Quantification of the Frequency and Multiplicity of Infection of Respiratory- and Lymph Node–Resident Dendritic Cells During Influenza Virus Infection

Background: Previous studies have demonstrated that DC differentially regulate influenza A virus (IAV)–specific CD8 T cell responses in vivo during high and low dose IAV infections. Furthermore, in vitro infection of DC with IAV at low versus high multiplicities of infection (MOI) results in altered cytokine production and a reduced ability to prime naïve CD8 T cell responses. Flow cytometric detection of IAV proteins within DC, a commonly used method for detection of cellular IAV infection, does not distinguish between the direct infection of these cells or their uptake of viral proteins from dying epithelial cells. Methods/Principal Findings: We have developed a novel, sensitive, single-cell RT-PCR–based approach to assess the infection of respiratory DC (rDC) and lymph node (LN)-resident DC (LNDC) following high and low dose IAV infections. Our results show that, while a fraction of both rDC and LNDC contain viral mRNA following IAV infection, there is little correlation between the percentage of rDC containing viral mRNA and the initial IAV inoculum dose. Instead, increasing IAV inoculums correlate with augmented rDC MOI. Conclusion/Significance: Together, our results demonstrate a novel and sensitive method for the detection of direct IAV infection at the single-cell level and suggest that the previously described ability of DC to differentially regulate IAV-specific T cell responses during high and low dose IAV infections could relate to the MOI of rDC within the LN rather than th

Hemozoin-mediated inflammasome activation limits long-lived anti-malarial immunity

During acute malaria, most individuals mount robust inflammatory responses that limit parasite burden. However, long-lived sterilizing anti-malarial memory responses are not efficiently induced, even following repeated Plasmodium exposures. Using multiple Plasmodium species, genetically modified parasites, and combinations of host genetic and pharmacologic approaches, we find that the deposition of the malarial pigment hemozoin directly limits the abundance and capacity of conventional type 1 dendritic cells to prime helper T cell responses. Hemozoin-induced dendritic cell dysfunction results in aberrant Plasmodium-specific CD4 T follicular helper cell differentiation, which constrains memory B cell and long-lived plasma cell formation. Mechanistically, we identify that dendritic cell-intrinsic NLRP3 inflammasome activation reduces conventional type 1 dendritic cell abundance, phagocytosis, and T cell priming functions in vivo. These data identify biological consequences of hemozoin deposition during malaria and highlight the capacity of the malarial pigment to program immune evasion during the earliest events following an initial Plasmodium exposure.Host-parasite interactio

The beta2 integrin CD11c distinguishes a subset of cytotoxic pulmonary T cells with potent antiviral effects in vitro and in vivo

BACKGROUND: The integrin CD11c is known as a marker for dendritic cells and has recently been described on T cells following lymphotropic choriomeningitis virus infection, a systemic infection affecting a multitude of organs. Here, we characterise CD11c bearing T cells in a murine model of localised pulmonary infection with respiratory syncytial virus (RSV). METHODS: Mice were infected intranasally with RSV and expression of β2 integrins and T lymphocyte activation markers were monitored by flow cytometry. On day 8 post RSV infection CD11c(+ )CD8(+ )and CD11c(- )CD8(+ )T cells were assessed for cytokine production, cytotoxic activity and migration. Expression of CD11c mRNA in CD8(+ )T cells was assessed by quantitative PCR. RESULTS: Following RSV infection CD11c(+ )CD8(+ )T cells were detectable in the lung from day 4 onwards and accounted for 45.9 ± 4.8% of CD8(+ )T cells on day 8 post infection, while only few such cells were present in mediastinal lymph nodes, spleen and blood. While CD11c was virtually absent from CD8(+ )T cells in the absence of RSV infection, its mRNA was expressed in CD8(+ )T cells of both naïve and RSV infected mice. CD11c(+), but not CD11c(-), CD8(+ )T cells showed signs of recent activation, including up-regulation of CD11a and expression of CD11b and CD69 and were recruited preferentially to the lung. In addition, CD11c(+ )CD8(+ )T cells were the major subset responsible for IFNγ production, induction of target cell apoptosis in vitro and reduction of viral titres in vivo. CONCLUSION: CD11c is a useful marker for detection and isolation of pulmonary antiviral cytotoxic T cells following RSV infection. It identifies a subset of activated, virus-specific, cytotoxic T cells that exhibit potent antiviral effects in vivo

Evasion by Stealth: Inefficient Immune Activation Underlies Poor T Cell Response and Severe Disease in SARS-CoV-Infected Mice

Severe Acute Respiratory Syndrome caused substantial morbidity and mortality during the 2002–2003 epidemic. Many of the features of the human disease are duplicated in BALB/c mice infected with a mouse-adapted version of the virus (MA15), which develop respiratory disease with high morbidity and mortality. Here, we show that severe disease is correlated with slow kinetics of virus clearance and delayed activation and transit of respiratory dendritic cells (rDC) to the draining lymph nodes (DLN) with a consequent deficient virus-specific T cell response. All of these defects are corrected when mice are treated with liposomes containing clodronate, which deplete alveolar macrophages (AM). Inhibitory AMs are believed to prevent the development of immune responses to environmental antigens and allergic responses by interacting with lung dendritic cells and T cells. The inhibitory effects of AM can also be nullified if mice or AMs are pretreated with poly I:C, which directly activate AMs and rDCs through toll-like receptors 3 (TLR3). Further, adoptive transfer of activated but not resting bone marrow–derived dendritic cells (BMDC) protect mice from lethal MA15 infection. These results may be relevant for SARS in humans, which is also characterized by prolonged virus persistence and delayed development of a SARS-CoV-specific immune response in individuals with severe disease

Endogenous antigen processing drives the primary CD4+ T cell response to influenza.

By convention, CD4+ T lymphocytes recognize foreign and self peptides derived from internalized antigens in combination with major histocompatibility complex class II molecules. Alternative pathways of epitope production have been identified, but their contributions to host defense have not been established. We show here in a mouse infection model that the CD4+ T cell response to influenza, critical for durable protection from the virus, is driven principally by unconventional processing of antigen synthesized within the infected antigen-presenting cell, not by classical processing of endocytosed virions or material from infected cells. Investigation of the cellular components involved, including the H2-M molecular chaperone, the proteasome and γ-interferon-inducible lysosomal thiol reductase revealed considerable heterogeneity in the generation of individual epitopes, an arrangement that ensures peptide diversity and broad CD4+ T cell engagement. These results could fundamentally revise strategies for rational vaccine design and may lead to key insights into the induction of autoimmune and anti-tumor responses

A Prominent Role for DC-SIGN+ Dendritic Cells in Initiation and Dissemination of Measles Virus Infection in Non-Human Primates

Measles virus (MV) is a highly contagious virus that is transmitted by aerosols. During systemic infection, CD150+T and B lymphocytes in blood and lymphoid tissues are the main cells infected by pathogenic MV. However, it is unclear which cell types are the primary targets for MV in the lungs and how the virus reaches the lymphoid tissues. In vitro studies have shown that dendritic cell (DC) C-type lectin DC-SIGN captures MV, leading to infection of DCs as well as transmission to lymphocytes. However, evidence of DC-SIGN-mediated transmission in vivo has not been established. Here we identified DC-SIGNhiDCs as first target cells in vivo and demonstrate that macaque DC-SIGN functions as an attachment receptor for MV. Notably, DC-SIGNhicells from macaque broncho-alveolar lavage and lymph nodes transmit MV to B lymphocytes, providing in vivo support for an important role for DCs in both initiation and dissemination of MV infection

Respiratory Dendritic Cell Subsets Differ in Their Capacity to Support the Induction of Virus-Specific Cytotoxic CD8+ T Cell Responses

Dendritic cells located at the body surfaces, e.g. skin, respiratory and gastrointestinal tract, play an essential role in the induction of adaptive immune responses to pathogens and inert antigens present at these surfaces. In the respiratory tract, multiple subsets of dendritic cells (RDC) have been identified in both the normal and inflamed lungs. While the importance of RDC in antigen transport from the inflamed or infected respiratory tract to the lymph nodes draining this site is well recognized, the contribution of individual RDC subsets to this process and the precise role of migrant RDC within the lymph nodes in antigen presentation to T cells is not clear. In this report, we demonstrate that two distinct subsets of migrant RDC - exhibiting the CD103+ and CD11bhi phenotype, respectively - are the primary DC presenting antigen to naïve CD4+ and CD8+ T lymphocytes in the draining nodes in response to respiratory influenza virus infection. Furthermore, the migrant CD103+ RDC subset preferentially drives efficient proliferation and differentiation of naive CD8+ T cells responding to infection into effector cells, and only the CD103+ RDC subset can present to naïve CD8+ T cells non-infectious viral vaccine introduced into the respiratory tract. These results identify CD103+ and CD11bhi RDC as critical regulators of the adaptive immune response to respiratory tract infection and potential targets in the design of mucosal vaccines

Both Conventional and Interferon Killer Dendritic Cells Have Antigen-Presenting Capacity during Influenza Virus Infection

Natural killer cells are innate effector cells known for their potential to produce interferon-γ and kill tumour and virus-infected cells. Recently, B220+CD11cintNK1.1+ NK cells were found to also have antigen-presenting capacity like dendritic cells (DC), hence their name interferon-producing killer DC (IKDC). Shortly after discovery, it has already been questioned if IKDC really represent a separate subset of NK cells or merely represent a state of activation. Despite similarities with DCs, in vivo evidence that they behave as bona fide APCs is lacking. Here, using a model of influenza infection, we found recruitment of both conventional B220− NK cells and IKDCs to the lung. To study antigen-presenting capacity of NK cell subsets and compare it to cDCs, all cell subsets were sorted from lungs of infected mice and co-cultured ex vivo with antigen specific T cells. Both IKDCs and conventional NK cells as well as cDCs presented virus-encoded antigen to CD8 T cells, whereas only cDCs presented to CD4 T cells. The absence of CD4 responses was predominantly due to a deficiency in MHCII processing, as preprocessed peptide antigen was presented equally well by cDCs and IKDCs. In vivo, the depletion of NK1.1-positive NK cells and IKDCs reduced the expansion of viral nucleoprotein-specific CD8 T cells in the lung and spleen, but did finally not affect viral clearance from the lung. In conclusion, we found evidence for APC function of lung NK cells during influenza infection, but this is a feature not exclusive to the IKDC subset

Viral Replication Rate Regulates Clinical Outcome and CD8 T Cell Responses during Highly Pathogenic H5N1 Influenza Virus Infection in Mice

Since the first recorded infection of humans with H5N1 viruses of avian origin in 1997, sporadic human infections continue to occur with a staggering mortality rate of >60%. Although sustained human-to-human transmission has not occurred yet, there is a growing concern that these H5N1 viruses might acquire this trait and raise the specter of a pandemic. Despite progress in deciphering viral determinants of pathogenicity, we still lack crucial information on virus/immune system interactions pertaining to severe disease and high mortality associated with human H5N1 influenza virus infections. Using two human isolates of H5N1 viruses that differ in their pathogenicity in mice, we have defined mechanistic links among the rate of viral replication, mortality, CD8 T cell responses, and immunopathology. The extreme pathogenicity of H5N1 viruses was directly linked to the ability of the virus to replicate rapidly, and swiftly attain high steady-state titers in the lungs within 48 hours after infection. The remarkably high replication rate of the highly pathogenic H5N1 virus did not prevent the induction of IFN-β or activation of CD8 T cells, but the CD8 T cell response was ineffective in controlling viral replication in the lungs and CD8 T cell deficiency did not affect viral titers or mortality. Additionally, BIM deficiency ameliorated lung pathology and inhibited T cell apoptosis without affecting survival of mice. Therefore, rapidly replicating, highly lethal H5N1 viruses could simply outpace and overwhelm the adaptive immune responses, and kill the host by direct cytopathic effects. However, therapeutic suppression of early viral replication and the associated enhancement of CD8 T cell responses improved the survival of mice following a lethal H5N1 infection. These findings suggest that suppression of early H5N1 virus replication is key to the programming of an effective host response, which has implications in treatment of this infection in humans

Interrelationship between Dendritic Cell Trafficking and Francisella tularensis Dissemination following Airway Infection

Francisella tularensis, the etiological agent of the inhalation tularemia, multiplies in a variety of cultured mammalian cells. Nevertheless, evidence for its in vivo intracellular residence is less conclusive. Dendritic cells (DC) that are adapted for engulfing bacteria and migration towards lymphatic organs could serve as potential targets for bacterial residence and trafficking. Here, we focus on the in vivo interactions of F. tularensis with DC following airway infection of mice. Lethal airway infection of mice with the live vaccine strain (LVS) results in trafficking of a CD11bhigh/CD11cmed/autofluorescencelow DC subset from the respiratory tract to the draining mediastinal lymph node (MdLN). Simultaneously, a rapid, massive bacterial colonization of the MdLN occurs, characterized by large bacterial foci formation. Analysis of bacteria in the MdLN revealed a major population of extracellular bacteria, which co-exists with a substantial fraction of intracellular bacteria. The intracellular bacteria are viable and reside in cells sorted for DC marker expression. Moreover, in vivo vital staining experiments indicate that most of these intracellular bacteria (∼75%) reside in cells that have migrated from the airways to the MdLN after infection. The correlation between DC and bacteria accumulation in the MdLN was further demonstrated by manipulating DC migration to the MdLN through two independent pathways. Impairment of DC migration to the MdLN, either by a sphingosine-1-phosphate receptor agonist (FTY720) or by the D prostanoid receptor 1 agonist (BW245C), resulted in reduced bacterial colonization of MdLN. Moreover, BW245C treatment delayed the onset of morbidity and the time to death of the infected mice. Taken together, these results suggest that DC can serve as an inhabitation niche for F. tularensis in the early stages of infection, and that DC trafficking plays a role in pathogen dissemination. This underscores the therapeutic potential of DC migration impairing drugs in tularemia treatment