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Journal of Virology, November 2008, p. 11016-11022, Vol. 82, No. 22
0022-538X/08/$08.00+0     doi:10.1128/JVI.00919-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

CD40 Engagement on Dendritic Cells, but Not on B or T Cells, Is Required for Long-Term Control of Murine Gammaherpesvirus 68{triangledown}

Francesca Giannoni,{dagger} Ashley Shea,{ddagger} Chandra Inglis,§ Lian Ni Lee, and Sally R. Sarawar*

Viral Immunology, Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121

Received 2 May 2008/ Accepted 25 August 2008


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ABSTRACT
 
CD4 T cells are not essential for primary clearance of replicating murine gammaherpesvirus 68 (MHV-68) but are required for effective long-term control. The virus reactivates in the lungs of major histocompatibility complex class II-deficient (CII–/–) mice that lack functional CD4 T cells. CD40 ligand (CD40L) is upregulated on activated CD4 T cells, and it is thought that CD40-CD40L interactions are an important component of CD4 T-cell help. Our previous studies have shown that agonistic antibodies to CD40 can substitute for CD4 T-cell function in the long-term control of MHV-68. In the present study, we sought to identify the CD40-positive cell type mediating this effect. To address this question, we adoptively transferred MHV-68 peptide-pulsed CII–/– dendritic cells (DC) that had been treated with an agonistic antibody to CD40 into MHV-68-infected CII–/– recipients. Viral reactivation was significantly lower in mice injected with anti-CD40-treated DC than in those injected with control DC or in mice that did not receive any DC. However, in similar experiments with B cells, anti-CD40 treatment had no effect. We also investigated the requirement for CD40 expression on T cells by adoptive transfer of T cells from CD40+/+ or CD40–/– mice into T-cell-deficient recipients that were subsequently infected with MHV-68. The results showed that CD40 expression on T cells is not necessary for preventing viral reactivation. Taken together, our data suggest that CD40 engagement on DC, but not on T or B cells, is essential for effective long-term control of MHV-68.


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INTRODUCTION
 
Murine gammaherpesvirus 68 (MHV-68) is a natural rodent pathogen (4) which is closely related to gammaherpesviruses of primates, Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, and herpesvirus saimiri (12, 13, 37). Intranasal inoculation of MHV-68 induces a primary infection characterized by acute viral replication in lung epithelial cells and a persistent latent infection in various cell types, including B lymphocytes, lung epithelial cells, dendritic cells (DC), and macrophages (14, 32-35, 38). Clearance of replicating virus occurs by days 10 to 13 after infection and is mediated by cytotoxic T cells (CTL) through a perforin- or Fas-dependent mechanism (34, 36).

CD4 T-cell help is dispensable for the clearance of infection by CTL activity but is required for long-term control. Thus, major histocompatibility complex (MHC) class II-deficient (CII–/–) mice, which lack functional CD4 T cells, or mice depleted of CD4 T cells by antibody treatment are still able to control the primary acute infection, but virus later reactivates in the lungs (8). Viral titers gradually increase in the lungs, resulting in chronic lung damage and death. The failure of CD8 T cells to control gammaherpesvirus replication in this mouse model in some ways parallels the situation in immunocompromised humans lacking effective CD4 T-cell function, such as AIDS patients or transplant recipients. In these patients, gammaherpesviruses are implicated in the development of diseases such as lymphoma, lymphoproliferative disease, and Kaposi's sarcoma, which are associated with declining CD4 T-cell help and DC function and a consequent loss of CD8 T-cell-mediated viral control (9, 10, 15, 22, 30, 39). It has been proposed that in CD4 T-cell-deficient mice, the absence of CD40 engagement could be responsible for defective long-term control of MHV-68. Thus, like CD4 T-cell-deficient mice, MHV-68-infected CD40–/– and CD40 ligand-deficient (CD40L–/–) mice are able to clear the primary infection but fail to maintain long-term control of the virus (6, 19). These data suggest that the interaction between CD40 and CD40L is a key costimulatory event during the development of the immune response to MHV-68.

In a previous report, we showed that treatment with an agonistic antibody to CD40 could substitute for CD4 T-cell help and was highly effective in preventing reactivation of murine gammaherpesvirus (MHV-68) in the lungs of CD4 T-cell-deficient mice. CD8+ T cells were essential for this effect, whereas virus-specific serum antibody was undetectable and gamma interferon production was unchanged (27). CD40 is expressed by a number of different cell types, such as mature DC and B cells and activated CD4 and CD8 T cells. CD40-stimulated DC or B cells have been shown to act as a conditioned bridge mediating CD8 T-cell activation in some models (2, 11, 17, 24, 28, 29), whereas in another published report, CD40 expression on CD8 T cells themselves was essential for activation of this cell type (5). Therefore, it was unclear which CD40+ cell type mediated the effect of agonistic antibody to CD40 on MHV-68 reactivation in CD4 T-cell-deficient mice.

In this study, to further our understanding of the role of CD40-CD40L interaction during MHV-68 infection, we sought to determine which cell types expressing CD40 were able to mediate the effect of anti-CD40 antibody in vivo in the long-term control of the virus.


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MATERIALS AND METHODS
 
Mice. Age-matched 6- to 12-week-old female mice were used in all experiments. Immunodeficient mice were housed under specific-pathogen-free conditions. C57BL/6 mice that were homozygous for a disruption in the H-IAb gene (CII–/– mice) (7) were obtained from breeding colonies maintained at the Torrey Pines Institute for Molecular Studies (TPIMS) or were purchased from Taconic Farms. C57BL/6J, Rag1–/– (Rag1tm1Mom), and CD40–/– (B6.129P2-Tnfrsf5tm1kik) mice were obtained from The Jackson Laboratory. Athymic nude mice (B6.Cg/NTac-Foxn1nuN9) were obtained from Taconic Farms. Mice were bred and housed under specific-pathogen-free conditions in the vivarium at TPIMS. Immunodeficient mice were also maintained in sterile caging. In order to reduce the occurrence of secondary bacterial infections, CII–/–, athymic nude, and Rag1–/– mice were treated with antibiotics (sulfamethoxazole and trimethoprim suspension [40 mg and 8 mg, respectively, per 100 ml in drinking water]) throughout the course of the experiment. We previously determined that this treatment does not affect viral reactivation.

Viral infection and sampling. MHV-68 was propagated in BHK-21 cells (ATCC CCL-10). Mice were infected intranasally with 2 x 104 PFU of the virus in phosphate-buffered saline (PBS). Mice were lightly anesthetized and bled via the retrobulbar sinus of the eye at various times after reconstitution and/or infection. At day 50 after infection, mice were terminally anesthetized with Avertin. The lungs were removed and homogenized in medium on ice prior to virus titration. The inflammatory cells infiltrating the lungs were harvested by bronchoalveolar lavage via the trachea, and single-cell suspensions were prepared from the spleen as previously described (1).

Generation of bone marrow-derived DC (BMDC) and treatment with agonistic anti-CD40 antibody. A hybridoma secreting FGK45, an agonistic antibody to CD40 (26), was kindly provided by F. Melchers and A. Rolink. FGK45 was purified from culture supernatants by protein G-Sepharose affinity chromatography. Immature DC were generated from bone marrow precursors as previously described (18). Briefly, DC precursors were isolated from the bone marrows of CII–/– mice by flushing femurs and tibias with ice-cold Click's medium supplemented with 10% fetal bovine serum, glutamine, penicillin, and streptomycin.

Red blood cells (RBC) were lysed by using Tris-ammonium chloride lysis buffer. White cells were washed three times and incubated with anti-CD4, anti-CD8, anti-CD19, anti-CD14, and anti-GR1 in complete Click's medium for 30 min at 4°C. The cells were then washed and resuspended in Hanks' buffered salt solution. CD11c+ DC were isolated by negative selection using anti-rat immunoglobulin G (IgG)-coated magnetic beads (Dynalbeads; Invitrogen).

DC were plated in 24-well plates at 5 x 105 cells/well in RPMI 1640, a 10% fetal calf serum complete medium, supplemented with 15 µg/ml granulocyte-macrophage colony-stimulating factor (GM-CSF). On days 2 and 4, supernatant containing nonadherent cells was replaced with fresh medium containing GM-CSF. By day 5, adherent immature DC had formed in culture. FGK45, an agonistic antibody to CD40, was added to the cultures at a concentration of 20 µg/ml and incubated for an additional 24 h. Other DC cultures were either left unstimulated or treated with 20 µg/ml rat Ig as negative controls. No difference was noted between the latter two treatments. On day 6, FGK45-treated and control DC were collected, washed, and pulsed with MHV-68 epitope peptides p56 and p79 (20, 31). DC were incubated for 2 h at 37°C with RPMI 1640 medium containing p56 and p79 at 1 µg/ml, washed three times, and resuspended in PBS at 106 cells/ml for injection. DC (5 x 105/mouse) were injected intraperitoneally into CII–/– mice on days 1 and 15 after infection with MHV-68.

Purification of resting B cells. Resting B cells were purified from the spleens of naive CII–/– mice. Single-cell suspensions were made by pressing tissue through a cell strainer. RBC were depleted by hypotonic lysis, using RBC lysis buffer as described above. Total B cells were isolated by incubating spleen cell suspensions with anti-CD8, anti-CD4, anti-CD14, and anti-CD11b antibodies (BD Pharmingen) for 30 min at 4°C. Cells were then washed twice in PBS and negatively selected using anti-rat Ig-coated magnetic beads (Dynalbeads; Invitrogen). Resting B cells were then isolated by removal of activated B cells by staining with anti-CD43 and negative selection using Dynalbeads. The B220+ CD43– phenotype of resting B cells was confirmed by fluorescence-activated cell sorter (FACS) analysis. At least 5 x 106 resting B cells were incubated for 48 h in the presence of 20 µg/ml FGK45 (anti-CD40). On day 1 after MHV-68 infection, FGK45-treated B cells and freshly isolated resting B cells were pulsed with p79 and p56 MHV-68 epitope peptides for 2 h at 37°C. After being washed, 106 cells were injected intravenously (i.v.) into CII–/– recipient mice.

Adoptive transfer of T cells to athymic nude or Rag1–/– mice. T cells were isolated from spleen suspensions from CD40+/+ or CD40–/– C57BL/6 donor mice and injected i.v. into athymic nude or Rag1–/– mice. RBC-depleted spleen cell suspensions were incubated with the following antibodies for 30 min at 4°C: anti-CD11c, anti-CD19, and anti-CD14 (BD Pharmingen). Cells were then washed twice in PBS, incubated with anti-rat Ig-coated magnetic beads (Dynalbeads; Invitrogen) for 30 min, and negatively selected using a magnet. CD8+ and CD4+ T-cell phenotypes were confirmed by FACS analysis. T cells (107) were then injected i.v. into recipient mice. Reconstitution of the T-cell pool was verified 2 weeks after T-cell injection by FACS analysis of blood cells. Mice were infected when the T-cell pool reached about 70% that of C57BL/6 wild-type (WT) mice.

Flow cytometry. Before injection into recipient mice, DC and B- and T-cell phenotypes were analyzed by FACS. Cells were stained by incubation with different fluorochrome-conjugated antibodies in PBS supplemented with 5% fetal calf serum for 30 min. Cell acquisition was performed using a FACSCalibur flow cytometer (Becton Dickinson), and data analysis was performed using Cellquest software (Becton Dickinson).

Quantification of lung viral titers. Titers of replicating virus in lung homogenates were determined by plaque assay on NIH 3T3 cells (ATCC CRL1658) as described previously (8). The detection limit of this assay is 10 PFU/ml of a 10% tissue homogenate, based on plaques recovered from homogenates of uninfected tissues spiked with known amounts of virus.

ELISA for virus-specific antibodies. Serum antibody titers were determined by enzyme-linked immunosorbent assay (ELISA) as described previously (27), using a peroxidase-labeled secondary reagent from Southern Biotechnology (Birmingham, AL). Sera from uninfected mice and positive control sera were included in each assay. The absorbance was measured at 405 nm.

Statistical analysis. Differences between experimental groups were evaluated by either Student's t test or the Mann-Whitney rank sum test, depending on whether the data were normally distributed. Differences were considered statistically significant for P values of <0.05.


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RESULTS
 
Requirement for CD40 expression on T cells in the long-term control of MHV-68. A study by Bourgeois et al. (5) showed that CD4 help for CD8 T cells could be provided by a direct interaction of CD4 and CD8 T cells through CD40L-CD40 ligation (5). Thus, CD40 expression on CD8 T cells was essential for the generation of memory cells to the male histocompatibility (HY) antigen. During MHV-68 infection, activated CD8 T cells in the mediastinal lymph node upregulate CD40 on their surfaces by day 4 after infection (F. Giannoni, unpublished data). Hence, we sought to determine whether CD40 engagement on the surfaces of CD8 T cells was necessary for effective long-term control of MHV-68.

To address this issue, we reconstituted the T-cell pool of athymic syngeneic nude mice (nude Foxn1nu mice) by using T cells from either CD40+/+ or CD40–/– C57BL/6 donor mice. The level of T-cell repopulation was tested in peripheral blood mononuclear cells (PBMC) at intervals after injection. When at least 70% of the T-cell number observed in WT mice was reached, recipient mice were infected with MHV-68. CD40–/– mice and unreconstituted nude mice were also infected as controls (the latter mice died 2 to 3 weeks after infection, as expected). At day 50 after infection, we examined viral reactivation by measuring the viral titers in lung homogenates.

As shown in Fig. 1A, there was no statistically significant difference in titer between the nude mice reconstituted with CD40+/+ and CD40–/– T cells. Both groups of mice showed very low viral titers, indicating that CD40 expression on T cells is not essential for long-term control of MHV-68. In contrast, control CD40–/– mice (which lacked CD40 on all cell types, not just the T cells) showed significantly higher viral titers than those of nude mice reconstituted with either CD40–/– or CD40+/+ T cells (P < 0.001).


Figure 1
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  		FIG. 1. Expression of CD40 on T cells is not required for effective long-term control of MHV-68 or for antiviral antibody production. Athymic C57BL/6 nude mice (nude Foxn1nu mice) were reconstituted with syngeneic CD4 and CD8 T cells from either CD40+/+ or CD40–/– C57BL/6 mice. Mice were then infected intranasally with 2 x 104 PFU of MHV-68. CD40–/– C57BL/6 mice were also infected as controls (CTR). (A) At day 50 after infection, viral titers in lung homogenates were measured by plaque assay. Data are expressed as PFU/0.1 g lung tissue for individual mice. The results shown are from two individual experiments with four mice per group. There was a statistically significant difference in viral titers of nude mice reconstituted with either CD40+/+ or CD40–/– T cells and those of CD40–/– control mice (P < 0.001; Student's t test). (B) Anti-MHV-68 specific antibody titers (total Ig) were determined by ELISA, using sera from infected animals and controls, at various times after the infection. Sera from four individual mice at each time point were tested. Data are expressed as mean absorbance values ± standard deviations. The results shown are representative of two independent experiments which gave similar results. There was a statistically significant difference in serum antibody titers of nude mice reconstituted with CD40–/– T cells and those of CD40–/– control mice (P < 0.001; Student's t test).

T-cell phenotypes were also analyzed by FACS on day 50 after infection. In PBMC of reconstituted nude mice, the percentage of CD8 T cells was slightly higher than that for the control mice, while the percentage of CD4 T cells was similar to that of control mice (data not shown).

Antiviral antibody production in T-cell-reconstituted athymic nude mice. Although nude mice have B cells, they do not make an effective antiviral antibody response due to a lack of CD4 T-cell help. To determine whether antiviral antibody responses were restored in nude mice reconstituted with CD40+/+ or CD40–/– T cells, anti-MHV-68 specific antibody titers (total Ig) were determined by ELISA, using sera from infected animals (and controls), at various intervals after infection. Nude mice reconstituted with either CD40+/+ or CD40–/– T cells showed a significant virus-specific antibody response to MHV-68 (Fig. 1B). There was no significant difference between antiviral antibody titers in the two groups of T-cell-reconstituted mice. CD40–/– T-cell-reconstituted nude mice showed significantly higher antiviral antibody titers than those of CD40–/– control mice (P < 0.001). The latter showed very low antibody titers that decreased progressively from day 15 to day 50 and were not significantly different from the titers in naive mice.

These results show that CD40 expression on T cells is not essential for antiviral antibody production. Thus, in the nude mice reconstituted with CD40–/– T cells, long-term control of MHV-68 could be mediated by either antibody or T-cell-dependent mechanisms.

CD40 expression on T cells is not required for long-term control of MHV-68 in the absence of an antiviral antibody response. To examine the capacity of CD40–/– T cells to mediate effective long-term control of MHV-68 in the absence of an effective antiviral antibody response, we used Rag1–/– mice, which lack both T and B cells. Rag1–/– mice were reconstituted with T cells from either CD40+/+ or CD40–/– C57BL/6 mice. As controls, we used CD40–/– and WT CD40+/+ C57BL/6 mice. Reconstitution was evaluated in PBMC at various times after injection, and mice were infected with MHV-68 when the T-cell pool reached at least 70% of the level in a WT mouse. Unreconstituted Rag1–/– mice were infected as controls. These mice died 2 to 3 weeks after infection. On day 50 after infection, viral reactivation was examined by measuring the viral titers in lung homogenates.

As shown in Fig. 2A, the majority of both CD40–/– and CD40+/+ T-cell-reconstituted mice showed low or undetectable levels of virus in the lung, comparable with the levels in C57BL/6 WT control mice. There was no significant difference in the lung virus titers for Rag–/– mice reconstituted with CD40–/– and CD40+/+ T cells, while in CD40–/– mice the viral titers were significantly higher than those in either group of reconstituted Rag1–/– mice or in WT mice (P < 0.01 for each group; Mann-Whitney rank sum test).


Figure 2
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  		FIG. 2. CD40 expression on T cells is not required for the long-term control of MHV-68 in the absence of an antiviral antibody response. (A) Rag1–/– mice were reconstituted with CD4 and CD8 T cells from either CD40+/+ or CD40–/– mice and then infected intranasally with MHV-68. Viral titers in lung homogenates were determined on day 50 after infection. Data are expressed as PFU/0.1 g lung tissue for individual mice. The results shown are from two individual experiments with four or five mice in each group. Groups of two or three WT mice were also infected as controls. There was a statistically significant difference between lung viral titers of Rag1–/– mice reconstituted with either CD40+/+ or CD40–/– T cells and those of CD40–/– control mice (P < 0.01; Mann-Whitney rank sum test). (B and C) Anti-MHV-68 specific antibody titers were determined by ELISA, using sera from infected animals and controls, on day 15 or 50 after infection. Data are expressed as mean absorbance values ± standard deviations (error bars). The results shown are representative of two individual experiments with four or five mice in each group.

To evaluate the level of T-cell repopulation upon infection, lymphocyte phenotypes were analyzed by FACS on day 50 after infection (data not shown). None of the Rag1–/– reconstituted mice showed any evidence of contaminating B cells (CD19+), either in the peripheral blood or in the spleens or bronchoalveolar lavage fluid, while the percentages of CD4 and CD8 T cells were not significantly different in Rag1–/– mice that received CD40+/+ or CD40–/– T cells (data not shown).

Although no B cells were detected in the T-cell-reconstituted Rag1–/– mice, in order to completely exclude the possibility that viral clearance was mediated by virus-specific antibody responses, anti-MHV-68 specific antibody titers were determined by ELISA, using sera from infected animals, at days 15 and 50 after infection. Fifteen days after infection, both CD40+/+ and CD40–/– T-cell-reconstituted Rag1–/– mice showed minimal virus-specific antibody titers comparable with those of naive WT C57BL/6 mice and infected CD40–/– mice (Fig. 2B). Similarly, no virus-specific antibody was observed in CD40–/– T-cell-reconstituted Rag mice when the sera were analyzed 50 days after infection (Fig. 2C). Thus, CD40 expression on T cells is not essential for long-term control of MHV-68 in the absence of an antiviral antibody response.

CD40 ligation on B cells does not restore the long-term control of MHV-68 or antiviral antibody production in MHC class II-deficient mice. Since we had determined that CD40 expression on T cells is not required for the long-term control of MHV-68 in either the presence or absence of an antibody response, we then investigated which other cell type could be the target of the anti-CD40 antibody in vivo. We postulated that treatment with anti-CD40 agonistic antibody acts via CD40 expressed on antigen-presenting cells (APCs).

We first analyzed the role of CD40 engagement on B cells in the control of viral reactivation, as anti-CD40-treated B cells have been shown to be highly effective APCs in other mouse models (25, 29). Resting splenic B cells were isolated and treated with FGK45 (anti-CD40) in vitro for 48 h. Anti-CD40-treated or control resting splenic B cells were pulsed with p56 and p79 MHV-68 epitope peptides and adoptively transferred to CII–/– mice 1 day after infection with MHV-68. T cells responding to the p56 and p79 epitopes are prominent during MHV-68 infection, and vaccination against the p56 epitope has been shown to induce a protective CD8 T-cell response (20, 31). A third group of control mice was infected but did not receive any cells. Immediately before injection into mice, the phenotypes of the treated and control B cells were analyzed by FACS staining. As shown in Fig. 3A, anti-CD40 treatment induced upregulation of activation markers and costimulatory molecules (CD69, CD80, and CD86) on CD19+ B cells, demonstrating the functionality of the anti-CD40 antibody. However, levels of CD70 were similar in control and anti-CD40-treated B cells.


Figure 3
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  		FIG. 3. Adoptive transfer of anti-CD40-treated B cells does not restore antiviral antibody production or long-term control of MHV-68 in CII–/– mice. Resting B cells (CD19+ CD43–) were purified from spleens of CII–/– donor mice and incubated with anti-CD40 antibody for 48 h. Freshly purified, unstimulated resting B cells were used as negative controls. (A) The expression of costimulatory molecules was analyzed by FACS. The percentage of CD19+ B cells that expressed CD80 and CD86 was significantly higher when B cells were incubated with anti-CD40 antibody than in untreated resting B cells (P < 0.05; Student's t test). CII–/– mice were injected i.v. with anti-CD40-treated or resting B cells and infected with 2 x 104 PFU of MHV-68. Untreated CII–/– mice were also infected as controls (CTR). (B) MHV-68 titers in lung homogenates were determined by plaque assay at day 50 after infection. Data are expressed as PFU/0.1 g lung tissue for individual mice. Error bars indicate standard deviations. The results shown are derived from two independent experiments with five mice in each group. There was no statistically significant difference between the groups of mice. (C) Anti-MHV-68 specific antibody titers were determined by ELISA at day 50 after infection. Data are expressed as mean absorbance values ± standard deviations. The results shown are representative of two individual experiments with five mice in each group.

The mice were sacrificed 50 days after infection, and MHV-68 viral titers were determined in lung homogenates. As shown in Fig. 3B, there was no statistically significant difference between lung virus titers in mice that received anti-CD40-treated B cells and those that received resting B cells. Similarly, there was no significant difference between mice that received anti-CD40-treated B cells and the untreated control CII–/– mice. These data show that B-cell maturation through CD40 signaling is not sufficient to prevent viral reactivation in MHV-68-infected CII–/– mice.

To determine whether the transferred B cells were able to produce virus-specific antibody in the donor mice, MHV-68-specific antibody titers were analyzed in mice that had received resting or anti-CD40-treated B cells. Sera were collected at day 50 postinfection, and antiviral antibody titers were determined by ELISA (Fig. 3C). In all three groups of mice, antibody titers were very low. There was no statistically significant difference between mice that received anti-CD40-treated B cells and those that received resting B cells or between mice that received anti-CD40-treated B cells and the untreated CII–/– mice. Neither the anti-CD40-treated B cells nor the resting B cells were able to restore anti-MHV-68 antibody production in CII–/– mice. Taken together with the data from the T-cell-reconstituted Rag1–/– mice, these results indicate that CD40 engagement on B cells does not play a major role in enabling CD8 T cells to prevent MHV-68 reactivation.

Adoptive transfer of anti-CD40-treated DC restores the long-term control of MHV-68 in CII–/– mice. We then investigated whether the effect of anti-CD40 antibody could be mediated through the activation of CD40 on types of APCs other than B cells. Among other CD40-expressing APCs, DC are the most capable of priming of naive CD8 T cells (reviewed in reference 3). Hence, to establish whether CD40 engagement by anti-CD40 antibody on BMDC could restore the capacity of CD8 T cells to control viral reactivation, CD40-conditioned DC and control DC were adoptively transferred into CII–/– mice previously infected with MHV-68.

CD11c+ DC precursors were isolated from bone marrows of donor CII–/– mice by negative selection using magnetic beads and were cultured for 5 days in the presence of GM-CSF. Immature DC were then harvested from cultures and incubated with anti-CD40 antibody for 24 h. Isolation and anti-CD40 treatment of DC, using a similar protocol, have been shown previously to convert DC into excellent stimulators of CTL responses against tumor cells and cells expressing the HY antigen (17, 24). After incubation, DC were pulsed with p56 and p79 peptides and injected into mice that had been infected with MHV-68 the day before. Another injection of DC was performed at day 15 after infection.

Before injection into recipient mice, the DC phenotypes were analyzed by FACS staining. After 6 days in culture, 80 to 90% of bone marrow cells were CD11c+. Compared with the untreated CD11c+ cells, the anti-CD40-treated CD11c+ cells showed upregulation of costimulatory molecules (CD80 and CD86) (Fig. 4A). The number of CD11+ cells expressing the CD70 costimulatory molecule was significantly higher when BMDC were treated with anti-CD40 than that for untreated CD11c+ cells (P < 0.001; Student's t test) (Fig. 4B). Furthermore, a population of CD11c+ cells that were doubly positive for CD86 and CD70 and for CD80 and CD70 was detected in anti-CD40-treated but not control cultures of BMDC. Thus, CD70 was upregulated only on cells that were CD86high and CD80high (data not shown).


Figure 4
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  		FIG. 4. Adoptive transfer of anti-CD40-treated DC restores the long-term control of MHV-68 in CII–/– mice. After 5 days in culture, BMDC were incubated with anti-CD40 antibody; unstimulated or rat Ig-treated BMDC were used as negative controls. After 24 h, the expression of maturation markers was analyzed by FACS. (A) Anti-CD40 treatment significantly increased the percentages of cells expressing high levels of CD80 and CD86 (P = 0.012 and 0.018, respectively). (B) The percentage of CD11c+ cells, expressing high levels of the CD70 costimulatory molecule, was significantly higher in the anti-CD40-treated group than in the control group (P < 0.001). (C) CII–/– mice were infected with 2 x 104 PFU of MHV-68. On days 1 and 15 after infection, mice were injected intraperitoneally with anti-CD40-conditioned or control BMDC. CII–/– mice that did not receive any DC were also infected as controls. On day 50 after infection, viral titers in lung homogenates were determined by plaque assay. Data are expressed as PFU/0.1 g of lung tissue. The chart shows the averages for five experiments with five mice in each group. Error bars indicate the standard errors. There was a statistically significant difference in lung viral titers of mice that received anti-CD40-treated DC and those of mice that received control DC (P = 0.032; Student's t test).

On day 50 after infection, mice were sacrificed and viral titers in the lung homogenates were determined. The results are shown in Fig. 4C. Viral titers were significantly lower in mice that were injected with anti-CD40-conditioned DC than in those that received control DC (P = 0.032). In contrast, although titers appeared to be slightly reduced in mice that received control DC, they were not statistically significantly different from those of mice that did not receive DC. The mean titers for the control group in Fig. 4 were slightly higher than those in Fig. 3. However the ranges of titers in the two groups overlapped. Thus, the failure of anti-CD40-treated B cells to reduce viral reactivation could not be explained by possible differences in viral burden.

These results show that the likely target of anti-CD40 treatment in stimulating effective long-term control of MHV-68 is the DC. Thus, CD40-induced maturation of DC is required to activate CD8 T cells in a specific way that enables them to mediate effective long-term control of MHV-68.


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DISCUSSION
 
Previously published studies have shown that CD4 T cells are not required for primary clearance of infectious MHV-68 but are essential for the prevention of viral reactivation from latency (8, 33). In a previous study, we demonstrated that during MHV-68 infection of CII–/– mice, CD4 T-cell help could be replaced by the in vivo use of an agonistic antibody to CD40 (27). Since CD40 is expressed on several cell types, in the present study we investigated which cells mediate the effect of anti-CD40 antibody in vivo in preventing viral reactivation from latency in mice that lack CD4 T-cell help.

CD40 is expressed on DC, B cells, and macrophages but also on activated CD4 and CD8 T cells. Bourgeois et al. (5) proposed a model in which a direct interaction between CD40L-expressing CD4 T cells and CD40-expressing CD8 T cells was required for full T-cell activation. They demonstrated that CD40 expression on CD8 T cells was essential for the generation of memory cells to the male histocompatibility (HY) antigen. In our studies of MHV-68 infection, nude mice reconstituted with CD40–/– CD4 and CD8 T cells were able maintain effective long-term control of the virus, demonstrating that CD40 expression on T cells is not essential for viral control in this model. Since in these reconstituted nude mice CD40-CD40L interactions can occur between endogenous B cells and adoptively transferred CD4 T cells, normal titers of neutralizing anti-MHV-68 antibodies were produced. Hence, antibody production requires CD40 ligation on B cells, but CD40 expression on T cells is dispensable for this function.

To examine the requirement for CD40 expression on T cells in the absence of an anti-MHV-68 antibody response, we repeated the T-cell reconstitution experiment in Rag1–/– recipient mice, which lack both endogenous T and B cells, thus precluding an antiviral antibody response. As in the T-cell-reconstituted nude mice, Rag1–/– mice reconstituted with either CD40-positive or -negative T cells showed little or no viral reactivation. Therefore, even in the absence of an antibody response, CD40–/– CD8 T cells are still able to control viral reactivation.

An alternative model of CD40-mediated T-cell priming involves CD40L+ CD4 T cells that provide help by interacting with CD40 on bone marrow-derived APCs, which in turn become "licensed" to stimulate naive CD8 T-cell responses (2, 17, 24, 28). In our model of viral infection, treatment with anti-CD40 antibody might substitute for CD4 T-cell help by interacting with CD40 expressed on other cells, such as B cells and/or DC. Our previous data showed that anti-CD40 treatment did not restore the production of antiviral antibody in CD4-deficient mice (27), suggesting that if B cells mediated the effect of anti-CD40, they were likely to act via their function as APCs. In accordance with this, CD40-activated B cells have been shown to be highly effective APCs for the generation of autologous antigen-specific T cells for adoptive immunotherapy (25, 29). Hence, we first investigated the role of CD40 expressed on B cells. However, in our system, although we used similar conditions for CD40 stimulation of B cells to those used in previous studies (25, 29), adoptive transfer of these cells did not restore control of viral reactivation. Furthermore, as mentioned above, Rag1–/– mice that had been reconstituted with either CD40+/+ or CD40–/– T cells (but not B cells) were still able to maintain effective long-term control of MHV-68. Thus, it appears that CD40 stimulation of B cells is neither sufficient nor essential to prevent viral reactivation in this model.

Several previous studies have found that activation of DC via CD40 mediates CD4 T-cell help for CD8 T-cell effector functions (16, 17, 24). These studies showed that DC from CII–/– mice could not present antigen to CD4 T cells, but when they were stimulated by an agonistic anti-CD40 antibody, they were able to induce effective CTL responses by CD8 T cells. Hence, during MHV-68 infection in CII–/– mice, CD8 T-cell priming by DC could be affected by the lack of CD40 engagement, thus rendering the CD8 T cells unable to mount an effective recall response to control viral reactivation. In agreement with these studies, our data show that the adoptive transfer of in vitro CD40-conditioned BMDC pulsed with MHV-68 peptides reduces viral reactivation in CD4 T-cell-deficient mice. Thus, it appears most likely that DC are the major cell type mediating the effect of anti-CD40 on the long-term control of MHV-68 in CD4 T-cell-deficient mice.

When anti-CD40 was injected directly into mice, viral titers were reduced to levels similar to those in WT mice (27). However, adoptive transfer of in vitro CD40-conditioned DC appeared to induce a slightly lower reduction in viral titers. This was confirmed in an experiment in which i.v. anti-CD40 treatment and transfer of anti-CD40-treated DC were performed in parallel (data not shown). One explanation for the difference could be that the CD40-activated DC have to traffic to the lungs or to the draining (mediastinal) lymph node following intraperitoneal injection and may be present in smaller numbers at these sites than when anti-CD40 antibody is injected into mice and activates DC in situ. In addition, the DC were pulsed with only two peptide epitopes, whereas the entire spectrum of viral epitopes would be available in anti-CD40-treated MHV-68-infected mice. Furthermore, culturing the DC may have slightly compromised their efficacy.

Our data suggest that, consistent with previous studies (17, 23, 24), DC act as a conditioned bridge between CD40L on CD4 T cells (replaced by an agonistic antibody to CD40 in our model) and a CD8 T cell. CD40-mediated activation of the DC enables it to present antigen to and program the CD8 T cell, rendering it capable of long-term control of the virus. Since both DC and B cells have been reported to present lytic cycle MHV-68 epitopes to T cells (20), it was somewhat surprising that anti-CD40-treated B cells were unable to rescue CD8 T-cell function in our model. As mentioned above, studies with other models have shown that B cells activated by CD40 stimulation are highly effective APCs for the generation of antigen-specific CD8 T cells (25, 29). A possible explanation is that because the function of CD8 T cells depends on the net effect of multiple signals mediated by interaction with APCs, CD40-activated B cells may lack the costimulatory molecules that are necessary for activating CD8 T cells such that they are able to mediate effective long-term control of MHV-68. Our previous data have shown that CD80 and CD86 are important for long-term control of MHV-68 (21). However, anti-CD40 treatment upregulated these molecules on both B cells and DC, showing that differential upregulation of these molecules could not explain the difference between B cells and DC in priming of CD8 T cells. In contrast, CD70 was upregulated on DC, but not on B cells, following anti-CD40 treatment, and it is possible that this or other costimulatory molecules on DC are important for T-cell activation in the long-term control of MHV-68.

In conclusion, our studies show that DC are the most likely CD40+ target of anti-CD40 treatment that enables effective long-term control of MHV-68 by CD8 T cells in CII–/– mice. We also demonstrated that in vivo immunization with CD40-conditioned DC can be utilized to control this persistent gammaherpesvirus infection in the absence of T-cell help.


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ACKNOWLEDGMENTS
 
We thank M. Iversen, P. Dias, and A. Franco for help and advice.

This work was supported by NIH grant AI50810 and by a grant from the Infectious Disease Science Center.


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FOOTNOTES
 
* Corresponding author. Mailing address: Viral Immunology, Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121. Phone: (858) 909-5139. Fax: (858) 909-5141. E-mail: ssarawar{at}tpims.org Back

{triangledown} Published ahead of print on 3 September 2008. Back

{dagger} Present address: Division of Research Immunology and Bone Marrow Transplantation, CHLA, 4650 Sunset Blvd., Los Angeles, CA 90027. Back

{ddagger} Present address: Novartis, 500 Technology Square, Cambridge, MA 02139. Back

§ Present address: Department of Neuroscience, UCSD, 9500 Gilman Drive, #0624, La Jolla, CA 92093-0624. Back


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Journal of Virology, November 2008, p. 11016-11022, Vol. 82, No. 22
0022-538X/08/$08.00+0     doi:10.1128/JVI.00919-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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