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.

Cutting edge: priming of CD8 T cell immunity to herpes simplex virus type 1 requires cognate TLR3 expression in vivo.

Despite its potential for involvement in viral immunity, little evidence links TLR3 to adaptive antiviral responses. Here we show that TLR3 is required for the generation of CD8 T cell immunity to HSV-1. The magnitude of the gB-specific CD8 T cell response after flank infection by HSV-1 was significantly reduced in mice lacking TIR domain-containing adaptor-inducing IFN-beta or TLR3, but not MyD88. Impaired CTL induction was evident in chimeric mice lacking TLR3 in bone marrow (BM)-derived cells. Among the dendritic cell subsets, TLR3 was expressed by CD8alpha(+) dendritic cells, known to be involved in priming HSV-1-specific CD8 T cells. Use of mixed BM chimeras revealed that TLR3 and the MHC class I-restriction element must be expressed by the same BM-derived cell for effective priming. These data imply that a cognate linkage between TLR3 and MHC class I is required for efficient CTL priming to HSV-1.

Depletion of Gr-1+, but not Ly6G+, immune cells exacerbates virus replication and disease in an intranasal model of herpes simplex virus type 1 infection.

In the absence of a viable 'knockout' mouse, researchers have relied extensively on monoclonal antibody (mAb) RB6-8C5 [anti-granulocyte receptor 1 (Gr-1)] to deplete neutrophils in murine models of inflammation and infection. Using an intranasal model of herpes simplex virus type 1 (HSV-1) infection, we demonstrate that mAb RB6-8C5 also binds to plasmacytoid dendritic cells, F4/80(+) macrophages/monocytes and CD8(+) T cells recovered from the airways of HSV-1-infected mice. In contrast, mAb 1A8 (anti-Ly6G) bound specifically to Ly6G(high) neutrophils. Following intranasal infection of C57BL/6 mice with HSV-1, few Ly6G(high) neutrophils were recruited to the airways and treatment of mice with purified mAb 1A8 induced systemic neutropenia, but did not alter virus replication or disease progression. In contrast, treatment of HSV-1-infected mice with mAb RB6-8C5 led to exacerbated virus replication, disease severity and mortality. These findings highlight the limitations associated with widespread use of antibody-mediated depletion of Gr-1(+) cells to define the role of neutrophils in vivo. Furthermore, we use mAb 1A8 to demonstrate that specific depletion of neutrophils does not modulate disease or alter virus replication following intranasal infection with HSV-1.

Gr-1+ cells, but not neutrophils, limit virus replication and lesion development following flank infection of mice with herpes simplex virus type-1.

Neutrophils are prominent in epidermal and dermal layers of human herpetic lesions and are rapidly recruited into the skin follow epidermal abrasion and infection of mice with herpes simplex virus type-1 (HSV-1). Herein, we demonstrate that early production of neutrophil-attracting chemokines KC/MIP-2 is associated with transient recruitment of neutrophils into the skin of HSV-1-infected mice in temporal association with the development of herpetic lesions. Treatment of HSV-1-infected mice with a Ly6G-specific mAb induced systemic neutropenia, but surprisingly did not alter virus replication or lesion development. In contrast, depletion of Gr-1(+) cells with mAb RB6-8C5 led to enhanced virus growth and lesion severity. Thus, while neutrophils are prominent in zosteriform lesions of HSV-1-infected mice, they do not appear to play a major role in controlling virus replication or lesion development and/or healing. In contrast, Gr-1(+) cells limit both virus replication and lesion development in the zosteriform model.

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-gamma 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.

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-gamma immediately following infection with herpes simplex virus type 1 (HSV-1). Activation of NK cells leading to synthesis of IFN-gamma 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-gamma 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-gamma, 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-gamma 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.

NK cells promote peritoneal xenograft rejection through an IFN-gamma-dependent mechanism.

BACKGROUND: Natural killer (NK) cells have emerged as major players in anti-viral and anti-tumour immune responses. Like cytotoxic T lymphocytes (CTL), they express perforin and are potent secretors of gamma-interferon (IFN-gamma). However, there is conflicting evidence about their role in mediating rejection of xenogeneic tissue. METHODS: A pig-to-mouse peritoneal cell model of xenotransplantation was used to investigate the effect of NK deficiency on xenograft recovery and the possible mechanisms behind this NK-mediated graft rejection. gamma c(-/-)RAG(-/-) mice were used as a model of NK deficiency. Additionally, NK cells were depleted in RAG(-/-) mice using anti-asialo GM1. The contributions of IFN-gamma, perforin and NKT cells were studied using knock-out mice that were depleted in vivo of T cells. Mice were injected with 10(7) pig cells intraperitoneally and peritoneal fluid was assessed 5 days later for xenograft recovery and phenotypic analysis. The requirement for NK cells for xenograft rejection was also assessed using luciferase-transfected porcine cells in a renal subcapsular model of transplantation. RESULTS: Pig cell recovery was enhanced in both gamma c(-/-)RAG(-/-) and NK-depleted RAG(-/-) mice when compared with RAG(-/-) control mice. IFN-gamma(-/-) mice depleted of T cells also demonstrated superior graft survival compared with their B6 counterparts. However, there were minimal graft survival differences between Pfp(-/-) and B6 control mice. Similarly, a deficiency in NKT cells did not improve pig xenograft recovery from the peritoneum of these mice. CONCLUSIONS: Therefore, we conclude that NK cells, but not NKT cells, are important mediators of xenograft rejection in the peritoneal cavity, and that their role may be unmasked in the absence of T cells. The mechanism for this xenorejection appears to involve IFN-gamma but is perforin independent.

Persistent expression of CD94/NKG2 receptors by virus-specific CD8 T cells is initiated by TCR-mediated signals.

Subsets of CD8 T cells express receptors that are critical in regulating the activity of NK cells. To characterize the expression of these receptors on CD8 T cells we made use of transgenic mice that express a H-2Kb restricted TCR specific for the immunodominant epitope located within the HSV-1 glycoprotein B (gB). Few naive gB-specific T cells express Ly49 or CD94/NKG2 receptors. Following acute infection of C57BL/6 mice with either HSV-1 or a recombinant influenza virus that encodes the gB determinant, gB-specific T cells showed a dramatic upregulation of CD94/NKG2 receptors. Moreover, gB-specific CD8 T cells that expressed CD94/NKG2 receptors were also found to express another NK receptor, KLRG1. We established that while Ag-stimulated gB-specific CD8 T cells primarily express inhibitory isoforms of CD94/NKG2 receptors, these cells remain capable of producing gammaIFN upon peptide stimulation. While peak CD94/NKG2 expression on gB-specific cells was reached 2-3 days following infection, it remained elevated beyond 60 days post-infection with either HSV-1 or a gB-expressing recombinant influenza virus. The data imply that the prolonged expression was not due to persistence of replicating virus and suggest that while recognition of the cognate Ag is necessary to trigger expression of CD94/NKG2 receptors, it is not required for their continued expression on memory T cells.