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The Journal of Immunology, 2006, 176: 2496-2504.
Copyright © 2006 by The American Association of Immunologists

Impaired Protection against Mycobacterium bovis Bacillus Calmette-Guérin Infection in IL-15-Deficient Mice1

Kimika Saito*, Toshiki Yajima*, Shino Kumabe*, Takehiko Doi*, Hisakata Yamada*, Subash Sad{dagger}, Hao Shen{ddagger} and Yasunobu Yoshikai2,*

* Division of Host Defense, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; {dagger} Laboratory of Cellular Immunology, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada; and {ddagger} Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
To investigate the potential role of endogenous IL-15 in mycobacterial infection, we examined protective immunity in IL-15-deficient (IL-15–/–) mice after infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG) or recombinant OVA-expressing BCG (rBCG-OVA). IL-15–/– mice exhibited an impaired protection in the lung on day 120 after BCG infection as assessed by bacterial growth. CD4+ Th1 response capable of producing IFN-{gamma} was normally detected in spleen and lung of IL-15–/– mice on day 120 after infection. Although Ag-specific CD8 responses capable of producing IFN-{gamma} and exhibiting cytotoxic activity were detected in the lung on day 21 after infection with rBCG-OVA, the responses were severely impaired on days 70 and 120 in IL-15–/– mice. The degree of proliferation of Ag-specific CD8+ T cells in IL-15–/– mice was similar to that in wild-type mice during the course of infection with rBCG-OVA, whereas sensitivity to apoptosis of Ag-specific CD8+ T cells significantly increased in IL-15–/– mice. These results suggest that IL-15 plays an important role in the development of long-lasting protective immunity to BCG infection via sustaining CD8 responses in the lung.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
It is widely accepted that protection against infection with mycobacteria such as Mycobacterium bovis and M. tuberculosis depends mainly on IFN-{gamma} produced by CD4+ Th1 cells (1, 2, 3). There are several lines of evidence that CD8+ T cells producing IFN-{gamma} and exhibiting cytotoxicity play a requisite role in resistance to mycobacterial infection (3, 4, 5). beta2-Microgloburin-deficient mice and TAP-deficient (TAP–/–) mice, which lack functional CD8+ T cells, are susceptible to infection with M. tuberculosis (6, 7). Adoptive transfer of immunized CD8+ T cells can confer protection against subsequent challenge with M. tuberculosis (8). It has been suggested that CD8+ CTLs releasing perforin and granulysin play a role in protection against M. tuberculosis infection via a cytolysis mechanism (9, 10). In contrast, the resistance to M. tuberculosis infection in both perforin- and Fas-deficient mice was unaltered, suggesting that the cytotoxic function of CD8+ T cells may not be critical in protection against tuberculosis (11). A recent study has suggested that CD8+ T cells are more important than CD4+ T cells in controlling the latent phase of tuberculosis infection, which comprises the majority of human infections (12, 13, 14). Thus, it appears that protection against chronic pulmonary tuberculosis requires a sustained cellular immunity mediated by CD8+ T cells and that Ag-specific CD8+ T cells are a major target for vaccine design.

IL-15 uses beta- and {gamma}-chains of the IL-2R for signal transduction and thus shares many properties of IL-2 despite having no sequence homology with IL-2 (15, 16). IL-15 has been reported to stimulate NK cells and TCR{gamma}{delta} intestinal intraepithelial lymphocytes to produce IFN-{gamma} and enhance their cytotoxicity (17, 18). Furthermore, IL-15 has an important function in the proliferation and survival of memory-phenotype CD8+ T cells. We previously found that IL-15 transgenic (Tg)3 mice expressing IL-15 cDNA encoding a secretable isoform had an increased number of memory CD8+ T cells in a naive state and showed enhanced protection against infection with Listeria monocytogenes and against infection with M. bovis bacillus Calmette-Guérin (BCG) via activation of NK cells and CD8+ T cells (19, 20, 21). In contrast, IL-15R{alpha}–/– and IL-15–/– mice have been reported to be deficient in memory-phenotype CD8+ T cells in addition to NK cells, NKT cells, and TCR{gamma}{delta} intestinal intraepithelial lymphocytes (22, 23). However, IL-15–/– mice have been reported not to be susceptible to infection with lymphocytic choriomeningitis virus (LCMV) (24) or Toxoplasma gondii (25), although soluble IL-15R{alpha} treatment exacerbated Toxoplasma infection (26). IL-15–/– mice may compensate for the defect in the host defense system by IL-15-dependent cell populations by alternative mechanisms.

With the aim of elucidating the roles of endogenous IL-15 in vivo, we examined susceptibility and T cell-mediated immunity against BCG and recombinant OVA-expressing BCG (rBCG-OVA) in IL-15–/– mice. We found that bacterial growth was increased in lungs of IL-15–/– mice on day 120 after i.p. infection with BCG. CD4+ Th1 response capable of producing IFN-{gamma} in response to purified protein derivative (PPD) was normally detected, but the model experiments performed in mice infected with rBCG-OVA suggest that Ag-specific CD8 responses capable of producing IFN-{gamma} and exhibiting cytotoxic activity were severely impaired in IL-15–/– mice on days 70 and 120 after rBCG-OVA infection. Although IL-15-dependent cell proliferation was important for maintenance of Ag-specific memory CD8+ T cells after acute infection with microbes, proliferation of memory CD8+ T cells in IL-15–/– mice was almost the same as that in wild-type (WT) mice during the course of rBCG-OVA infection. In contrast, Ag-specific CD8+ T cells from infected IL-15–/– mice were more susceptible to apoptosis than were those from infected WT mice. Thus, these results suggest that IL-15 plays an important role in the development of long-lasting protective immunity to BCG infection via sustaining T cytotoxic 1 (Tc1) responses in the lung.


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

C57BL/6-background IL-15–/– mice were purchased from Taconic. In each experiment, age- and sex-matched C57BL/6 mice, purchased from Charles River Japan, were used as controls. Mice were maintained under specific pathogen-free conditions and offered food and water ad libitum. All mice were used at 6–8 wk of age.

Microorganisms

Lyophilized M. bovis BCG (Tokyo strain) was purchased from Kyowa Pharmaceuticals. rBCG-OVA was previously described (27, 28). Briefly, BCG (Pasteur strain) was grown on Middlebrook 7H10 solid medium (Difco). A partial sequence of the OVA gene 230–359, which encodes the SIINFEKL epitope and its flanking sequences, was cloned in the pMV261 vector, downstream of the Ag 85B secretion signal and under the control of heat shock protein 60 promoter. BCG and rBCG-OVA were dissolved in 7H9 medium (Difco) supplemented with albumin-dextrose-catalase enrichment (Difco). The viable bacterial numbers were determined by a 7H10 (Difco) plate supplemented with oleic acid-albumin-dextrose-catalase enrichment (Difco). Small aliquots of BCG suspended in 7H9 medium containing 10% glycerol were stored at –80°C until use. The concentration of bacteria was quantified by plate counting. Before use, the bacteria were washed three times with PBS and resuspended in PBS. Mice were inoculated i.p. with 2–10 x 106 CFU (depending on the experiment) of BCG or rBCG-OVA in a volume of 200 µl of PBS.

Abs and reagents

FITC-conjugated anti-CD3{epsilon} (145-2C11), CD44 (IM7), and IFN-{gamma} (XMG1.2) mAbs; PE-conjugated anti-NK1.1 (PK136), TCR{gamma}{delta} (UC7), and CD8{alpha} (53-6.7) mAbs; CyChrome-conjugated anti-CD4 (GK1.5), CD8{alpha} (53-6.7), and TCRbeta (H57-597) mAbs; biotin-conjugated anti-CD44 (IM7) mAb; allophycocyanin-conjugated streptavidin; and biotin-conjugated anti-Ly5.1 mAb (A20) were purchased from BD Pharmingen. FITC-conjugated hamster anti-mouse activated form of caspase-3 (C92-605), Bcl-2 mAb (3F11), and hamster anti-mouse isotype control were obtained from BD Pharmingen. Annexin VFITC apoptosis detection kit was purchased from Sigma-Aldrich. OVA257–264 H-2Kb tetramers were purchased from MBL.

Cell preparation

Splenocytes from BCG-infected mice were prepared by centrifugation and resuspended in RPMI 1640 containing 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 mM HEPES. Lung mononuclear cells (MNCs) were prepared as described previously (29). Briefly, lung tissue was minced and incubated with stirring at 37°C for 30 min in HBSS with 1.3 mM EDTA, followed by treatment at 37°C for 1 h with collagenase (150 U/ml; Invitrogen Life Technologies) in RPMI 1640 with 10% FBS. The resulting suspension was pelleted by centrifugation, resuspended in 44% Percoll (Pharmacia) layered on 66.6% Percoll, and centrifuged at 600 x g. Cells at the gradient interface were harvested and washed extensively before use.

Flow cytometric analysis

Splenocytes or lung MNCs were preincubated with a culture supernatant from 2.4 G2 to prevent nonspecific staining. After washing, cells were stained with various combinations of mAbs. Staining with biotin-conjugated mAb was followed by treatment with streptavidin-CyChrome or -allophycocyanin. The stained cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences). The data were analyzed with CellQuest software (BD Biosciences).

In vitro culture and cytokine ELISA

Spleen cells were subjected to an Ag stimulation assay for cytokine production. Nylon-wool-passed spleen cells from BCG-infected mice on days 14 and 120 after infection were resuspended in RPMI 1640 and added to 96-well plates at a concentration of 2 x 105 cells/well. Cells were cultured without any stimulation or with 5 µg/ml PPD (Japan BCG Association) in the presence of mitomycin C-treated splenocytes (1 x 106) from naive mice for 48 h at 37°C. Supernatants were collected and stored at –80°C until the cytokine assay. IFN-{gamma}, IL-10, and IL-4 concentrations in the culture supernatant were measured using ELISA development kits (Genzyme Diagnostics).

Intracellular cytokine, caspase-3, or Bcl-2 staining

Splenocytes or lung MNCs were incubated without any stimulation or with 5 µg/ml PPD or 5 µg/ml OVA257–264 peptide and 100 pg/ml rIL-2 (Takeda Chemical) for 6 h at 37°C and 5% CO2 with 10 µg/ml brefeldin A (Sigma-Aldrich) added for the last 2 h in 48-well flat-bottom plates at a concentration of 5 x 106 cells/well (Falcon; BD Biosciences) in a volume of 0.5 ml of RPMI 1640 containing 10% FCS. After 6 h of culture, the cells were harvested and surface stained in staining buffer with CyChrome-conjugated anti-CD4 mAb, PE-conjugated anti-CD8 mAb, and allophycocyanin anti-CD44 mAb. After surface staining, cells were subjected to intracellular cytokine staining using a Fast Immune Cytokine System (BD Biosciences) according to the manufacturer’s instructions. The cells were washed and fixed in 1000 µl of FACS lysing solution (BD Biosciences) for 10 min at room temperature and were then washed again, resuspended in 500 µl of FACS permeabilizing solution (BD Biosciences), and incubated for 10 min at room temperature. After washing, the cells were stained with FITC-conjugated IFN-{gamma} mAb or FITC-conjugated isotype control rat IgG (BD Pharmingen).

Before staining for intracellular activated caspase-3 and Bcl-2, splenocytes or lung MNCs from infected mice were stained for surface markers in staining buffer with CyChrome-conjugated anti-CD8 mAb and PE-conjugated OVA257–264 H-2Kb tetramers for 60 min at 4°C. After that, half of the cells were fixed and permeabilized with the above solution and then were stained with FITC-conjugated hamster anti-mouse Bcl-2 or its isotype control. The other half of the cells were washed, fixed, and permeabilized using the Cytofix/Cytoperm intracellular staining kit (BD Biosciences). The cells were incubated with anti-caspase-3 at 1/100 dilution in perm/wash buffer for 30 min at room temperature. After intracellular staining, fluorescence of the cells was analyzed using a flow cytometer.

Annexin V staining

Splenocytes and lung MNCs from uninfected WT and IL-15–/– mice were stained for surface markers in staining buffer with CyChrome-conjugated anti-CD8 mAb and PE-conjugated OVA257–264 H-2Kb tetramers for 60 min. Then the amount of apoptosis was determined by staining with Annexin VFITC conjugate according to the manufacturer’s instructions. Briefly, cells were suspended in 500 µl/tube 1x binding buffer and incubated with 5 µl/tube Annexin VFITC for 10 min in the dark at room temperature. Cells were washed twice with 1x binding buffer to remove any unbound Annexin VFITC. Samples were analyzed within 30 min.

In vivo cytotoxicity assay

Analysis of in vivo cytolytic activity was carried by a protocol similar to those previously reported (29). B6-Ly5.1+ splenocytes were divided into two populations and labeled with a high concentration (5 µM) and a low concentration (0.5 µM) of CFSE. Next, CFSEhigh cells were pulsed with 5 µg/ml OVA257–264 peptide for 1 h at 37°C, whereas CFSElow cells remained nonpulsed. After washing, these groups were mixed in equal proportions and then injected i.v. into mice infected with rBCG-OVA 21, 70, or 120 days previously. Spleens or lungs were obtained from recipients 24 h later for flow cytometric analysis to measure in vivo killing activities. Percent specific lysis was calculated according to the following formula: 1 – (ratio primed/ratio unprimed) x 100, where the ratio unprimed = % CFSElow/% CFSEhigh cells remaining in noninfected recipients and ratio primed = % CFSElow/% CFSEhigh cells remaining in infected recipients.

Analysis of T cell proliferation after rBCG-OVA infection in vivo

Mice infected with rBCG-OVA 14 or 113 days previously were given water containing 0.8 mg/ml BrdU for 7 days. Splenocytes or lung MNCs were stained with OVA257–264 Kb tetramer and anti-CD8 mAb for 30 min at 4°C and then were subjected to intracellular BrdU staining using a BrdU flow kit according to the instructions of the manufacturer (BD Biosciences).

Statistical analysis

The statistical significance of the data was determined by Student’s t test; a value of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Bacterial growth in IL-15–/– mice after BCG infection

To elucidate the roles of endogenous IL-15 in protection against BCG infection, we examined the kinetics of bacterial growth in the peritoneal cavity, liver, lung, and spleen of IL-15–/– mice after i.p. infection with BCG. As shown in Fig. 1, number of bacteria in the spleen and peritoneal cavity decreased with time both in WT and IL-15–/– mice. However, the number of bacteria in the lung on day 120 after BCG infection was significantly higher in IL-15–/– mice than in WT mice (p < 0.005). A similar tendency in bacterial growth was observed in the liver (p < 0.05). Thus, IL-15–/– mice were susceptible to BCG infection, especially in the lung and liver at the later stage after infection as assessed by bacterial growth.


Figure 1
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  		FIGURE 1. Bacterial growth in IL-15–/– mice after infection with BCG. IL-15–/– mice and age-matched WT mice were infected i.p. with 1.0 x 106 CFU of BCG (Tokyo strain). The numbers of bacteria recovered from peritoneal cavity, liver, spleen, or lung of infected mice were determined at the indicated days. Data of a representative are shown from three separate experiments and are expressed as means ± SD of five mice of each group. ND, Not detectable. *, p < 0.05; **, p < 0.005.

 
Lymphocyte populations in the spleen and lung of IL-15–/– mice after BCG infection

We next examined the kinetics of lymphocytes in the spleen and lung of IL-15–/– mice and WT mice after BCG infection. Flow cytometry analysis for expression of CD3{epsilon}, TCR{alpha}beta, TCR{gamma}{delta}, NK1.1, CD4, and CD8 was conducted on cells of the spleen and lung on days 0, 14, 70, and 120 after infection. The numbers of CD8+cells, CD3+NK1.1+ cells, and {gamma}{delta} T cells in the spleen and lung were significantly decreased in IL-15–/– mice before infection, whereas the spleen and lung of IL-15–/– mice were almost devoid of CD3–NK1.1+ cells (Fig. 2 and data not shown). CD4+ T cells had increased until day 14 and gradually decreased by day 70 after infection in the spleen. The number of CD4+ T cells in lung showed slower time kinetics. There were no significant differences between WT and IL-15–/– mice. In contrast, the numbers of CD8+ T cells in the spleen and lung were increased in WT mice on day 14 and remained at increased levels on day 120. Similar to WT mice, the numbers of CD8+ T cells were substantially increased in the spleen of IL-15–/– mice on day 14 after BCG infection but, in contrast with WT mice, the numbers of CD8+ T cells were gradually decreased by day 70 after infection. The number of CD3–NK1.1+ cells was increased in the spleens of WT mice on day 14 after infection and then decreased by day 120 after infection, whereas CD3–NK1.1+ cells were undetectable in the spleens and lungs of IL-15–/– mice during the course of BCG infection. The numbers of CD3+{gamma}{delta}+ and CD3+NK1.1+ cells were increased in the spleens of IL-15–/– mice and WT mice on day 14, and the increase was more prominent in WT mice (data not shown). Taken together, the results indicate that only a few, if any, CD8+ T cells remained in lung and spleen of IL-15–/– mice for a long time after BCG infection.


Figure 2
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  		FIGURE 2. Kinetics of absolute number of lymphocyte subsets in IL-15–/– mice after BCG infection. The cells of spleen and the lung on day 0, 14, 70, or 120 after BCG infection were stained with various mAbs, and the absolute numbers of CD4+, CD8+, or NK1.1+CD3– cells were calculated by multiplying total splenocytes or lung MNCs by the percentage of each subset in spleen or lung. Data of a representative are shown from three separate experiments and are expressed as means ± SD of three mice of each group. *, p < 0.05; **, p < 0.01.

 
Cytokine production by Ag-stimulated T cells in the spleen and lung of IL-15–/– mice after BCG infection

To investigate whether Ag-specific T cells were able to be generated in IL-15–/– mice during the course of BCG infection, T cells were isolated from spleens of the mice on days 14 and 120 after BCG infection and were cultured with or without PPD in the presence of APC, and the culture supernatants were examined by ELISA for IFN-{gamma}, IL-4, or IL-10 release. T cells from IL-15–/– mice on day 14 after infection produced higher levels of IFN-{gamma} in response to PPD than did those from WT mice (Fig. 3A; p < 0.05). The same tendency was observed in T cells from IL-15–/– mice infected with BCG 120 days previously, although the difference was not statistically significant. These results suggest that BCG-specific Th1 cell responses are normally generated in IL-15–/– mice after BCG infection. We further examined the production of IL-4 and IL-10 by CD4+ T cells from WT and IL-15–/– mice infected with BCG. Neither T cells from WT mice nor those from IL-15–/– mice produced IL-4 or IL-10 in response to PPD, suggesting that PPD-specific Th2 or regulatory T cells are not generated in IL-15–/– mice.


Figure 3
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  		FIGURE 3. Cytokine production of T cells from IL-15–/– mice infected with BCG. A, T cells on day 14 or 120 after BCG infection were cultured with PPD for 48 h, and IFN-{gamma}, IL-4, or IL-10 production in the supernatants was assayed by ELISA. The data are representative of two separate experiments and are expressed as means of triplicates ± SD; ND, not detectable; *, p < 0.05. B, Intracellular expression of IFN-{gamma} by CD4+ T cells and CD8+ T cells from IL-15–/– mice infected with BCG. Lung MNCs were pooled from three mice of each group on day 14 or 120 after BCG infection and were cultured with PPD and surface-stained with anti-CD4, -CD8, and -CD44 mAbs. Intracellular cytokine-producing cells were examined using a flow cytometer and were analyzed by gating on CD4+ or CD8+ T cells. Data are representative of two independent experiments and are shown as typical two-color profiles.

 
To determine CD4+ Th1 and CD8+ Tc1 responses in the spleen and lung from infected IL-15–/– mice, we used cytokine FACS analysis for determination of expression of CD4 or CD8 and CD44 and for determination of intracellular IFN-{gamma} after stimulation with PPD. As shown in Fig. 3B, CD4+ Th1 response capable of producing IFN-{gamma} in response to PPD was substantially detected in the lungs of IL-15–/– mice on days 14 and 120 after BCG infection. In contrast, IFN-{gamma} production in response to PPD was not detected in the CD8+ T cell population from either IL-15–/– mice or WT mice infected with BCG 14 or 120 days previously. Similar results were obtained for the spleen, although the differences were less pronounced (data not shown).

Ag-specific CD8+ T cells in IL-15–/– mice infected with rBCG-OVA

Ag-specific CD8+ T cells are an important T cell subset for controlling the late phase of tuberculosis infection (13, 14). Unfortunately, immunostimulatory BCG-derived peptides recognized by CD8+ T cells have not been defined. Therefore, to examine more carefully the kinetics of the Ag-specific CD8+ T cell response after BCG infection, we decided to use rBCG-OVA for detection of Ag-specific CD8+ T cells in IL-15–/– mice. OVA expressed by rBCG-OVA was at ~0.01% of total protein of rBCG-OVA. The timing of OVA expression was as early as 7 days, because OVA-specific immune responses were detected at this stage after infection (data not shown). The numbers of OVA-specific CD8+ T cells were assessed by staining with an H-2Kb tetramer coupled with an OVA-derived SIINFEKL peptide on days 21, 70, and 120 after rBCG-OVA infection (27). The kinetics of bacterial growth of rBCG-OVA were similar to those of BCG Tokyo strain (data not shown). As shown in Fig. 4A, the numbers of OVA-specific CD8+ T cells in both the spleens and lungs of IL-15–/– mice on day 21 after infection were comparable with those in WT mice, whereas the numbers were significantly lower in IL-15–/– mice, especially in the lung, on days 70 and 120 after infection (p < 0.05). Thus, Ag-specific effector CD8+ T cells can be generated in the spleen and lung at the early stage after infection, but they do not remain for a long time after BCG infection in IL-15–/– mice.


Figure 4
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  		FIGURE 4. Generation of OVA257–264-specific CD8+ T cells in IL-15–/– mice infected with rBCG-OVA. A, Splenocytes and lung MNCs on day 21, 70, or 120 after rBCG-OVA infection were stained with anti-CD8 mAb, anti-CD44 mAb, and OVA257–264 MHC class I tetramer. Samples were analyzed by flow cytometry and analyzed by gating on CD8+ T cells. Data were obtained from three separate experiments, and each value shown is the mean ± SD for three experiments. Statistically significant differences between IL-15–/– mice and WT mice are shown (*, p < 0.05). B, Intracellular expression of IFN-{gamma} in the OVA257–264-specific CD8+ T cells from IL-15–/– mice after rBCG-OVA infection. Cells pooled from three mice of each group on day 21, 70, or 120 after infection were cultured with OVA257–264 peptide and then were subjected to intercellular cytokine staining. Analysis gate was set on CD8+ T cells. Data were obtained from three separate experiments, and each value shown is the mean ± SD for three experiments. Statistically significant differences between IL-15–/– mice and WT mice are shown (*, p < 0.05).

 
To further confirm the degree of generation of Ag-specific CD8+ T cells in IL-15–/– mice infected with rBCG-OVA, we used cytokine FACS analysis for determination of the expression of CD4 or CD8 and CD44 and for determination of the intracellular IFN-{gamma} after stimulation with OVA257–264 peptide. The levels of CD44+CD8+ T cells producing IFN-{gamma} in response to OVA peptide in the spleens and lungs from IL-15–/– mice on day 21 were comparable with those in the spleens and lungs from WT mice (Fig. 4B). An appreciable number of CD8+ T cells in the spleens and lungs from WT mice on day 120 produced IFN-{gamma} in response to OVA peptide, whereas the numbers of such CD8+ T cells were greatly reduced in the spleens and lungs from IL-15–/– mice (p < 0.05). Thus, Ag-specific CD8+ T cells may not be able to be sustained for a long time in the lung in the absence of IL-15.

In vivo cytotoxicity of CD8+ T cells in IL-15–/– mice infected with rBCG-OVA

To directly detect cytotoxic activity of CD8+ T cells in vivo, we measured the ability of CD8+ T cells to eliminate fluorescent-labeled spleen cells pulsed with OVA257–264 peptides after rBCG-OVA infection. B6-Ly5.1+ splenocytes were divided into two populations and labeled with a high concentration and a low concentration of CFSE. Next, CFSEhigh cells were pulsed with OVA257–264 peptide, whereas CFSElow cells remained nonpulsed. These groups were mixed in equal proportions and then injected i.v. into mice infected with rBCG-OVA 21, 70, or 120 days previously. Splenocytes or lung MNCs were obtained from recipients 24 h later for flow cytometric analysis to measure in vivo killing activities. As shown in Fig. 5, OVA-pulsed target cells had been eliminated equally in the spleen or lung of IL-15–/– mice and WT mice on day 21 after infection, indicating that CD8 effector CTLs can be generated in IL-15–/– mice at the early stage after BCG infection. In contrast, the elimination of OVA-pulsed target cells was severely impaired in the spleen and lung of IL-15–/– mice on days 70 and 120 after infection. Thus, in vivo CTL activity was severely reduced in IL-15–/– mice at the late stage after BCG infection.


Figure 5
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  		FIGURE 5. In vivo cytotoxic activities in IL-15–/– mice infected with rBCG-OVA. Histograms are gated on Ly5.1+ cells in the spleen or lung 24 h after coinjection with equal numbers of CFSEhigh-labeled and OVA257–264-pulsed and CFSElow-labeled and nonpulsed Ly5.1+ splenocytes into mice infected with rBCG-OVA 21, 70, or 120 days previously. The values at the upper right of each panel represent the percentage of specific killing compared with nonpulsed cells. Data were obtained from three separate experiments, and each value shown is the mean ± SD for three experiments. Statistically significant differences between IL-15–/– mice and WT mice are shown (*, p < 0.05).

 
Cell division of OVA-specific CD8+ T cells in IL-15–/– mice infected with rBCG-OVA

The number of memory CD8+ T cells is maintained by a balance among cell survival, apoptosis, and proliferation. It is well established that Ag-specific memory CD8+ T cells undergo homeostatic proliferation, which is believed to be an important mechanism promoting survival by avoiding cell attribution over time. To elucidate whether the proliferation is involved in the decreases in OVA-specific memory CD8+ T cells in IL-15–/– mice, we examined the cell proliferation of Ag-specific CD8+ T cells in IL-15–/– mice at the early and late stages after rBCG-OVA infection. IL-15–/– mice and WT mice were infected with rBCG-OVA, and then they were given BrdU in their drinking water during the early stage (days 14–21) or late stage (days 113–120) after infection. On day 21 or 120 after infection, these mice were sacrificed and BrdU incorporation was examined in OVA-specific CD8+ T cells from the lung and spleen. As shown in Fig. 6B, proliferation of OVA-specific CD8+ T cells in the spleen and lung of IL-15–/– mice was comparable with that in the spleen and lung of WT mice on day 21 after rBCG-OVA infection, indicating that IL-15 is not essential for expansion of Ag-specific effector CD8+ T cells at the early stage of BCG infection. Surprisingly, we found that OVA-specific memory CD8+ T cells in the spleen and lung were also able to undergo cell division in the absence of IL-15 at the later stage of rBCG-OVA infection. Cell division of whole CD8+ T cells in IL-15–/– mice was also equivalently increased compared with that in WT mice on days 21 and 120 after infection (Fig. 6A). Taken together, the results suggest that Ag-specific CD8+ T cells generated in IL-15–/– mice persist by cell division after BCG infection in an IL-15-independent manner.


Figure 6
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  		FIGURE 6. Analysis of cell proliferation of Ag-specific CD8+ T cells in IL-15–/– mice infected with rBCG-OVA. WT or IL-15–/– mice infected with rBCG-OVA 14 or 113 days previously were given water containing 0.8 mg/ml BrdU for 7 days. On days 21 or 120 after infection, splenocytes or lung MNCs were stained with OVA257–264 Kb tetramer and anti-CD8 mAb for 30 min at 4°C and then subjected to intracellular BrdU staining. Histograms are gated on CD8+ (A) or OVA Kb + CD8+ (B) in the spleen or lung, and the number indicated is the percentage of whole CD8+ T cells (CD8+) or OVA-specific CD8+ T cells (OVA257–264 Kb tet+ CD8+) stained positive for anti-BrdU mAb.

 
Sensitivity to apoptosis and Bcl-2 protein in OVA-specific CD8+ T cells in IL-15–/– mice after rBCG-OVA infection

It is well known that antiapoptotic molecules play a critical role in the regulation of survival of memory CD8+ T cells. Based on the finding of a gradual decline in the number of OVA-specific CD8+ T cells during the late stage of rBCG-OVA infection, we hypothesized that Ag-specific CD8+ T cells might be more sensitive to apoptosis in an IL-15-deficient environment. To address this, we examined their binding of annexin V, an early marker of apoptotic cells, and expression of active caspase-3, a terminal effector for apoptosis. Splenocytes or lung MNCs from IL-15–/– and WT mice infected with rBCG-OVA were stained with anti-CD8 and OVA257–264 Kb tetramer and then with annexin V and active caspase-3 mAb. As shown in Fig. 7, A and B, the frequency of annexin V-positive cells in OVA-specific CD8+ T cells from the lung was markedly increased in infected IL-15–/– mice compared with those in WT mice on days 21 and 120 after infection, whereas the frequency of active caspase-3-positive in OVA-specific CD8+ T cells from the spleen and lung was markedly increased on day 70. We also evaluated intracellular Bcl-2 levels in OVA-specific CD8+ T cells on days 21 and 120 after rBCG-OVA infection. As shown in Fig. 7C, the levels of Bcl-2 expression in the spleen and lung of WT mice are the same as those of IL-15–/– mice on day 21 after rBCG-OVA infection. However, the expression levels were significantly lower in the lung of IL-15–/– mice compared with those in WT mice on day 120 after infection (p < 0.05; Fig. 7C). These results indicate that Ag-specific CD8+ T cells generated in IL-15–/– mice are more sensitive to apoptosis due to decreased Bcl-2 levels in those cells.


Figure 7
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  		FIGURE 7. Sensitivity to apoptosis and Bcl-2 expression in OVA-specific CD8+ T cells in IL-15–/– mice infected with rBCG-OVA. Splenocytes or lung MNCs from IL-15–/– and WT mice infected with rBCG-OVA were stained with anti-CD8 and OVA257–264 Kb tetramer, followed by annexin V (A), anti-active caspase-3 Ab (B), or Bcl-2 with its isotype control staining (C). The number indicated in A and B is the percentage of OVA-specific CD8+ T cells (OVA Kb tet+ CD8+) stained negative for anti-annexin V mAb or positive for anti-active caspase-3 Ab, and in C it is the mean fluorescence intensity. The level of isotype control was drawn on each histogram of Bcl-2 as a vertical dotted line. Data of representative are shown from two separate experiments and are expressed as means ± SD of three mice of each group. Statistically significant differences between IL-15–/– mice and WT mice are shown (*, p < 0.05; **, p < 0.01).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It is widely accepted that CD8+ T cells play a requisite role in resistance to mycobacterial infection (3, 4, 5). In this study, we investigated cell-mediated immunity against primary infection with an avirulent strain of BCG (Tokyo strain) and rBCG-OVA in IL-15–/– mice and found that the IL-15–/– mice showed impaired resistance in the lung at the late stage of primary BCG infection accompanied by marked decreases in Ag-specific CD8+ T cells producing IFN-{gamma} and exhibiting cytotoxicity. Although the degree of proliferation of Ag-specific memory CD8+ T cells was similar to that in WT mice, sensitivity to apoptosis of OVA-specific CD8+ T cells significantly increased in IL-15–/– mice during the course of BCG infection. These results suggest that IL-15 plays an important role in the development of long-lasting protective immunity to BCG infection via sustaining CD8 responses in the lung. The i.p. route of BCG administration we used in the present study does not model for mycobacterial infections, which usually enter the body through respiratory tract, or for BCG vaccination in human, which is s.c. delivered. However, there is evidence that CD8+ CTL cells might not be able to migrate to lung efficiently after s.c. vaccination (30). Furthermore, i.p. BCG vaccination is reported to be able to elicit CD8+ CTL cells in lung more effectively than the s.c. vaccination (31). The role of IL-15 in sustaining CD8 responses in the lung may be conspicuous only in i.p. route for BCG infection; however, Lazarevic et al. (32) have recently reported the induction of M. tuberculosis-specific primary and secondary T cell responses in IL-15–/– mice, which shows a significant increase in bacterial number in lung but not in spleen of IL-15–/– mice at late stage (12 wk) after aerosol infection with M. tuberculosis. These results are consistent with our results, although they did not elucidate the mechanisms for impaired protection in lung of IL-15–/– mice. Our findings showing the roles of IL-15 in protection in lung against BCG infection may be able to apply to infection with all mycobacteria species usually penetrating through respiratory tract.

A notable finding in the present study is that Ag-specific CD8+ T cells could not be sustained for a long time in lung in the absence of IL-15. Upon encounter with a pathogenic microbe, Ag-specific T cells proliferate and differentiate into activated effector T cells at the expansion phase. Most of the activated T cells die by apoptosis at the contraction phase, but the few that survive become memory cells and persist for a long period of time at the maintenance stage, sometimes throughout the life of an animal (33, 34, 35). A key issue is at which stage IL-15 is involved in determining the size of Ag-driven CD8+ T cells in the lung after BCG infection. IL-15 is important in maturation of dendritic cells (DCs) for Ag presentation (36), suggesting that generation of effector CD8+ T cells may be impaired at the expansion phase after BCG infection, resulting in a decrease in the number of Ag-specific CD8+ T cells in the lung at the late stage of infection. Recent studies have demonstrated that primary responses to LCMV and OVA were readily generated in IL-15–/– mice or IL-15R{alpha}–/– mice to a level equal to that in control mice (24, 37). We have also reported that generation of Ag-specific CD8+ T cells in IL-15 Tg mice and IL-15–/– mice normally occurred after primary infection with L. monocytogenes (38, 39). In contrast with acute infection such as infection with L. monocytogenes or LCMV, in which effector CD8+ T cell response peaks at ~5–7 days postinfection, BCG causes chronic infection, in which the bacterial burden persists for a long time. Peak response by Ag-specific CD8+ T cells is delayed at ~21–30 days after BCG infection compared with that in the case of acute infection (27). We have shown in this study that levels of effector CD8+ T cells in the spleen and lung of IL-15–/– mice were normal on day 21 after rBCG-OVA infection. This may explain why IL-15–/– mice showed the same level of resistance in the lung at the early stage of BCG infection. IL-15 is not mandatory for the expansion of CD8+ T cells in the immune response against BCG.

IL-15 has been shown to be a potent inhibitor of several apoptosis pathways in several lymphocytes via induction of antiapoptotic molecules (40, 41, 42, 43). Similar to acute infection with L. monocytogenes and LCMV, the number of effector CD8+ T cells was gradually decreased by apoptosis from day 21 to day 120 after BCG infection. In the present study, we found that the levels of annexin V expression in OVA-specific CD8+ T cells of IL-15–/– mice were significantly higher on days 21 and 120 after rBCG-OVA infection than those in WT mice. We further confirmed that the number of apoptotic cells in Ag-specific CD8+ T cells was increased in lung of IL-15–/– mice at the late stage after infection as assessed by active caspase-3 expression. Therefore, it is possible that IL-15 protects effector CD8+ T cells from apoptosis during the contraction phase of immune responses after BCG infection. Most of CD8+ effector T cells down-regulate the expression of Bcl-2 as compared with those in naive CD8+ T cells in the contraction phase. Bcl-2 expression is induced via signaling from the common cytokine receptor {gamma}-chain, which is used by IL-15 (15, 38, 39, 40) and prevents apoptosis by withdrawal of growth factors. We found that the expression level of Bcl-2 in Ag-specific CD8+ T cells was significantly decreased in IL-15–/– mice compared with that in WT mice on day 120 after rBCG-OVA infection. This indicates that the absence of IL-15 makes the Ag-specific CD8+ T cells more sensitive to apoptosis due to decreased Bcl-2 levels in those cells. However, recent studies have demonstrated that Fas-Fas ligand signaling is responsible for apoptosis induced by repetitive Ag exposure, high doses of a persistent Ag, or an Ag expressed systemically (44, 45, 46). Fas-Fas ligand signaling may also be involved in the death of activated T cells after BCG infection, because BCG causes chronic infection in which the bacterial burden persists for a long time. It has been shown that IL-15 blocks TNFR1-mediated cell death of fibroblasts by inhibition of an early step in the apoptosis signal cascade (47). Therefore, IL-15 may protect Ag-specific effector CD8+ T cells from Fas-mediated activation-induced cell death in response to a persistent BCG Ag. Further investigation is needed to elucidate these possibilities. We have recently reported that IL-15 Tg mice showed augmented Tc1 responses against BCG infection (21), and these augmented Tc1 responses may be explained by inhibition of apoptosis of Ag-specific CD8+ T cells after BCG immunization. The decrease in the number of Ag-specific CD8+ T cells at this stage may also be due to T cell exhaustion caused by the persistence of high Ag levels. IL-15 may prevent Ag-specific CD8+ T cells from T cell exhaustion. Alternatively, DCs infected with mycobacteria often sequester Ag or production of suppressive cytokines such as IL-10 (48). DCs in IL-15–/– mice may have such characteristics during BCG infection, resulting in impaired APC activity to sustain the CD8+ T cells.

T cells undergo two distinct types of proliferation: Ag-driven (Ag-dependent) proliferation and homeostatic (Ag-independent) proliferation. Ag-independent proliferation is thought to be required for the long-term maintenance of Ag-specific memory CD8+ T cells after acute infection with microbes (33, 34, 35). IL-15 may play a role in the long-term survival of T cells in vivo by inducing proliferation of memory CD8+ T cells in addition to protecting CD8+ T cells against activation-induced apoptosis. We have demonstrated that the number of listeriolysin O 91–99-positive memory CD8+ T cells was significantly higher in IL-15 Tg mice 6 wk after primary infection with L. monocytogenes, which resulted in Ag-independent cell proliferation (38). Thus, IL-15 plays an important role in long-term maintenance of Ag-specific memory CD8+ T cells in an Ag-independent manner. However, we found in the present study that Ag-specific memory CD8+ T cells were still able to proliferate in the absence of IL-15, unlike what was found for acute infection with microbes that are completely cleared. We previously reported that IL-15 is not required for Ag-dependent proliferation of memory CD8+ T cells after secondary infection with L. monocytogenes (39). Wherry et al. reported that Ag-specific CD8+ T cells failed to acquire the cardinal memory T cell properties of long-term Ag-independent persistence during chronic infection with LCMV (49). A recent study has demonstrated that Ag-specific memory CD8+ T cells in IL-15–/– mice were able to undergo cell proliferation in an Ag-dependent manner during latent gammaherpesvirus infection (50). Therefore, during chronic infection with BCG, Ag-dependent, but not IL-15-dependent, proliferation may be important in the maintenance of Ag-specific CD8+ T cells due to the persistent low levels of Ags. Two subsets of memory CD8+ T cells based on their anatomical location, expression of cell surface markers, and effector functions have been described (51). Memory CD8+ T cells expressing homing receptors such as CD62 ligand (CD62L) and CCR7, which allow efficient homing to lymph nodes, are termed central memory cells, whereas memory T cells lacking these lymph node homing receptors, which are located in nonlymphoid tissues, are termed effector memory cells. During chronic infection, Ag-specific CD8+ T cells often retain a CD62LlowCCR7low phenotype that favors homing to nonlymphoid tissues (52, 53). We found in this study that the numbers of Ag-specific memory CD8+ T cells in IL-15–/– mice were more decreased in nonlymphoid tissue than were those in lymphoid tissue on day 120 after rBCG-OVA infection. In correlation with the number of memory CD8+ T cells, the number of annexin V-negative OVA-specific CD8+ T cells of IL-15–/– mice was significantly decreased in the lung compared with that in the spleen on day 120 after rBCG-OVA infection. We recently found that IL-15 affects mainly the survival of Ag-specific CD62Llow CD8+ T cells during the contraction phase after Listeria infection (our unpublished data). Therefore, IL-15 may affect mainly the survival of effector cells or effector memory cells, which reside mainly in nonlymphoid tissues such as the lung and serve as the fist line of host defense against microbial invasion.

In addition, there are several lines of evidence that IL-15 is capable of stimulating CD8+ CTLs to exhibit increased cytotoxicity (54, 55). IL-15 has been reported to directly up-regulate the expression of cytotoxic molecules such as granzyme B and perforin, mimicking TCR cross-linking in the induction of cytotoxic molecules and cytotoxicity of effector CD8+ T cells (56). Recent studies have suggested that CD8+ CTLs releasing perforin and granulysin play a role in protection against M. tuberculosis infection via a cytolysis mechanism (9, 10, 11). We showed in this study that in vivo killer activity of Ag-specific CD8+ T cells was significantly impaired in the lung at the late stage of rBCG-OVA infection. These findings raise the possibility that IL-15 plays an important role in rapid elicitation of cytotoxic functions in effector CD8+ T cells in microbial invasion, providing robust protection against chronic infection; however, at present we do not know the relative contribution of cytotoxicity by CD8+ T cells to protection against BCG infection.

Infection of mice with less virulent BCG consistently showed that CD8+ T cells made no contribution to immunity in normal mice (57, 58). Although CD8+ T cells play a crucial role in protection in the lung at the late stage of BCG infection, the relative contribution of cells other than CD8+ T cells to protection against BCG infection remains to be elucidated. IL-15 is known to play important roles in proliferation, accumulation, and maintenance of NK cells (22, 23). The results of the present study revealed that IL-15–/– mice have greatly reduced numbers of NK cells in peripheral lymphoid tissues and that NK cells remained at undetectable levels on day 120 after BCG infection. We previously reported that in vivo administration of either anti-Asialo GM1Ab or anti-CD8 mAb abrogated antibacterial activity, suggesting that both NK cells and CD8+ T cells are required for protection against BCG infection in IL-15 Tg mice (21). Therefore, NK cells may also contribute to protection in the lung at the late stage of infection in WT mice; however, in WT mice, in vivo depletion of NK cells did not have an obvious effect on the growth of bacteria at the early stage of BCG infection (21, 59). In the present study, we found that IL-15–/– mice lacking NK cells exhibited the same level of resistance as that shown by WT mice by day 14 after BCG infection. Therefore, NK cells do not appear to be important for the control of BCG infection in WT mice. Taken together, it appears that IL-15 may serve to induce proliferation and/or accumulation of NK cells during BCG infection and that an increase in the number of NK cells is not essential for enhanced resistance against BCG infection in WT mice.

IL-15 also plays important roles in proliferation, accumulation, and maintenance of NKT cells and a subset of {gamma}{delta} T cells (18, 22, 23). The results of the present study revealed that the numbers of NKT cells and {gamma}{delta} T cells were remarkably increased in WT mice after BCG infection (data not shown). It has been reported that host defense and delayed-type hypersensitivity response to M. bovis BCG in NKT–/– mice were not different from those in WT mice after pulmonary infection (60). Studies with TCR{gamma}{delta} gene-knockout mice suggested that TCR{gamma}{delta} T cells play a role in granuloma formation to Mycobacteria but not in protection as assessed by bacterial growth (61). These results suggest that V{alpha}14 NKT cells and {gamma}{delta} T cells play only a marginal role, if any, in host resistance to mycobacterial infection.

Th1 cells secreting IFN-{gamma} and TNF-{alpha} play a crucial role in protection against mycobacterial infection (1, 2, 3). The results of the present study reveal that there is no difference in Th1 responses in the spleen and lung of IL-15–/– mice and WT mice. This may explain why no difference was found between numbers of bacteria in the spleen at the early and late stages of BCG infection. CD8+ T cells are more important for protection in the lung at the late stage of BCG infection. This speculation warrants further examination with deletion of Ag-specific CD4+ T cells in IL-15–/– mice infected with BCG.

In conclusion, we found that the IL-15–/– mice showed impaired resistance in the lung at the late stage of primary BCG infection accompanied by marked decreases in Ag-specific CD8+ T cells producing IFN-{gamma} and exhibiting cytotoxicity. Although BCG has been used as a vaccine, it confers incomplete protection against tuberculosis, at least in adults. Because the results of this study using IL-15–/– mice indicate that IL-15 is important for long-lasting protective immunity in the lung mediated by CD8+ Tc1/CTL, it is thought that IL-15 can be used as an immune adjuvant to increase the efficacy of BCG vaccination via enhancing CD8 response in the lung. Additional experiments with aerosol challenge with M. tuberculosis might enable us to determine conclusively whether IL-15 is useful for the development of new immunoprotective approaches against mycobacterial infection.


   Acknowledgments
 
We thank Yohko Kobayashi and Kazue Kaneda for providing excellent technical support.


   Disclosures
Top
 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 supported by a Grant-in-Aid for Scientific Research on Priority Areas and Young Scientists (B) from the Japan Society for Promotion of Science and by grants from the Japanese Ministry of Education, Science and Culture (to Y.Y.), Yakult Bioscience Foundation (to Y.Y.), Uehara Memorial Foundation (to Y.Y.), and Nakamura Jishirou Foundation (to T.Y.). Back

2 Address correspondence and reprint requests to Dr. Yasunobu Yoshikai, Division of Host Defense, Medical Institute of Bioregulation, Kyushu University, Fukuoka, 812-8582, Japan. E-mail address: yoshikai{at}bioreg.kyushu-u.ac.jp Back

3 Abbreviations used in this paper: Tg, transgenic; BCG, bacillus Calmette-Guérin; LCMV, lymphocytic choriomeningitis virus; rBCG-OVA, recombinant OVA-expressing BCG; PPD, purified protein derivative; WT, wild type; Tc1, T cytotoxic 1; MNC, mononuclear cell; DC, dendritic cell; CD62L, CD62 ligand. Back

Received for publication March 21, 2005. Accepted for publication December 7, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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