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Keywords:

  • HSV-1;
  • Innate;
  • NK cell and lung

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

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.

Abbreviations:
AAGM:

anti-asialo-GM1 rabbit antisera

B6:

C57BL/6

BALF:

bronchoalveolar lavage fluid

gB:

glycoprotein B

TG:

trigeminal ganglion

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Herpes simplex virus type 1 (HSV-1) is a ubiquitous human herpesvirus that infects epidermal or epithelial cells of mucosal surfaces prior to the establishment of latent infection in sensory neurons. Protective immunity to HSV-1 requires a coordinated response by both the innate and adaptive immune systems. The initial stages of HSV-1 infection are influenced by innate immune mechanisms, such as the activity of type I interferons (IFN), macrophages and natural killer (NK) cells, which serve to limit early virus replication and spread. As the adaptive immune response develops, antigen-specific T cells and B cells play critical roles in the resolution of infection and there is evidence to suggest that these cells, in particular T cells, play a critical role in maintaining latency (reviewed in 1).

In addition to the skin, HSV-1 can infect a variety of mucosal tissues, including the respiratory tract. Herpetic respiratory infections have been reported in patients with underlying immunosuppression and in neonates 2, 3, and can lead to a range of pathologic conditions including tracheobronchitits and pneumonia 47. Although generally a self-limiting infection, viral dissemination can lead to further complications such as sporadic fatal meningoencephalitits 8. Despite its clinical significance, the pathogenesis of HSV-1 infection of the respiratory tract is not well understood.

We have used the intranasal inoculation route to examine the pathogenesis of HSV-1 infection in the respiratory tract of mice. To this end, Adler et al.9 reported the induction of HSV-1 pneumonia to be due to aspects of the inflammatory response, in particular nitric oxide, rather than the direct cytopathic effects of the virus itself. Another study has demonstrated that mice deficient in both NK and T cells succumb to intranasal infection with HSV-1, whereas mice lacking only T cells survive 10. However, studies defining the nature and magnitude of the host response to HSV-1 infection of the lung are lacking. We have performed a thorough characterization of the cellular inflammatory response induced in the respiratory tract following intranasal HSV-1 infection. NK cells, and subsequently T cells, are recruited to the airways and NK cells restrict early virus replication in the lung. However, adaptive immunity was critical for controlling spread of HSV-1 in the CNS and its replication in the brain.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Intranasal infection of mice with HSV-1 strain KOS

C57BL/6 (B6) mice were infected via the intranasal route with 106 PFU of HSV-1 and were weighed daily and monitored for signs of disease over a 14-day period. Fig. 1A shows the modest weight loss observed during the first week of infection; however, mice recovered rapidly and at no time displayed evidence of pneumonia or neurological abnormalities. At various times after infection, lung homogenates were prepared and assayed for infectious virus. As shown in Fig. 1B, little virus was detected in lungs at 4 h post-infection, but 100- to 1000-fold more virus was observed at day 1, indicating replication of HSV-1 in the lung rather than persistence of viral inoculum. Maximum virus titres were observed at day 1 and declined thereafter, with clearance achieved 5–7 days post-infection, the rapid decline being consistent with control by innate mechanisms. At no time point tested (days 1–14) was infectious virus detected in homogenates prepared from brains, livers or kidneys of HSV-1-infected animals.

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Figure 1. Virulence of HSV-1 strain KOS following intranasal infection of mice. Groups of five to ten male B6 mice were mock-infected with VERO lysate (○) or infected with 106 PFU of HSV-1 (▪) and monitored for 14 days. (A) Mice were weighed daily and results expressed as the mean percentage weight loss of each group ± SD, compared with the weight immediately prior to infection. (B) Growth and replication of HSV-1 in the lungs of mice. At indicated times after infection lung homogenates were prepared and assayed for infectious HSV-1 by plaque assay on VERO cells. The dashed line indicates the minimum detection limit of the plaque assay.

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Cell influx in BAL fluid after intranasal infection with HSV-1

To assess the cellular inflammatory response to HSV-1 infection, the number of viable cells recruited into the airways of the lung was determined. BAL fluid (BALF) cell numbers increased over the course of HSV-1 infection, reaching a peak at day 7 and subsiding by day 14 post-infection (Fig. 2A). BALF cells were predominantly leukocytes, with 85–95% expressing the common leukocyte antigen CD45 (data not shown).

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Figure 2. Characterization of cells recovered from the BALF of HSV-1-infected mice. B6 mice were infected intranasally with 106 PFU of HSV-1 and cells infiltrating into the lungs were collected at various times via BAL. (A) Total numbers of viable cells were determined by trypan blue exclusion. Data are mean ± SD of cell counts for three to five mice at each time point. (B) BALF cells were recovered, stained with Diff Quick and inflammatory cells classified (MAC, macrophages; LYMPH, lymphocytes; NEUT, neutrophils) based on morphological characteristics. Columns represent the mean percent BALF cells from groups of three to five mice. (C) Lymphocyte subsets in BALF from HSV-1-infected mice. BALF cells were stained with FITC-, PE- or allophycocyanin-conjugated monoclonal antibodies. Cells were analyzed by flow cytometry, and classified as NK cells (CD3 NK1.1+), B cells (CD3 B220+), CD4+ T cells (CD3+ CD4+) or CD8+ T cells (CD3+ CD8+). The data shown are from pooled BALF cells of three to five mice at each time point, and are representative of three experiments; un, uninfected mice.

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BALF cells were differentially stained to examine the cell types present over the course of infection (Fig. 2B). Whilst the low numbers (<4×105 cells per total BALF) of leukocytes in the airways of naive mice were predominantly alveolar macrophages, HSV-1 infection led to a steady increase in the proportion of infiltrating lymphocytes, peaking at day 7 post-infection. Neutrophils represented less than 10% of BALF cells at any time point tested.

Flow cytometry was used to characterize BALF lymphocytes in more detail. An early influx of NK cells (NK1.1+ CD3) was followed by the subsequent recruitment of T cells (CD3+ NK1.1) to the airspaces of the lung (Fig. 2C). The majority of recruited T cells expressed α/β TCR, as γ/δ TCR+ lymphocytes were <1% of BALF cells at all time points tested (data not shown). CD8+ T cells predominated over CD4+ T cells in BALF reaching a CD8:CD4 ratio of approximately 3:1 by day 7, the peak of the T cell response, and 45–60% of CD8+ T cells also stained with tetramer containing a peptide corresponding to the immunodominant epitope of HSV-1 glycoprotein B (gB498–505) at this time. The proportions of B cells (CD3 B220+) and NKT cells (TCR β+ CD1d tetramer+) remained low (<5% and <2% of BALF cells, respectively) at all time points tested (data not shown).

Activation of NK cells in the lungs of HSV-1-infected mice

Following intranasal HSV-1 infection, NK cells were rapidly recruited to the airways (Fig. 2C). To investigate NK cell activation, lung cells prepared from HSV-1-infected mice were assessed for their ability to lyse YAC-1 target cells (Fig. 3A). Cytotoxic activity peaked at day 3 post-infection and was not observed in lungs from naive mice. Cytotoxicity was mediated by NK cells, as lung cells taken 3 days after infection with HSV-1 and depleted of asialo-GMmath image cells in the presence of guinea pig complement failed to lyse YAC-1 targets (untreated lung cells: 23%, complement alone: 23%, and complement plus anti-asialo-GMmath image: 2% cytolysis; at effector-to-target ratio of 100:1).

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Figure 3. Cytolytic activity of NK cells in the lung following intranasal HSV-1 infection. Groups of four to five B6 mice were infected with 106 PFU of HSV-1. Specific lysis of YAC-1 targets by lung cells was assessed by 51Cr-release assay. (A) Data are expressed as the mean percent lysis ± SD from individual mice at a total lung cell-to-target ratio of 50:1. (B) Pooled lung cell suspensions (three to five mice for each time point) were stained for expression of NK1.1 and CD3, and NK1.1+ CD3 cells were quantitated and used to express cytotoxicity results as NK cell-to-target ratio. Shown is the mean percent lysis from triplicate cultures and all SD were less than 15%. The dashed line represents a NK cell-to-target ratio of 7:1 and enables comparison of cytotoxicity at an equivalent NK cell number; un, uninfected mice.

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Effector cell populations were also stained for expression of the NK cell marker NK1.1 to ascertain whether the enhanced NK cell-mediated cytotoxic activity was due to an increase in the cytotoxic potential of NK cells or simply an accumulation of NK cells at the site of infection. The cytotoxicity of lung cells taken 1, 3, 5, 7 and 10 days after intranasal infection, expressed as NK cell-to-target ratio, showed that the lytic capacity of NK cells increased through the initial stages of the infection peaking at day 3 and declined thereafter (Fig. 3B). Consequently, the data indicate that the cytotoxicity of lung NK cells is influenced by both NK cell number and their activation state.

We also used flow cytometry to examine the ability of NK cells to produce IFN-γ following ex vivo culture in the presence of Brefeldin A (Fig. 4A–C). While few NK cells from the BALF of naive mice expressed intracellular IFN-γ, there was a marked increase in both the proportion and numbers of NK cells that expressed IFN-γ peaking at 3 days post-infection and declining to basal levels by day 7 (Fig. 4D). We also determined the concentration of IFN-γ present in cell-free BALF (Fig. 4E). Whilst IFN-γ was detected early after infection, a striking peak was noted at day 5 post-infection, a time at which few NK cells stained IFN-γ+. Thus, whilst NK cells are early producers of IFN-γ in the respiratory tract, other cell types, presumably activated T cells, produce high levels of the cytokine by day 5.

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Figure 4. Activation of lung NK cells to produce IFN-γ in response to intranasal HSV-1 infection. Mice were infected with HSV-1, and at specific time points post-infection BALF NK cells were assessed for intracellular IFN-γ. Shown are representative dot plots of (A) NK1.1+ CD3 cells from day 1 HSV-1-infected mice stained for (B) intracellular expression of IFN-γ or (C) with an isotype control. (D) Numbers of IFN-γ+ NK cells after HSV-1 infection. (E) IFN-γ levels in cell-free BALF at various times after intranasal HSV-1 infection. The detection limit of the ELISA was 0.06 ng/mL. Data shown in (D) and (E) are the means ± SD from groups of three to five mice and are representative of three independent experiments.

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Depletion of NK cells leads to an increase in early HSV-1 replication in the lung but does not alter viral clearance

To determine whether the rapid accumulation and activation of NK cells following HSV-1 infection had any consequences for viral replication, the levels of virus in the lungs were compared between normal mice and those that had been depleted of NK cells. Mice depleted of NK cells had elevated viral loads at day 1 and particularly at day 3 post-infection (Fig. 5A), indicating a role in the control of early HSV-1 replication. However, NK cells were not required for effective viral clearance, as viral titres were similar between all infected groups at day 5. BALF IFN-γ levels from anti-asialo-GM1 rabbit antisera (AAGM)-treated mice were particularly low 1 and 3 days after infection, consistent with a role for NK cells in production of early IFN-γ (Fig. 5B). AAGM treatment did not affect peak IFN-γ levels observed in BALF 5 days after intranasal HSV-1 infection, consistent with a role for other cell types in producing IFN-γ at this time.

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Figure 5. NK cell depletion enhances early viral growth and reduces IFN-γ production in HSV-1-infected mice. Mice were treated with AAGM (□), normal rabbit sera (NRS, ▮) or PBS (▪), and infected with 106 PFU of HSV-1. (A) Titres of infectious virus in lung homogenates 1, 3 and 5 days after intranasal infection with HSV-1. Data represent mean titres ± SD from groups of four to five mice. The dashed line indicates the minimum detection limit of the plaque assay. (B) Levels of IFN-γ in cell-free BALF following NK cell depletion of HSV-1-infected mice. IFN-γ levels in were determined in individual mice by ELISA, and data are expressed as the mean ± SD from groups of four to five mice. The detection limit of the ELISA was 0.06 ng/mL. The experiment was repeated twice with similar results.

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Activation of T cells in the lung after intranasal HSV-1 infection

The massive increase in IFN-γ levels in the BALF of normal mice and NK cell-depleted mice 5 days after HSV-1 infection suggested that activated T cells were entering the lung at this time. We therefore defined T cell recruitment and activation in the airways by assessment of the ability of lung CD8+ T cells to (1) produce IFN-γ following in vitro stimulation with immunodominant peptide gB498–505 (Fig. 6A), and (2) to kill target cells expressing gB498–505in vitro (Fig. 6B). In both assays, gB-specific T cells were first observed in the lung at day 5 post-infection, peaked at day 7, and declined thereafter.

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Figure 6. Kinetics of CD8+ T cell activation in the lung after intranasal HSV-1 infection. Groups of B6 mice were infected with 106 PFU of HSV-1 and on days 1, 3, 5, 7 and 10 lung cell suspensions were prepared. (A) For analysis of intracellular IFN-γ production, lung cells were stimulated with gB498–505 and stained for surface CD8 and intracellular IFN-γ expression. Shown is the mean number of lung CD8+ T lymphocytes producing IFN-γ per mouse (± SD) from groups of three to five mice. (B) Specific lysis of gB498–505-coated EL4 target cells was assessed in a standard 51Cr-release assay. Effector-to-target ratios were assayed in triplicate and the data shown represent the mean percent lysis (+ SD) from three to four mice at a lung cell-to-target ratio of 50:1. (C) Levels of gB-specific cytolytic activity present in the draining mediastinal lymph node (MLN) and in spleen, lung and the non-draining brachial LN (BLN) were determined at various times after intranasal HSV-1 infection using a 4 h in vivo CTL assay. Results are expressed as mean percent specific lysis ± SD.

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To understand the role of NK cells in mediating early protection against HSV-1 infection it was important to determine at which time point the first T cell responses could be detected. Given the limited sensitivity of ex vivo assays, we next took advantage of an in vivo CTL assay to assess the development of HSV-1-specific effector CD8+ T cells. CTL activity was first detected in the mediastinal lymph node draining the lung 3 days post-infection (Fig. 6C), consistent with the expansion and activation of HSV-1-specific CD8+ T cells at this site. However, there was no detectable CTL activity in the lung, spleen or non-draining lymph nodes at this time. By 5 days post-infection, gB-specific T cells had been expanded and disseminated throughout infected mice as high levels of killing were observed in spleen, draining and non-draining lymph nodes as well as the lung.

Role of adaptive immunity in controlling HSV-1 infection of the respiratory tract

The timing of viral clearance from the lung (Fig. 1B) correlated with the first detection of in vivo CTL activity in the periphery (Fig. 6C), consistent with an important role for CD8+ T cells in clearance of HSV-1 from the lung. To explore this further, immunodeficient mice lacking particular components of the adaptive immune response were infected with HSV-1 (Fig. 7).

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Figure 7. Virulence of HSV-1 in immunodeficient mice after intranasal infection. Groups of B6 mice, B6 mice treated with antibodies specific for CD4 and CD8, and B6.TAP–/–, B6.Aβo and B6.RAG-1–/– mice were infected with 106 PFU of HSV-1. (A) At various times post-infection, virus titres were determined in lung homogenates by plaquing on Vero cells. (B) Mice were observed daily and assessed for signs of illness over a 20-day period. Results are expressed as percent survival. (C) Lungs and brains were removed from RAG-1–/– mice at time of sacrifice and titres of infectious HSV-1 were determined by plaque assay on Vero cells.

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B6 mice treated with monoclonal antibodies specific for CD4 and CD8 showed no impairment in ability to clear HSV-1 infection from the lung (Fig. 7A). Moreover, B6 mice, B6.Aβo mice, which lack MHC class II molecules, and B6.TAP–/– mice, which have defective CD8+ T cell-mediated immunity, showed similar kinetics of HSV-1 replication and clearance from the lung. Whilst there was no obvious impairment in the ability of B6.RAG-1–/– mice, lacking both T and B cells, to clear HSV-1 from the lung (Fig. 7A), all mice succumbed to disease 10–15 days after intranasal infection (Fig. 7B). Critically, B6.RAG-1–/– mice were unable to control viral spread and replication through the CNS (Fig. 7C) and high titres of virus were detected in the brains, but not the lungs, at the time of death. Thus, adaptive immunity is not required to clear virus from the primary site of infection but is required to inhibit HSV-1 replication in the CNS and brain.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Despite the availability of potent antiviral agents, HSV-1 induced pneumonia and/or encephalitis remain of significant public health concern and surprisingly little is known about the pathogenesis of HSV-1 infections of the respiratory tract. We have examined the inflammatory response in the airways following intranasal HSV-1 infection. This response was characterized by an early expansion of the NK cell population and the subsequent recruitment of T cells, in particular CD8+ T cells, to the lung following intranasal HSV-1 infection. Whilst both NKT cells and/or γδ T cells have been reported to respond to HSV-1 following skin and ocular infections 1113, we found no significant expansion of either population in the airways, suggesting that these cells do not play a major role in the pulmonary response to HSV-1.

Lymphocytes, in particular NK and T cells, predominate in the airways during the course of pulmonary HSV-1 infection (Fig. 2B). Of interest, neutrophils represented <10% of inflammatory cells in the airways at all time points tested (Fig. 2B), but are the major cell type recruited following corneal 14 or skin scarification 15, and neutrophil depletion in these models has been shown to modulate disease progression 15, 16. The responses observed in the lung are more similar to those noted in the trigeminal ganglion (TG) after corneal infection, where macrophages and lymphocytes predominate 17. These studies indicate that there are markedly different patterns of leukocyte infiltration following HSV-1 infection of distinct anatomical sites.

NK cells were the major cell type recruited to the airways early after HSV-1 infection, and lung NK cells were rapidly activated, as demonstrated by up-regulated cytotoxic capacity and production of IFN-γ. NK cell depletion was associated with increased replication of HSV-1 in the lung at days 1 and 3 post-infection, but had no effect on viral titres at day 5. In contrast, HSV-1-specific CD8+ T cells were first detected in the lung at day 5 and coincided with the enhanced expression of inhibitory receptor KLRG1 by NK cells (data not shown) and the down-regulation of NK cell effector mechanisms. Together these observations suggest a tightly coordinated interaction between innate and adaptive responses, and highlight the importance of NK cells in innate control of HSV-1 infection in the respiratory tract.

NK cells and CD8+ T cells can respond to viral infection via production of cytokines such as IFN-γ or TNF-α, or through killing of target cells by perforin/granzyme or Fas/FasL-mediated mechanisms 18. The early IFN-γ production in the lung was mediated by NK cells, as IFN-γ levels in BALF were significantly reduced in AAGM-treated mice 1 and 3 days after infection. Peak BALF IFN-γ levels observed at day 5 post-infection were unaffected by AAGM treatment but were reduced more than tenfold in mice depleted of T cells (data not shown), suggesting that T cells were the major contributors to IFN-γ production at this time. Previous studies in a variety of mouse models have examined the effects of IFN-γ on HSV infection 1923, and suggested that while IFN-γ had minimal effects on viral replication or neuroinvasiveness, it plays a critical role in protecting mice from fatal HSV-1 encephalitis. In particular the suppression of HSV-1 replication in neurons by CD8+ T cells is thought to be dependent on the production of IFN-γ 2123.

Exocytosis of perforin is accompanied by the release of serine proteases and activation of caspases that trigger programmed cell death in target cells, and plays a crucial role in clearance of viruses such as LCMV 24. After corneal HSV-1 infection, viral clearance is unaffected in perforin-deficient mice, and perforin-mediated cytotoxicity may actually contribute to the chronic inflammatory condition known as herpetic stromal keratitis 25. Analysis of the NK cell and T cell responses in mice deficient in either IFN-γ and/or perforin would be required to delineate the relative contribution of IFN-γ production and cell-mediated cytotoxicity to control of HSV-1 in the lung and nervous system following intranasal inoculation.

HSV-1 pneumonia and/or encephalitis in humans are typically associated with immunosuppression or stress 26. In murine models of HSV-1 infection, the absence of T cell immunity is typically associated with viral dissemination and replication in the CNS, leading to fatal encephalitis. A notable exception to this has been reported in a study by Adler et al.10, where B6.Tcrb-Tcrd mice, which lack T cells, were found to control acute HSV-1 replication following intranasal infection and to resist mortality associated with HSV-1-induced encephalitis. We report that B6.RAG-1–/– mice, lacking both T cell and B cell immunity, readily succumb to HSV-1-induced encephalitis whilst mice lacking effective CD4+ or CD8+ T cell-mediated immunity do not. Consistent with these findings, Ghiasi et al.27 reported that following ocular infection, absence of either CD4+ or CD8+ T cells did not alter HSV-1 clearance from the TG; however, absence of both led to establishment of chronic TG infection.

The results presented here characterize the inflammatory response to HSV-1 infection of the respiratory tract, and demonstrate that NK cells and subsequently T cells are major components of the inflammatory infiltrate. Furthermore, we define the kinetics of recruitment and activation for both NK cells and HSV-1-specific CD8+ T cells, and demonstrate that NK cells play a role in controlling acute HSV-1 infection of the lung. Components of adaptive immunity were, however, critical for protection against HSV-1-induced encephalitis following intranasal infection.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Mice, viruses and peptide

B6 mice, and RAG-1-knockout (B6.RAG-1–/–), TAP-knockout (B6.TAP–/–) and MHC class II-knockout (B6.Aβo) mice on a B6 background were bred and housed in specific pathogen-free conditions at the Department of Microbiology and Immunology, The University of Melbourne. Adult male mice (6–12 wk old) were used in all experiments. The KOS strain of HSV-1 was propagated and titred using Vero cells. The gB498–505 peptide with the sequence SSIEFARL was obtained from Auspep. Kb tetramers containing the gB peptide were prepared as described elsewhere 28.

Infection and treatments of mice

Mice were anaesthetized and infected via the intranasal route with 106 PFU of HSV-1 in 50 μL of PBS. Each day mice were weighed and monitored for signs of illness, and those suffering a severe infection or having lost >20% of their original body weight were euthanized. To determine virus titres in organs, mice were euthanized, and lung, brains, livers and spleens were removed, homogenized and clarified by centrifugation. Samples were assayed for infectious virus by plaque assay on Vero cells.

In some experiments mice were treated with PBS, normal rabbit sera or AAGM (Wako Pure Chemicals, Osaka, Japan) to deplete NK cells. NK cell depletion was achieved using combination of intraperitoneal (200 μL) and intranasal (50 μL) treatments with AAGM every second day. This treatment did not affect numbers of CD3+ NK1.1 T cells, Gr-1high neutrophils or Mac-1+ macrophages/monocytes but did reduce NK cell numbers by >90% in the lungs. To deplete CD4/CD8 cells, mice received an intraperitoneal injection of 1.0 mg purified anti-CD4 (GK1.5) and anti-CD8 (YTS 169) antibodies or 2.0 mg control rat IgG. CD4/CD8 depletion in spleen and lung was monitored by flow cytometry using anti-CD8 (clone 53.6.7, BD Pharmingen) and anti-CD4 (clone H129.19, BD Pharmingen) antibodies and found to be >90%.

Recovery of immune cells for flow cytometry and cytotoxicity assays

BALF and lung cells were obtained from uninfected and HSV-1-infected mice at various times post-infection. For collection of BALF cells, mice were sacrificed and the lungs were flushed three times with a 1-mL volume of PBS through a blunted 23-gauge needle inserted into the trachea. The samples were pooled and cells treated with Tris-NH4Cl (0.14 M NH4Cl in 17 mM Tris, adjusted to pH 7.2) to lyse erythrocytes, washed twice and resuspended in cold RPMI 1640 medium supplemented with 10% FCS. Aliqouts of approximately 5×104 BAL cells were cytocentrifuged onto microscope slides, dried and stained with Diff-Quick (Lab Aids, Victoria, Australia) for differential cell counts. Single-cell suspensions of lung cells were prepared by mincing lung tissue prior to incubation with 2 mg/mL collagenase A (Roche Diagnostics, Mannheim, Germany) in serum-free RPMI 1640 at 37°C for 30 min. After treatment, lungs were sieved through wire mesh followed by hypotonic lysis of erythrocytes. Cell viability was assessed using trypan blue exclusion.

Flow cytometric analysis of cell surface and intracellular antigens

Single-cell suspensions of BALF or lung cells were incubated on ice for 20 min with supernatant from hybridoma 2.4G2 to block Fc receptors and then stained with appropriate combinations of fluorescein isothiocynate (FITC), phycoerythrin (PE) or allophycocyanin antibodies to NK1.1 (PK136), CD3ϵ (145-2C11), CD4 (GK1.5), CD8α (53.6.7), CD45R/B220 (RA3-6B2), TCR β chain (H57-597) and γ/δ TCR (GL3).

In some experiments, cells were stained with PE-labelled α-galactosylceramide-loaded mCD1d tetramers (a gift from Prof. Dale Godfrey, Department of Microbiology and Immunology, The University of Melbourne). To detect intracellular IFN-γ from NK cells, BALF cells were incubated without stimulation in 10 μg/mL Brefeldin A (Sigma-Aldrich, St Louis, MO) for 4 h at 37°C. To detect intracellular IFN-γ from HSV-1-specific CD8 T cells, lung cell suspensions were stimulated with gB498–505 peptide in the presence of 10 μg/mL Brefeldin A for 6 h at 37°C.

Cells were then washed, incubated in 2.4G2 supernatant and stained with anti-CD3ϵ-allophycocyanin and anti-NK1.1-FITC or with anti-CD8-allophycocyanin, respectively. Samples were fixed, permeabilized and stained for intracellular IFN-γ (XMG1.2, BD Pharmingen). Cells were analyzed on a FACS Calibur flow cytometer collecting data from at least 10 000 lymphocytes.

ELISA for IFN-γ in BALF

Levels of IFN-γ in BALF were determined by sandwich ELISA using matched antibody pairs from BD Pharmingen according to manufacturer's instructions. BALF samples were clarified and the concentration of IFN-γ protein was determined relative to a standard curve.

Cytotoxicity assays

NK cell cytotoxicity and HSV-1-specific CTL activity in lung cell suspensions was assayed with a standard 51Cr-release assay. NK cell-mediated cytotoxicity was tested on YAC-1 cells, while EL4 cells (H-2b, thymoma) pulsed with 1 μM gB498–505 peptide were used as targets for HSV-1-specific CTL killing. The percentage of specific 51Cr release was calculated as: % specific lysis = [(experimental release – spontaneous release) / (total detergent release – spontaneous release)] × 100%. The spontaneous release values were always <15% of total cytolysis.

In vivo CTL

Splenocyte target cell suspensions from B6 mice were divided into two populations. One was pulsed with 1 μM gB498–505 peptide for 45 min at 37°C and then labelled with a high concentration (5 μM) of 5-carboxyfluorescein diacetate succinimidyl ester (CFSE; CFSEhi population). The other was incubated for 45 min at 37°C without peptide and labelled with a low concentration (0.5 μM) of CFSE (CFSElo population). An equal number of cells from each population (107) was mixed together and adoptively transferred into HSV-1-infected mice that were then killed 4 h later. Cell suspensions were analyzed by flow cytometry and each population distinguished by their different fluorescent intensities. Percent specific lysis was determined by loss of the peptide-pulsed CFSEhi population compared with control CFSElo population using the formula described previously 29.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

The authors wish to thank Prof. Frank Carbone for assistance and helpful discussion. We also thank Shannon Griffiths (Peter Mac) for the breeding and maintenance of the gene-targeted mice. This work was supported by Program Grants from The National Health and Medical Research Council of Australia (NHMRC). P.C.R. is a C. R. Roper Fellow. M.J.S. is an NHMRC Principal Research Fellow.

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