Cecilia Johansson

The response of natural killer T cells to glycolipid antigens is characterized by surface receptor down-modulation and expansion

CD1d-restricted natural killer T (NKT) cells are a subset of regulatory T cells that react with glycolipid antigens. Although preclinical studies have effectively targeted NKT cells for immunotherapy, little is known regarding the early in vivo response of these cells to antigenic stimulation. We have analyzed the early response of NKT cells to glycolipid antigens and bacterial infection by using specific reagents for tracking these cells. Our results demonstrate dramatic in vivo expansion and surface phenotype alterations after NKT cell activation with α-galactosylceramide. In addition, we show significant NK1.1 down-modulation on NKT cells in the setting of oral Salmonella infection. Our results indicate that in vivo activation of NKT cells leads to a dynamic response characterized by surface receptor down-modulation and expansion. These findings alter current understanding of NKT cell biology and should aid in the rational design of NKT cell-based immunotherapies.

CD25+ Natural Regulatory T Cells Are Critical in Limiting Innate and Adaptive Immunity and Resolving Disease following Respiratory Syncytial Virus Infection

ABSTRACT Regulatory CD4+ T cells have been shown to be important in limiting immune responses, but their role in respiratory viral infections has received little attention. Here we observed that following respiratory syncytial virus (RSV) infection, CD4+ Foxp3+ CD25+ natural regulatory T-cell numbers increased in the bronchoalveolar lavage fluid, lung, mediastinal lymph nodes, and spleen. The depletion of CD25+ natural regulatory T cells prior to RSV infection led to enhanced weight loss with delayed recovery that was surprisingly accompanied by increased numbers of activated natural killer cells in the lung and bronchoalveolar lavage fluid on day 8 postinfection. Increased numbers of neutrophils were also detected within the bronchoalveolar lavage fluid and correlated with elevated levels of myeloperoxidase as well as interleukin-6 (IL-6) and gamma interferon (IFN-γ). CD25+ natural regulatory T-cell depletion also led to enhanced numbers of proinflammatory T cells producing IFN-γ and tumor necrosis factor alpha (TNF-α) in the lung. Despite these increases in inflammatory responses and disease severity, the viral load was unaltered. This work highlights a critical role for natural regulatory T cells in regulating the adaptive and innate immune responses during the later stages of lung viral infections.

Regulatory T cells expressing granzyme B play a critical role in controlling lung inflammation during acute viral infection

The inflammatory response to lung infections must be tightly regulated, enabling pathogen elimination while maintaining crucial gas exchange. Using recently described “depletion of regulatory T cell” (DEREG) mice, we found that selective depletion of regulatory T cells (Tregs) during acute respiratory syncytial virus (RSV) infection enhanced viral clearance but increased weight loss, local cytokine and chemokine release, and T-cell activation and cellular influx into the lungs. Conversely, inflammation was decreased when Treg numbers and activity were boosted using interleukin-2 immune complexes. Unexpectedly, lung (but not draining lymph node) Tregs from RSV-infected mice expressed granzyme B (GzmB), and bone marrow chimeric mice with selective loss of GzmB in the Treg compartment displayed markedly enhanced cellular infiltration into the lung after infection. A crucial role for GzmB-expressing Tregs has not hitherto been described in the lung or during acute infections, but may explain the inability of children with perforin/GzmB defects to regulate immune responses to infection. The effects of RSV infection in mice with defective immune regulation closely parallel the observed effects of RSV in children with bronchiolitis, suggesting that the pathogenesis of bronchiolitis may involve an inability to regulate virus-induced inflammation.

IL-10 Regulates Viral Lung Immunopathology during Acute Respiratory Syncytial Virus Infection in Mice

Interleukin (IL-) 10 is a pleiotropic cytokine with broad immunosuppressive functions, particularly at mucosal sites such as the intestine and lung. Here we demonstrate that infection of BALB/c mice with respiratory syncytial virus (RSV) induced IL-10 production by CD4+ and CD8+ T cells in the airways at later time points (e.g. day 8); a proportion of these cells also co-produced IFN-γ. Furthermore, RSV infection of IL-10−/− mice resulted in more severe disease with enhanced weight loss, delayed recovery and greater cell infiltration of the respiratory tract without affecting viral load. In addition, IL-10−/− mice had a pronounced airway neutrophilia and heightened levels of pro-inflammatory cytokines and chemokines in the bronchoalveolar lavage fluid. Notably, the proportion of lung T cells producing IFN-γ was enhanced, suggesting that IL-10 may act in an autocrine manner to dampen effector T cell responses. Similar findings were made in mice treated with anti-IL-10R antibody and infected with RSV. Therefore, IL-10 inhibits disease and inflammation in mice infected with RSV, especially during recovery from infection.

Regulation of human papillomavirus gene expression by splicing and polyadenylation

Human papillomaviruses (HPVs) are small DNA tumour viruses that are present in more than 99% of all cervical cancers. The ability of these viruses to cause disease is partly attributed to the strict coordination of viral gene expression with the differentiation stage of the infected cell. HPV gene expression is regulated temporally at the level of RNA splicing and polyadenylation, and a dysregulated gene expression programme allows some HPV types to establish long-term persistence, which is a risk factor for cancer. In this Review, we summarize the role of splicing and polyadenylation in the regulation of HPV gene expression and discuss the viral and cellular factors that control these processes.

The role of CD1d-restricted NK T lymphocytes in the immune response to oral infection with Salmonella typhimurium

CD1d‐restricted natural killer T (NKT) cells belong to the innate‐like lymphocytes which respond rapidly to stress and infectious challenge. We have studied murine CD1d‐restricted NKT cells in the early immune response to virulent Salmonella enterica serovar Typhimurium after oral infection. In the liver and spleen, neutrophil and macrophage numbers had increased several‐fold by day 5 post‐infection, while the frequency of B and T lymphocytes decreased. These cellular changes occurred independently of CD1d‐restricted NKT cells, and further, CD1d‐restricted T cells did not influence the bacterial load. However, in CD1d+ mice NK1.1+ T cells and invariant CD1d‐restricted T cells were activated by the infection, as demonstrated by an increase in size, up‐regulation of CD69 and production of IFN‐γ. The NK1.1 antigen was down‐modulated on these cells during the course of infection, while TCR levels were unaffected. While dendritic cells (DC) up‐regulated CD1d‐levels upon 24 h of in vitro exposure to the bacteria, increased CD1d expression was not evident on DC in vivo during infection. Furthermore, in vitro re‐stimulation of CD1d‐restricted T cells isolated from infected mice demonstrated a significant skewing of the cytokine profile, with suppressed IL‐4 and increased IFN‐γ production.

Salmonella enterica Serovar Typhimurium-Induced Maturation of Bone Marrow-Derived Dendritic Cells

ABSTRACT Murine bone marrow-derived dendritic cells (DC) can phagocytose and process Salmonella enterica serovar Typhimurium for peptide presentation on major histocompatibility complex class I (MHC-I) and MHC-II molecules. To investigate if a serovar Typhimurium encounter with DC induces maturation and downregulates their ability to present antigens from subsequently encountered bacteria, DC were pulsed with serovar Typhimurium 24 h prior to coincubating withEscherichia coli expressing the model antigen Crl-OVA. Quantitating presentation of OVA epitopes contained within Crl-OVA showed that Salmonella-pulsed DC had a reduced capacity to process Crl-OVA-expressing E. colifor OVA(257-264)/Kb and OVA(265-277)/I-Abpresentation. In addition, time course studies of DC pulsed with Crl-OVA-expressing serovar Typhimurium showed that OVA(257-264)/Kb complexes could stimulate CD8OVA T-hybridoma cells for <24 h following a bacterial pulse, while OVA(265-277)/I-Ab complexes could stimulate OT4H T-hybridoma cells for >24 but <48 h. The phoP-phoQvirulence locus of serovar Typhimurium also influenced the ability of DC to process Crl-OVA-expressing serovar Typhimurium for OVA(265-277)/I-Ab presentation but not for OVA(257-264)/Kb presentation. Furthermore, pulsing of DC with serovar Typhimurium followed by incubation for 24 or 48 h altered surface expression of MHC-I, MHC-II, CD40, CD54, CD80, and CD86, generating a DC population with a uniform, high expression level of these molecules. Finally, neither the serovar TyphimuriumphoP-phoQ locus nor lipopolysaccharides (LPS) containing lipid A modifications purified from phoP mutant strains had a different effect on DC maturation from that of wild-type serovar Typhimurium or purified wild-type LPS. Thus, these data show thatSalmonella or Salmonella LPS induces maturation of DC and that this process is not altered by the Salmonella phoP virulence locus. However, phoP did influence OVA(265-277)/I-Ab presentation by DC infected with Crl-OVA-expressing serovar Typhimurium when quantitated after 2 h of bacterial infection.

Alveolar macrophage–derived type I interferons orchestrate innate immunity to RSV through recruitment of antiviral monocytes

Goritzka et al. describe a role for recruited inflammatory monocytes in antiviral immunity and protection from RSV infection in mice. The authors demonstrate that this is critically dependent on the production of type I IFNs by alveolar macrophages triggered via RIG-I–like receptors, thus highlighting an important cell-extrinsic mechanism of type I IFN–mediated antiviral activity.

Type I interferons produced by hematopoietic cells protect mice against lethal infection by mammalian reovirus

We defined the function of type I interferons (IFNs) in defense against reovirus strain type 1 Lang (T1L), which is a double-stranded RNA virus that infects Peyers patches (PPs) after peroral inoculation of mice. T1L induced expression of mRNA for IFN-α, IFN-β, and Mx-1 in PPs and caused localized intestinal infection that was cleared in 10 d. In contrast, T1L produced fatal systemic infection in IFNαR1 knockout (KO) mice with extensive cell loss in lymphoid tissues and necrosis of the intestinal mucosa. Studies of bone-marrow chimeric mice indicated an essential role for hematopoietic cells in IFN-dependent viral clearance. Dendritic cells (DCs), including conventional DCs (cDCs), were the major source of type I IFNs in PPs of reovirus-infected mice, whereas all cell types expressed the antiviral protein Mx-1. Neither NK cells nor signaling via Toll-like receptor 3 or MyD88 were essential for viral clearance. These data demonstrate a requirement for type I IFNs in the control of an intestinal viral infection and indicate that cDCs are a significant source of type I IFN production in vivo. Therefore, innate immunity in PPs is an essential component of host defense that limits systemic spread of pathogens that infect the intestinal mucosa.

HPV-16 E2 contributes to induction of HPV-16 late gene expression by inhibiting early polyadenylation

We provide evidence that the human papillomavirus (HPV) E2 protein regulates HPV late gene expression. High levels of E2 caused a read‐through at the early polyadenylation signal pAE into the late region of the HPV genome, thereby inducing expression of L1 and L2 mRNAs. This is a conserved property of E2 of both mucosal and cutaneous HPV types. Induction could be reversed by high levels of HPV‐16 E1 protein, or by the polyadenylation factor CPSF30. HPV‐16 E2 inhibited polyadenylation in vitro by preventing the assembly of the CPSF complex. Both the N‐terminal and hinge domains of E2 were required for induction of HPV late gene expression in transfected cells as well as for inhibition of polyadenylation in vitro. Finally, overexpression of HPV‐16 E2 induced late gene expression from a full‐length genomic clone of HPV‐16. We speculate that the accumulation of high levels of E2 during the viral life cycle, not only turns off the expression of the pro‐mitotic viral E6 and E7 genes, but also induces the expression of the late HPV genes L1 and L2.