hot spot on interferon α β receptor subunit 1 ifnar1 underpins its interaction with interferon β and dictates signaling
Journal of Biological Chemistry, 2017Co-Authors: Antony Yaron Matthews, Phillip R. Pattie, Nollaig M. Bourke, San Sui Lim, Nicole A De Weerd, Julian P VivianAbstract:The interaction of IFN-β with its receptor IFNAR1 (interferon α/β receptor subunit 1) is vital for host-protective anti-viral and anti-proliferative responses, but signaling via this interaction can be detrimental if dysregulated. Whereas it is established that IFNAR1 is an essential component of the IFNAR signaling complex, the key residues underpinning the IFN-β-IFNAR1 interaction are unknown. Guided by the crystal structure of the IFN-β-IFNAR1 complex, we used truncation variants and site-directed mutagenesis to investigate domains and residues enabling complexation of IFN-β to IFNAR1. We have identified an interface on IFNAR1-subdomain-3 that is differentially utilized by IFN-β and IFN-α for signal transduction. We used surface plasmon resonance and cell-based assays to investigate this important IFN-β binding interface that is centered on IFNAR1 residues Tyr240 and Tyr274 binding the C and N termini of the B and C helices of IFN-β, respectively. Using IFNAR1 and IFN-β variants, we show that this interface contributes significantly to the affinity of IFN-β for IFNAR1, its ability to activate STAT1, the expression of interferon stimulated genes, and ultimately to the anti-viral and anti-proliferative properties of IFN-β. These results identify a key interface created by IFNAR1 residues Tyr240 and Tyr274 interacting with IFN-β residues Phe63, Leu64, Glu77, Thr78, Val81, and Arg82 that underlie IFN-β-IFNAR1-mediated signaling and biological processes.
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structural basis of a unique interferon beta signaling axis mediated via the receptor ifnar1
Nature Immunology, 2013Co-Authors: Julian P Vivian, Thao Kim Thi Nguyen, Niamh E Mangan, Nicole A De Weerd, Jodee A Gould, Susie-jane BraniffAbstract:Type I interferons regulate immune responses by signaling via heterodimeric IFNAR1-IFNAR2 complexes. Hertzog and colleagues reveal a unique IFN-β–IFNAR1 signaling complex that is IFNAR2-independent and modulates expression of a distinct set of interferon-inducible genes.
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structural basis of a unique interferon beta signaling axis mediated via the receptor ifnar1
Nature Immunology, 2013Co-Authors: Nicole Anne De Weerd, Jodee Gould, Julian P Vivian, Thao Kim Thi Nguyen, Niamh E Mangan, Susie-jane Braniff, Leyla ZakertabriziAbstract:Type I interferons are important in regulating immune responses to pathogens and tumors. All interferons are considered to signal via the heterodimeric IFNAR1-IFNAR2 complex, yet some subtypes such as interferon-β (IFN-β) can exhibit distinct functional properties, although the molecular basis of this is unclear. Here we demonstrate IFN-β can uniquely and specifically ligate to IFNAR1 in an IFNAR2-independent manner, and we provide the structural basis of the IFNAR1-IFN-β interaction. The IFNAR1-IFN-β complex transduced signals that modulated expression of a distinct set of genes independently of Jak-STAT pathways. Lipopolysaccharide-induced sepsis was ameliorated in Ifnar1(-/-) mice but not IFNAR2(-/-) mice, suggesting that IFNAR1-IFN-β signaling is pathologically relevant. Thus, we provide a molecular basis for understanding specific functions of IFN-β.
