Philippa Hillyer

Respiratory syncytial virus interferon antagonist NS1 protein suppresses and skews the human T lymphocyte response

We recently demonstrated that the respiratory syncytial virus (RSV) NS1 protein, an antagonist of host type I interferon (IFN-I) production and signaling, has a suppressive effect on the maturation of human dendritic cells (DC) that was only partly dependent on released IFN-I. Here we investigated whether NS1 affects the ability of DC to activate CD8+ and CD4+ T cells. Human DC were infected with RSV deletion mutants lacking the NS1 and/or NS2 genes and assayed for the ability to activate autologous T cells in vitro, which were analyzed by multi-color flow cytometry. Deletion of the NS1, but not NS2, protein resulted in three major effects: (i) an increased activation and proliferation of CD8+ T cells that express CD103, a tissue homing integrin that directs CD8+ T cells to mucosal epithelial cells of the respiratory tract and triggers cytolytic activity; (ii) an increased activation and proliferation of Th17 cells, which have recently been shown to have anti-viral effects and also indirectly attract neutrophils; and (iii) decreased activation of IL-4-producing CD4+ T cells - which are associated with enhanced RSV disease - and reduced proliferation of total CD4+ T cells. Except for total CD4+ T cell proliferation, none of the T cell effects appeared to be due to increased IFN-I signaling. In the infected DC, deletion of the NS1 and NS2 genes strongly up-regulated the expression of cytokines and other molecules involved in DC maturation. This was partly IFN-I-independent, and thus might account for the T cell effects. Taken together, these data demonstrate that the NS1 protein suppresses proliferation and activation of two of the protective cell populations (CD103+ CD8+ T cells and Th17 cells), and promotes proliferation and activation of Th2 cells that can enhance RSV disease.

Effects of human respiratory syncytial virus, metapneumovirus, parainfluenza virus 3 and influenza virus on CD4+ T cell activation by dendritic cells.

Background Human respiratory syncytial virus (HRSV), and to a lesser extent human metapneumovirus (HMPV) and human parainfluenza virus type 3 (HPIV3), re-infect symptomatically throughout life without antigenic change, suggestive of incomplete immunity. One causative factor is thought to be viral interference with dendritic cell (DC)-mediated stimulation of CD4+ T cells. Methodology, Principal Findings We infected human monocyte-derived DC with purified HRSV, HMPV, HPIV3, or influenza A virus (IAV) and compared their ability to induce activation and proliferation of autologous CD4+ T cells in vitro. IAV was included because symptomatic re-infection without antigenic change is less frequent, suggesting that immune protection is more complete and durable. We examined virus-specific memory responses and superantigen-induced responses by multiparameter flow cytometry. Live virus was more stimulatory than inactivated virus in inducing DC-mediated proliferation of virus-specific memory CD4+ T cells, suggesting a lack of strong suppression by live virus. There were trends of increasing proliferation in the order: HMPV<HRSV<HPIV3<IAV, and greater production of interferon-γ and tumor necrosis factor-α by proliferating cells in response to IAV, but differences were not significant. Exposure of DC to HRSV, HPIV3, or IAV reduced CD4+ T cell proliferation in response to secondary stimulus with superantigen, but the effect was transitory and greatest for IAV. T cell cytokine production was similar, with no evidence of Th2 or Th17 skewing. Conclusions, Significance Understanding the basis for the ability of HRSV in particular to symptomatically re-infect without significant antigenic change is of considerable interest. The present results show that these common respiratory viruses are similar in their ability to induce DC to activate CD4+ T cells. Thus, the results do not support the common model in which viral suppression of CD4+ T cell activation and proliferation by HRSV, HMPV, and HPIV3 is a major factor in the difference in re-infectability compared to IAV.

Low CCR7-Mediated Migration of Human Monocyte Derived Dendritic Cells in Response to Human Respiratory Syncytial Virus and Human Metapneumovirus

Human respiratory syncytial virus (HRSV) and, to a lesser extent, human metapneumovirus (HMPV) and human parainfluenza virus type 3 (HPIV3), can re-infect symptomatically throughout life without significant antigenic change, suggestive of incomplete or short-lived immunity. In contrast, re-infection by influenza A virus (IAV) largely depends on antigenic change, suggestive of more complete immunity. Antigen presentation by dendritic cells (DC) is critical in initiating the adaptive immune response. Antigen uptake by DC induces maturational changes that include decreased expression of the chemokine receptors CCR1, CCR2, and CCR5 that maintain DC residence in peripheral tissues, and increased expression of CCR7 that mediates the migration of antigen-bearing DC to lymphatic tissue. We stimulated human monocyte-derived DC (MDDC) with virus and found that, in contrast to HPIV3 and IAV, HMPV and HRSV did not efficiently decrease CCR1, 2, and 5 expression, and did not efficiently increase CCR7 expression. Consistent with the differences in CCR7 mRNA and protein expression, MDDC stimulated with HRSV or HMPV migrated less efficiently to the CCR7 ligand CCL19 than did IAV-stimulated MDDC. Using GFP-expressing recombinant virus, we showed that the subpopulation of MDDC that was robustly infected with HRSV was particularly inefficient in chemokine receptor modulation. HMPV- or HRSV-stimulated MDDC responded to secondary stimulation with bacterial lipopolysaccharide or with a cocktail of proinflammatory cytokines by increasing CCR7 and decreasing CCR1, 2 and 5 expression, and by more efficient migration to CCL19, suggesting that HMPV and HRSV suboptimally stimulate rather than irreversibly inhibit MDDC migration. This also suggests that the low concentration of proinflammatory cytokines released from HRSV- and HMPV-stimulated MDDC is partly responsible for the low CCR7-mediated migration. We propose that inefficient migration of HRSV- and HMPV-stimulated DC to lymphatic tissue contributes to reduced adaptive responses to these viruses.

Human Metapneumovirus SH and G Glycoproteins Inhibit Macropinocytosis-Mediated Entry into Human Dendritic Cells and Reduce CD4+ T Cell Activation

ABSTRACT Human metapneumovirus (HMPV) is a major etiologic agent of respiratory disease worldwide. HMPV reinfections are common in healthy adults and children, suggesting that the protective immune response to HMPV is incomplete and short-lived. We used gene-deletion viruses to evaluate the role of the attachment G and small hydrophobic SH glycoproteins on virus uptake by primary human monocyte-derived dendritic cells (MDDC) in vitro and on subsequent MDDC maturation and activation of autologous T cells. HMPV with deletion of G and SH (ΔSHG) exhibited increased infectivity but had little effect on MDDC maturation. However, MDDC stimulated with ΔSHG induced increased proliferation of autologous Th1-polarized CD4+ T cells. This effect was independent of virus replication. Increased T cell proliferation was strictly dependent on contact between virus-stimulated MDDC and CD4+ T cells. Confocal microscopy revealed that deletion of SH and G was associated with an increased number of immunological synapses between memory CD4+ T cells and virus-stimulated MDDC. Uptake of HMPV by MDDC was found to be primarily by macropinocytosis. Uptake of wild-type (WT) virus was reduced compared to that of ΔSHG, indicative of inhibition by the SH and G glycoproteins. In addition, DC-SIGN-mediated endocytosis provided a minor alternative pathway that depended on SH and/or G and thus operated only for WT. Altogether, our results show that SH and G glycoproteins reduce the ability of HMPV to be internalized by MDDC, resulting in a reduced ability of the HMPV-stimulated MDDC to activate CD4+ T cells. This study describes a previously unknown mechanism of virus immune evasion. IMPORTANCE Human metapneumovirus (HMPV) is a major etiologic agent of respiratory disease worldwide. HMPV reinfections are common in healthy adults and children, suggesting that the protective immune response to HMPV is incomplete and short-lived. We found that HMPV attachment G and small hydrophobic SH glycoproteins reduce the ability of HMPV to be internalized by macropinocytosis into human dendritic cells (DC). This results in a reduced ability of the HMPV-stimulated DC to activate Th1-polarized CD4+ T cells. These results contribute to a better understanding of the nature of incomplete protection against this important human respiratory virus, provide new information on the entry of HMPV into human cells, and describe a new mechanism of virus immune evasion.

Subtypes of type I IFN differentially enhance cytokine expression by suboptimally stimulated CD4+ T cells

Human type I interferons (IFNs) include IFN‐β and 12 subtypes of IFN‐α. During viral infection, infiltrating memory CD4+ T cells are exposed to IFNs, but their impact on memory T‐cell function is poorly understood. To address this, we pretreated PBMCs with different IFNs for 16 h before stimulation with Staphylococcus aureus enterotoxin B and measured cytokine expression by flow cytometry. IFN‐α8 and ‐α10 most potently enhanced expression of IFN‐γ, IL‐2, and IL‐4. Potency among the subtypes differed most at doses between 10 and 100 U/mL. While enhancement of IL‐2 and IL‐4 correlated with the time of preincubation with type I IFN, IFN‐γ production was enhanced best when IFN‐α was added immediately preceding or simultaneously with T‐cell stimulation. Comparison of T‐cell responses to multiple doses of Staphylococcus aureus enterotoxin B and to peptide libraries from RSV or CMV demonstrated that IFN‐α best enhanced cytokine expression when CD4+ T cells were suboptimally stimulated. We conclude that type I IFNs enhance Th1 and Th2 function with dose dependency and subtype specificity, and best when T‐cell stimulation is suboptimal. While type I IFNs may beneficially enhance CD4+ T‐cell memory responses to vaccines or viral pathogens, they may also enhance the function of resident Th2 cells and exacerbate allergic inflammation.

Respiratory syncytial virus infection induces a subset of types I and III interferons in human dendritic cells

Whether respiratory syncytial virus (RSV) induces severe infantile pulmonary disease may depend on viral strain and expression of types I and III interferons (IFNs). These IFNs impact disease severity by inducing expression of many anti-viral IFN-stimulated genes (ISGs). To investigate the impact of RSV strain on IFN and ISG expression, we stimulated human monocyte-derived DCs (MDDCs) with either RSV A2 or Line 19 and measured expression of types I and III IFNs and ISGs. At 24h, A2 elicited higher ISG expression than Line 19. Both strains induced MDDCs to express genes for IFN-β, IFN-α1, IFN-α8, and IFN-λ1-3, but only A2 induced IFN-α2, -α14 and -α21. We then show that IFN-α8 and IFN-α14 most potently induced MDDCs and bronchial epithelial cells (BECs) to express ISGs. Our findings demonstrate that RSV strain may impact patterns of types I and III IFN expression and the magnitude of the ISG response by DCs and BECs.