Huawei Mao

Cytotoxic T Lymphocytes Established by Seasonal Human Influenza Cross-React against 2009 Pandemic H1N1 Influenza Virus

ABSTRACT While few children and young adults have cross-protective antibodies to the pandemic H1N1 2009 (pdmH1N1) virus, the illness remains mild. The biological reasons for these epidemiological observations are unclear. In this study, we demonstrate that the bulk memory cytotoxic T lymphocytes (CTLs) established by seasonal influenza viruses from healthy individuals who have not been exposed to pdmH1N1 can directly lyse pdmH1N1-infected target cells and produce gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Using influenza A virus matrix protein 1 (M158-66) epitope-specific CTLs isolated from healthy HLA-A2+ individuals, we further found that M158-66 epitope-specific CTLs efficiently killed both M158-66 peptide-pulsed and pdmH1N1-infected target cells ex vivo. These M158-66-specific CTLs showed an effector memory phenotype and expressed CXCR3 and CCR5 chemokine receptors. Of 94 influenza A virus CD8 T-cell epitopes obtained from the Immune Epitope Database (IEDB), 17 epitopes are conserved in pdmH1N1, and more than half of these conserved epitopes are derived from M1 protein. In addition, 65% (11/17) of these epitopes were 100% conserved in seasonal influenza vaccine H1N1 strains during the last 20 years. Importantly, seasonal influenza vaccination could expand the functional M158-66 epitope-specific CTLs in 20% (4/20) of HLA-A2+ individuals. Our results indicated that memory CTLs established by seasonal influenza A viruses or vaccines had cross-reactivity against pdmH1N1. These might explain, at least in part, the unexpected mild pdmH1N1 illness in the community and also might provide some valuable insights for the future design of broadly protective vaccines to prevent influenza, especially pandemic influenza.

Phosphoantigen-Expanded Human γδ T Cells Display Potent Cytotoxicity against Monocyte-Derived Macrophages Infected with Human and Avian Influenza Viruses

Abstract BackgroundInfluenza virus is a cause of substantial annual morbidity and mortality worldwide. The potential emergence of a new pandemic strain (eg, avian influenza virus) is a major concern. Currently available vaccines and anti-influenza drugs have limited effectiveness for influenza virus infections, especially for new pandemic strains. Therefore, there is an acute need to develop alternative strategies for influenza therapy. γδ T cells have potent antiviral activities against different viruses, but no data are available concerning their antiviral activity against influenza viruses MethodsIn this study, we used virus-infected primary human monocyte-derived macrophages (MDMs) to examine the antiviral activity of phosphoantigen isopentenyl pyrophosphate (IPP)–expanded human Vγ9Vδ2 T cells against influenza viruses ResultsVγ9Vδ2 T cells were selectively activated and expanded by IPP from peripheral blood mononuclear cells. IPP-expanded Vγ9Vδ2 T cells efficiently killed MDMs infected with human (H1N1) or avian (H9N2 or H5N1) influenza virus and significantly inhibited viral replication. The cytotoxicity of Vγ9Vδ2 T cells against influenza virus–infected MDMs was dependent on NKG2D activation and was mediated by Fas–Fas ligand and perforin–granzyme B pathways ConclusionOur findings suggest a potentially novel therapeutic approach to seasonal, zoonotic avian, and pandemic influenza—the use of phosphoantigens to activate γδ T cells against influenza virus infections

Efficient generation of human alloantigen-specific CD4+ regulatory T cells from naive precursors by CD40-activated B cells

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) play an important role in the induction and maintenance of immune tolerance. Although adoptive transfer of bulk populations of Treg can prevent or treat T cell-mediated inflammatory diseases and transplant allograft rejection in animal models, optimal Treg immunotherapy in humans would ideally use antigen-specific rather than polyclonal Treg for greater specificity of regulation and avoidance of general suppression. However, no robust approaches have been reported for the generation of human antigen-specific Treg at a practical scale for clinical use. Here, we report a simple and cost-effective novel method to rapidly induce and expand large numbers of functional human alloantigen-specific Treg from antigenically naive precursors in vitro using allogeneic nontransformed B cells as stimulators. By this approach naive CD4(+)CD25(-) T cells could be expanded 8-fold into alloantigen-specific Treg after 3 weeks of culture without any exogenous cytokines. The induced alloantigen-specific Treg were CD45RO(+)CCR7(-) memory cells, and had a CD4(high), CD25(+), Foxp3(+), and CD62L (L-selectin)(+) phenotype. Although these CD4(high)CD25(+)Foxp3(+) alloantigen-specific Treg had no cytotoxic capacity, their suppressive function was cell-cell contact dependent and partially relied on cytotoxic T lymphocyte antigen-4 expression. This approach may accelerate the clinical application of Treg-based immunotherapy in transplantation and autoimmune diseases.

Influenza Virus Directly Infects Human Natural Killer Cells and Induces Cell Apoptosis

ABSTRACT Influenza is an acute respiratory viral disease that is transmitted in the first few days of infection. Evasion of host innate immune defenses, including natural killer (NK) cells, is important for the viruss success as a pathogen of humans and other animals. NK cells encounter influenza viruses within the microenvironment of infected cells and are important for host innate immunity during influenza virus infection. It is therefore important to investigate the direct effects of influenza virus on NK cells. In this study, we demonstrated for the first time that influenza virus directly infects and replicates in primary human NK cells. Viral entry into NK cells was mediated by both clathrin- and caveolin-dependent endocytosis rather than through macropinocytosis and was dependent on the sialic acids on cell surfaces. In addition, influenza virus infection induced a marked apoptosis of NK cells. Our findings suggest that influenza virus can directly target and kill NK cells, a potential novel strategy of influenza virus to evade the NK cell innate immune defense that is likely to facilitate viral transmission and may also contribute to virus pathogenesis.

Inhibition of Human Natural Killer Cell Activity by Influenza Virions and Hemagglutinin

ABSTRACT Natural killer (NK) cells keep viral infections under control at the early phase by directly killing infected cells. Influenza is an acute contagious respiratory viral disease transmitted from host-to-host in the first few days of infection. The evasion of host innate immune defenses including NK cells is important for its success as a viral pathogen of humans and animals. NK cells encounter influenza virus within the microenvironment of infected cells. It therefore is important to investigate the direct effects of influenza virus on NK cell activity. Recently we demonstrated that influenza virus directly infects human NK cells and induces cell apoptosis to counter their function (H. Mao, W. Tu, G. Qin, H. K. W. Law, S. F. Sia, P.-L. Chan, Y. Liu, K.-T. Lam, J. Zheng, M. Peiris, and Y.-L. Lau, J. Virol. 83:9215-9222, 2009). Here, we further demonstrated that both the intact influenza virion and free hemagglutinin protein inhibited the cytotoxicity of fresh and interleukin-2 (IL-2)-activated primary human NK cells. Hemagglutinin bound and internalized into NK cells via the sialic acids. This interaction did not decrease NKp46 expression but caused the downregulation of the ζ chain through the lysosomal pathway, which caused the decrease of NK cell cytotoxicity mediated by NKp46 and NKp30. The underlying dysregulation of the signaling pathway involved ζ chain downregulation, leading to decreased Syk and ERK activation and granule exocytosis upon target cell stimulation, finally causing reduced cytotoxicity. These findings suggest that influenza virus developed a novel strategy to evade NK cell innate immune defense that is likely to facilitate viral transmission and also contribute to virus pathogenesis.

Type 1 Responses of Human Vγ9Vδ2 T Cells to Influenza A Viruses

ABSTRACT γδ T cells are essential constituents of antimicrobial and antitumor defenses. We have recently reported that phosphoantigen isopentenyl pyrophosphate (IPP)-expanded human Vγ9Vδ2 T cells participated in anti-influenza virus immunity by efficiently killing both human and avian influenza virus-infected monocyte-derived macrophages (MDMs) in vitro. However, little is known about the noncytolytic responses and trafficking program of γδ T cells to influenza virus. In this study, we found that Vγ9Vδ2 T cells expressed both type 1 cytokines and chemokine receptors during influenza virus infection, and IPP-expanded cells had a higher capacity to produce gamma interferon (IFN-γ). Besides their potent cytolytic activity against pandemic H1N1 virus-infected cells, IPP-activated γδ T cells also had noncytolytic inhibitory effects on seasonal and pandemic H1N1 viruses via IFN-γ but had no such effects on avian H5N1 or H9N2 virus. Avian H5N1 and H9N2 viruses induced significantly higher CCL3, CCL4, and CCL5 production in Vγ9Vδ2 T cells than human seasonal H1N1 virus. CCR5 mediated the migration of Vγ9Vδ2 T cells toward influenza virus-infected cells. Our findings suggest a novel therapeutic strategy of using phosphoantigens to boost the antiviral activities of human Vγ9Vδ2 T cells against influenza virus infection.

Penicillium marneffei infection and impaired IFN-γ immunity in humans with autosomal-dominant gain-of-phosphorylation STAT1 mutations

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Mannose-Binding Lectin Contributes to Deleterious Inflammatory Response in Pandemic H1N1 and Avian H9N2 Infection

Abstract Background. Mannose-binding lectin (MBL) is a pattern-recognition molecule, which functions as a first line of host defense. Pandemic H1N1 (pdmH1N1) influenza A virus caused massive infection in 2009 and currently circulates worldwide. Avian influenza A H9N2 (H9N2/G1) virus has infected humans and has the potential to be the next pandemic virus. Antiviral function and immunomodulatory role of MBL in pdmH1N1 and H9N2/G1 virus infection have not been investigated. Methods. In this study, MBL wild-type (WT) and MBL knockout (KO) murine models were used to examine the role of MBL in pdmH1N1 and H9N2/G1 virus infection. Results. Our study demonstrated that in vitro, MBL binds to pdmH1N1 and H9N2/G1 viruses, likely via the carbohydrate recognition domain of MBL. Wild-type mice developed more severe disease, as evidenced by a greater weight loss than MBL KO mice during influenza virus infection. Furthermore, MBL WT mice had enhanced production of proinflammatory cytokines and chemokines compared with MBL KO mice, suggesting that MBL could upregulate inflammatory responses that may potentially worsen pdmH1N1 and H9N2/G1 virus infections. Conclusions. Our study provided the first in vivo evidence that MBL may be a risk factor during pdmH1N1 and H9N2/G1 infection by upregulating proinflammatory response.

H5N1 Influenza Virus–Induced Mediators Upregulate RIG-I in Uninfected Cells by Paracrine Effects Contributing to Amplified Cytokine Cascades

Highly pathogenic avian influenza H5N1 viruses cause severe disease in humans, and dysregulation of cytokine responses is believed to contribute to the pathogenesis of human H5N1 disease. However, mechanisms leading to the increased induction of proinflammatory cytokines by H5N1 viruses are poorly understood. We show that the innate sensing receptor RIG-I is involved in interferon regulatory factor 3 (IRF3), NF-κB nuclear translocation, p38 activation, and the subsequent interferon (IFN) β, IFN-λ1, and tumor necrosis factor α induction during H5N1 infection. Soluble mediators from H5N1-infected human macrophages upregulate RIG-I, MDA5, and TLR3 to much higher levels than those from seasonal H1N1 in uninfected human macrophages and alveolar epithelial cells via paracrine IFNAR1/JAK but not IFN-λ receptor signaling. Compared with H1N1 virus-induced mediators, H5N1 mediators markedly enhance the cytokine response to PolyIC and to both seasonal and H5N1 virus infection in a RIG-I-dependent manner. Thus, sensitizing neighboring cells by upregulation of RIG-I contributes to the amplified cytokine cascades during H5N1 infection.

Successive influenza virus infection and Streptococcus pneumoniae stimulation alter human dendritic cell function

BackgroundInfluenza virus is a major cause of respiratory disease worldwide and Streptococcus pneumoniae infection associated with influenza often leads to severe complications. Dendritic cells are key antigen presenting cells but its role in such co-infection is unclear.MethodsIn this study, human monocyte derived-dentritic cells were either concurrently or successively challenged with the combination of live influenza virus and heat killed pneumococcus to mimic the viral pneumococcal infection. Dendritic cell viability, phenotypic maturation and cytokine production were then examined.ResultsThe challenge of influenza virus and pneumococcus altered dendritic cell functions dependent on the time interval between the successive challenge of influenza virus and pneumococcus, as well as the doses of pneumococcus. When dendritic cells were exposed to pneumococcus at 6 hr, but not 0 hr nor 24 hr after influenza virus infection, both virus and pneumococcus treated dendritic cells had greater cell apoptosis and expressed higher CD83 and CD86 than dendritic cells infected with influenza virus alone. Dendritic cells produced pro-inflammatory cytokines: TNF-α, IL-12 and IFN-γ synergistically to the successive viral and pneumococcal challenge. Whereas prior influenza virus infection suppressed the IL-10 response independent of the timing of the subsequent pneumococcal stimulation.ConclusionsOur results demonstrated that successive challenge of dendritic cells with influenza virus and pneumococcus resulted in synergistic up-regulation of pro-inflammatory cytokines with simultaneous down-regulation of anti-inflammatory cytokine, which may explain the immuno-pathogenesis of this important co-infection.