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The Journal of Immunology, 2007, 179, 7506 -7513
Copyright © 2007 by The American Association of Immunologists, Inc.
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Dendritic Cells Present Lytic Antigens and Maintain Function throughout Persistent {gamma}-Herpesvirus Infection1

Fiona Kupresanin*, Jonathan Chow2,*, Adele Mount*, Christopher M. Smith{dagger}, Philip G. Stevenson{dagger} and Gabrielle T. Belz3,*

* The Walter and Eliza Hall Institute of Medical, Melbourne, Victoria, Australia; and {dagger} Virology, Department of Pathology, Addenbrooke’s Hospital, University of Cambridge, Cambridge, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The activation and maintenance of Ag-specific CD8+ T cells is central to the long-term control of persistent infections. These killer T cells act to continuously scan and remove reservoirs of pathogen that have eluded the acute immune response. Acutely cleared viral infections depend almost exclusively on dendritic cells (DC) to present Ags to, and to activate, the CD8+ T cell response. Paradoxically, persistent pathogens often infect professional APCs such as DC, in addition to infecting a broad range of nonprofessional APC, raising the possibility that many cell types could present viral Ags and activate T cells. We addressed whether in persistent viral infection with murine gammaherpesviruses, DC or non-DC, such as B cells and macrophages, were required to maintain the continued activation of Ag-specific CD8+ T cells. We found that presentation of the surrogate Ag, OVA, expressed under a lytic promoter to CD8+ T cells during persistent infection was largely restricted to DC, with little contribution from other lymphoid resident cells, such as B cells. This is despite the fact that B cells harbor a very large reservoir of latent virus. Our results support that, during persistent viral infection, continual presentation of lytic Ags by DC leads to T cell activation critical for maintaining CD8+ T cells capable of limiting persistent viral infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD8+ killer T cells play an important role in controlling persistent viral infections (1, 2). In acutely cleared pathogen infections, dendritic cells (DC)4 are critical in driving the activation and amplification of naive CD8+ T cell populations expressing T cell receptors specific for virus-derived antigenic peptides (3, 4, 5, 6). Persistent viral infections often display wide tissue trophisms allowing multiple cell types to harbor quiescent virus, so it is feasible that non-DC, such as B cells harboring latent reservoirs of virus, play an important role in maintaining CD8+ T cell responses that limit viral spread in the host (7). Therefore, it remains to be addressed whether, during persistent infections, DC are the exclusive cell type required for activating CD8+ T cells during periods of viral reactivation (8).

Gammaherpesviruses ({gamma}-HV) characteristically establish persistent, asymptomatic infections, but can re-emerge after immune suppression to cause overt disease (9). Murine {gamma}-HV 68 (MHV-68) is an important murine experimental model that that is genetically highly similar to the human pathogens such as Karposi’s sarcoma-associated virus and Epstein-Barr virus (10, 11). Intranasal inoculation of mice with MHV-68 results in the development of an acute lytic respiratory infection localized initially to the lung, but which disseminates to other tissues of the body, such as the spleen, later in infection (12). Viral latency is established predominantly in lymphoid cells, such as B cells, macrophages, and DC, and can also be found in lung epithelial cells (13, 14, 15).

Viruses use a variety of mechanisms to elude the immune system. MHV-68 is indeed no exception, employing an armory of virally encoded molecules to deter viral detection by the immune system. In MHV-68, these include murine K3, an E3 ubiquitin ligase that catalyzes the destruction of MHC I heavy chains (16, 17); the transporter associated with Ag processing (18), ORF 73, an episome maintenance protein that impairs Ag presentation by inhibiting translation of cis-encoded antigenic peptides (19); and M3, a chemokine-binding protein (20). Several of these genes target components of the Ag presentation machinery, potentially impairing the development of CD8+ T cell responses. In MHV-68 infection, Ag-specific CD8+ T cell responses are maintained against viral gene products throughout the life of the animal, yet it is not clear whether persistent viral Ag presentation plays a role in this maintenance (21, 22).

Precisely how T cell populations are maintained during persistent infections is unclear, although several important mechanisms have been proposed. These include roles for the following: 1) persistent inflammation promoting continuous cytokine production that may drive CD8+ T cell expansion, 2) homeostatic division of virus-specific CD8+ T cells during viral persistence (23), 3) continuous supply of newly emerged naive Ag-specific CD8+ T cells from the thymus (24), and 4) persistent viral Ag presentation to T cells.

During chronic lymphocytic choriomeningitis viral (LCMV) infection, memory T cells have been shown to be depleted from the total T cell pool. In this infection, continuous recruitment of thymic emigrants appears to be required to replenish functionally impaired T cells and to avoid rapid onset of immunological exhaustion characteristic of LCMV infection (24). In contrast, in {gamma}-HV infection, thymectomy to eliminate a continual supply of naive T cells did not result in loss of T cell responses or dysregulation of viral control, suggesting that the two viruses may use different mechanisms or signals to maintain CD8+ T cells during viral persistence (25). Both LCMV and {gamma}-HV appear to promote high rates of T cell proliferation in an effort to maintain T cell populations (23, 26, 27).

Despite previous studies, the precise mechanism(s) by which Ag is supplied to naive or memory T cells during long-term persistent infection are poorly defined. A recent study suggests that MHV-68 infection functionally modulates DC by impairing their ability to present encountered Ags (14). Although these DC produced the cytokine IL-10, which is known for its suppressive effects, whether the generation of such cytokines impinge on DC ability to present MHC class I Ags to CD8+ T cells in vivo, is unknown. Such dysregulation, if it exists, would have important implications for effective Ag presentation by virally infected cells and cross-presentation of exogenous Ags. Preservation of cross-presentation during persistent viral infections was first proposed by Bevan and colleagues (28), but maintenance of this pathway during infection has not been investigated.

In this study, we addressed the question whether persistent Ag presentation during murine {gamma}-HV infection could amplify CD8+ T cells and whether this was dependent on presentation of Ags by DC or non-DC populations. In addition, we tested whether persistent viral infection modulated the capacity of DC to cross-present exogenous Ags that could be important in maintaining the immune response to MHV-68.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Mice used were C57BL/6 (H-2b), bm-1, and the transgenic strains Ly5.1.OT-I (H-2Kb-restricted anti-OVA257–264) (29), Ly5.1.gBT-I (H-2Kb-restricted anti-glycoprotein B498–505) (30), and CD11c-diptheria toxin receptor (DTR) (6). Mice were used between 6 and 12 wk of age, and were maintained in specific pathogen-free conditions. C57BL/6 and transgenic mice were obtained from The Walter and Eliza Hall Institute breeding facility and were kept in accordance with the ethics license under the Melbourne Health Research Directorate.

Viruses

Mice were infected intranasally with 3 x 104 plaque-forming units (PFU) of MHV-68 or recombinant MHV-OVA under brief halothane anesthesia.

The generation of MHV-68 engineered to express OVA from an intergenic expression cassette under a lytic cycle promoter has been previously described (2). This approach incorporates an ectopic copy of the 500 bp upstream of the MHV-68 M3 open reading frame (ORF) as a strong, ORF50-dependent lytic cycle promoter (31). The full-length OVA-encoding sequence was PCR-cloned between the M3 promoter and a bovine growth hormone poly-A site derived from pcDNA3 (Invitrogen Life Technologies). The expression construct was then subcloned into MHV-68 genomic clone (75338–78717), and subcloned with its genomic flanks into pST76K-SR. This expression cassette was then recombined into the MHV-68 BAC, between the 30 ends of ORF57 and ORF58. Infectious virus was reconstituted from BAC DNA by transfection into BHK-21 cells with Fugene-6 (Roche Diagnostics). The BAC cassette was removed by serial viral passage through NIH-3T3-CRE cells. Virus stocks were grown and titered on BHK-21 cells. All the introduced mutations were confirmed as correct by viral DNA sequencing (2).

For some experiments, recombinant influenza WSN-gB was used. This virus contains a H-2Kb-restricted CD8+ T cell determinant (SSIEFARL) derived from the glycoprotein B (gB, gB498–505) of herpes simplex virus inserted into the neuraminidase stalk (32).

Purification and preparation of CFSE-labeled transgenic T cells

Lymphocyte suspension from lymph nodes (inguinal, axillary, sacral, cervical, and mesenteric) or spleen were enriched for CD8+ T cells by incubation with predetermined optimal concentrations of Abs specific for Mac-1 (M1/70), Mac-3 (F4/80), erythrocytes (Ter-119), B220 (RA3–6B2), MHC class II (M5/114), and CD4 (GK1.5). Ab-coated cells were removed using anti-rat IgG magnetic beads (Qiagen). Purity was verified to be ~95%. Enriched CD8+ T cell preparations were resuspended at 107 cells/ml in 0.1% BSA/PBS and incubated with 5 µM CFSE stock solution (5 mM in DMSO; Molecular Probes) for 10 min at 37°C. Cells were washed twice, resuspended in complete medium, and plated at 5 x 104 T cells/well.

Generation of memory CD8+ T cell populations

Memory CD8+ T cells were created using an established model for the in vitro differentiation of central memory T cells (33, 34, 35, 36, 37). In brief, naive OT-I transgenic CD8+ T cells were coated with 1 µM SIINFEKL (OVA257–264) peptide for 1 h at 37°C. Cells were then washed twice in HEPES Earl’s medium containing 2.5% FCS before culture at 1.7 x 105 cells/ml in complete mouse tonicity RPMI 1640 medium (RPMI 1640 containing 10% FCS, 50 µM 2-ME, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin, complete medium). After 2 days, cells were washed and supplemented with recombinant hIL15 (20 ng/ml) (R&D Systems). Complete medium containing hIL15 was replaced every 3–4 days and cells were used between 14 and 20 days after initiation of the culture.

CD11c-DTR bone marrow chimeras

Recipient C57BL/6 mice were irradiated with two doses of 550 cGy 3 h apart, and were reconstituted with 3–5 x 106 T cell-depleted bone marrow cells extracted from the femurs and tibias of CD11c-DTR transgenic mice. Mature T cells were depleted using predetermined optimal concentrations of anti-CD4 (RL172), anti-CD8 (3.168), and anti-Thy1 (J1j) Abs, followed by treatment with rabbit complement. The day following reconstitution, mice were injected i.p. with 100 µg anti-Thy1 (T24) Ab to deplete radioresistant host T cells. Mice were rested for 5–7 wk before use. For systemic DC depletion, chimeras were injected i.p. with 4 ng/g body weight diptheria toxin (DT) (in PBS; CSL) every 2 days for the duration of the experiment.

Ag presentation assays

DC were purified from the spleens of mice infected with MHV-68 as previously described (38, 39). In brief, spleen fragments were digested for 20 min at room temperature with collagenase/DNase, and then treated with EDTA for 5 min to disrupt T cell-DC complexes. Cells bearing non-DC lineage markers were depleted using optimal concentrations of Abs specific for CD19 (ID3), CD3 (KT31.1), Thy1 (T24/31.7), and erythrocytes (TER-119) followed by anti-rat IgG magnetic beads (Dynabeads; Dynal). Enriched DC preparations were fluorescently labeled with CD11c (N418-fluorescein isothiocynate), CD8{alpha} (53–6.7-allophycocyanin) and CD4 (RM4–5-PE), and sorted by flow cytometry using a MoFlo instrument (Dako Cytomation). All preparations were >98% pure as determined by reanalysis of sorted populations. Two-fold serial dilutions of purified DC were cocultured in vitro with 5 x 104 CFSE-labeled transgenic T cells in U-bottom 96-well plates.

Crosspresentation of soluble Ag by virally infected DC

Purified DC were exposed to 5 PFU of either {gamma}-HV or influenza virus (WSN-gB), or were not exposed to virus for 2 h then washed twice before exposure to 30 ng/ml OVA for 45 min at 37°C in complete medium. After incubation, DC were washed twice and cocultured with 5 x 104 CFSE-labeled OVA-specific or glycoprotein B-specific CD8+ transgenic T cells specific for OVA-specific T cells. Proliferation was measured by flow cytometry after 60 h of culture.

OVA-coated spleen cells

To prepare cell-associated OVA (40) bm1 spleen cells were irradiated for 1500 cGy, washed, and incubated with 10 mg/ml OVA protein in mouse tonicity RPMI 1640 for 10 min at 37°C and washed three times with RPMI 1640 supplemented with 3% FCS. The amount of OVA associated with the cells was quantitated to be 8 ng OVA/106 cells (41).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Persistent Ag presentation drives amplification of naive and memory CD8+ T cells

{gamma}-HV, such as Karposi’s sarcoma-associated virus or MHV-68, persist for the life of an animal. MHV-68 infection results in a biphasic immune response. The first phase is an acute respiratory infection resulting in the amplification of virus-specific CD8+ T cells, dominated by H-2Db p56 and H-2Kb p79 CTLs. Following the acute localized respiratory infection, the virus is disseminated throughout the body and the established CD8+ T cell populations persist and remain at relatively constant levels (21, 26). Moreover, these populations maintain their capacity to secrete IFN-{gamma} and retain cytolytic activity for months to years (21, 22). The pathways by which these T cells are maintained during persistent infection are largely unknown. This is particularly important in light of the pleiotropic capacity of such viruses to infect a wide variety of cell types; both hemopoietic and nonhemopoietic cells could potentially drive proliferation of T cells.

To gain some insight into the capacity of persistent {gamma}-HV infection to drive continued proliferation of naive or memory T cells, we generated a recombinant {gamma}-HV that coexpressed full-length OVA from an intergenic expression cassette driven by an ectopic MHV-68 lytic promoter (MHV-OVA) (2). This virus expresses both secreted and cytoplasmic OVA. In addition to mimicking the expression pattern of {gamma}-HV viral proteins, such as M3, M4, and ORF4 (42, 43, 44, 45), this virus allows us to track the immune response to the surrogate Ag, OVA, using MHC class I tetramers and transgenic T cells. CFSE-labeled naive or memory OVA-specific CD8+ T cells were transferred into acute or persistently infected mice. Three days after transfer, the capacity to recruit these OVA-specific T cells to sites of viral Ag expression and induce their proliferation was monitored by the dilution of the fluorescent dye (Fig. 1). Both naive and memory T cells proliferated in acute (days 3–6) or persistently infected recipients (days 59–62) in the mediastinal LN and spleen (Fig. 1). Persistence of MHV-68 virus has been previously been detected in spleen, and in the mediastinal LN and our data support that persistent Ag presentation at these sites drives activation of CD8+ T cells (14, 46).


Figure 1
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  		FIGURE 1. Ag expression driven off a lytic promoter during persistent infection activates naive and memory CD8+ OVA-specific T cells. Ag presentation during acute (top panels) and persistent (middle and lower panels) MHV-OVA infection drives proliferation of naive and memory CD8+ T cells in vivo. A total of 1 x 106 CFSE-labeled naive (top and middle panels) and memory (lower panels) CD8+ T cells specific for OVA were adoptively transferred into mice on day 0 or 59 after intranasal infection with MHV-OVA. Proliferation was examined by monitoring the dilution of CFSE intensity of OT-I cells isolated from spleen (center panels), mediastinal LN (right panels), or spleen of uninfected control mice (left panel) 3 days after transfer. Data are representative of three independent experiments.

 
Multiple DC subsets present viral Ag during persistent infection

Several studies have assessed the persistence of MHV-68 in latency and shown that this virus infects a broad range of immune cells, including B lymphocytes, DC, and macrophages. As nonhemopoietic cells have recently been shown to contribute significantly to T cell expansion in LCMV infection (7), it was first important to ascertain the cell types contributing to T cell activation during the persistent phase of infection. To assess the capacity of different cell types to present virus-derived epitopes to naive Ag-specific CD8+ T cell, we purified B cells (CD19+), macrophages (CD11b+F4/80+), and different subsets of conventional DC (CD8{alpha} DC, CD8–CD4– double negative (DN) DC) from persistently infected mice and cocultured them with CFSE-labeled naive OVA-specific CD8+ T cells. After 60 h, the proliferation of the T cells was assessed. As shown in Fig. 2, the major component of Ag presentation was restricted to DC (Fig. 2). Although CD8{alpha} DC were most efficient in presenting OVA expressed by the recombinant {gamma}-HV virus, DN DC, but not CD4+ DC (data not shown), also presented Ags to naive OVA-specific CD8+ T cells. DC purified from spleen of naive mice did not induce proliferation of OT-I CD8+ T cells (data not shown).


Figure 2
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  		FIGURE 2. DC are the predominant cell type responsible for induction of proliferation of naive T cells during persistent infection. Different populations of splenic DC sorted as shown in A. CD11c+CD8{alpha}+ DC and DN (CD8{alpha}–CD4–) DC and F4/80+CD11b+ macrophages as shown in B were purified from spleens of mice infected with MHV-OVA at least 35 days earlier. C and D, Lymphoid cell subsets were cocultured in vitro with 5 x 104 OT-I cells labeled with CFSE. After 60 h of culture, the number of proliferating OT-I cells were analyzed by flow cytometry. The total number of proliferating cells was determined by inclusion of 2 x 104 6 µm unlabeled counting beads (Spherobeads; BD Pharmingen) in each sample and 2000 beads were collected for each sample. Data are representative of three independent experiments with similar results.

 
Lack of Ag presentation by CD11c+ cells ablates MHV-specific CD8+ T cell proliferation in vivo

To test whether Ag presentation in persistently infected mice depends on CD11c-expressing cells in vivo, C57BL/6 recipient mice were lethally irradiated and reconstituted with bone marrow from CD11c-DTR transgenic mice (6). The bone marrow of these donor mice express the monkey diptheria toxin receptor under the control of the CD11c promoter, thereby rendering DTR-expressing CD11c+ DC sensitive to treatment with DT. Effective depletion could be traced by the loss of GFP-tagged CD11c+ DC. After 5–7 wk of reconstitution, bone marrow-chimeric mice were infected with MHV-OVA and allowed at least a further 4 wk for the virus to progress to latency. Chimeric mice were injected i.p. every second day with DT, to deplete DC, or PBS control. Twenty-four hours after the first DT treatment, naive OVA-specific CD8+ T cells were adoptively transferred into MHV-OVA-infected mice and proliferation of the cells was measured in the spleen and mediastinal LN 3 days later (Fig. 3, A–D). In mice that did not receive DT, robust OVA-specific T cell proliferation was apparent (Fig. 3, C and E). In the absence of CD11c+ DC (Fig. 3, D and F), OVA-specific T cell proliferation was substantially impaired, showing that {gamma}-HV infection depends on hemopoietically-derived DC, and that stromal cells are not sufficient to drive continued proliferation of CD8+ T cells in persistently infected mice.


Figure 3
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  		FIGURE 3. CD11c+ cells are the major cells presenting Ag during persistent MHV-OVA infection. A, Schematic representation of experimental approach for adoptive transfer of CD8+ T cells. CD11c-DTR->C57BL/6 bone marrow chimeric mice were generated and after at least 5 wk, mice were infected intranasally with MHV-OVA. Mice were then allowed a further 40 days to establish latent infection. One day before adoptive transfer of CFSE-labeled naive Ly5.1+ CD8+ OT-I cells (106), mice were treated i.p. with diptheria toxin to deplete CD11c+ DC. At 96 h after transfer of cells, tissues were removed, a single cell suspension was created, and cells were stained with anti-CD8{alpha}-allophycocyanin and anti-Ly5.1-PE Abs. Cells were analyzed for proliferation of CD8+ T cells by loss of CFSE fluorescence in the FL1 channel of a BD LSR flow cytometer. B–D, A total of 1 x 106 CFSE-labeled naive CD8+ OT-I T cells were adoptively transferred into CD11c-DTR->C57BL/6 bone marrow chimeras infected with MHV-OVA 40 days previously. Chimeras were treated with DT, or control PBS, commencing 1 day before T cell transfer. The proliferation of OVA-specific CD8+ T cells was monitored in the spleen 3 days after transfer. E and F, Scatter plots showing the presence of CD11c+ DC (monitored by expression of GFP) in undepleted (PBS treated) mice (E), and the efficiency of depletion of CD11c+ cells in DT treated mice (F). Data represent a total of four mice per group from two experiments.

 
Absence of DC activation during persistent infection maintains DC ability to respond to exogenous Ags during {gamma}-HV infection

One notable feature of {gamma}-HV is that they have a lower frequency of CpG dinucleotides than would be predicted from analysis of the composition of the viral genome (47). This low frequency of CpG dinucleotides may limit inflammatory activation of lymphoid cell, such as DC and B cells. Such a scenario would allow covert persistence of the virus and ensure DC are maintained in an immature form. To test whether MHV-OVA infection matures DC, DC were isolated from naive mice, mice persistently infected with MHV-OVA, and mice treated with 1 µg LPS 24 h before harvesting the spleen. DC were then stained with Abs to the surface molecules CD40, CD80, CD86, and MHC class II to determine whether persistent infection resulted in overt DC activation (Fig. 4A). Interestingly, the levels of expression of these molecules were similar to that shown for DC isolated from naive mice. Similar levels of activation were observed when bone marrow cells were infected in vitro with MHV-OVA. In contrast, influenza-infected DC efficiently up-regulated activation markers and more closely resembling LPS-treated DC (Fig. 4B).


Figure 4
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  		FIGURE 4. Persistent infection with MHV-OVA does not induce maturation of splenic DC. A, Surface expression of MHC class II (MHC II), MHC class I (H-2Db), CD80, CD86, and CD40 in splenic CD8{alpha} and DN DC from naive (black dotted line), MHV-OVA infected (40 days, red line), and LPS-treated (24 h before, gray line) mice. Results are representative of three independent experiments. B, Surface expression of activation markers in bone marrow-derived DC on day 8 of stimulation with FLT3L. DC were left uninfected (black dotted line), treated with LPS (gray line), or infected with 5 plaque-forming units (PFU) MHV-OVA (red line) or influenza virus (blue line). Profiles are representative of two independent FLT3L-bone marrow cultures.

 
Flano et al. (48) have previously reported that murine {gamma}-HV may have developed strategies similar to other pathogens for disrupting DC function (49, 50). They suggest that murine {gamma}-HV infected DC have an increased propensity to secrete IL10, a cytokine known to suppress cellular responses. Although abrogation of CD8+ T cell responses can occur in response to IL10 in vitro, in Epstein-Barr viral infection CD8+ T cell function is unaffected by this cytokine in vivo (51). In separate studies, we have shown that stimulation of the activation program in DC, such as might occur during acute or persistent viral infection, down-regulates the ability of those cells to cross-present subsequently encountered Ags (52). Despite this, we have found little evidence that T cell priming is impaired in {gamma}-HV (22). In fact, our current findings show that persistent Ag presentation appears to be important for maintaining proliferation of T cells during persistent infection. This raises the question of whether common immune evasion programs, such as enhanced IL10 production (53), or impaired Ag presentation induced by DC activation (52) that might be activated during {gamma}-HV infection, impair other immune responses in vivo. To examine this question, we determined whether {gamma}-HV-infected mice could respond to a model exogenous Ag, such as OVA, during persistent infection. Such Ags are presented by cross-presentation and require functionally intact DC to elicit CD8+ T cell responses (6). Mice that had been infected with {gamma}-HV 40 to 50 days previously were injected with H-2bm–1 splenocytes (that have the incorrect MHC molecule to present the OVA peptide) loaded with OVA and CFSE-labeled CD8+ OT-I cells. After 72 h, the proliferation of the transferred T cells was analyzed by flow cytometry (Fig. 5). These data show that even in the face of widespread viral colonization, the ability to cross-present Ags is maintained.


Figure 5
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  		FIGURE 5. Cross-presentation is maintained during persistent {gamma}-HV viral infection in vivo. Mice infected with {gamma}-HV for 60 days (top panels) or left uninfected (lower panels) were given cell-associated OVA without CpG or with CpG (12 h before Ag) and 1 x 106 CFSE-labeled OT-I T cells. Sixty hours after transfer of OT-I cells, proliferation was assessed by flow cytometry. Data are representative of three experiments with at least two mice per group.

 
Despite the relatively high colonization of B cells in spleen by the virus, a trivial explanation for this outcome could be that only a relatively small number of DC, the main drivers of CD8+ T cell proliferation in this infection, are infected in the spleen at any time during viral persistence (54). Thus, the maintenance of cross-presentation of exogenous Ag during latency may not reflect the direct impact of viral infection on splenocytes. To circumvent such a problem, and to test more rigorously the ability of {gamma}-HV to impair Ag crosspresentation, we assessed the ability of DC isolated from spleens of naive mice to cross-present Ags following infection with {gamma}-HV or influenza. CD4 DC, a non-crosspresenting DC subset, and CD8 DC, the classical cross-presenting subset, were infected with either {gamma}-HV or influenza WSN-gB for 2 h, after which they were coated with OVA protein. Their capacity to present exogenous Ag was monitored by proliferation of CFSE-labeled OVA-specific CD8+ T cells (Fig. 6). In these experiments, the parent {gamma}-HV not expressing OVA was used to allow us to probe cross-presentation in infected cells exposed to exogenous OVA, while the recombinant MHV-OVA virus was used to confirm infectivity of DC. Similarly, influenza WSN-gB-infected DC were cocultured with OT-I cells to analyze cross-presentation, or alternately, gBT-I cells, to confirm the cells were infected and any lack of response could not be attributed to viral-induced death of DC. Strikingly, influenza virus rapidly down-modulated the ability of the CD8{alpha} DC, the prototypical crosspresenting DC, to present exogenous Ags, while {gamma}-HV infection did not significantly impair this process, at least under conditions of very short-term infection. As expected, CD4 DC did not cross-present exogenous OVA, but as with CD8 DC, they were able to present endogenously synthesized viral Ags showing that a loss of crosspresentation in influenza infection was mediated by the virus and not other factors.


Figure 6
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  		FIGURE 6. Crosspresentation is maintained in DC infected with {gamma}-HV, but is rapidly down-modulated in DC infected with influenza WSN-gB. Constitutively crosspresenting CD8{alpha} DC and non-crosspresenting CD4 DC were flow cytometrically purified from the spleens of naive mice. DC were infected with various combinations of {gamma}-herpesvirus (5 PFU/DC), influenza virus (5 PFU/DC), or no exposure to virus for 2 h before ± exposure to 30 ng/ml OVA for 40 min. Cells were washed twice in complete medium before coculture with 5 x 104 CFSE-labeled OVA or glycoprotein B specific CD8+ transgenic T cells. Data are representative of two similar experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
A defining property of protective CD8+ T cell immunity after acute viral infection or vaccination is the long-term persistence of Ag-specific T cells independent of Ag that can be rapidly recruited into an immune response on subsequent encounter with an Ag (55). The transition of naive T cells to a memory state after Ag encounter is thought to occur gradually as effector T cells develop the qualities of self-renewing memory T cells (56). In contrast, chronic infections often lead to the generation of CD8+ T cells that are characterized by varying degrees of functional impairment that sometimes lead to complete exhaustion of the T cell response (57, 58, 59). Similarly, adoptive transfer of T cells educated in chronically infected hosts into naive mice is poorly maintained and fails to undergo homeostatic proliferation (60). Interestingly, some persistent infections, such as murine {gamma}-HV infection, generate CD8+ T cells that appear to be completely functional throughout viral persistence (22, 61, 62). These Ag-specific CD8+ T cells frequently persist for prolonged periods at high levels and are maintained by increased division compared with rapidly cleared virus infections such as influenza (23).

Although prevention of signaling through molecules such as programmed death 1 have been shown to be important in generating protective CD8+ T cell memory, the mechanism(s) by which T cells are suboptimally signaled in persistent infection during the priming process in response to Ag remain poorly understood (59). Recently, we and others have demonstrated that professional APCs are critical to drive effective priming of T cells to acutely cleared viral and microbial infections (34, 63). Persistent pathogens, in contrast to acutely cleared pathogens, are often harbored in diverse cell types within the body. This raises the possibility that MHC class I molecules loaded with viral peptides expressed on nonprofessional APC could be sufficient to drive amplification of the more promiscuous memory cells. Indeed, in LCMV infection, non-DC, possibly stromal cells, carrying Ag drive significant expansion of adoptively transferred Ag-specific T cells suggesting that they provide a very significant source of Ag important in the maintenance of CD8+ T cell populations in chronic infection (23, 60, 64). To gain a clearer understanding of how Ag is delivered during a persistent infection, we examined whether CD11c+ DC and non-CD11c+ cells were involved in activating T cells during persistent {gamma}-HV infection. Our studies show that during persistent {gamma}-HV infection presentation of a lytic Ag relies largely on Ag presentation by DC to activate T cells. This is somewhat surprising given the high viral load present in non-DC, particularly B cells, in spleen. However, overall the frequency of infected cells (particularly DC) that are detected after 21 days of infection is low (54). Thus, it seems most likely that a significant fraction of the Ag presentation demonstrated in our assays must be attributable to cross-presentation of soluble or cell-associated Ag. The feature of DC to efficiently capture and cross-present Ags, together with our depletion studies (Fig. 3), highlights the importance of the crosspresentation pathway to the activation and maintenance of T cells during viral persistence. The expression of viral molecules, such as murine K3, that specifically target MHC class I Ag presentation, could also bias long-term Ag presentation toward cross-presentation of acquired Ags. Further studies will be required to clarify the role of B cells and macrophages in driving ongoing T cell and B cell immune responses during persistent infection (65).

During persistent {gamma}-HV infection activation of "newly" recruited T cells is maintained by DC in the secondary lymphoid tissues associated with the lung, kidney, and spleen, that harbor persistent virus. It is presentation by these DC that provides the source of Ag likely to be responsible for driving extensive cellular division during infection (26). Despite this, the number of virus-specific CD8+ T cells remains relatively stable, and these cells retain effector functions (22, 66). However, poor responses were elicited when persistently infected mice were challenged with a second virus, namely vaccinia virus (which itself encodes evasion molecules) expressing {gamma}-HV Ags (22, 66). This suggests that dysregulation of the recall response may be a feature of the challenge virus, potentially impairing DC to significantly impact on the development and maintenance of CD8+ T cells even when Ag is presented on a professional APC.

The cardinal features of DC that can provide the signals necessary to induce efficient activation of T cells are denoted by the high cell surface expression of MHC molecules, CD80, CD86 and CD40. The expansion of Ag-specific T cells in the absence of appropriate costimulation could underlie CD8+ T cells functional impairment or exhaustion seen during persistent LCMV infection (67). Some evidence for {gamma}-HV interference with costimulatory molecules has already been observed in DC infected with {gamma}-HV in vitro (48). Infection with murine {gamma}-herpesvirus in vivo results in virtually undetectable activation of DC. However, this infection does not culminate in loss of virus-specific CD8+ T cell during persistence (68). This apparent absence of activation contrasts markedly with the rapid induction of maturation markers when an acutely cleared pathogen, such as influenza virus, is used as the inoculum. These dynamics were further emphasized by the rapid shutdown of exogenous Ag uptake induced by short-term in vitro influenza, but not persistent {gamma}-HV, infection allowing the cross-presentation of exogenous Ags to be maintained to a significant degree in the latter infection. These data show that the limited capacity of MHV-68 to disturb DC function in persistent viral infection also enables the maintenance of the ability to respond effectively to exogenous Ags.


   Acknowledgments
 
We thank the staff of the Walter and Eliza Hall Institute Flow Cytometry Facility and Mary Camilleri for technical assistance.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was funded by the National Health and Medical Research Council (NHMRC) of Australia, a University of Melbourne Research Scholarship (to A.M. and C.M.S.), and an NHMRC C.J. Martin Fellowship (to C.M.S), a Harvard College and Howard Hughes Medical Institute Scholarship (to J.C), a Wellcome Trust Senior Overseas Fellowship (to G.T.B.), and Howard Hughes Medical Institute International Fellowship (to G.T.B.). Back

2 Current address: Harvard College, Cambridge, MA 02138, E-mail address: chow2{at}fas.harvard.edu Back

3 Address correspondence and reprint requests to Dr. Gabrielle T. Belz, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria, 3050, Australia. E-mail address: belz{at}wehi.edu.au Back

4 Abbreviations used in this paper: DC, dendritic cell; {gamma}-HV, gammaherpesviruses; MHV-68, murine {gamma}-HV 68; LCMV, lymphocytic choriomeningitis viral; PFU, plaque-forming units; ORF, open reading frame; DTR, diptheria toxin receptor; DN, double negative. Back

Received for publication July 23, 2007. Accepted for publication September 24, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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S. S. Cush and E. Flano
Protective Antigen-Independent CD8 T Cell Memory Is Maintained during {gamma}-Herpesvirus Persistence
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