Paul G. Whitney

Cross-presentation of viral and self antigens by skin-derived CD103 + dendritic cells

Skin-derived dendritic cells (DCs) include Langerhans cells, classical dermal DCs and a langerin-positive CD103+ dermal subset. We examined their involvement in the presentation of skin-associated viral and self antigens. Only the CD103+ subset efficiently presented antigens of herpes simplex virus type 1 to naive CD8+ T cells, although all subsets presented these antigens to CD4+ T cells. This showed that CD103+ DCs were the migratory subset most efficient at processing viral antigens into the major histocompatibility complex class I pathway, potentially through cross-presentation. This was supported by data showing only CD103+ DCs efficiently cross-presented skin-derived self antigens. This indicates CD103+ DCs are the main migratory subtype able to cross-present viral and self antigens, which identifies another level of specialization for skin DCs.

Different patterns of peripheral migration by memory CD4+ and CD8+ T cells

Infections localized to peripheral tissues such as the skin result in the priming of T-cell responses that act to control pathogens. Activated T cells undergo migrational imprinting within the draining lymph nodes, resulting in memory T cells that provide local and systemic protection. Combinations of migrating and resident memory T cells have been implicated in long-term peripheral immunity, especially at the surfaces that form pathogen entry points into the body. However, T-cell immunity consists of separate CD4+ helper T cells and CD8+ killer T cells, with distinct effector and memory programming requirements. Whether these subsets also differ in their ability to form a migrating pool involved in peripheral immunosurveillance or a separate resident population responsible for local infection control has not been explored. Here, using mice, we show key differences in the migration and tissue localization of memory CD4+ and CD8+ T cells following infection of the skin by herpes simplex virus. On resolution of infection, the skin contained two distinct virus-specific memory subsets; a slow-moving population of sequestered CD8+ T cells that were resident in the epidermis and confined largely to the original site of infection, and a dynamic population of CD4+ T cells that trafficked rapidly through the dermis as part of a wider recirculation pattern. Unique homing-molecule expression by recirculating CD4+ T effector-memory cells mirrored their preferential skin-migratory capacity. Overall, these results identify a complexity in memory T-cell migration, illuminating previously unappreciated differences between the CD4+ and CD8+ subsets.

A Protective Vaccine Delivery System for In Vivo T Cell Stimulation Using Nanoengineered Polymer Hydrogel Capsules

Successful delivery of labile vaccine antigens, such as peptides and proteins, to stimulate CD4 and CD8 T cell immunity could improve vaccine strategies against chronic infections such as HIV and Hepatitis C. Layer-by-layer (LbL)-assembled nanoengineered hydrogel capsules represent a novel and promising technology for the protection and delivery of labile vaccine candidates to antigen-presenting cells (APCs). Here we report on the in vitro and in vivo immunostimulatory capabilities of LbL-assembled disulfide cross-linked poly(methacrylic acid) (PMA(SH)) hydrogel capsules as a delivery strategy for protein and peptide vaccines using robust transgenic mice models and ovalbumin (OVA) as a model vaccine. We demonstrate that OVA protein as well as multiple OVA peptides can be successfully encapsulated within nanoengineered PMA(SH) hydrogel capsules. OVA-containing PMA(SH) capsules are internalized by mouse APCs, resulting in presentation of OVA epitopes and subsequent activation of OVA-specific CD4 and CD8 T cells in vitro. OVA-specific CD4 and CD8 T cells are also activated to proliferate in vivo following intravenous vaccination of mice with OVA protein- and OVA peptide-loaded PMA(SH) hydrogel capsules. Furthermore, we show that OVA encapsulated within the PMA(SH) capsules resulted in at least 6-fold greater proliferation of OVA-specific CD8 T cells and 70-fold greater proliferation of OVA-specific CD4 T cells in vivo compared to the equivalent amount of OVA protein administered alone. These results highlight the potential of nanoengineered hydrogel capsules for vaccine delivery.

NLRC4 inflammasomes in dendritic cells regulate noncognate effector function by memory CD8 + T cells

Memory T cells exert antigen-independent effector functions, but how these responses are regulated is unclear. We discovered an in vivo link between flagellin-induced NLRC4 inflammasome activation in splenic dendritic cells (DCs) and host protective interferon-γ (IFN-γ) secretion by noncognate memory CD8+ T cells, which could be activated by Salmonella enterica serovar Typhimurium, Yersinia pseudotuberculosis and Pseudomonas aeruginosa. We show that CD8α+ DCs were particularly efficient at sensing bacterial flagellin through NLRC4 inflammasomes. Although this activation released interleukin 18 (IL-18) and IL-1β, only IL-18 was required for IFN-γ production by memory CD8+ T cells. Conversely, only the release of IL-1β, but not IL-18, depended on priming signals mediated by Toll-like receptors. These findings provide a comprehensive mechanistic framework for the regulation of noncognate memory T cell responses during bacterial immunity.

NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase‐4 and caspase‐5

Humans encode two inflammatory caspases that detect cytoplasmic LPS, caspase‐4 and caspase‐5. When activated, these trigger pyroptotic cell death and caspase‐1‐dependent IL‐1β production; however the mechanism underlying this process is not yet confirmed. We now show that a specific NLRP3 inhibitor, MCC950, prevents caspase‐4/5‐dependent IL‐1β production elicited by transfected LPS. Given that both caspase‐4 and caspase‐5 can detect cytoplasmic LPS, it is possible that these proteins exhibit some degree of redundancy. Therefore, we generated human monocytic cell lines in which caspase‐4 and caspase‐5 were genetically deleted either individually or together. We found that the deletion of caspase‐4 suppressed cell death and IL‐1β production following transfection of LPS into the cytoplasm, or in response to infection with Salmonella typhimurium. Although deletion of caspase‐5 did not confer protection against transfected LPS, cell death and IL‐1β production were reduced after infection with Salmonella. Furthermore, double deletion of caspase‐4 and caspase‐5 had a synergistic effect in the context of Salmonella infection. Our results identify the NLRP3 inflammasome as the specific platform for IL‐1β maturation, downstream of cytoplasmic LPS detection by caspase‐4/5. We also show that both caspase‐4 and caspase‐5 are functionally important for appropriate responses to intracellular Gram‐negative bacteria.

Loss of a single N-linked glycan from the hemagglutinin of influenza virus is associated with resistance to collectins and increased virulence in mice

BackgroundGlycosylation on the globular head of the hemagglutinin (HA) protein of influenza virus acts as an important target for recognition and destruction of virus by innate immune proteins of the collectin family. This, in turn, modulates the virulence of different viruses for mice. The role of particular oligosaccharide attachments on the HA in determining sensitivity to collectins has yet to be fully elucidated.MethodsWhen comparing the virulence of H3N2 subtype viruses for mice we found that viruses isolated after 1980 were highly glycosylated and induced mild disease in mice. During these studies, we were surprised to find a small plaque variant of strain A/Beijing/353/89 (Beij/89) emerged following infection of mice and grew to high titres in mouse lung. In the current study we have characterized the properties of this small plaque mutant both in vitro and in vivo.ResultsSmall plaque mutants were recovered following plaquing of lung homogenates from mice infected with influenza virus seed Beij/89. Compared to wild-type virus, small plaque mutants showed increased virulence in mice yet did not differ in their ability to infect or replicate in airway epithelial cells in vitro. Instead, small plaque variants were markedly resistant to neutralization by murine collectins, a property that correlated with the acquisition of an amino acid substitution at residue 246 on the viral HA. We present evidence that this substitution was associated with the loss of an oligosaccharide glycan from the globular head of HA.ConclusionA point mutation in the gene encoding the HA of Beij/89 was shown to ablate a glycan attachment site. This was associated with resistance to collectins and increased virulence in mice.

NK cells contribute to the early clearance of HSV-1 from the lung but cannot control replication in the central nervous system following intranasal infection

While infection of the respiratory tract with herpes simplex virus type 1 (HSV‐1) can have severe clinical complications, little is known of the immune mechanisms that control both the replication and spread of HSV‐1 in this site. To better understand the contribution of innate immunity and in particular natural killer (NK) cells to the control of infection at this site, we have utilized a mouse model of intranasal HSV‐1 infection. NK cell numbers increased in the lung following intranasal infection and they produced IFN‐γ and acquired an enhanced cytotoxic capacity. While depletion of NK cells resulted in increased HSV‐1 titres in the lung, the time taken to clear the virus was unaffected. Interestingly, HSV‐1 was also effectively cleared from the lungs of RAG‐1–/– mice that lack both B and T cells. However, RAG‐1–/– mice could not control the spread of virus to the central nervous system and its subsequent replication in the brain. Together, these data demonstrate that NK cells are recruited, activated and contribute to early protection of the lung during acute HSV‐1 infection of the respiratory tract, but in the absence of adaptive immunity are unable to control the replication and spread of virus in the nervous system.

A role for plasmacytoid dendritic cells in the rapid IL-18-dependent activation of NK cells following HSV-1 infection.

Natural killer (NK) cells play a crucial role in the initial response to viral infections but the mechanisms controlling their activation are unclear. We show a rapid and transient activation of NK cells that results in the production of IFN‐γ immediately following infection with herpes simplex virus type 1 (HSV‐1). Activation of NK cells leading to synthesis of IFN‐γ was not mediated by a direct interaction with virus but required the presence of additional cell types and was largely dependent on the cytokine IL‐18, but not IL‐12. HSV‐1‐induced IFN‐γ expression by NK cells in vitro was impaired in spleen cultures depleted of CD11c+ cells. Conversely, coculture of NK cells with virus‐exposed conventional DC or plasmacytoid (p)DC restored the production of IFN‐γ, indicating that multiple DC subsets could mediate NK cell activation. While conventional DC populations stimulated NK cells independently of IL‐18, they were less effective than pDC in promoting NK cell IFN‐γ expression. In contrast, the potent stimulation of NK cells by pDC was dependent on IL‐18 as pDC from IL‐18‐deficient mice only activated a similar proportion of NK cells as conventional DC. These data identify IL‐18 as a crucial factor for pDC‐mediated NK cell regulation.

IL-18, but not IL-12, Regulates NK Cell Activity following Intranasal Herpes Simplex Virus Type 1 Infection

Infection of the respiratory tract with HSV type 1 (HSV-1) can have severe clinical complications, yet little is known of the immune mechanisms that control the replication and spread of HSV-1 in this site. The present study investigated the protective role of IL-12 and IL-18 in host defense against intranasal HSV-1 infection. Both IL-12 and IL-18 were detected in lung fluids following intranasal infection of C57BL/6 (B6) mice. IL-18-deficient (B6.IL-18−/−) mice were more susceptible to HSV-1 infection than wild-type B6 mice as evidenced by exacerbated weight loss and enhanced virus growth in the lung. IL-12-deficient (B6.IL-12−/−) mice behaved similarly to B6 controls. Enhanced susceptibility of B6.IL-18−/− mice to HSV-1 infection correlated with a profound impairment in the ability of NK cells recovered from the lungs to produce IFN-γ or to mediate cytotoxic activity ex vivo. The weak cytotoxic capacity of NK cells from the lungs of B6.IL-18−/− mice correlated with reduced expression of the cytolytic effector molecule granzyme B. Moreover, depletion of NK cells from B6 or B6.IL-12−/− mice led to enhanced viral growth in lungs by day 3 postinfection; however, this treatment had no effect on viral titers in lungs of B6.IL-18−/− mice. Together these studies demonstrate that IL-18, but not IL-12, plays a key role in the rapid activation of NK cells and therefore in control of early HSV-1 replication in the lung.

Influenza viruses differ in ability to infect macrophages and to induce a local inflammatory response following intraperitoneal injection of mice

Strains of influenza A virus show marked differences in their ability to infect murine macrophages (MΦ) such that strain A/PR/8/34 (PR8; H1N1) infects MΦ poorly while strain BJx109 (H3N2) infects MΦ to high levels. Given the central role of MΦ in initiating and regulating inflammatory responses, we hypothesized that virus strains that infect MΦ poorly may also be poor at initiating inflammatory responses. Studies to compare the inflammatory response of mice after intranasal inoculation with either BJx109 or PR8 were confounded by the rapid growth of the PR8 virus in lung tissues. Consequently, we have characterized the cellular inflammatory response following inoculation into the peritoneal cavity, as influenza viruses do not replicate at this site. Herein, we report marked differences in the local inflammatory response to BJx109 or PR8 in the peritoneal cavity with strain PR8 being particularly poor in its ability to recruit and activate peritoneal leukocytes, including NK cells and MΦ. In vitro BJx109, but not PR8, stimulated release of high levels of type I IFNs and TNF‐α from PEC MΦ, and treatment of mice with neutralizing antibodies to either cytokine inhibited the ability of BJx109 to recruit and activate NK cells and MΦs in the peritoneal cavity. Together, these data suggest that the ability of influenza virus strains to infect MΦ and stimulate cytokine release is an important factor governing the nature of the acute inflammatory response.