Showing papers on "Interferon published in 2003"

Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway.

Abstract: Stimulation of Toll-like receptors (TLRs) triggers activation of a common MyD88-dependent signaling pathway as well as a MyD88-independent pathway that is unique to TLR3 and TLR4 signaling pathways leading to interferon (IFN)-beta production. Here we disrupted the gene encoding a Toll/IL-1 receptor (TIR) domain-containing adaptor, TRIF. TRIF-deficient mice were defective in both TLR3- and TLR4-mediated expression of IFN-beta and activation of IRF-3. Furthermore, inflammatory cytokine production in response to the TLR4 ligand, but not to other TLR ligands, was severely impaired in TRIF-deficient macrophages. Mice deficient in both MyD88 and TRIF showed complete loss of nuclear factor kappa B activation in response to TLR4 stimulation. These findings demonstrate that TRIF is essential for TLR3- and TLR4-mediated signaling pathways facilitating mammalian antiviral host defense.

Interferon and Granulopoiesis Signatures in Systemic Lupus Erythematosus Blood

Abstract: Systemic lupus erythematosus (SLE) is a prototype systemic autoimmune disease characterized by flares of high morbidity. Using oligonucleotide microarrays, we now show that active SLE can be distinguished by a remarkably homogeneous gene expression pattern with overexpression of granulopoiesis-related and interferon (IFN)-induced genes. Using the most stringent statistical analysis (Bonferroni correction), 15 genes were found highly up-regulated in SLE patients, 14 of which are targets of IFN and one, defensin DEFA-3, a major product of immature granulocytes. A more liberal correction (Benjamini and Hochberg correction) yielded 18 additional genes, 12 of which are IFN-regulated and 4 granulocyte-specific. Indeed immature neutrophils were identified in a large fraction of SLE patients white blood cells. High dose glucocorticoids, a standard treatment of disease flares, shuts down the interferon signature, further supporting the role of this cytokine in SLE. The expression of 10 genes correlated with disease activity according to the SLEDAI. The most striking correlation (P < 0.001, r = 0.55) was found with the formyl peptide receptor-like 1 protein that mediates chemotactic activities of defensins. Therefore, while the IFN signature confirms the central role of this cytokine in SLE, microarray analysis of blood cells reveals that immature granulocytes may be involved in SLE pathogenesis.

Triggering the Interferon Antiviral Response Through an IKK-Related Pathway

Abstract: Rapid induction of type I interferon expression, a central event in establishing the innate antiviral response, requires cooperative activation of numerous transcription factors. Although signaling pathways that activate the transcription factors nuclear factor kappaB and ATF-2/c-Jun have been well characterized, activation of the interferon regulatory factors IRF-3 and IRF-7 has remained a critical missing link in understanding interferon signaling. We report here that the IkappaB kinase (IKK)-related kinases IKKepsilon and TANK-binding kinase 1 are components of the virus-activated kinase that phosphorylate IRF-3 and IRF-7. These studies illustrate an essential role for an IKK-related kinase pathway in triggering the host antiviral response to viral infection.

TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-beta induction.

Abstract: Human Toll-like receptor (TLR) 3 recognizes double-stranded (ds) RNA and induces production of interferon (IFN)-beta independent of the adaptor molecules MyD88 and TIRAP. Thus, another adaptor must exist that preferentially mediates TLR3-dependent production of IFN-beta. We have identified an alternative adaptor, designated Toll-interleukin 1 receptor domain (TIR)-containing adaptor molecule (TICAM)-1, that can physically bind the TIR domain of TLR3 and activate the IFN-beta promoter in response to poly(I):poly(C). Thus, dsRNA-TLR3-dependent production of IFN-beta is mediated mainly by TICAM-1. This TICAM-1-dependent pathway may have a role in other TLR-IFN-beta pathways, which form part of the MyD88-independent cellular immune response.

Induction of an interferon response by RNAi vectors in mammalian cells

Abstract: DNA vectors that express short hairpin RNAs (shRNAs) from RNA polymerase III (Pol III) promoters are a promising new tool to reduce gene expression in mammalian cells. shRNAs are processed to small interfering RNAs (siRNAs) of 21 nucleotides (nt) that guide the cleavage of the cognate mRNA by the RNA-induced silencing complex. Although siRNAs are thought to be too short to induce interferon expression, we report here that a substantial number of shRNA vectors can trigger an interferon response.

Regulation of Interferon Regulatory Factor-3 by the Hepatitis C Virus Serine Protease

Abstract: Persistent infections with hepatitis C virus (HCV) are likely to depend on viral inhibition of host defenses. We show that the HCV NS3/4A serine protease blocks the phosphorylation and effector action of interferon regulatory factor-3 (IRF-3), a key cellular antiviral signaling molecule. Disruption of NS3/4A protease function by mutation or a ketoamide peptidomimetic inhibitor relieved this blockade and restored IRF-3 phosphorylation after cellular challenge with an unrelated virus. Furthermore, dominant-negative or constitutively active IRF-3 mutants, respectively, enhanced or suppressed HCV RNA replication in hepatoma cells. Thus, the NS3/4A protease represents a dual therapeutic target, the inhibition of which may both block viral replication and restore IRF-3 control of HCV infection.

Cross-priming of CD8 + T cells stimulated by virus-induced type I interferon

Abstract: CD8+ T cell responses can be generated against antigens that are not expressed directly within antigen-presenting cells (APCs), through a process known as cross-priming. To initiate cross-priming, APCs must both capture extracellular antigen and receive specific activation signals. We have investigated the nature of APC activation signals associated with virus infection that stimulate cross-priming. We show that infection with lymphocytic choriomeningitis virus induces cross-priming by a mechanism dependent on type I interferon (IFN-alpha/beta). Activation of cross-priming by IFN-alpha/beta was independent of CD4+ T cell help or interaction of CD40 and CD40 ligand, and involved direct stimulation of dendritic cells. These data identify expression of IFN-alpha/beta as a mechanism for the induction of cross-priming during virus infections.

Apoptosis and interferons: role of interferon-stimulated genes as mediators of apoptosis.

Abstract: IFNs are a family of cytokines with pleiotropic biological effects mediated by scores of responsive genes. IFNs were the first human proteins to be effective in cancer therapy and were among the first recombinant DNA products to be used clinically. Both quality and quantity of life has been improved in response to IFNs in various malignancies. Despite its beneficial effects, unraveling the mechanisms of the anti-tumor effects of IFN has proven to be a complex task. IFNs may mediate anti-tumor effects either indirectly by modulating immunomodulatory and anti-angiogenic responses or by directly affecting proliferation or cellular differentiation of tumor cells. Both direct or indirect effects of IFNs result from induction of a subset of genes, called IFN stimulated genes (ISGs). In addition to the ISGs implicated in anti-viral, anti-angiogenic, immunomodulatory and cell cycle inhibitory effects, oligonucleotide microarray studies have identified ISGs with apoptotic functions. These include TNF-alpha related apoptosis inducing ligand (TRAIL/Apo2L), Fas/FasL, XIAP associated factor-1 (XAF-1), caspase-4, caspase-8, dsRNA activated protein kinase (PKR), 2'5'A oligoadenylate synthetase (OAS), death activating protein kinases (DAP kinase), phospholipid scramblase, galectin 9, IFN regulatory factors (IRFs), promyelocytic leukemia gene (PML) and regulators of IFN induced death (RIDs). In vitro IFN-alpha, IFN-beta and IFN-gamma induced apoptosis in multiple cell lines of varied histologies. This review will emphasize possible mechanisms and the role of ISGs involved in mediating apoptotic function of IFNs.

Stroke-induced immunodeficiency promotes spontaneous bacterial infections and is mediated by sympathetic activation reversal by poststroke T helper cell type 1-like immunostimulation.

Abstract: Infections are a leading cause of death in stroke patients. In a mouse model of focal cerebral ischemia, we tested the hypothesis that a stroke-induced immunodeficiency increases the susceptibility to bacterial infections. 3 d after ischemia, all animals developed spontaneous septicemia and pneumonia. Stroke induced an extensive apoptotic loss of lymphocytes and a shift from T helper cell (Th)1 to Th2 cytokine production. Adoptive transfer of T and natural killer cells from wild-type mice, but not from interferon (IFN)-γ–deficient mice, or administration of IFN-γ at day 1 after stroke greatly decreased the bacterial burden. Importantly, the defective IFN-γ response and the occurrence of bacterial infections were prevented by blocking the sympathetic nervous system but not the hypothalamo-pituitary-adrenal axis. Furthermore, administration of the β-adrenoreceptor blocker propranolol drastically reduced mortality after stroke. These data suggest that a catecholamine-mediated defect in early lymphocyte activation is the key factor in the impaired antibacterial immune response after stroke.

Activation of Natural Killer T Cells by α-Galactosylceramide Rapidly Induces the Full Maturation of Dendritic Cells In Vivo and Thereby Acts as an Adjuvant for Combined CD4 and CD8 T Cell Immunity to a Coadministered Protein

Abstract: The maturation of dendritic cells (DCs) allows these antigen-presenting cells to initiate immunity. We pursued this concept in situ by studying the adjuvant action of α-galactosylceramide (αGalCer) in mice. A single i.v. injection of glycolipid induced the full maturation of splenic DCs, beginning within 4 h. Maturation was manifest by marked increases in costimulator and major histocompatibility complex class II expression, interferon (IFN)-γ production, and stimulation of the mixed leukocyte reaction. These changes were not induced directly by αGalCer but required natural killer T (NKT) cells acting independently of the MyD88 adaptor protein. To establish that DC maturation was responsible for the adjuvant role of αGalCer, mice were given αGalCer together with soluble or cell-associated ovalbumin antigen. Th1 type CD4+ and CD8+ T cell responses developed, and the mice became resistant to challenge with ovalbumin-expressing tumor. DCs from mice given ovalbumin plus adjuvant, but not the non-DCs, stimulated ovalbumin-specific proliferative responses and importantly, induced antigen-specific, IFN-γ producing, CD4+ and CD8+ T cells upon transfer into naive animals. In the latter instance, immune priming did not require further exposure to ovalbumin, αGalCer, NKT, or NK cells. Therefore a single dose of αGalCer i.v. rapidly stimulates the full maturation of DCs in situ, and this accounts for the induction of combined Th1 CD4+ and CD8+ T cell immunity to a coadministered protein.

Impaired response to interferon-alpha/beta and lethal viral disease in human STAT1 deficiency.

Abstract: The receptors for interferon-alpha/beta (IFN-alpha/beta) and IFN-gamma activate components of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway, leading to the formation of at least two transcription factor complexes. STAT1 interacts with STAT2 and p48/IRF-9 to form the transcription factor IFN-stimulated gene factor 3 (ISGF3). STAT1 dimers form gamma-activated factor (GAF). ISGF3 is induced mainly by IFN-alpha/beta, and GAF by IFN-gamma, although both factors can be activated by both types of IFN. Individuals with mutations in either chain of the IFN-gamma receptor (IFN-gammaR) are susceptible to infection with mycobacteria. A heterozygous STAT1 mutation that impairs GAF but not ISGF3 activation has been found in other individuals with mycobacterial disease. No individuals with deleterious mutations in the IFN-alpha/beta signaling pathway have been described. We report here two unrelated infants homozygous with respect to mutated STAT1 alleles. Neither IFN-alpha/beta nor IFN-gamma activated STAT1-containing transcription factors. Like individuals with IFN-gammaR deficiency, both infants suffered from mycobacterial disease, but unlike individuals with IFN-gammaR deficiency, both died of viral disease. Viral multiplication was not inhibited by recombinant IFN-alpha/beta in cell lines from the two individuals. Inherited impairment of the STAT1-dependent response to human IFN-alpha/beta thus results in susceptibility to viral disease.

Human Cytomegalovirus Activates Inflammatory Cytokine Responses via CD14 and Toll-Like Receptor 2

Abstract: Human cytomegalovirus (CMV) is a ubiquitous opportunistic pathogen. Clinically, CMV disease correlates with immune suppression in which severe presentations are evident in neonates, persons with AIDS, and other immune-suppressed patient groups (38). CMV infection of neonates is associated with deafness, mental retardation, and mortality, whereas AIDS patients often suffer a blinding CMV retinitis, as well as pneumonia and gastrointestinal inflammation. In organ transplant recipients, a patient group hard hit by CMV infection, disease is associated with an increased frequency of graft rejection and is a major cause of posttransplant infection. The varied array of clinical disease correlates with the exceptionally broad tropism of this virus. Indeed, histological analysis of autopsy tissues obtained from patients with CMV disease has demonstrated infected cells in virtually all organs. At the cellular level, CMV can infect monocytes/macrophages, endothelial cells, epithelial cells, smooth muscle cells, fibroblasts, stromal cells, neuronal cells, neutrophils, and hepatocyes (13, 17, 35, 44, 48, 49, 61). In fact, CMV is a suspected pathogenetic agent in cardiovascular disease due to its ability to persist in large-vessel endothelial cells and to infect all cell types involved in cardiovascular lesions (24). Cells exposed to CMV undergo a number of physiological changes that are rendered upon the cell with extremely rapid kinetics (15). These events include changes in Ca2+ homeostasis (1) and activation of phospholipase C and phospholipase A2, as well as increased release of arachidonic acid and its metabolites (1, 59). All of these changes can be triggered by UV-inactivated virions, suggesting that structural components of the virus are responsible for the alterations in cell physiology and intracellular signaling that occur during virus-cell contact and/or virus entry. Virus-cell contact also results in the activation of the transcription factors NF-κB and SP-1, as well as mitogen-activated protein (MAP) kinase, ERK1/2, and p38 (7, 26, 63). Activation of transcription factors by CMV suggests that alterations in cellular transcription should occur in CMV-infected cells, and this is precisely the case. Several transcriptional-profiling studies reveal that cells infected with CMV exhibit profound reprogramming of gene expression (8, 47, 64, 65). Interestingly, the most strongly induced genes were indicators of innate immune activation. Antiviral genes belonging to the interferon-stimulated gene family (ISGs) and inflammatory genes, such as those for RANTES, interleukin 6 (IL-6), IL-7, IL-11, and cyclooxygenase 2 (COX-2), were all robustly induced in CMV-infected fibroblasts (8, 47, 64, 65). Induction of these innate immune markers did not require virus replication. Indeed, cells treated with only the primary ligand of CMV, glycoprotein B (gB) (6), exhibited a response very similar, but not wholly identical, to that of cells treated with intact virus (47). In particular, cells treated with gB strongly induced ISGs. Taken together, the findings suggest that a signal transduction pathway is activated by cell contact of CMV envelope proteins, resulting in numerous physiological changes that culminate, in part, with innate immune activation. The innate immune system is an ancient, universal host defense system. A limited number of evolutionarily conserved germ line receptors found in plants, Drosophila, and humans mediate certain innate immune responses. Termed pattern recognition receptors, these molecules form the basis of the primary host alarm system in response to pathogen-associated molecular patterns (PAMPs) (23). Toll-like receptors (TLRs) are now understood to play a major role in pathogen recognition. Stimulation of TLRs by pathogens activates signal transduction pathways that lead to induction of a range of antimicrobial genes and inflammatory cytokines (2, 23, 29). In addition, key costimulatory molecules, such as CD80 and CD86, which are important for activation of adaptive immunity, are also induced as a consequence of TLR signaling. At present, 10 TLR molecules have been described in humans and mice. Ligands ranging from lipopolysaccharide (LPS) of gram-negative bacteria, peptidoglycan of gram-positive bacteria, flagellin, CpG DNA, and various components from mycobacteria, yeast, and parasitic pathogens, are all detected by TLRs. Although the precise molecular patterns are not completely characterized, PAMPs are hypothesized to be macromolecular modifications unique to these organisms. Viruses have long been known to activate innate immune responses characterized by the induction of inflammatory cytokines and a comprehensive set of ISGs (45, 53). Until recently, the only identified molecular trigger of host innate responses was double-stranded RNA, a common replicative intermediate in the life cycle of many viruses. Double-stranded RNA can activate a key interferon transcription factor, interferon regulatory factor 3 (IRF-3), and induce synthesis of many antiviral genes. Generally, however, the mechanisms by which viruses activate innate immunity remain largely undefined. Since viruses are obligate intracellular parasites, virus-encoded proteins are synthesized by host machinery and ultimately bear protein modifications reflective of the host. Thus, it is not immediately obvious what PAMPs are displayed on viruses. Currently, however, data are clearly emerging showing that TLRs detect viruses and trigger inflammatory responses. Respiratory syncytcial virus (RSV) and mouse mammary tumor virus (MMTV) both signal through TLR4 (28, 42), the well-described LPS receptor. In the case of RSV, TLR4-deficient mice challenged with RSV exhibited a variety of impaired innate immune functions and an inability to clear the virus (20). These findings strongly suggest that Toll signaling pathways have an important role in the innate immunity to RSV. More recently, measles virus was reported to be detected by TLR2, a TLR with broad ligand recognition properties (5). The most striking common feature of these first three reports of virus detection by TLRs is that TLR responses were triggered by viral envelope glycoproteins. Specifically, the fusion protein of RSV, the envelope (env) protein of MMTV, and the hemagglutinin protein of measles virus were the identified triggers of the TLR responsiveness in their respective systems. Interestingly, all of these proteins play key roles in the virus entry pathway, such as attachment and fusion. These studies plainly point to a heretofore-unknown ability of the host to detect viruses during entry but prior to the onset of any replication events. Here, we report that TLR2 and CD14 form the central basis of the membrane detection machinery for CMV.

Inhibition of interferon signaling by dengue virus

Abstract: Dengue virus is a worldwide-distributed mosquito-borne flavivirus with a positive strand RNA genome. Its transcribed polyprotein is cleaved by host- and virus-encoded peptidases into 10 proteins, some of which are of unknown function. Although dengue virus-infected cells seem to be resistant to the antiviral action of IFN, the viral products that mediate this resistance are unknown. Therefore, we have analyzed the ability of the 10 dengue virus-encoded proteins to antagonize the IFN response. We found that expression in human A549 cells of the dengue virus nonstructural proteins NS2A, NS4A, or NS4B enhances replication of an IFN-sensitive virus. Moreover, expression of NS4B and, to a lesser extent, of NS2A and NS4A proteins results in down-regulation of IFN-β-stimulated gene expression. Cells expressing NS4B or infected with dengue virus do not exhibit nuclear signal transducer and activator of transcription (STAT) 1 on treatment with IFN-β or IFN-γ, indicating that NS4B might be involved in blocking IFN signaling during dengue virus infections. This protein, encoded by a positive strand RNA virus, is implicated as an IFN-signaling inhibitor.

Viral infection switches non-plasmacytoid dendritic cells into high interferon producers

Abstract: Type I interferons (IFN-I) are important cytokines linking innate and adaptive immunity. Plasmacytoid dendritic cells make high levels of IFN-I in response to viral infection and are thought to be the major source of the cytokines in vivo. Here, we show that conventional non-plasmacytoid dendritic cells taken from mice infected with a dendritic-cell-tropic strain of lymphocytic choriomeningitis virus make similarly high levels of IFN-I on subsequent culture. Similarly, non-plasmacytoid dendritic cells secrete high levels of IFN-I in response to double-stranded RNA (dsRNA), a major viral signature, when the latter is introduced into the cytoplasm to mimic direct viral infection. This response is partially dependent on the cytosolic dsRNA-binding enzyme protein kinase R and does not require signalling through toll-like receptor (TLR) 3, a surface receptor for dsRNA. Furthermore, we show that sequestration of dsRNA by viral NS1 (refs 6, 7) explains the inability of conventional dendritic cells to produce IFN-I on infection with influenza. Our results suggest that multiple dendritic cell types, not just plasmacytoid cells, can act as specialized interferon-producing cells in certain viral infections, and reveal the existence of a TLR-independent pathway for dendritic cell activation that can be the target of viral interference.

Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans.

Abstract: In animals, effective immune responses against malignancies and against several infectious pathogens, including malaria, are mediated by T cells. Here we show that a heterologous prime-boost vaccination regime of DNA either intramuscularly or epidermally, followed by intradermal recombinant modified vaccinia virus Ankara (MVA), induces high frequencies of interferon (IFN)-gamma-secreting, antigen-specific T-cell responses in humans to a pre-erythrocytic malaria antigen, thrombospondin-related adhesion protein (TRAP). These responses are five- to tenfold higher than the T-cell responses induced by the DNA vaccine or recombinant MVA vaccine alone, and produce partial protection manifest as delayed parasitemia after sporozoite challenge with a different strain of Plasmodium falciparum. Such heterologous prime-boost immunization approaches may provide a basis for preventative and therapeutic vaccination in humans.

Inhibition of Hepatitis B Virus Replication by Drug-Induced Depletion of Nucleocapsids

Abstract: Chronic hepatitis B virus (HBV) infection is a major cause of liver disease. Only interferon-alpha and the nucleosidic inhibitors of the viral polymerase, 3TC and adefovir, are approved for therapy. However, these therapies are limited by the side effects of interferon and the substantial resistance of the virus to nucleosidic inhibitors. Potent new antiviral compounds suitable for monotherapy or combination therapy are highly desired. We describe non-nucleosidic inhibitors of HBV nucleocapsid maturation that possess in vitro and in vivo antiviral activity. These inhibitors have potential for future therapeutic regimens to combat chronic HBV infection.

Peginterferon alpha-2a (40 kDa): an advance in the treatment of hepatitis B e antigen-positive chronic hepatitis B.

Abstract: Current therapies for chronic hepatitis B (CHB) have a number of limitations, and better treatment options are needed. Peginterferon alpha-2a (40 kDa) is superior to conventional interferon alpha-2a in the treatment of chronic hepatitis C. This is the first report on peginterferon alpha-2a (40 kDa) in the treatment of CHB. In this phase II study, 194 patients with CHB not previously treated with conventional interferon-alpha were randomized to receive weekly subcutaneous doses of peginterferon alpha-2a (40 kDa) 90, 180 or 270 microg, or conventional interferon alpha-2a 4.5 MIU three times weekly. Twenty-four weeks of therapy were followed by 24 weeks of treatment-free follow-up. All subjects were assessed for loss of hepatitis B e antigen (HBeAg), presence of hepatitis B antibody (anti-HBe), suppression of hepatitis B virus (HBV) DNA, and normalization of serum alanine transaminase (ALT) after follow-up. At the end of follow-up, HBeAg was cleared in 37, 35 and 29% of patients receiving peginterferon alpha-2a (40 kDa) 90, 180 and 270 microg, respectively, compared with 25% of patients on conventional interferon alpha-2a. The combined response (HBeAg loss, HBV DNA suppression, and ALT normalization) of all peginterferon alpha-2a (40 kDa) doses combined was twice that achieved with conventional interferon alpha-2a (24%vs 12%; P = 0.036). All treatment groups were similar with respect to frequency and severity of adverse events. These results indicate that peginterferon alpha-2a (40 kDa) is superior in efficacy to conventional interferon alpha-2a in chronic hepatitis B based on clearance of HBeAg, suppression of HBV DNA, and normalization of ALT.

HIV-1 Viremia Prevents the Establishment of Interleukin 2–producing HIV-specific Memory CD4+ T Cells Endowed with Proliferative Capacity

Abstract: CD4+ T cell responses are associated with disease control in chronic viral infections. We analyzed human immunodeficiency virus (HIV)-specific responses in ten aviremic and eight viremic patients treated during primary HIV-1 infection and for up to 6 yr thereafter. Using a highly sensitive 5-(and-6)-carboxyfluorescein diacetate-succinimidyl ester-based proliferation assay, we observed that proliferative Gag and Nef peptide-specific CD4+ T cell responses were 30-fold higher in the aviremic patients. Two subsets of HIV-specific memory CD4+ T cells were identified in aviremic patients, CD45RA- CCR7+ central memory cells (Tcm) producing exclusively interleukin (IL)-2, and CD45RA- CCR7- effector memory cells (Tem) that produced both IL-2 and interferon (IFN)-gamma. In contrast, in viremic, therapy-failing patients, we found significant frequencies of Tem that unexpectedly produced exclusively IFN-gamma. Longitudinal analysis of HIV epitope-specific CD4+ T cells revealed that only cells that had the capacity to produce IL-2 persisted as long-term memory cells. In viremic patients the presence of IFN-gamma-producing cells was restricted to periods of elevated viremia. These findings suggest that long-term CD4+ T cell memory depends on IL-2-producing CD4+ T cells and that IFN-gamma only-producing cells are short lived. Our data favor a model whereby competent HIV-specific Tcm continuously arise in small numbers but under persistent antigenemia are rapidly induced to differentiate into IFN-gamma only-producing cells that lack self-renewal capacity.

The Ebola Virus VP35 Protein Inhibits Activation of Interferon Regulatory Factor 3

Abstract: The Ebola virus VP35 protein was previously found to act as an interferon (IFN) antagonist which could complement growth of influenza delNS1 virus, a mutant influenza virus lacking the influenza virus IFN antagonist protein, NS1. The Ebola virus VP35 could also prevent the virus- or double-stranded RNA-mediated transcriptional activation of both the beta IFN (IFN-β) promoter and the IFN-stimulated ISG54 promoter (C. Basler et al., Proc. Natl. Acad. Sci. USA 97:12289-12294, 2000). We now show that VP35 inhibits virus infection-induced transcriptional activation of IFN regulatory factor 3 (IRF-3)-responsive mammalian promoters and that VP35 does not block signaling from the IFN-α/β receptor. The ability of VP35 to inhibit this virus-induced transcription correlates with its ability to block activation of IRF-3, a cellular transcription factor of central importance in initiating the host cell IFN response. We demonstrate that VP35 blocks the Sendai virus-induced activation of two promoters which can be directly activated by IRF-3, namely, the ISG54 promoter and the ISG56 promoter. Further, expression of VP35 prevents the IRF-3-dependent activation of the IFN-α4 promoter in response to viral infection. The inhibition of IRF-3 appears to occur through an inhibition of IRF-3 phosphorylation. VP35 blocks virus-induced IRF-3 phosphorylation and subsequent IRF-3 dimerization and nuclear translocation. Consistent with these observations, Ebola virus infection of Vero cells activated neither transcription from the ISG54 promoter nor nuclear accumulation of IRF-3. These data suggest that in Ebola virus-infected cells, VP35 inhibits the induction of antiviral genes, including the IFN-β gene, by blocking IRF-3 activation.

A reversible defect in natural killer T cell function characterizes the progression of premalignant to malignant multiple myeloma.

Abstract: We studied the function of antitumor T and natural killer T (NKT) cells from the blood and tumor bed in 23 patients with premalignant gammopathy, nonprogressive myeloma, or progressive multiple myeloma. We show that antitumor killer T cells can be detected in patients with both progressive or nonprogressive myeloma. Vα24+Vβ11+ invariant NKT cells are detectable in the blood and tumor bed of all cohorts. However, freshly isolated NKT cells from both the blood and tumor bed of patients with progressive disease, but not nonprogressive myeloma or premalignant gammopathy, have a marked deficiency of ligand-dependent interferon-γ production. This functional defect can be overcome in vitro using dendritic cells pulsed with the NKT ligand, α-galactosylceramide (α-GalCer). Fresh myeloma cells express CD1d, and can be efficiently killed by autologous NKT cells. We hypothesize that presentation of tumor derived glycolipids by myeloma cells leads to NKT dysfunction in vivo. These data demonstrate that clinical progression in patients with monoclonal gammopathies is associated with an acquired but potentially reversible defect in NKT cell function and support the possibility that these innate lymphocytes play a role in controlling the malignant growth of this incurable B cell tumor in patients.

Cytokines and transcription factors that regulate T helper cell differentiation: new players and new insights.

Abstract: The differentiation of naive CD4+ T cells into subsets of T helper cells is a pivotal process with major implications for host defense and the pathogenesis of immune-mediated diseases. Though the basic paradigm was discovered more than 15 years ago, new discoveries continue to be made that offer fresh insights into the regulation of this process (1). T helper (TH)1 cells produce interferon (IFN)-γ, promoting cell-mediated immunity and control of intracellular pathogens. We now know that TH1 differentiation is regulated by transcription factors such as T-bet, Stat1, and Stat4, as well as cytokines such as IL-12, IL-23, IL-27, type I IFNs, and IFN-γ. In contrast, TH2 cells produce IL-4, which promotes allergic responses and is important in host defense against helminths. The transcription factors Stat6, GATA-3, c-Maf, NFATs, and the cytokine IL-4 promote TH2 differentiation. These key regulators of TH differentiation are the subject of this review.

Superior Protection against Malaria and Melanoma Metastases by a C-glycoside Analogue of the Natural Killer T Cell Ligand α-Galactosylceramide

Abstract: α-Galactosylceramide (α-GalCer) is a glycolipid that stimulates natural killer T cells to produce both T helper (Th) 1 and Th2 cytokines. This property enables α-GalCer to ameliorate a wide variety of infectious, neoplastic, and autoimmune diseases; however, its effectiveness against any one disease is limited by the opposing activities of the induced Th1 and Th2 cytokines. Here, we report that a synthetic C-glycoside analogue of α-GalCer, α-C-galactosylceramide (α-C-GalCer), acts as natural killer T cell ligand in vivo, and stimulates an enhanced Th1-type response in mice. In two disease models requiring Th1-type responses for control, namely malaria and melanoma metastases, α-C-GalCer exhibited a 1,000-fold more potent antimalaria activity and a 100-fold more potent antimetastatic activity than α-GalCer. Moreover, α-C-GalCer consistently stimulated prolonged production of the Th1 cytokines interferon-γ and interleukin (IL)-12, and decreased production of the Th2 cytokine IL-4 compared with α-GalCer. Finally, α-C-GalCer's enhanced therapeutic activity required the presence of IL-12, which was needed to stimulate natural killer cells for optimal interferon-γ production, but did not affect IL-4. Overall, our results suggest that α-C-GalCer may one day be an excellent therapeutic option for diseases resolved by Th1-type responses.

γδ T Cells Provide an Early Source of Interferon γ in Tumor Immunity

Abstract: Interferon (IFN)-γ is necessary for tumor immunity, however, its initial cellular source is unknown. Because γδ T cells primarily produce this cytokine upon activation, we hypothesized that they would provide an important early source of IFN-γ in tumor immunosurveillance. To address this hypothesis, we first demonstrated that γδ T cell–deficient mice had a significantly higher incidence of tumor development after challenge with a chemical carcinogen methylcholanthrene (MCA) or inoculation with the melanoma cell line B16. In wild-type mice, γδ T cells were recruited to the site of tumor as early as day 3 after inoculation, followed by αβ T cells at day 5. We then used bone marrow chimeras and fetal liver reconstitutions to create mice with an intact γδ T cell repertoire but one that was specifically deficient in the capacity to produce IFN-γ. Such mice had a higher incidence of tumor development, induced either with MCA or by inoculation of B16 melanoma cells, compared with mice with IFN-γ–competent γδ T cells. Moreover, genetic deficiency of γδ T cells resulted in impaired IFN-γ production by tumor antigen-triggered αβ T cell upon immunization with tumor lysate. These results demonstrate that γδ T cells can play a necessary role in tumor immunity through provision of an early source of IFN-γ that in turn may regulate the function of tumor-triggered αβ T cells.

Dendritic Cell Responses to Early Murine Cytomegalovirus Infection Subset Functional Specialization and Differential Regulation by Interferon α/β

Abstract: Differentiation of dendritic cells (DCs) into particular subsets may act to shape innate and adaptive immune responses, but little is known about how this occurs during infections. Plasmacytoid dendritic cells (PDCs) are major producers of interferon (IFN)-α/β in response to many viruses. Here, the functions of these and other splenic DC subsets are further analyzed after in vivo infection with murine cytomegalovirus (MCMV). Viral challenge induced PDC maturation, their production of high levels of innate cytokines, and their ability to activate natural killer (NK) cells. The conditions also licensed PDCs to efficiently activate CD8 T cells in vitro. Non-plasmacytoid DCs induced T lymphocyte activation in vitro. As MCMV preferentially infected CD8α+ DCs, however, restricted access to antigens may limit plasmacytoid and CD11b+ DC contribution to CD8 T cell activation. IFN-α/β regulated multiple DC responses, limiting viral replication in all DC and IL-12 production especially in the CD11b+ subset but promoting PDC accumulation and CD8α+ DC maturation. Thus, during defense against a viral infection, PDCs appear specialized for initiation of innate, and as a result of their production of IFN-α/β, regulate other DCs for induction of adaptive immunity. Therefore, they may orchestrate the DC subsets to shape endogenous immune responses to viruses.

Newcastle disease virus (NDV)-based assay demonstrates interferon-antagonist activity for the NDV V protein and the Nipah virus V, W, and C proteins.

Abstract: We have generated a recombinant Newcastle disease virus (NDV) that expresses the green fluorescence protein (GFP) in infected chicken embryo fibroblasts (CEFs). This virus is interferon (IFN) sensitive, and pretreatment of cells with chicken alpha/beta IFN (IFN-α/β) completely blocks viral GFP expression. Prior transfection of plasmid DNA induces an IFN response in CEFs and blocks NDV-GFP replication. However, transfection of known inhibitors of the IFN-α/β system, including the influenza A virus NS1 protein and the Ebola virus VP35 protein, restores NDV-GFP replication. We therefore conclude that the NDV-GFP virus could be used to screen proteins expressed from plasmids for the ability to counteract the host cell IFN response. Using this system, we show that expression of the NDV V protein or the Nipah virus V, W, or C proteins rescues NDV-GFP replication in the face of the transfection-induced IFN response. The V and W proteins of Nipah virus, a highly lethal pathogen in humans, also block activation of an IFN-inducible promoter in primate cells. Interestingly, the amino-terminal region of the Nipah virus V protein, which is identical to the amino terminus of Nipah virus W, is sufficient to exert the IFN-antagonist activity. In contrast, the anti-IFN activity of the NDV V protein appears to be located in the carboxy-terminal region of the protein, a region implicated in the IFN-antagonist activity exhibited by the V proteins of mumps virus and human parainfluenza virus type 2.