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Journal of Virology, October 2005, p. 12798-12806, Vol. 79, No. 20
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.20.12798-12806.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

T-bet Is Required for Protection against Vaccinia Virus Infection

Masanori Matsui,1* Osamu Moriya,1 Takayuki Yoshimoto,2 and Toshitaka Akatsuka1

Department of Microbiology, Saitama Medical School, Saitama,1 Intractable Immune System Disease Research Center, Tokyo Medical University, Tokyo, Japan2

Received 13 June 2005/ Accepted 29 July 2005


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ABSTRACT
 
The transcription factor T-bet regulates the differentiation of CD4+ T-helper type 1 (Th1) cells and represses Th2 lineage commitment. Since Th1 cells are crucial in the defense against pathogens, several studies addressed the role of T-bet in immunity to infection using T-bet knockout (T-bet–/–) mice. Nevertheless, it is still unclear whether T-bet is required for defense. Although vaccinia virus (VV) has extensively been used as an expression vector and the smallpox vaccine, there is only limited knowledge about immunity to VV infection. The urgency to understand the immune responses has been increased because of concerns about bioterrorism. Here, we show that T-bet is critical in the defense against VV infection as follows: (i) the survival rate of T-bet–/– mice was lower than that of control littermates postinfection; (ii) T-bet–/– mice lost more weight postinfection; and (iii) control mice cleared VV faster than T-bet–/– mice. As expected, a significant Th2 shift was observed in CD4+ T cells of T-bet–/– mice. Furthermore, absence of T-bet impaired VV-specific CD8+ cytotoxic T-lymphocyte (CTL) function, including cytolytic activity, antiviral cytokine production, and proliferation. Cytolytic capacity of natural killer (NK) cells was also diminished in T-bet–/– mice, whereas anti-VV antibody production was not impaired. These data reveal that the enhanced susceptibility to VV infection in T-bet–/– mice was at least partially due to the Th2 shift of CD4+ T cells and the diminished function of VV-specific CTLs and NK cells but not due to downregulation of antibody production.


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INTRODUCTION
 
Naïve CD4+ T-helper precursor cells differentiate into either T-helper type 1 (Th1) or T-helper type 2 (Th2) cells, as judged by functional capacities and cytokine profiles (8). Th1 cells mainly produce gamma-interferon (IFN-{gamma}) and direct cell-mediated protective immunity against viruses and intracellular pathogens. Th2 cells secrete interleukin-4 (IL-4), IL-5, IL-10, and IL-13 and promote humoral responses against extracellular pathogens. Certain cytokines lead to the differentiation of these two T-cell subsets. The differentiation of Th2 is mediated by IL-4, which activates a signaling pathway of signal transducer and activator of transcription 6 (STAT-6). This signal induces the expression of the transcription factor GATA-3, which appears to be a master regulator in Th2 cells (36). On the other hand, the differentiation of Th1 cells is directed mainly by IFN-{gamma} and IL-12, which activate signaling pathways involving STAT-1 and STAT-4, respectively (32).

Recently, a T-box transcription factor, T-box expressed in T cells (T-bet), has been defined to transactivate the gene of the hallmark Th1 cytokine, IFN-{gamma} (30). T-bet is expressed in Th1 cells but not in Th2 cells, and ectopic expression of T-bet in Th2 cells results in IFN-{gamma} production and suppression of Th2-specific cytokines (30). In addition, T-bet knockout (T-bet–/–) mice are deficient in Th1 cells (31) and spontaneously develop multiple physiological and inflammatory features characteristic of asthma, which is a Th2-mediated disease (11). Furthermore, T-bet–/– mice are resistant to the development of Th1-mediated, experimental autoimmune encephalomyelitis (5) and Th1-mediated colitis (21), whereas these mice are more susceptible to Th2-mediated colitis than control littermates (21). Therefore, it is believed that T-bet is the master regulator of the Th1 development, similar to GATA-3 in Th2 cells. The expression of T-bet is dependent on the STAT-1 signaling pathway but independent of STAT-4 activation (1, 17).

Since Th1 cells along with IFN-{gamma} are essential for the defense against various pathogens, several studies addressed the role of T-bet in protective immune responses to infection using T-bet–/– mice. It was reported that T-bet–/– mice were more susceptible to infection with Leishmania major (L. major) (31), lymphocytic choriomeningitis virus (LCMV) (14, 28), and herpes simplex virus type 2 (HSV-2) (29) than control littermates. In contrast, T-bet was not necessary for host resistance to infection with Listeria monocytogenes (34) or murine cytomegalovirus (MCMV) (33). Thus, the requirement of T-bet is likely to vary in host defense to primary infection between various pathogens.

In addition to Th1 cells, CD8+ cytotoxic T lymphocytes (CTLs) play a crucial role in the course of adaptive Th1-mediated protective immunity. CTLs can also be divided into IFN-{gamma}-producing (Tc1) and IL-4-producing (Tc2) subsets (9). Therefore, it was previously investigated whether T-bet regulated the production of IFN-{gamma} in CD8+ CTLs. Surprisingly, the initial study demonstrated that the absence of T-bet did not alter IFN-{gamma} production in CD8+ T cells under polyclonal stimulation (31), indicating distinct effects of T-bet on IFN-{gamma} production within CD4+ and CD8+ T cells. It was then proposed that the other transcription factor, Eomesodermin, was likely to complement the actions of T-bet in the differentiation of CD8+ T cells (23). On the other hand, under antigen-specific stimulation, IFN-{gamma} production by virus-specific CD8+ T cells was greatly impaired in T-bet–/– mice infected with LCMV (14, 28) but not at all in T-bet–/– mice infected with Listeria monocytogenes (34) or HSV-2 (29). Furthermore, CD8+ T cells from T-bet–/– mice had a significantly higher specific killing capacity than CD8+ T cells from control mice in HSV-2 infection (29). Accordingly, it is still unclear whether T-bet regulates the function of antigen-driven effector CD8+ T cells.

Vaccinia virus (VV) is a large DNA virus and a member of the Orthopoxvirus genus in the Poxviridae family. VV has been used as an effective vaccine against variola virus, which is the cause of smallpox. VV has also been used as an expression vector for foreign genes in a great number of experimental systems (4). Recently, the threat of bioterrorism has raised concerns over the reemergence of smallpox, and therefore it is urgent to understand the mechanism of protective immunity and develop a new more effective vaccine against smallpox. It has been thought that both cellular and humoral immunity play a significant role in protection against VV infection (4). It was reported that innate immunity containing natural killer (NK) cells and {gamma}{delta} T cells was important in resistance to VV infection (15, 25). Furthermore, it was demonstrated that antivirus neutralizing antibodies (Abs) were more important in clearing VV than CTLs following acute infection, while in the absence of Abs, CTLs contribute to protection against VV infection (3, 35). However, there is still only limited knowledge about the protective immune responses against VV infection.

In the current study, we investigated whether T-bet was required for protection against VV infection using T-bet–/– mice. We report here that T-bet–/– mice are more susceptible to primary VV infection than control littermates. The increased susceptibility of T-bet–/– mice is supposed to be, at least in part, due to the impairment of both VV-specific CD8+ CTL function and NK activity and the Th2 shift of CD4+ T cells.


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MATERIALS AND METHODS
 
Cell lines. The mouse mastocytoma cell line P815 (H-2d), the Moloney leukemia virus-induced lymphoma YAC-1, the African green monkey-derived kidney cell lines CV-1 and BS-C-1, and the anti-mouse IL-4 hybridoma 11B11 (22) were obtained from the American Type Culture Collection (Rockville, MD). These cell lines were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum (FCS).

Vaccinia virus. The VV Western Reserve (WR) strain was kindly provided by T. Shioda (Osaka University, Osaka, Japan). Virus was propagated in CV-1 cells, and the titers were measured by standard plaque assays on BS-C-1 cells.

Mice and infection. T-bet–/– mice generated as described previously (31) were kindly provided by L. Glimcher (Harvard Medical School, Boston, Mass.) and were backcrossed eight generations to BALB/c (H-2d) mice. BALB/c mice were purchased from Japan Charles River (Yokohama, Japan) and were also used as controls. Six- to 8-week-old mice were used for all experiments. Mice were housed in appropriate animal care facilities at Saitama Medical School, Japan, and were handled according to international guidelines for experiments with animals.

Mice were intraperitoneally (i.p.) infected with a sublethal dose (5 x 106 PFU) of VV for all of the experiments except for those monitoring survival rates. To plot survival curves, eight mice in each group were infected i.p. with 5 x 107 PFU of VV and were monitored daily for mortality. Changes in body weight were calculated as the percentage of the mean weight of 8 to 12 mice per group in comparison with starting body weight. For neutralization of IL-4, mice were given 1 mg of either the anti-mouse IL-4 monoclonal antibody (MAb) (11B11) or control rat immunoglobulin G (IgG) (SIGMA, St. Louis, MO) in 0.5 ml of PBS by the i.p. route on days 2, 4, 6, 10, and 14 after infection as described previously (10). The 11B11 MAb was purified from ascites fluid by using ammonium sulfate precipitation.

Ovary VV titer assay. Mice were sacrificed on certain days after infection with 5 x 106 PFU of VV, and viral titers in mice were measured as described before (19). In brief, two ovaries of each mouse were homogenized and resuspended in 0.5 ml of phosphate-buffered saline (PBS) containing 1% FCS and 1 mM MgCl2. Virus was released from the cells by three freeze-thaw cycles followed by sonication. Virus titers were measured by plating serial 10-fold dilutions on BS-C-1 indicator cells in 6-well plates and staining with 0.1% crystal violet after 48 h. Six to 12 mice were used in each group. All titrations were performed in duplicate, and the average PFU per mouse was calculated.

Cytotoxic assay. At the indicated time points following infection with 5 x 106 PFU of VV, mice were sacrificed and spleen cells were prepared for effector cells. In some experiments, CD8+ T cells were isolated to a purity of more than 95% from the spleen cells by negative selection using the magnetic cell separation system (MACS; Miltenyi Biotec, Aubum, CA) according to the manufacturer's instructions and were used for effector cells. Cytotoxic activities of VV-specific CTLs and NK cells were measured in standard 51Cr release assays. For preparation of virus-infected targets in CTL assays, P815 cells were infected with VV at a multiplicity of infection (MOI) of 3 for 90 min at 37°C, washed three times, and incubated in RPMI 1640 containing 10% FCS (R-10) overnight at 37°C. For detection of NK cell-mediated lysis, YAC-1 cells were employed as targets. Target cells (1 x 106 cells) were labeled with 100 µCi of Na251CrO4 for 30 min at 37°C. After being washed three times, the labeled target cells were plated in wells of a round-bottom 96-well plate at 1 x 104 cells/well with or without effector cells at various effector-to-target (E:T) ratios. After a 4-h incubation at 37°C, supernatant from each well was harvested and the radioactivity was counted. The results were calculated as the mean of a triplicate assay. Percent specific lysis was calculated according to the following formula: % specific lysis = [(cpmsample – cpmspontaneous)/(cpmmaximum – cpmspontaneous)] x 100, where cpm is counts per minute. Spontaneous release represents the radioactivity released by target cells in the absence of effectors, and maximum release represents the radioactivity released by target cells lysed with 5% Triton X-100. At least three mice per group were used in each experiment. The experiment was repeated three times.

Detection of VV-specific antibodies. Titers of VV-specific serum antibodies were determined by a solid-phase enzyme-linked immunosorbent assay (ELISA) as described previously (2, 35) with slight modifications. In brief, VV-infected CV-1 cell lysate was diluted in 0.05 M carbonate-bicarbonate buffer (pH 9.6) at 5 x 107 PFU/ml. Each well of 96-well flat-bottom plates (Costar #3590; Corning, Corning, NY) was coated with 100 µl of the diluted cell lysate by incubation overnight at 4°C. The plates were then fixed with 2% paraformaldehyde and washed three times with PBS containing 0.05% Tween-20 (PBS-Tween). After blocking with 5% bovine serum albumin in PBS, 100 µl of diluted mouse serum was added to each well, and the plates were incubated for 1 h at 37°C. After being washed three times with PBS-Tween, horseradish peroxidase-conjugated goat anti-mouse IgM (SIGMA) or IgG (SIGMA), diluted 1:5,000 in blocking buffer, was added, and the plates were incubated for 1 h at 37°C. After being washed five times with PBS-Tween, 100 µl of o-phenylenediamine dihydrochloride (OPD) substrate (SIGMA) was added to each well. The plates were incubated at room temperature for 15 to 30 min. The reaction was stopped with 50 µl/well of 6 N H2SO4, and the plates were read at 492 nm. Four to six mice per group were used in the experiments.

Cytokine ELISA. Mice were sacrificed at day 8 following infection with 5 x 106 PFU of VV. CD4+ T cells and CD8+ T cells were purified to a purity of more than 95% from spleen cells of either naive mice or infected mice by negative selection using the CD4+ T-cell isolation kit and the CD8{alpha}+ T-cell isolation kit (MACS; Miltenyi Biotec) according to the manufacturer's instructions, respectively. Purified T cells were activated in vitro by either polyclonal stimulation or antigen-specific stimulation. For polyclonal stimulation, each well of a 96-well round-bottom plate was coated with hamster anti-mouse CD3{varepsilon} (clone 145-2C11; BD Biosciences, San Jose, CA) and anti-mouse CD28 (clone 37.51; BD Biosciences) MAbs at a final concentration of 1 µg/ml for 90 min at 37°C. After being washed with PBS three times, 4 x 105 purified T cells were added into each well and incubated for 2 days at 37°C. For antigen-specific stimulation, spleen cells of naive BALB/c mice were infected with VV at an MOI of 3 for 1 h at 37°C, irradiated at 20 Gy, and used as stimulator cells. Purified T cells (4 x 105/well) were then incubated with stimulator cells (1 x 106/well) in wells of a 96-well round-bottom plate for 2 days at 37°C. Culture supernatants in both polyclonal activation and antigen-specific activation were then harvested and were screened for the presence of various cytokines by ELISA. Capture Abs, biotinylated detection Abs, and recombinant cytokines were purchased from BD Biosciences. Quantitative ELISA for IFN-{gamma}, IL-4, IL-5, and IL-10 was performed using paired MAbs specific for corresponding cytokines according to the manufacturer's instructions. Briefly, ELISA plates were coated with capture Abs for the respective cytokines and incubated overnight at 4°C. The plates were washed with PBS-Tween and blocked with 10% calf serum in PBS for 2 h at room temperature. After being washed, serially diluted samples or recombinant standards were added to the plates and incubated at 4°C overnight. The plates were washed four times followed by the addition of cytokine-specific detection Abs. After 1 h of incubation at room temperature, horseradish peroxidase-conjugated avidin (BD Biosciences) was added. The color was developed by adding OPD substrate, and the reaction was stopped with H2SO4. The concentration of each cytokine was calculated by reading the plates at 492 nm. Data represent three to five mice per group and are given as mean values ± standard errors of the means (SEM).

Intracellular cytokine staining. Spleen cells of three to five mice per group infected with VV were pooled and were resuspended in R-10. For preparation of stimulator cells, P815 cells were infected with VV at an MOI of 3 for 90 min at 37°C, washed three times, and incubated in R-10 overnight at 37°C. In each well of a 96-well round-bottom plate, 2 x 106 spleen cells were incubated with 1 x 105 cells of either VV-infected P815 or naïve P815 in the presence of 0.2 µl/well brefeldin A (GolgiPlug; BD Biosciences) for 5 h at 37°C. The cells were then washed once with ice-cold fluorescence-activated cell-sorting buffer composed of PBS containing 1% FCS and 15 mM sodium azide and incubated for 10 min at 4°C with the rat anti-mouse CD16/CD32 MAb (Fc Block; BD Biosciences) at a concentration of 1 µg/well. Following incubation, cells were stained with fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD8{alpha} MAb (clone 53-6.7; BD Biosciences) at a concentration of 0.5 µg/well for 30 min at 4°C. After being washed twice with fluorescence-activated cell-sorting buffer, the cells were fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences) and stained with phycoerythrin (PE)-conjugated rat anti-mouse IFN-{gamma} (clone XMG1.2; BD Biosciences), anti-mouse tumor necrosis factor alpha (TNF-{alpha}) (clone MP6-XT22; BD Biosciences), or anti-mouse IL-2 (clone JES6-5H4; BD Biosciences) MAb according to the manufacturer's instructions. Isotype-matched control Abs were added to control wells to confirm the specificity of the staining. After removal of unbound antibody by washing with 1x Perm/Wash solution from the kit, flow cytometric analyses were performed. The experiment was repeated three times.

In vivo proliferation assay. Bromodeoxyuridine (BrdU) staining was performed using the FITC BrdU Flow kit (BD Biosciences) according to the manufacturer's instructions. Briefly, mice infected with VV were injected i.p. with 200 µl of BrdU solution in PBS (5 mg/ml) 1 day before sacrifice. After spleen cells were prepared, surface staining was performed as described above by incubation with PE-conjugated rat anti-mouse CD4 MAb (clone RM4-5; BD Biosciences) or rat anti-mouse CD8{alpha} MAb (clone 53-6.7; BD Biosciences) at a concentration of 0.5 µg/well for 15 min at 4°C. After being washed, spleen cells were fixed and permeabilized by Cytofix/Cytoperm solution and Cytoperm Plus buffer from the kit. After treatment with DNase to expose BrdU, cells were stained with FITC-conjugated anti-BrdU Ab for 20 min at room temperature. After removal of unbound antibody by washing with 1x Perm/Wash solution from the kit, flow cytometric analyses were performed. Three mice per group were used in the experiments. The experiment was repeated twice.

Statistical analyses. Statistical analyses were performed with Student's t tests. P < 0.05 was considered statistically significant.


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RESULTS
 
T-bet–/– mice are more susceptible to infection with VV than BALB/c mice. To assess whether the absence of T-bet has any effect on resistance to VV infection, T-bet–/– mice and control BALB/c mice were infected i.p. with 5 x 107 PFU of VV and were monitored daily for mortality. As shown in Fig. 1A, all T-bet–/– mice (n = 8) succumbed to the infection by day 3 postinfection, while five out of eight BALB/c mice survived the infection with a lethal dose by day 8. We next examined changes in body weight of mice injected with a sublethal dose (5 x 106 PFU) of VV. T-bet–/– mice lost an average of 7.5% of starting body weight at day 4 postinfection, whereas BALB/c mice did not show significant weight loss after infection (Fig. 1B). Furthermore, we determined viral load in ovaries of mice at days 7, 14, 21, and 28 after infection with 5 x 106 PFU of VV (Fig. 1C). T-bet–/– mice as well as BALB/c mice showed high viral titers at day 7 postinfection. T-bet–/– mice, however, harbored 10-fold more PFU than BALB/c mice at day 14. Furthermore, T-bet–/– mice still retained high virus titers at day 21 postinfection, whereas BALB/c mice completely cleared virus from ovaries by the same day. By day 28 postinfection, T-bet–/– mice cleared virus from ovaries as well. Taken together, these data demonstrate that T-bet–/– mice are more susceptible to VV infection than control BALB/c mice.



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  		FIG. 1. T-bet–/– mice are more susceptible to infection with VV than BALB/c mice. T-bet–/– mice and BALB/c mice were infected i.p. with either 5 x 107 PFU (A) or 5 x 106 PFU (B and C) of VV. (A) Eight mice per group were monitored daily for mortality. (B) Changes in body weight of T-bet–/– (solid symbols) and BALB/c (open symbols) mice were calculated as the percentage of the mean weight of 8 to 12 mice per group in comparison with starting body weight. The data are shown as the means ± SEM. (C) T-bet–/– (solid bars) and BALB/c (open bars) mice were sacrificed at days 7, 14, 21, and 28 postinfection, and virus titers in ovaries of mice were measured. Eight to 12 mice were used in each group, and all titrations were performed in duplicates. The data are shown as the mean PFU ± SEM. ND, not detected. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to BALB/c mice.

Cellular and humoral immunity to VV in T-bet–/– mice. Because CTLs play a role in protection against VV (3, 35), we tested whether lack of T-bet altered cytolytic activity of VV-specific CTLs in mice infected with 5 x 106 PFU of VV. As shown in Fig. 2A to C, it was found that VV-specific CTL activity in spleen cells of T-bet–/– mice was significantly less than that of BALB/c mice at days 7, 21, and 35 postinfection. To exclude the possibility of inhibitory effects by non-CD8+ cells, CD8+ T cells were purified from spleen cells by using the magnetic cell sorting system (MACS; Miltenyi Biotec) and were subjected to 51Cr release assays. Consistent with the data of bulk spleen cells (Fig. 2A to C), MACS-purified CD8+ T cells derived from T-bet–/– mice revealed lower VV-specific CTL activity than those from BALB/c mice at any time point after infection (Fig. 2D).



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  		FIG. 2. VV-specific CTL activity in T-bet–/– and BALB/c mice after VV infection. (A to C) At days 7 (A), 21 (B), and 35 (C) after infection with 5 x 106 PFU of VV, spleen cells were isolated from T-bet–/– mice (inverted triangles) and BALB/c mice (circles). 51Cr release assays were then performed to detect VV-specific CTL activity in bulk spleen cells at various E:T ratios, using P815 cells infected with (open symbols) or without (solid symbols) VV as targets. (D) CD8+ T cells were purified from spleen cells of T-bet–/– mice (solid bars) and BALB/c mice (open bars) at the indicated time points following infection with 5 x 106 PFU of VV. 51Cr release assays were performed to detect VV-specific CTL activity in CD8+ T cells at an E:T ratio of 50, using P815 cells infected with VV as targets. Significant CTL activity in CD8+ T cells was not detected when noninfected P815 cells were used as targets (data not shown). (A to D) The data are representative of one of three independent experiments and are shown as the means ± SEM. At least three mice per group were used in each experiment. *, P < 0.05; **, P < 0.01 compared to BALB/c mice.

It has recently been reported that the cytolytic capacity of NK cells was reduced in T-bet–/– mice after infection with MCMV (33). Therefore, NK cell-mediated lysis was compared between the two groups of mice infected with 5 x 106 PFU of VV. The cytolytic capacity of splenic NK cells in T-bet–/– mice was almost equal to that in BALB/c mice at day 1 postinfection (Fig. 3B), whereas a significant reduction of splenic NK cell activity was observed in T-bet–/– mice at day 2 postinfection (Fig. 3C). In spite of the discrepancy in NK cell-mediated lysis, there was no difference of viral titers in ovaries between the two groups of mice at days 1 and 2 postinfection (Fig. 3D).



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  		FIG. 3. NK cell-mediated cytolytic activity and virus titers in T-bet–/– mice at early days postinfection. (A to C) T-bet–/– (solid symbols) and BALB/c mice (open symbols) were infected i.p. with or without 5 x 106 PFU of VV, and at the indicated days postinfection spleen cells were prepared and used as effector cells in standard 51Cr YAC-1 cell-killing assays. The data are shown as the means ± SEM. The experiment was repeated three times with similar results, and at least three mice per group were used in each experiment. (D) T-bet–/– (solid bars) and BALB/c (open bars) mice were sacrificed at the indicated days pre- and postinfection, and viral titers in ovaries of mice were measured. Six to 10 mice were used in each group, and all titrations were performed in duplicate. The data are shown as the mean PFU ± SEM. ND, not detected. *, P < 0.05; **, P < 0.01 compared to BALB/c mice.

It was indicated that humoral immunity was essential to protect against VV infection (3, 35). In the following experiments, antiviral IgM and IgG antibodies were monitored in both T-bet–/– mice and BALB/c mice after infection with a sublethal dose of VV. As shown in Fig. 4A, the level of VV-specific IgM production in T-bet–/– mice was similar to but slightly higher than that in control BALB/c mice at day 7 postinfection. Induction of anti-VV IgG Ab in T-bet–/– mice was almost equal to that in BALB/c mice at days 7, 14, and 28 postinfection (Fig. 4B to D). These data indicate that absence of T-bet does not significantly alter the production of VV-specific IgG and IgM Abs in mice.



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  		FIG. 4. VV-specific IgM and IgG Ab production in T-bet–/– mice following VV infection. T-bet–/– mice (inverted triangles) and BALB/c mice (circles) were infected i.p. with (open symbols) or without (solid symbols) 5 x 106 PFU of VV, and serum of each mouse was collected at days 7 (A and B), 14 (C), and 28 (D) postinfection. Titers of anti-VV IgM (A) and IgG (B-D) Abs in serially diluted serum were determined by a solid-phase ELISA. Data are shown as the average of four to six mice per group ± SEM. The experiment was repeated twice with similar results.

Taken together, these data suggest that the decreased resistance to VV infection in the absence of T-bet could be explained, at least in part, by impaired CTL activity specific for VV and diminished NK-mediated cytolysis but not by humoral immunity.

Cytokine production by CD4+ and CD8+ T cells in T-bet–/– mice after VV infection. To examine whether T-bet controls CD4+ T cells and CD8+ T cells for their cytokine production, both of the T-cell subsets were purified by MACS (Miltenyi Biotec) from spleen cells of T-bet–/– mice and BALB/c mice at day 8 after infection with VV. Purified T cells were then stimulated in vitro either with plate-bound anti-CD3 and anti-CD28 MAbs or with irradiated BALB/c spleen cells infected with VV. After 2 days of incubation, culture supernatants were collected and cytokine production in the supernatants was measured by ELISA. As expected, T-bet–/– CD4+ T cells produced much smaller amounts of the Th1 type cytokine IFN-{gamma} than T-bet+/+ CD4+ T cells under both polyclonal activation (Fig. 5A) and antigen-specific stimulation (Fig. 5I), indicating that T-bet plays an essential role for CD4+ T cells to produce IFN-{gamma}. In contrast, in agreement with the previous report (31), T-bet–/– CD8+ T cells secreted IFN-{gamma} as much as T-bet+/+ CD8+ T cells after polyclonal stimulation with anti-CD3 and anti-CD28 MAbs (Fig. 5B). However, T-bet–/– CD8+ T cells produced significantly smaller amounts of IFN-{gamma} than T-bet+/+ CD8+ T cells under antigen-specific stimulation with VV-infected antigen-presenting cells (APCs) (Fig. 5J). These data suggest that T-bet is required for efficient IFN-{gamma} production by CD8+ T cells under VV-specific stimulation but not under polyclonal activation.



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  		FIG. 5. Cytokine production by CD4+ and CD8+ T cells in T-bet–/– mice following VV infection. T-bet–/– mice (KO) and BALB/c mice (WT) were infected i.p. with (gray bars) or without (black bars) 5 x 106 PFU of VV, and spleen cells were prepared at day 8 postinfection. CD4+ T cells (A, C, E, G, I, K, and M) and CD8+ T cells (B, D, F, H, J, L, and N) were purified from spleen cells by MACS (Miltenyi Biotec) and were activated in vitro with either plate-bound anti-CD3 and anti-CD28 MAbs ({alpha}CD3/{alpha}CD28 Abs) or irradiated BALB/c spleen cells infected with VV (VV-infected APCs). After 2 days of incubation, amounts of IFN-{gamma} (A, B, I, and J), IL-4 (C and D), IL-5 (E, F, K, and L), and IL-10 (G, H, M, and N) in the culture supernatants were quantitated by ELISA. Data are shown as the means of three to five mice per group ± SEM. The experiment was repeated twice with similar results. ND, not detected. *, P < 0.05; **, P < 0.01 compared to BALB/c mice.

In contrast, T-bet–/– CD4+ and CD8+ T cells produced larger amounts of the Th2 type cytokines IL-4, IL-5, and IL-10 than T-bet+/+ CD4+ and CD8+ T cells, respectively (Fig. 5C to H and K to N). IL-4 production by T-bet–/– CD8+ T cells (Fig. 5D), IL-5 production by T-bet–/– CD4+ T cells (Fig. 5E) and CD8+ T cells (Fig. 5F), and IL-10 production by T-bet–/– CD8+ T cells (Fig. 5H) were remarkably augmented relative to T-bet-expressing T cells from control littermates under polyclonal stimulation. Furthermore, T-bet–/– CD4+ T cells secreted larger amounts of IL-5 (Fig. 5K) and IL-10 (Fig. 5 M) than T-bet+/+ CD4+ T cells in the VV-specific stimulation. T-bet–/– CD8+ T cells nonspecifically produced a small amount of IL-5 (Fig. 5L) and IL-10 (Fig. 5N) under antigen-specific stimulation (Fig. 5L and N). IL-4 was not detected in the culture supernatants of either purified CD4+ T cells or CD8+ T cells under antigen-specific stimulation (data not shown).

To further analyze the cytokine production by T-bet–/– CD8+ T cells, we stained antigen-induced intracellular cytokines in splenic CD8+ T cells prepared from T-bet–/– mice and BALB/c mice at days 7, 21, and 35 postinfection (Fig. 6). Since the cells were stimulated in vitro with VV-infected APCs for only 5 h, the possibility of substantial in vitro expansion of responder cells is precluded (7, 20). At day 7 postinfection, the frequency of IFN-{gamma}-producing CD8+ T cells in T-bet–/– mice (8.56%) was lower than that in BALB/c mice (18.02%) (Fig. 6C and D). This is in agreement with the data in Fig. 5J and supports the idea that T-bet is required for efficient IFN-{gamma} production by CD8+ T cells under VV-specific stimulation. A similar pattern was observed in the case of the other major antiviral cytokine, TNF-{alpha}. As shown in Fig. 6K and L, the frequency of TNF-{alpha}-producing CD8+ T cells in T-bet–/– mice (5.37%) was diminished in comparison to that in BALB/c mice (12.89%) at day 7 postinfection. On the other hand, at days 21 and 35 postinfection, the numbers of IFN-{gamma}- and TNF-{alpha}-producing CD8+ T cells were comparable between T-bet-deficient and control mice (Fig. 6E to H and M to P). Thus, these data suggest that diminished numbers of IFN-{gamma}- and TNF-{alpha}-producing CD8+ T cells at the early stage of infection might be related to enhanced susceptibility to VV infection in T-bet–/– mice. In contrast, in accordance with the previous report (30) the frequency of IL-2-producing CD8+ T cells in T-bet–/– mice was considerably higher than that in BALB/c mice at days 21 and 35 postinfection (Fig. 6U to X), although there was no significant difference in percentages of IL-2-producing CD8+ T cells between the two groups of mice at day 7 postinfection (Fig. 6S and T).



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  		FIG. 6. Intracellular cytokine staining of VV-specific CD8+ T cells in spleen cells of T-bet–/– mice following infection with VV. T-bet–/– mice (D, F, H, L, N, P, T, V, and X) and BALB/c mice (C, E, G, K, M, O, S, U, and W) were infected i.p. with 5 x 106 PFU of VV, and spleen cells were prepared at days 7 (C, D, K, L, S, and T), 21 (E, F, M, N, U, and V), and 35 (G, H, O, P, W, and X) postinfection. Nonimmunized (Naive) T-bet–/– (B, J, and R) and BALB/c mice (A, I, and Q) were used as negative controls. Spleen cells were stimulated with VV-infected P815 (A to X) and noninfected P815 cells (data not shown) for 5 h. After stimulation, cells were stained for their surface expression of CD8 (x axis) with FITC-conjugated MAb and for their intracellular expression of either IFN-{gamma} (A to H), TNF-{alpha} (I to P), or IL-2 (Q to X) with PE-conjugated MAbs. All lymphocytes were gated and analyzed on a FACScan flow cytometer using CellQuest software (BD Biosciences). Numbers shown indicate the percentages of CD8+ cells that are positive for intracellular cytokine. When spleen cells were stimulated with noninfected P815 cells for 5 h, percentages of CD8+ cells that were positive for intracellular IFN-{gamma}, TNF-{alpha}, and IL-2 were less than 0.41%, 0.54%, and 0.18%, respectively. Three to five mice per group were used in each experiment, and spleen cells of mice per group were pooled. Data shown are representative of three independent experiments. We analyzed data of the three experiments and concluded that the difference between panels C and D (P = 0.004), K and L (P = 0.008), U and V (P = 0.01), or W and X (P = 0.004) was statistically significant as determined by applying the Student's t test.

In vivo proliferation of CD4+ and CD8+ T cells in T-bet–/– mice after VV infection. To obtain direct in vivo evidence for VV-specific responses in T-bet–/– mice, in vivo proliferation of CD4+ and CD8+ T cells was measured by the BrdU incorporation assay. Both groups of mice infected with 5 x 106 PFU of VV were pulsed with BrdU 1 day before sacrifice. Spleen cells were then analyzed at days 7 and 21 postinfection by flow cytometry. At day 7 postinfection, T-bet–/– CD8+ T cells (1.99%) proliferated much less than T-bet+/+ CD8+ T cells (3.91%) (Fig. 7I and J), although T-bet–/– CD4+ T cells (0.97%) expanded marginally less than T-bet+/+ CD4+ T cells (1.10%) (Fig. 7C and D). On the other hand, at day 21 postinfection the percentages of CD4+ (0.65%) and CD8+ (0.54%) T cells in T-bet–/– mice were slightly greater than those of CD4+ (0.38%) and CD8+ (0.39%) T cells in control littermates, respectively (Fig. 7E, F, K, and L). It is interesting that CD4– and/or CD8– splenic cells in T-bet–/– mice vigorously proliferated compared with those in BALB/c mice at day 7 postinfection (Fig. 7C, D, I, and J).



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  		FIG. 7. In vivo proliferation of CD4+ and CD8+ T cells in T-bet–/– mice after VV infection. T-bet–/– mice (D, F, J, and L) and BALB/c mice (C, E, I, and K) were infected i.p. with 5 x 106 PFU of VV. Nonimmunized (Naive) T-bet–/– (B and H) and BALB/c mice (A and G) were used as negative controls. Mice were injected with BrdU 1 day before sacrifice, and spleen cells were prepared at days 0 (A, B, G, and H), 7 (C, D, I, and J), and 21 (E, F, K, and L) postinfection. Spleen cells were stained for their surface expression of either CD4 (A to F) or CD8 (G to L) with PE-conjugated MAbs and for incorporated BrdU with FITC-conjugated anti-BrdU MAb. Numbers shown at the upper right corner and the lower right corner in each panel indicate percentages of CD4+ (or CD8+) cells and CD4– (or CD8–) cells that are positive for incorporated BrdU, respectively. Three mice per group were used in each experiment, and spleen cells of mice per group were pooled. Data shown are representative of two independent experiments.


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DISCUSSION
 
In the current study, we show that T-bet plays a critical role in the defense against VV infection based on the following data: (i) the survival rate of T-bet–/– mice was obviously lower than that of BALB/c mice after infection with a high dose of VV (Fig. 1A); (ii) T-bet–/– mice lost weight after infection with a sublethal dose of VV, although BALB/c mice did not (Fig. 1B); and (iii) BALB/c mice cleared VV much faster than T-bet–/– mice. In accordance with the current data, it was previously reported that T-bet–/– mice exhibited poor protection against LCMV (28) and HSV-2 (29) infection. In contrast, it was demonstrated that T-bet was not required for host resistance to Listeria monocytogenes infection (34) or MCMV infection (33). Overall, the current data confirm that the requirement of T-bet appears to vary in the defense against pathogens, probably because distinct and unique immune responses are elicited for protection against various infectious pathogens.

In the case of VV infection, the absence of T-bet impaired VV-specific CTL activity (Fig. 2) and production of the two major antiviral cytokines, IFN-{gamma} (Fig. 5 and 6) and TNF-{alpha} (Fig. 6), by VV-specific CD8+ T cells. In addition, CD8+ T cells showed reduced proliferation in T-bet–/– mice at day 7 postinfection (Fig. 7). Furthermore, as expected, a significant Th2 shift in terms of cytokine production was observed in CD4+ T cells of T-bet–/– mice under both polyclonal stimulation (Fig. 5A, C, E, and G) and VV-specific stimulation (Fig. 5I, K, and M). These data strongly suggest that the increased susceptibility to VV infection in T-bet–/– mice could be explained, at least in part, by the diminished function of VV-specific CD8+ CTLs and the Th2 shift of CD4+ T cells. The impaired CTL function in T-bet–/– mice could be caused directly by T-bet deficiency or could be a secondary effect due to the inadequate CD4+ Th1 help to VV-specific CTLs in the T-bet deficiency. It has been shown that the insertion of IL-4 into vaccinia virus significantly increases the efficiency of the recombinant virus as a pathogen by directly inhibiting the development of Th1 immunity (27). In mice, the expression of IL-4 from the recombinant virus results in a decrease in CTL levels and a delay in viral clearance (26, 27). In the current study, however, it seems unlikely that the absence of T-bet allowed more IL-4 production, which had a direct effect on CTL function and virus clearance, because IL-4 was not detected by cytokine ELISA in the culture supernatants of either purified CD4+ T cells or CD8+ T cells of VV-infected T-bet–/– mice under antigen-specific stimulation with VV-infected APCs (data not shown). Furthermore, T-bet–/– mice treated with anti-IL-4 MAb during VV infection lost body weight in a manner similar to T-bet–/– mice injected with control rat IgG, and T-bet–/– mice in both of the groups harbored around 10-fold more PFU in their ovaries than BALB/c mice at day 14 postinfection (data not shown).

It was previously reported that T-bet was required for the control of IFN-{gamma} production in CD4+ T cells and NK cells but not in CD8+ T cells under polyclonal stimulation (31). The data show distinct effects of T-bet on IFN-{gamma} production within the T-cell lineage. These findings are consistent with the current results under stimulation with anti-CD3 and anti-CD28 MAbs (Fig. 5A and B). In the context of antigen-specific activation, however, T-bet was necessary for IFN-{gamma} production by CD8+ T cells following LCMV infection (14, 28), although T-bet deficiency did not spoil IFN-{gamma} production by CD8+ T cells during Listeria monocytogenes infection (34) or HSV-2 infection (29). In the current study, CD8+ T cells in T-bet–/– mice produced modest but significantly smaller amounts of IFN-{gamma} than those in BALB/c mice under VV-specific activation (Fig. 5J). In support of this, the frequency of intracellular IFN-{gamma}-positive CD8+ T cells in T-bet–/– mice was about twofold less than that in BALB/c mice under VV-specific stimulation at day 7 postinfection (Fig. 6G and D). These data indicate that T-bet is essential for the optimal production of IFN-{gamma} by CD8+ T cells in VV infection and suggest that the impairment of IFN-{gamma} production by CD8+ T cells might be related to the diminished resistance to VV infection in T-bet–/– mice. It is, however, noteworthy that the production of IFN-{gamma} by CD8+ T cells was not entirely abolished in T-bet deficiency under VV-specific stimulation (Fig. 5J), while VV-specific CD4+ T cells almost failed to produce IFN-{gamma} in the absence of T-bet (Fig. 5I). This finding strongly suggests that multiple transcription factors are involved in the regulation of IFN-{gamma} in VV-specific CD8+ T cells. It is possible that the CD8+ T-cell-specific transcription factor, Eomesodermin (23), complements the action of T-bet in IFN-{gamma} production by VV-specific CD8+ T cells.

In addition to secretion of IFN-{gamma}, the other major function of CD8+ CTLs is the ability to lyse target cells by using perforin and granzymes. As shown in Fig. 2, the cytolytic activity of VV-specific CTLs was significantly reduced in the absence of T-bet. Pearce et al. found that T-bet deficiency resulted in a modest but reproducible defect in granzyme B induction (23). Furthermore, gene expression profiling of CD8+ T cells from T-bet–/– mice has revealed that the expression of genes encoding perforin and granzyme B is reduced (13). Hence, the impaired killing activity of VV-specific T-bet–/– CTLs (Fig. 2) might result from insufficient amounts of perforin and granzyme B in CD8+ T cells.

On the other hand, T-bet–/– CD8+ T cells secreted much higher levels of the Th2/Tc2-specific cytokines IL-4, IL-5, and IL-10 under polyclonal stimulation (Fig. 5D, F, and H), indicating that the deficiency of T-bet results in the type 1-to-type 2 shift of CD8+ T cells as well as CD4+ T cells. When stimulated with VV-infected APCs, T-bet–/– CD4+ T cells produced larger amounts of IL-5 and IL-10 in an antigen-specific manner (Fig. 5K and M), whereas T-bet–/– CD8+ T cells secreted marginal amounts of IL-5 and IL-10 in an antigen-independent manner (Fig. 5L and N). These data also pointed out distinct effects of T-bet within the two major T-cell subsets. Interestingly, the frequency of IL-2-producing VV-specific CD8+ T cells was considerably increased in the absence of T-bet at days 21 and 35 postinfection (Fig. 6U to X). Similar results were obtained by others (15, 29, 31). It was previously shown that T-bet repressed the IL-2 promoter (30), but it remains unclear why T-bet represses IL-2 production. Although IL-2 is defined as a positive growth factor for T cells and enhances antiviral T-cell responses in vivo (6), IL-2 also has negative effects on T cells, including activation-induced cell death (16). This might cause the reduced proliferation of CD8+ T cells in T-bet–/– mice (Fig. 7I and J) under increased IL-2 production by CD8+ T cells (Fig. 6U to X).

NK cells play a critical role early in host defense. In fact, it was found that NK cells contributed to the recovery of immunodeficient mice from infection with VV (15). In the current study, NK activity was substantially diminished in T-bet–/– mice at day 2 after VV infection (Fig. 3C). However, any difference was not seen in viral titers between the two groups of mice at the early days postinfection (Fig. 3D), indicating that early in infection the extent of virus replication is based on the availability of susceptible target cells more than the innate immune response. Furthermore, we found that the deficiency of T-bet did not have any impact on the total production of anti-VV IgM and IgG Abs (Fig. 4). These data collectively reveal that the enhanced susceptibility to VV infection in T-bet–/– mice could be partially due to downregulation of NK activity but not due to insufficient production of anti-VV Abs. It has been shown that T-bet is implicated as a critical mediator of class-switch recombination to IgG2a (12, 24). Therefore, it is supposed that the level of anti-VV IgG2a Ab production in T-bet–/– mice was less than that in control mice. If anti-VV IgG2a Ab is important in the clearance of VV (18), it might be possible to explain that the manner of Ab production in T-bet–/– mice is associated with the enhanced susceptibility to VV infection. In the absence of prior immunity, primary infection must generate both T-cell-mediated immunity and humoral immunity, but VV-specific T cells arise faster than anti-VV Abs. Therefore, CTLs start clearing virus before Abs neutralize virus. Because VV was not cleared at day 21 postinfection in T-bet–/– mice which generated anti-VV Abs as much as BALB/c did, the complete clearance of VV in BALB/c mice at day 21 postinfection should be mainly due to T-cell-mediated immunity. It is interesting that virus was suddenly cleared in ovaries of T-bet–/– mice as well as control mice at day 28 postinfection (Fig. 1C). It is most likely that this clearance was owing to anti-VV IgG Abs, because it was recently shown that anti-VV Ab production was important in clearing virus following acute infection with VV (3, 35).

In conclusion, we investigated whether T-bet was required for protection against infection with VV using T-bet–/– mice. It was found that T-bet–/– mice were more susceptible to primary VV infection than control littermates. These results were supposed to be, at least in part, due to the Th2 shift of CD4+ T cells and the impairment of both NK-mediated lysis and VV-specific CD8+ CTL function, including killing activity, IFN-{gamma} and TNF-{alpha} production, and in vivo proliferation, but not due to the impairment of anti-VV Ab production. Overall, these data indicate that T-bet plays a crucial role in the defense against VV infection.


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ACKNOWLEDGMENTS
 
This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

We are grateful to L. H. Glimcher (Harvard Medical School) and T. Shioda (Osaka University, Japan) for providing T-bet–/– mice and vaccinia virus (WR strain), respectively.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Saitama Medical School, Moroyama-Cho, Iruma-Gun, Saitama 350-0495, Japan. Phone: 81-49-276-1166. Fax: 81-49-295-9107. E-mail: mmatsui{at}saitama-med.ac.jp. Back


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Journal of Virology, October 2005, p. 12798-12806, Vol. 79, No. 20
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.20.12798-12806.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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