Induction of Mycobacterium tuberculosis-Specific Primary and Secondary T-Cell Responses in Interleukin-15-Deficient Mice
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感染与免疫杂志 2005年第5期
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
ABSTRACT
Several studies have provided evidence that interleukin-15 (IL-15) can enhance protective immune responses against Mycobacterium tuberculosis infection. However, the effects of IL-15 deficiency on the functionality of M. tuberculosis-specific CD4 and CD8 T cells are unknown. In this study, we investigated the generation and maintenance of effector and memory T-cell responses following M. tuberculosis infection of IL-15–/– mice. IL-15–/– mice had slightly higher bacterial numbers during chronic infection, which were accompanied by an increase in gamma interferon (IFN-)-producing CD4 and CD8 T cells. There was no evidence of increased apoptosis or a defect in proliferation of CD8 effector T cells following M. tuberculosis infection. The induction of cytotoxic and IFN- CD8 T-cell responses was normal in the absence of IL-15 signaling. The infiltration of CD4 and CD8 T cells into the lungs of "immune" IL-15–/– mice was delayed in response to M. tuberculosis challenge. These findings demonstrate that efficient effector CD4 and CD8 T cells can be developed following M. tuberculosis infection in the absence of IL-15 but that recall T-cell responses may be impaired.
INTRODUCTION
Interleukin-15 (IL-15) exerts its biological effects on multiple cell types as a result of wide distribution of its receptor (IL-15R). IL-15–/– and IL-15R–/– mice exhibit lymphopenia due to marked reductions of thymic and peripheral NK, NK T cells, and T-cell receptor intraepithelial lymphocytes (7, 13). Furthermore, in the absence of IL-15 signaling, the population of CD8 T cells with a memory phenotype (CD8+ CD44hi) was significantly diminished (7, 13).
Zhang et al. identified alpha/beta interferon to be a potent inducer of IL-15, which strongly and selectively stimulated proliferation of memory CD8 T cells both in vivo and in vitro (31). IL-2/IL-15R (a common receptor subunit for these two cytokines) is strongly expressed on memory CD8+ CD44hi T cells but only at low levels on nave CD8 T cells and memory CD4 T cells (11, 31). Treatment of cells with the blocking anti-IL-2/IL-15R antibody, but not with anti-IL-2 antibody, markedly reduced the numbers of proliferating memory CD8 T cells (11). Additional evidence for the role of IL-15 in regulating homeostasis of memory CD8 T cells came from IL-15 transgenic mice, which have markedly increased numbers of memory CD8 T cells (19, 29).
IL-15 signaling is also essential for optimal antigen-presenting functions of dendritic cells (17). Dendritic cells and macrophages from c–/–, IL-2/IL-15R–/–, and IL-15–/– mice but not from IL-2–/– mice showed impaired production of IL-12, gamma interferon (IFN-), and NO and reduced levels of antigen-presenting and costimulatory molecules (20). IL-15 stimulated human monocytes to produce IL-12 upon contact with CD4 T cells via CD40-CD40L interaction and thus contributed to IL-12-mediated induction of IFN- secretion by CD4 T cells (1).
Due to pleiotropic effects of IL-15 on multiple cell types of innate immunity and the CD8 T-cell compartment, it was not surprising that IL-15–/– and IL-15R–/– mice exhibited compromised host defense responses against viral and bacterial pathogens. Although the generation of lymphocytic choriomeningitis virus (LCMV)-specific effector CD8 T-cell responses was unimpaired in IL-15-deficient mice (2), the absence of IL-15 had a profound effect on the maintenance of LCMV-specific memory CD8 T-cell responses (2). The generation of primary and memory CD8 T-cell responses against vesicular stomatitis virus was dependent on IL-15 signaling, as longitudinal analysis revealed a slow decline in virus-specific memory CD8 T cells in IL-15–/– and IL-15R–/– mice (22).
Treatment of Toxoplasma gondii-immune mice with soluble IL-15R markedly reduced the ability of mice to control infection (10). CD8 T-cell responses in soluble IL-15R-administered mice demonstrated reduced IFN- production, cytolytic activity, and replicative capacity in response to T. gondii infection (10). In contrast, treatment of T. gondii-infected mice with IL-15 augmented and prolonged the duration of CD8 T-cell-mediated immunity against T. gondii infection (8, 9). Furthermore, IL-15 transgenic mice had significantly increased numbers of memory CD8 T cells and higher levels of resistance to Listeria and Salmonella infections (19, 29, 30). Collectively, these studies demonstrate the importance of IL-15 in protective immunity against viral, parasitic, and bacterial infections.
Several lines of evidence indicate that IL-15 may play a role in protective immunity to mycobacterial infections. Although Jullien et al. detected increased levels of IL-15 mRNA in patients with resistant tuberculoid lesions versus patients with susceptible lepromatous lesions (6, 15), immunohistochemical staining of skin biopsy specimens revealed similar levels of IL-15 protein in both forms of the disease (15). In vivo and in vitro studies support the idea that Mycobacterium tuberculosis induces IL-15 expression in murine and human macrophages (4, 15, 18).
In murine studies, IL-15 transgenic mice exhibited increased resistance to Mycobacterium bovis BCG infection, which in part could be attributed to increased numbers of NK cells and augmented IFN- production by CD8 T cells (26). BCG-vaccinated IL-15 transgenic mice had significantly lower bacterial burdens following M. tuberculosis challenge than BCG-vaccinated wild-type mice (27). The increased protective effect observed in IL-15 transgenic BCG-vaccinated mice was accompanied by enhanced IFN- CD8 T-cell responses (27). IL-15 administration also protected BALB/c mice against virulent M. tuberculosis intravenous infection when given as a treatment at 3 weeks postinfection (16), where timing of administration appeared to be crucial to this response.
Although these studies collectively show that IL-15 is expressed following mycobacterial infections and that IL-15 can enhance protective immunity against M. tuberculosis infection, there is no information about the functionality of M. tuberculosis-specific primary and secondary T-cell responses in the absence of IL-15. In this study, we evaluated the quality of CD4 and CD8 effector and memory T-cell responses against M. tuberculosis infection in IL-15–/– mice. Our data indicate IL-15–/– mice were slightly impaired in the ability to control chronic infection, but this was not due to defects in the frequency of IFN--producing CD4 and CD8 T-cell responses. However, there were significantly fewer CD4 and CD8 T cells in the lungs of "immune" IL-15–/– mice than in wild-type mice early after challenge. These findings suggest that the generation of M. tuberculosis-specific effector CD4 and CD8 T-cell responses is unimpaired in IL-15–/– mice but that memory cells may not be maintained properly.
MATERIALS AND METHODS
Mice and infections. Three independent studies were performed using IL-15–/– mice. The first study was carried out with IL-15–/– mice that were bred at the University of Pittsburgh Biotech Center Animal Facility. An IL-15–/– breeding pair was obtained from Jacques Pechon, Immunex. The IL-15–/– breeding pairs were deposited at Taconic (Germantown, NY) by Immunex, and we purchased mice for two additional experiments from Taconic. C57BL/6 wild-type mice were obtained from Charles River Laboratories (Wilmington, MA). All mice were kept under specific-pathogen-free conditions in a biosafety level 3 facility. Animal protocols used in this study were approved by the University Institutional Animal Care and Use Committee. For the primary infection and secondary challenge, mice were infected with a low dose of M. tuberculosis (Erdman strain; Trudeau Institute, Saranac Lake, NY) at 5 x 105/ml using a nose-exposure-only aerosolizer unit (Intox Inc., Moriarty, NM). The dose received was estimated by plating whole-lung homogenates of two mice 24 h following each aerosol infection (30 CFU/mouse).
Secondary infection of IL-15–/– mice. Mice were infected with a low dose of M. tuberculosis (30 CFU/mouse) via the aerosol route. From 4 weeks postinfection, mice were treated with isoniazid (0.1 g/liter) and pyrazinamide (15 g/liter) in drinking water twice weekly for 8 weeks to clear the infection. As IL-15–/– mice were unable to clear M. tuberculosis with this regimen, the treatment was changed to isoniazid (0.1g/liter) and rifampin (0.15 g/liter) for an additional 12 weeks. At the end of antibiotic treatment, mice were sacrificed and several organ homogenates (lung and spleen) were plated on 7H10 plates (Difco) to confirm the absence of viable mycobacteria. Mice were then challenged with a low dose of M. tuberculosis via the aerosol route, and the quality of CD8 memory T-cell responses was investigated after secondary M. tuberculosis infection.
Exogenous IL-15 administration to wild-type mice. Mice were injected intraperitoneally with 1 μg/mouse of recombinant human IL-15 daily starting from day 5 postinfection until day 26 postinfection. Control mice were injected with phosphate-buffered saline (PBS) daily for the same duration of time. Recombinant human IL-15 was generously provided by Immunex.
CFU determination. Lung homogenates were serially diluted in PBS-0.05% Tween 80 and plated on 7H10 agar plates (Difco). Plates were incubated at 37°C and 5% CO2 for 21 days prior to counting colonies.
Bone marrow-derived macrophages and dendritic cell cultures. For ex vivo stimulation assays, such as enzyme-linked immunospot (ELISPOT) and limiting dilution analysis (LDA), bone marrow-derived dendritic cells were cultured in the presence of granulocyte-macrophage colony-stimulating factor supernatant at a 1:200 dilution (a generous gift from Binfeng Lu, University of Pittsburgh) and 20 ng/ml of IL-4 (PeproTech Inc, Rocky Hill, NJ), and macrophages were cultured in the presence of L cell supernatant as a source of CSF-1 as described previously (24).
Flow cytometry. Lung single-cell suspensions were stained as described previously (24). Cells were stained with anti-CD4 (clone H129.19), anti-CD8 (clone 53-6.7), and anti-CD69 (clone H1.2F3) fluorescently conjugated antibodies. All antibodies were purchased from BD Pharmingen (San Diego, CA) and used at a concentration of 0.2 μg/ml. Cells were collected on a FACSCaliber (Becton Dickinson, San Jose, CA) and analyzed by CellQuest (Becton Dickinson) or FlowJo (Tree Star Inc, San Carlos, CA) software.
Proliferation of T cells in the lungs of infected mice. Sixteen hours prior to each experimental time point, mice were injected intraperitoneally with saline containing 1 mg of 5-bromo-2'-deoxyuridine (BrdU; Sigma-Aldrich, St. Louis, MO). Lung cells were stained for cell surface markers CD4 and CD8 at room temperature for 20 min prior to a fixation step with 4% paraformaldehyde (PFA) (200 μl/tube) on ice for 20 min. Cells were washed with tissue culture PBS and centrifuged at 470 x g, and cell pellets were suspended in ice-cold 0.15 M NaCl (100 μl/tube), followed immediately by dropwise fixation with ice-cold 95% ethanol (200 μl/tube) on ice for 30 min. Following a PBS wash, cells were permeabilized and fixed with 200 μl/tube of 0.4% saponin and 2% PFA for 1 h at room temperature. Cells were washed with PBS and suspended in 200 μl/tube of 0.15 M NaCl-4.2 mM MgCl2 (pH 5) containing 250 U/ml of DNase I (Roche, Indianapolis, IN) for 30 min in a 37°C water bath. Cells were washed with PBS and incubated with anti-BrdU antibody or the respective isotype control (fluorescein isothiocyanate [FITC]-conjugated antibody set; BD Pharmingen) diluted at 1:3 in 0.5% Tween 20 and 0.5% bovine serum albumin (50 μl/tube). Following 30 min of incubation at room temperature, cells were washed with PBS and fixed with 4% PFA prior to acquisition on the flow cytometer.
Apoptosis staining. Lung cells were stained for the expression of CD4 and CD8 molecules for 20 min at room temperature. The amount of apoptosis was determined by staining lung cells with annexin V-FITC and 7-amino-actinomycin D (7-AAD) reagents (BD Pharmingen) according to the manufacturer's instructions. Briefly, cells were suspended in 100 μl/tube of 1x binding buffer and incubated with 5 μl/tube of annexin V-FITC and 5 μl/tube of 7-AAD for 15 min in the dark at room temperature. Cells were washed with 2 ml/tube of 1x binding buffer to remove any unbound annexin V-FITC and 7-AAD and fixed with 4% PFA in 1x binding buffer. Samples were analyzed within 30 min.
IFN- production. Cytokine production by T cells isolated from the lungs of M. tuberculosis-infected mice was evaluated by ELISPOT as described previously (14, 25). Briefly, lung and lymph node cells were plated in anti-IFN- antibody (clone R4-6A2; BD Pharmingen)-coated plates (MAIPS4510; Millipore Corp., Bedford, MA) at 80,000 cells/well and 150,000 cells/well, respectively. Cells were incubated in duplicate wells with medium, concanavalin A (10 μg/ml; Sigma-Aldrich), and uninfected and M. tuberculosis-infected dendritic cells (multiplicity of infection of 3; incubated overnight) to estimate the total number of IFN--producing T cells, and M. tuberculosis-infected dendritic cells were incubated with the blocking anti-major histocompatibility complex class I (anti-MHC-I) (clone 8F12; BD Pharmingen) or anti-MHC-II (clone M5/114.15.2; BD Pharmingen) antibodies at 10 μg/ml to estimate the number of IFN--producing CD4 and CD8 T cells, respectively. All dendritic cells were added to lung and lymph nodes cells at a 1:2 ratio, and the cultures were supplemented with IL-2 (PeproTech) at a final concentration of 20 U/ml. Following 40 h of incubation, the IFN--producing T cells were visualized after stepwise incubation of plates with biotinylated anti-IFN- antibody (clone XMG 1.2; BD Pharmingen), streptavidin-conjugated enzyme (PK-6100; Vector Laboratories), and AEC substrate (SK-4200; Vector Laboratories). The spot-forming units (SFU) were enumerated by using an ELISpot reader (Cellular Technology Ltd, Cleveland, OH).
LDA. The cytotoxic potential of CD8 T cells was estimated using LDA (14, 25). Effector cells were derived from the lungs and lung-draining lymph nodes of M. tuberculosis-infected mice at designated time points. Freshly isolated cells were plated in twofold serial dilutions starting from 40,000 cells/well to 1,250 cells/well in V-bottom 96-well plates (24 replicates/input number) supplemented with IL-2 at 20 U/ml. Lung and lymph node cells were incubated with M. tuberculosis-infected dendritic cells (500 dendritic cells/well) for 7 days. Following incubation, 100 μl of spent medium was removed from each well, and lung cells were cultured for another round of stimulation with M. tuberculosis-infected macrophages (1,000 macrophages/100 μl/well) and IL-2 (20 U/ml) to allow for the expansion of cytotoxic T lymphocyte precursors (CTLp). Flow cytometry analysis revealed that 75 to 95% of cells were CD8 T cells after 2 weeks of stimulation. Cytotoxicity was determined in each well by a standard chromium-51 release assay with M. tuberculosis-infected macrophages as targets. M. tuberculosis-infected macrophages were labeled with 51Cr (100 μl of 51Cr per 3 x 106 macrophages) for 1 h at 37°C and added to lung and lymph node T-cell cultures at 4,000 cells/well. Following 4 h of incubation, 100 μl of supernatant was collected (Skatron SCS System; Skatron, Sterling, VA), and radioactivity was quantified using a gamma counter. Positive wells were defined as having greater than the mean plus 3 standard deviations of spontaneous target cell release. The frequency of CTLp was determined by using a zero-order Poisson equation (ln Y = –Fx + ln A; where x is the number of effector cells per well, Y is the percentage of negative wells, A is the y axis intercept, and F is the CTLp frequency defined by the negative slope of the line). All calculations were performed using a software program fitted to the equation by 2 minimization analysis (a generous gift from Carolyn A. Keever-Taylor, Medical College of Wisconsin).
Quantitative reverse transcription (RT)-PCR. Total lung RNA was extracted with Trizol (Life Technology, Green Island, NY) and an RNA extraction kit as directed by the manufacturer (QIAGEN, Valencia, CA). cDNA synthesis was performed using the Superscript II enzyme system according to the manufacturer's instructions (QIAGEN). We adopted a relative gene expression method, as described previously (12), using the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA). In our assay, we used RNA isolated from the lungs of uninfected wild-type mice as a calibrator, since uninfected IL-15–/– mice exhibited higher baseline levels of cytokine gene expression. Hypoxanthine phosphoribosyltransferase (HPRT) was used as a normalizer gene. Relative gene expression was calculated as 2–Ct, where Ct = Ct (gene of interest) -Ct (normalizer) and Ct = Ct (sample) – Ct (calibrator). We used published sequences for the IL-2, IL-7, IL-10, and IL-12 primer and probe sets (5), which were used at 400 and 250 nM concentrations, respectively. The efficiency of each primer pair and probe was tested, and all showed greater than 99% efficiency.
Statistics. Statistically significant differences in the numbers of effector T cells between IL-15–/– and wild-type mice were determined using an unpaired, two-tailed Student t test. Differences in the relative expression of cytokine genes as determined by RT-PCR were analyzed by a two-tailed Mann-Whitney test. Bacterial numbers were log transformed and compared using an unpaired, two-tailed Student t test. A P value of <0.05 was defined as being significant.
RESULTS
IL-15–/– mice did not have increased susceptibility to M. tuberculosis. IL-15–/– and wild-type mice were infected with a low dose of M. tuberculosis via the aerosol route. At 3-week intervals, lung, spleen, and lymph node homogenates were plated to determine the number of CFU. IL-15–/– and wild-type mice were equally capable of controlling M. tuberculosis infection in the lungs during acute infection; however, IL-15–/– mice harbored a slightly higher bacterial burden during chronic infection (reproducible in two separate experiments) (Fig. 1A). There were no significant differences in bacterial numbers in the spleens and lymph nodes of IL-15–/– and wild-type mice at any time point (Fig. 1A).
CD4 and CD8 T-cell responses during primary M. tuberculosis infection. Overall numbers of cells infiltrating the lungs and within the lymph nodes were lower in IL-15–/– mice than in wild-type mice after the initial phase of infection. Although reproducible, differences were not always statistically significant (Fig. 2A). IL-15–/– and wild-type mice had similar numbers of CD4 T cells in the lungs and lymph nodes (Fig. 2B). In contrast, uninfected IL-15–/– mice had 50% fewer CD8 T cells in the lungs and 70% fewer CD8 T cells in the lymph nodes than uninfected wild-type mice (data not shown). A twofold difference in the numbers of CD8 T cells was maintained throughout the infection (Fig. 2C). There was a similar percentage of activated CD69+ CD4 T cells, while significantly more CD8 T cells were activated in IL-15–/– mice during chronic infection (Fig. 2D).
Proliferation and apoptosis of CD8 T cells during M. tuberculosis infection. IL-15–/– or IL-15R–/– mice are deficient in peripheral CD8 but not CD4 T cells (7, 13, 28) (Fig. 2B and C). IL-15 signaling promotes the survival of nave CD8 T cells by inducing expression of antiapoptotic proteins, such as Bcl-2 (3, 28). The lack of IL-15 signaling likely explains the initial CD8 T-cell deficiency in uninfected IL-15–/– mice. Since IL-15 is also important for stimulating the proliferation and survival of antigen-specific memory CD8 T cells (CD8+ CD44hi) (2, 7, 13), we sought to determine whether proliferation of CD8 T cells was impaired in the absence of IL-15 and whether CD8 T cells were more prone to apoptosis after M. tuberculosis infection in an IL-15-deficient environment.
Proliferation of CD8 T cells in IL-15–/– and wild-type mice was determined by flow cytometry. Sixteen hours prior to each experiment, mice were injected with BrdU intraperitoneally, and the percentage of BrdU+ cells within the CD8 gate was determined (Fig. 3A). In IL-15–/– mice, CD8 T cells did not show any defects in proliferation in response to M. tuberculosis infection. In fact, a significantly higher percentage of proliferating CD8 T cells in IL-15–/– mice was detected during acute and chronic infection, suggesting that even in the face of persistent exposure to M. tuberculosis antigens in IL-15-deficient mice, CD8 T cells did not lose their capacity to proliferate (Fig. 3A). There were no differences in proliferation of CD8 T cells from the lymph nodes of IL-15–/– and wild-type mice (data not shown).
Since CD8 T cells may be more sensitive to activation-induced cell death in the absence of IL-15, we measured the amount of apoptosis in the CD8 T-cell population of IL-15–/– and wild-type mice; apoptotic CD8 T cells were defined as being annexin V and 7-AAD positive. Similar percentages of lung CD8 T cells from IL-15–/– and wild-type mice underwent apoptosis following M. tuberculosis infection (Fig. 3B). The dynamic T-cell responses in the lungs over time, with waxing and waning of proliferation and apoptosis, have been observed previously (V. Lazarevic et al., submitted).
Collectively, these findings suggest that the remaining CD8 T cells in IL-15–/– mice were capable of proliferating and were not more susceptible to apoptosis than wild-type CD8 T cells following M. tuberculosis infection.
IFN- production by CD4 and CD8 T cells in IL-15–/– mice. The number of IFN- producing CD4 and CD8 T cells was determined after ex vivo stimulation of lung cells with M. tuberculosis-infected dendritic cells in the presence of blocking anti-MHC-I and anti-MHC-II antibodies, respectively. During acute infection, there were no differences in the numbers of IFN--producing CD4 and CD8 T cells in the lungs of IL-15–/– and wild-type mice (Fig. 3C and D). In chronic infection, the number of IFN--producing CD4 and CD8 T cells increased in IL-15–/– mice, which could be due in part to a slightly higher bacterial burden in the lungs of IL-15–/– mice. Although there were significantly fewer CD8 T cells overall in the IL-15–/– mice (50% fewer), the number of IFN--producing CD8 T cells was actually higher in IL-15–/– mice than in wild-type mice during chronic infection as a result of the increased frequency of IFN--producing T cells within the CD8 T-cell population (data not shown). These findings indicate that long-lasting effector CD8 T cells can develop in the absence of IL-15 during M. tuberculosis infection.
Cytotoxic activity of M. tuberculosis-specific CD8 T cells in the absence of IL-15. The cytotoxic potential of IL-15–/– and wild-type CD8 T cells was determined by LDA using isolated lung T cells as effector cells and M. tuberculosis-infected antigen-presenting cells as stimulators. The data are summarized as the mean number of CTLp per lung for each group of mice (Fig. 3E). Our results demonstrate that the cytotoxic CD8 T cells develop and function normally in an IL-15-deficient environment during acute infection. Both groups of mice followed the same kinetics of cytotoxic activity, which in the murine model of tuberculosis is characterized by high cytotoxic activity during acute infection and loss of cytolytic CD8 T cells during chronic infection (V. Lazarevic et al., submitted). Although there were significantly more CTLp in the lungs of IL-15–/– mice 9 weeks postinfection, the cytotoxicity was negligible in both groups of mice by 12 weeks postinfection (Fig. 3E).
Cytokine profile of IL-15–/– mice. Since effector T-cell responses developed normally under IL-15-deficient conditions, we next investigated whether another cytokine(s) could replace IL-15 in providing signals for survival, proliferation, and maintenance of CD4 and CD8 T-cell effector functions. We determined the relative expression levels of IL-2, IL-7, IL-10, and IL-12 cytokine genes in the lungs of IL-15–/– and wild-type mice by quantitative RT-PCR. IL-2, IL-7, and IL-15 belong to the same c family of cytokines. IL-10 and IL-12 cytokines were of interest due to their anti-inflammatory and proinflammatory effects, respectively. We postulated that increased frequency and numbers of IFN--producing CD4 and CD8 T cells in IL-15–/– mice during chronic M. tuberculosis infection (Fig. 3C and D) could be due to either decreased IL-10 or increased IL-12 gene expression. Our results show there was significantly less IL-2 mRNA in the lungs of IL-15–/– than in wild-type mice, while there were no detectable differences in the amounts of IL-7, IL-10, and IL-12 mRNA (Fig. 4).
Effects of exogenous IL-15 administration on the development of T-cell responses and control of M. tuberculosis infection. Initial studies in which the potential of IL-15 as an immunotherapeutic agent against M. tuberculosis was investigated suggested that overexpression of IL-15 can augment T-cell-mediated immune responses to M. tuberculosis (16, 26, 27). We administered recombinant IL-15 (1 μg/mouse) to wild-type (C57BL/6) mice daily from day 5 to day 26 after low-dose aerosol infection and examined the development of T-cell effector functions and protection against M. tuberculosis infection. Daily administration of IL-15 for 21 days had no significant effect on the control of bacterial growth in the lungs of treated mice (Fig. 5A). Both IL-15-treated and control wild-type mice had similar percentages and numbers of CD4 and CD8 T cells in the lungs (Fig. 5B and C and data not shown). No differences in survival or in the numbers of IFN--producing CD4 and CD8 T cells were detected in the lungs of IL-15- and PBS-treated wild-type mice (data not shown).
IL-15–/– mice successfully control secondary infection with M. tuberculosis. C57BL/6 wild-type mice control M. tuberculosis infection, but they are unable to eliminate the bacteria, even with a robust immune response. To study memory responses, previously infected mice were cleared of mycobacteria by treating IL-15–/– and wild-type mice with a combination of pyrazinamide and isoniazid beginning 4 weeks postinfection for 2 months. Prolonged treatment with this combination of antibiotics was previously shown to have sterilizing activity in wild-type mice (21). After completion of a 2-month antibiotic treatment, two IL-15–/– mice and one wild-type mouse were sacrificed and whole-lung homogenates were plated to determine whether antibiotic treatment cleared the bacteria. Surprisingly, IL-15–/– mice were unable to clear mycobacteria and harbored 6 and 63 CFU, respectively. In contrast, no bacteria were recovered from the lungs of wild-type mice. At this stage, the antibiotic regimen was changed to isoniazid (0.1 g/liter) and rifampin (0.15 g/liter). Three months after antibiotic treatment commenced, one mouse from each experimental group was sacrificed to check for the presence of viable bacteria. The IL-15–/– mouse harbored 24 CFU, while again no bacteria were detected in wild-type mice. It was not until mice underwent 5 months of antibiotic treatment that no bacteria were detected in the lungs of IL-15–/– mice. After 2 months of rest, mice were challenged with a low dose of M. tuberculosis via the aerosol route, and the ability of "immune" IL-15–/– and wild-type mice to control secondary M. tuberculosis infection was evaluated. Both "immune" IL-15–/– and wild-type mice were equally efficient at controlling bacterial burden after M. tuberculosis challenge (Fig. 1B).
Memory CD4 and CD8 T-cell responses in IL-15–/– mice. Previous reports have demonstrated that IL-15–/– or IL-15R–/– mice have reduced numbers of memory CD8 T cells (7, 13), mainly due to decreased proliferation and decreased homing of IL-15R–/– lymphocytes to peripheral lymph nodes (13). We investigated the importance of IL-15 in the development of M. tuberculosis-specific T-cell memory after challenging immune mice with a low dose of M. tuberculosis. Infiltration of immune cells into the lungs of IL-15–/– "immune" mice was delayed at 3 weeks postchallenge, but by 6 weeks postchallenge infiltrations of cells into the lungs of IL-15–/– and wild-type mice were similar (Fig. 6A). No apparent differences were observed in lymph node cell numbers between the two groups of mice throughout the secondary infection (Fig. 6A). Although there were similar numbers of CD4 T cells in the lymph nodes, infiltration of CD4 T cells into the lungs of IL-15–/– mice initially lagged behind wild-type mice (Fig. 6B). The absence of IL-15 resulted in a significantly lower percentage of activated CD69+ CD4 T cells after challenge (Fig. 6D). IL-15–/– mice had significantly fewer CD8 T cells in the lungs and lymph nodes initially after challenge. In the lungs, it was not until 9 weeks postinfection that CD8 T cells in IL-15–/– mice finally reached wild-type numbers (Fig. 6C). In the lymph nodes, it appeared that CD8 T cells never expanded in numbers after secondary infection (Fig. 6C). Similar percentages of activated CD8 T cells were observed in the lungs of IL-15–/– and wild-type mice after secondary M. tuberculosis infection (Fig. 6D).
Proliferation of memory CD4 and CD8 T-cell responses in IL-15–/– mice. In contrast to previous findings where numbers of virus-specific memory CD8 T cells slowly declined in IL-15-deficient mice (2, 22), the most dramatic difference in memory T-cell responses of IL-15–/– and wild-type mice was observed early after secondary M. tuberculosis infection. "Immune" IL-15–/– mice had significantly fewer CD4 and CD8 T cells until 6 weeks postchallenge (Fig. 6B and C). This difference in total numbers of CD4 and CD8 T cells during early recall response was not due to impaired proliferation, as similar percentages of proliferating CD4 and CD8 T cells were detected in the lungs of "immune" IL-15–/– and wild-type mice (Fig. 7A). These differences are most likely due to delayed infiltration of memory CD4 and CD8 T cells into the infected lungs following secondary M. tuberculosis infection. It is important to note that any effect of IL-15 deficiency could be masked by de novo priming of effector T cells later in infection. Therefore, similar numbers of lung cells at 6 weeks postchallenge could be attributed to the influx of primary effector T cells rather than memory T cells.
IFN- production by memory CD4 and CD8 T cells is not dependent on IL-15. We assessed the ability of memory CD4 and CD8 T cells from IL-15–/– and wild-type mice to produce IFN- in response to M. tuberculosis-infected dendritic cells. Equal numbers of IFN--producing CD4 and CD8 memory T cells were present in the lungs of "immune" IL-15–/– and wild-type mice (Fig. 7B), suggesting that IFN- production by memory T cells is not dependent on IL-15.
DISCUSSION
The main goal of this study was to investigate whether IL-15 is required for the generation and maintenance of effector and memory T-cell responses following M. tuberculosis infection. The results of this study indicate that IL-15–/– mice were not substantially impaired in their ability to control primary and secondary M. tuberculosis infection. Similar numbers of mycobacteria were detected in the lungs, lymph nodes, and spleens of IL-15–/– and wild-type mice. There was a tendency toward a slightly higher bacterial burden in the lungs of IL-15–/– mice during chronic M. tuberculosis infection, and IL-15–/– mice were less able to clear the infection in the presence of antibiotics. CD4 and CD8 T-cell effector functions were not affected by IL-15 deficiency after primary and secondary M. tuberculosis infections. In view of recent studies, the most relevant finding is that CD8 T cells were not more prone to apoptosis following M. tuberculosis infection, and there was no sign of reduced proliferation of effector CD8 T cells in IL-15-deficient mice. The most dramatic difference in "immune" IL-15–/– and wild-type mice was characterized by delayed infiltration of memory cells into the lungs of "immune" mice after secondary challenge. We did not find a beneficial effect of IL-15 treatment on the ability of wild-type mice to control M. tuberculosis infection. However, the route or dose of infection, the timing of administration, or the background of the mouse strains may be confounding factors.
In this study, we sought to determine whether IL-15 was required for the generation of primary and recall T-cell responses against M. tuberculosis. The baseline number of CD8 T cells in the lungs and lymph nodes in uninfected IL-15–/– mice was at least twofold lower than in uninfected wild-type mice. Initial studies with IL-15–/– and IL-15R–/– mice revealed that in the absence of IL-15 signaling there was a 30 to 50% reduction in peripheral CD8 T cells (7, 13). IL-15 signaling is important for the survival of peripheral nave CD8 T cells, which is most likely mediated by increased expression of antiapoptotic proteins, such as Bcl-2 (3, 28). Although the initial CD8 T-cell deficiency in IL-15–/– mice could be explained by reduced survival of nave T cells associated with IL-15-deficient mice, there was still a possibility that a reduction in the overall CD8 effector population after primary infection could be due to reduced proliferation or enhanced apoptosis of remaining CD8 T cells in the absence of IL-15. We tested these hypotheses, and our data indicate that a significantly higher percentage of effector CD8 T cells proliferated in IL-15–/– mice during acute and chronic M. tuberculosis infection. Therefore, the proliferative ability of CD8 T cells in the face of persistent stimulation with M. tuberculosis antigens was not undermined in the absence of IL-15. Since CD8 T cells may be more prone to undergo apoptosis after antigenic stimulation in the absence of IL-15, we determined the percentage of apoptotic CD8 T cells during acute and chronic M. tuberculosis infections. There were similar percentages of apoptotic CD8 T cells in the lungs of IL-15–/– and wild-type mice. Therefore, the low-magnitude CD8 T-cell responses during primary M. tuberculosis infection of IL-15–/– mice is not a result of reduced proliferation or enhanced apoptosis but more likely due to the reduced numbers of CD8 T cells inherent in IL-15–/– mice. Furthermore, the cytokine production and cytotoxic activity of the remaining CD8 T cells were normal or even higher in IL-15–/– mice, supporting the idea that IL-15 is not required for the priming or effector functions of M. tuberculosis-specific CD8 T cells.
In accordance with previous studies (7, 13), CD4 T-cell responses were not affected by the absence of IL-15 signaling. Similar percentages of proliferating and apoptotic CD4 T cells were detected in the lungs and lymph nodes of IL-15–/– and wild-type mice (data not shown). The effector function of CD4 T cells was normal in IL-15–/– mice, as evidenced by a high percentage of activated CD69+ CD4 T cells and potent IFN- CD4 T-cell responses.
Although there were significant differences in the absolute numbers of CD8 T cells between IL-15–/– and wild-type mice, the increased frequency of IFN--producing CD8 T cells in the lungs of IL-15–/– mice resulted in equivalent or even significantly higher numbers of IFN--producing CD8 T cells in IL-15–/– mice during chronic infection. We postulated that enhanced IFN- T-cell responses in IL-15–/– mice could be due to either decreased IL-10 or increased IL-12 gene expression. Since there were no significant differences in IL-10 and IL-12 mRNA in the lungs of IL-15–/– and wild-type mice, it is possible that this boosted IFN- response may be due to increased bacterial burden in the lungs of IL-15–/– mice during the chronic stage of infection. Notably, the number of IFN--producing CD4 and CD8 T cells did not decline in the absence of IL-15.
Using the aerosol route of infection, we have previously demonstrated that after primary M. tuberculosis infection a pool of memory T cells is generated in the lungs of immune mice (23). In the first 2 weeks after challenge with M. tuberculosis, more than 30% of both CD4 and CD8 T cells in the lungs of immune mice expressed activation marker CD69 and produced IFN- upon restimulation. In contrast, less than 5% of T cells in the lungs of nave mice were CD69+ and produced IFN- during the first 2 weeks postinfection (23). These results suggest that M. tuberculosis infection leads to the development of memory CD4 and CD8 T-cell populations capable of rapid activation and effector function in the lungs upon rechallenge (23). However, these memory T-cell responses do not protect mice from reinfection. In fact, protective immunity due to previous exposure to M. tuberculosis generally results in a 10-fold-lower bacterial burden after rechallenge compared to nave mice. The secondary infection is established and bacterial burden is stringently controlled at 105 to 106 CFU per lung over 6 months. Therefore, a log difference in bacterial numbers is a level of protection conferred by memory T-cell responses upon secondary infection of mice with M. tuberculosis.
The inability of memory T cells in the lungs to control initial M. tuberculosis replication and to prevent reinfection remains the largest obstacle that immunologists face, and better understanding of lung-specific immunity is required if we are to design more-effective vaccines against M. tuberculosis.
There are no phenotypic markers that reproducibly represent the functional characteristics of memory CD4 and CD8 T cells in a murine model of tuberculosis. A combination of CD44, Ly6C, and CD62L markers failed to clearly delineate memory cells from effector cells after M. tuberculosis infection of nave or memory mice (unpublished data). The only distinguishing feature of memory response in a murine model of tuberculosis is a higher percentage of activated CD69+ T cells in the lungs of "immune" mice compared with nave mice within the first 3 weeks postinfection (reference 23 and unpublished data). Our data indicate that protective immune responses were generated in the absence of IL-15, as evidenced by the similar control of bacterial loads in the lungs and spleens of challenged IL-15–/– and wild-type mice. Several groups reported that in the absence of IL-15 or IL-15R signaling, the homeostatic proliferation of memory CD8 T cells was significantly diminished, leading to a slow decline in virus-specific memory CD8 T cells (2, 22). There was no indication of reduced proliferation or a decline in memory CD4 and CD8 T-cell responses in IL-15–/– mice in our studies. The most dramatic difference between "immune" IL-15–/– and wild-type mice was observed early after secondary challenge with M. tuberculosis. There was a significant delay in the infiltration of CD4 and CD8 T cells into the lungs of IL-15–/– "immune" mice at 3 weeks postchallenge. By 6 weeks postchallenge, the kinetics and magnitude of memory T-cell responses were similar for IL-15–/– and wild-type mice. As M. tuberculosis is not cleared by secondary immune response, and reinfection is established, it is important to note that any effect of IL-15–/– deficiency on maintenance of memory T-cell responses could be masked by the influx of de novo primed effector T cells after 3 weeks postinfection.
Although development of primary T-cell responses occurred normally in IL-15–/– mice, these mice had difficulty clearing M. tuberculosis infection when treated with antibiotics. We observed previously that CD4–/– mice were unable to eliminate M. tuberculosis during antibiotic treatment (unpublished data). Since CD4 T cells play a pivotal role in controlling acute and persistent M. tuberculosis infection, the efficacy of antibiotics was undermined in the absence of this important T-cell subset. These findings suggest antibiotic treatment must be prolonged in cases of immunodeficiency, such as the absence of CD4 T cells. That IL-15–/– mice had a similar difficulty in eliminating mycobacteria during antibiotic treatment suggested that this cytokine may have other unidentified roles in modulating the "immune" system that may be overlooked using standard immunological techniques. Flow cytometric analysis revealed similar numbers of dendritic cells, neutrophils, and macrophages in the lungs of IL-15–/– and wild-type mice (data not shown), and cytokine profile analysis revealed no significant differences in IL-7, IL-10, and IL-12 mRNA expression in the lungs. Since no major differences in the functionality of T cells, composition of innate immune cells, and cytokine profile (except for IL-2 mRNA) were detected in IL-15–/– mice, it remains unresolved why IL-15–/– mice were unable to clear M. tuberculosis infection in the presence of a standard course of antibiotics and why chronic infection is less well controlled in these mice.
Collectively, our results indicate that IL-15 is not essential for the generation and maintenance of effector CD4 and CD8 T-cell responses. The magnitude of the recall response in "immune" IL-15–/– mice was significantly smaller than in "immune" wild-type mice, which could be due to delayed infiltration or impaired maintenance of the memory cells or a consequence of a smaller burst size of effector CD8 T-cell responses during primary infection of IL-15–/– mice. Development of tetramer staining reagents for M. tuberculosis antigens and better phenotypic definition of M. tuberculosis-specific memory T cells will enable a detailed investigation of development and maintenance of memory T-cell responses following secondary M. tuberculosis infection.
ACKNOWLEDGMENTS
We are grateful to Jacques Pechon at Immunex for providing IL-15–/– breeding pairs as well as recombinant IL-15. We acknowledge the technical assistance of Amy Myers and Carolyn Bigbee.
These studies were supported by the National Institutes of Health grants AI37859 and AI50732 (J.L.F.) and the American Lung Association (CL-016; J.L.F.).
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ABSTRACT
Several studies have provided evidence that interleukin-15 (IL-15) can enhance protective immune responses against Mycobacterium tuberculosis infection. However, the effects of IL-15 deficiency on the functionality of M. tuberculosis-specific CD4 and CD8 T cells are unknown. In this study, we investigated the generation and maintenance of effector and memory T-cell responses following M. tuberculosis infection of IL-15–/– mice. IL-15–/– mice had slightly higher bacterial numbers during chronic infection, which were accompanied by an increase in gamma interferon (IFN-)-producing CD4 and CD8 T cells. There was no evidence of increased apoptosis or a defect in proliferation of CD8 effector T cells following M. tuberculosis infection. The induction of cytotoxic and IFN- CD8 T-cell responses was normal in the absence of IL-15 signaling. The infiltration of CD4 and CD8 T cells into the lungs of "immune" IL-15–/– mice was delayed in response to M. tuberculosis challenge. These findings demonstrate that efficient effector CD4 and CD8 T cells can be developed following M. tuberculosis infection in the absence of IL-15 but that recall T-cell responses may be impaired.
INTRODUCTION
Interleukin-15 (IL-15) exerts its biological effects on multiple cell types as a result of wide distribution of its receptor (IL-15R). IL-15–/– and IL-15R–/– mice exhibit lymphopenia due to marked reductions of thymic and peripheral NK, NK T cells, and T-cell receptor intraepithelial lymphocytes (7, 13). Furthermore, in the absence of IL-15 signaling, the population of CD8 T cells with a memory phenotype (CD8+ CD44hi) was significantly diminished (7, 13).
Zhang et al. identified alpha/beta interferon to be a potent inducer of IL-15, which strongly and selectively stimulated proliferation of memory CD8 T cells both in vivo and in vitro (31). IL-2/IL-15R (a common receptor subunit for these two cytokines) is strongly expressed on memory CD8+ CD44hi T cells but only at low levels on nave CD8 T cells and memory CD4 T cells (11, 31). Treatment of cells with the blocking anti-IL-2/IL-15R antibody, but not with anti-IL-2 antibody, markedly reduced the numbers of proliferating memory CD8 T cells (11). Additional evidence for the role of IL-15 in regulating homeostasis of memory CD8 T cells came from IL-15 transgenic mice, which have markedly increased numbers of memory CD8 T cells (19, 29).
IL-15 signaling is also essential for optimal antigen-presenting functions of dendritic cells (17). Dendritic cells and macrophages from c–/–, IL-2/IL-15R–/–, and IL-15–/– mice but not from IL-2–/– mice showed impaired production of IL-12, gamma interferon (IFN-), and NO and reduced levels of antigen-presenting and costimulatory molecules (20). IL-15 stimulated human monocytes to produce IL-12 upon contact with CD4 T cells via CD40-CD40L interaction and thus contributed to IL-12-mediated induction of IFN- secretion by CD4 T cells (1).
Due to pleiotropic effects of IL-15 on multiple cell types of innate immunity and the CD8 T-cell compartment, it was not surprising that IL-15–/– and IL-15R–/– mice exhibited compromised host defense responses against viral and bacterial pathogens. Although the generation of lymphocytic choriomeningitis virus (LCMV)-specific effector CD8 T-cell responses was unimpaired in IL-15-deficient mice (2), the absence of IL-15 had a profound effect on the maintenance of LCMV-specific memory CD8 T-cell responses (2). The generation of primary and memory CD8 T-cell responses against vesicular stomatitis virus was dependent on IL-15 signaling, as longitudinal analysis revealed a slow decline in virus-specific memory CD8 T cells in IL-15–/– and IL-15R–/– mice (22).
Treatment of Toxoplasma gondii-immune mice with soluble IL-15R markedly reduced the ability of mice to control infection (10). CD8 T-cell responses in soluble IL-15R-administered mice demonstrated reduced IFN- production, cytolytic activity, and replicative capacity in response to T. gondii infection (10). In contrast, treatment of T. gondii-infected mice with IL-15 augmented and prolonged the duration of CD8 T-cell-mediated immunity against T. gondii infection (8, 9). Furthermore, IL-15 transgenic mice had significantly increased numbers of memory CD8 T cells and higher levels of resistance to Listeria and Salmonella infections (19, 29, 30). Collectively, these studies demonstrate the importance of IL-15 in protective immunity against viral, parasitic, and bacterial infections.
Several lines of evidence indicate that IL-15 may play a role in protective immunity to mycobacterial infections. Although Jullien et al. detected increased levels of IL-15 mRNA in patients with resistant tuberculoid lesions versus patients with susceptible lepromatous lesions (6, 15), immunohistochemical staining of skin biopsy specimens revealed similar levels of IL-15 protein in both forms of the disease (15). In vivo and in vitro studies support the idea that Mycobacterium tuberculosis induces IL-15 expression in murine and human macrophages (4, 15, 18).
In murine studies, IL-15 transgenic mice exhibited increased resistance to Mycobacterium bovis BCG infection, which in part could be attributed to increased numbers of NK cells and augmented IFN- production by CD8 T cells (26). BCG-vaccinated IL-15 transgenic mice had significantly lower bacterial burdens following M. tuberculosis challenge than BCG-vaccinated wild-type mice (27). The increased protective effect observed in IL-15 transgenic BCG-vaccinated mice was accompanied by enhanced IFN- CD8 T-cell responses (27). IL-15 administration also protected BALB/c mice against virulent M. tuberculosis intravenous infection when given as a treatment at 3 weeks postinfection (16), where timing of administration appeared to be crucial to this response.
Although these studies collectively show that IL-15 is expressed following mycobacterial infections and that IL-15 can enhance protective immunity against M. tuberculosis infection, there is no information about the functionality of M. tuberculosis-specific primary and secondary T-cell responses in the absence of IL-15. In this study, we evaluated the quality of CD4 and CD8 effector and memory T-cell responses against M. tuberculosis infection in IL-15–/– mice. Our data indicate IL-15–/– mice were slightly impaired in the ability to control chronic infection, but this was not due to defects in the frequency of IFN--producing CD4 and CD8 T-cell responses. However, there were significantly fewer CD4 and CD8 T cells in the lungs of "immune" IL-15–/– mice than in wild-type mice early after challenge. These findings suggest that the generation of M. tuberculosis-specific effector CD4 and CD8 T-cell responses is unimpaired in IL-15–/– mice but that memory cells may not be maintained properly.
MATERIALS AND METHODS
Mice and infections. Three independent studies were performed using IL-15–/– mice. The first study was carried out with IL-15–/– mice that were bred at the University of Pittsburgh Biotech Center Animal Facility. An IL-15–/– breeding pair was obtained from Jacques Pechon, Immunex. The IL-15–/– breeding pairs were deposited at Taconic (Germantown, NY) by Immunex, and we purchased mice for two additional experiments from Taconic. C57BL/6 wild-type mice were obtained from Charles River Laboratories (Wilmington, MA). All mice were kept under specific-pathogen-free conditions in a biosafety level 3 facility. Animal protocols used in this study were approved by the University Institutional Animal Care and Use Committee. For the primary infection and secondary challenge, mice were infected with a low dose of M. tuberculosis (Erdman strain; Trudeau Institute, Saranac Lake, NY) at 5 x 105/ml using a nose-exposure-only aerosolizer unit (Intox Inc., Moriarty, NM). The dose received was estimated by plating whole-lung homogenates of two mice 24 h following each aerosol infection (30 CFU/mouse).
Secondary infection of IL-15–/– mice. Mice were infected with a low dose of M. tuberculosis (30 CFU/mouse) via the aerosol route. From 4 weeks postinfection, mice were treated with isoniazid (0.1 g/liter) and pyrazinamide (15 g/liter) in drinking water twice weekly for 8 weeks to clear the infection. As IL-15–/– mice were unable to clear M. tuberculosis with this regimen, the treatment was changed to isoniazid (0.1g/liter) and rifampin (0.15 g/liter) for an additional 12 weeks. At the end of antibiotic treatment, mice were sacrificed and several organ homogenates (lung and spleen) were plated on 7H10 plates (Difco) to confirm the absence of viable mycobacteria. Mice were then challenged with a low dose of M. tuberculosis via the aerosol route, and the quality of CD8 memory T-cell responses was investigated after secondary M. tuberculosis infection.
Exogenous IL-15 administration to wild-type mice. Mice were injected intraperitoneally with 1 μg/mouse of recombinant human IL-15 daily starting from day 5 postinfection until day 26 postinfection. Control mice were injected with phosphate-buffered saline (PBS) daily for the same duration of time. Recombinant human IL-15 was generously provided by Immunex.
CFU determination. Lung homogenates were serially diluted in PBS-0.05% Tween 80 and plated on 7H10 agar plates (Difco). Plates were incubated at 37°C and 5% CO2 for 21 days prior to counting colonies.
Bone marrow-derived macrophages and dendritic cell cultures. For ex vivo stimulation assays, such as enzyme-linked immunospot (ELISPOT) and limiting dilution analysis (LDA), bone marrow-derived dendritic cells were cultured in the presence of granulocyte-macrophage colony-stimulating factor supernatant at a 1:200 dilution (a generous gift from Binfeng Lu, University of Pittsburgh) and 20 ng/ml of IL-4 (PeproTech Inc, Rocky Hill, NJ), and macrophages were cultured in the presence of L cell supernatant as a source of CSF-1 as described previously (24).
Flow cytometry. Lung single-cell suspensions were stained as described previously (24). Cells were stained with anti-CD4 (clone H129.19), anti-CD8 (clone 53-6.7), and anti-CD69 (clone H1.2F3) fluorescently conjugated antibodies. All antibodies were purchased from BD Pharmingen (San Diego, CA) and used at a concentration of 0.2 μg/ml. Cells were collected on a FACSCaliber (Becton Dickinson, San Jose, CA) and analyzed by CellQuest (Becton Dickinson) or FlowJo (Tree Star Inc, San Carlos, CA) software.
Proliferation of T cells in the lungs of infected mice. Sixteen hours prior to each experimental time point, mice were injected intraperitoneally with saline containing 1 mg of 5-bromo-2'-deoxyuridine (BrdU; Sigma-Aldrich, St. Louis, MO). Lung cells were stained for cell surface markers CD4 and CD8 at room temperature for 20 min prior to a fixation step with 4% paraformaldehyde (PFA) (200 μl/tube) on ice for 20 min. Cells were washed with tissue culture PBS and centrifuged at 470 x g, and cell pellets were suspended in ice-cold 0.15 M NaCl (100 μl/tube), followed immediately by dropwise fixation with ice-cold 95% ethanol (200 μl/tube) on ice for 30 min. Following a PBS wash, cells were permeabilized and fixed with 200 μl/tube of 0.4% saponin and 2% PFA for 1 h at room temperature. Cells were washed with PBS and suspended in 200 μl/tube of 0.15 M NaCl-4.2 mM MgCl2 (pH 5) containing 250 U/ml of DNase I (Roche, Indianapolis, IN) for 30 min in a 37°C water bath. Cells were washed with PBS and incubated with anti-BrdU antibody or the respective isotype control (fluorescein isothiocyanate [FITC]-conjugated antibody set; BD Pharmingen) diluted at 1:3 in 0.5% Tween 20 and 0.5% bovine serum albumin (50 μl/tube). Following 30 min of incubation at room temperature, cells were washed with PBS and fixed with 4% PFA prior to acquisition on the flow cytometer.
Apoptosis staining. Lung cells were stained for the expression of CD4 and CD8 molecules for 20 min at room temperature. The amount of apoptosis was determined by staining lung cells with annexin V-FITC and 7-amino-actinomycin D (7-AAD) reagents (BD Pharmingen) according to the manufacturer's instructions. Briefly, cells were suspended in 100 μl/tube of 1x binding buffer and incubated with 5 μl/tube of annexin V-FITC and 5 μl/tube of 7-AAD for 15 min in the dark at room temperature. Cells were washed with 2 ml/tube of 1x binding buffer to remove any unbound annexin V-FITC and 7-AAD and fixed with 4% PFA in 1x binding buffer. Samples were analyzed within 30 min.
IFN- production. Cytokine production by T cells isolated from the lungs of M. tuberculosis-infected mice was evaluated by ELISPOT as described previously (14, 25). Briefly, lung and lymph node cells were plated in anti-IFN- antibody (clone R4-6A2; BD Pharmingen)-coated plates (MAIPS4510; Millipore Corp., Bedford, MA) at 80,000 cells/well and 150,000 cells/well, respectively. Cells were incubated in duplicate wells with medium, concanavalin A (10 μg/ml; Sigma-Aldrich), and uninfected and M. tuberculosis-infected dendritic cells (multiplicity of infection of 3; incubated overnight) to estimate the total number of IFN--producing T cells, and M. tuberculosis-infected dendritic cells were incubated with the blocking anti-major histocompatibility complex class I (anti-MHC-I) (clone 8F12; BD Pharmingen) or anti-MHC-II (clone M5/114.15.2; BD Pharmingen) antibodies at 10 μg/ml to estimate the number of IFN--producing CD4 and CD8 T cells, respectively. All dendritic cells were added to lung and lymph nodes cells at a 1:2 ratio, and the cultures were supplemented with IL-2 (PeproTech) at a final concentration of 20 U/ml. Following 40 h of incubation, the IFN--producing T cells were visualized after stepwise incubation of plates with biotinylated anti-IFN- antibody (clone XMG 1.2; BD Pharmingen), streptavidin-conjugated enzyme (PK-6100; Vector Laboratories), and AEC substrate (SK-4200; Vector Laboratories). The spot-forming units (SFU) were enumerated by using an ELISpot reader (Cellular Technology Ltd, Cleveland, OH).
LDA. The cytotoxic potential of CD8 T cells was estimated using LDA (14, 25). Effector cells were derived from the lungs and lung-draining lymph nodes of M. tuberculosis-infected mice at designated time points. Freshly isolated cells were plated in twofold serial dilutions starting from 40,000 cells/well to 1,250 cells/well in V-bottom 96-well plates (24 replicates/input number) supplemented with IL-2 at 20 U/ml. Lung and lymph node cells were incubated with M. tuberculosis-infected dendritic cells (500 dendritic cells/well) for 7 days. Following incubation, 100 μl of spent medium was removed from each well, and lung cells were cultured for another round of stimulation with M. tuberculosis-infected macrophages (1,000 macrophages/100 μl/well) and IL-2 (20 U/ml) to allow for the expansion of cytotoxic T lymphocyte precursors (CTLp). Flow cytometry analysis revealed that 75 to 95% of cells were CD8 T cells after 2 weeks of stimulation. Cytotoxicity was determined in each well by a standard chromium-51 release assay with M. tuberculosis-infected macrophages as targets. M. tuberculosis-infected macrophages were labeled with 51Cr (100 μl of 51Cr per 3 x 106 macrophages) for 1 h at 37°C and added to lung and lymph node T-cell cultures at 4,000 cells/well. Following 4 h of incubation, 100 μl of supernatant was collected (Skatron SCS System; Skatron, Sterling, VA), and radioactivity was quantified using a gamma counter. Positive wells were defined as having greater than the mean plus 3 standard deviations of spontaneous target cell release. The frequency of CTLp was determined by using a zero-order Poisson equation (ln Y = –Fx + ln A; where x is the number of effector cells per well, Y is the percentage of negative wells, A is the y axis intercept, and F is the CTLp frequency defined by the negative slope of the line). All calculations were performed using a software program fitted to the equation by 2 minimization analysis (a generous gift from Carolyn A. Keever-Taylor, Medical College of Wisconsin).
Quantitative reverse transcription (RT)-PCR. Total lung RNA was extracted with Trizol (Life Technology, Green Island, NY) and an RNA extraction kit as directed by the manufacturer (QIAGEN, Valencia, CA). cDNA synthesis was performed using the Superscript II enzyme system according to the manufacturer's instructions (QIAGEN). We adopted a relative gene expression method, as described previously (12), using the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA). In our assay, we used RNA isolated from the lungs of uninfected wild-type mice as a calibrator, since uninfected IL-15–/– mice exhibited higher baseline levels of cytokine gene expression. Hypoxanthine phosphoribosyltransferase (HPRT) was used as a normalizer gene. Relative gene expression was calculated as 2–Ct, where Ct = Ct (gene of interest) -Ct (normalizer) and Ct = Ct (sample) – Ct (calibrator). We used published sequences for the IL-2, IL-7, IL-10, and IL-12 primer and probe sets (5), which were used at 400 and 250 nM concentrations, respectively. The efficiency of each primer pair and probe was tested, and all showed greater than 99% efficiency.
Statistics. Statistically significant differences in the numbers of effector T cells between IL-15–/– and wild-type mice were determined using an unpaired, two-tailed Student t test. Differences in the relative expression of cytokine genes as determined by RT-PCR were analyzed by a two-tailed Mann-Whitney test. Bacterial numbers were log transformed and compared using an unpaired, two-tailed Student t test. A P value of <0.05 was defined as being significant.
RESULTS
IL-15–/– mice did not have increased susceptibility to M. tuberculosis. IL-15–/– and wild-type mice were infected with a low dose of M. tuberculosis via the aerosol route. At 3-week intervals, lung, spleen, and lymph node homogenates were plated to determine the number of CFU. IL-15–/– and wild-type mice were equally capable of controlling M. tuberculosis infection in the lungs during acute infection; however, IL-15–/– mice harbored a slightly higher bacterial burden during chronic infection (reproducible in two separate experiments) (Fig. 1A). There were no significant differences in bacterial numbers in the spleens and lymph nodes of IL-15–/– and wild-type mice at any time point (Fig. 1A).
CD4 and CD8 T-cell responses during primary M. tuberculosis infection. Overall numbers of cells infiltrating the lungs and within the lymph nodes were lower in IL-15–/– mice than in wild-type mice after the initial phase of infection. Although reproducible, differences were not always statistically significant (Fig. 2A). IL-15–/– and wild-type mice had similar numbers of CD4 T cells in the lungs and lymph nodes (Fig. 2B). In contrast, uninfected IL-15–/– mice had 50% fewer CD8 T cells in the lungs and 70% fewer CD8 T cells in the lymph nodes than uninfected wild-type mice (data not shown). A twofold difference in the numbers of CD8 T cells was maintained throughout the infection (Fig. 2C). There was a similar percentage of activated CD69+ CD4 T cells, while significantly more CD8 T cells were activated in IL-15–/– mice during chronic infection (Fig. 2D).
Proliferation and apoptosis of CD8 T cells during M. tuberculosis infection. IL-15–/– or IL-15R–/– mice are deficient in peripheral CD8 but not CD4 T cells (7, 13, 28) (Fig. 2B and C). IL-15 signaling promotes the survival of nave CD8 T cells by inducing expression of antiapoptotic proteins, such as Bcl-2 (3, 28). The lack of IL-15 signaling likely explains the initial CD8 T-cell deficiency in uninfected IL-15–/– mice. Since IL-15 is also important for stimulating the proliferation and survival of antigen-specific memory CD8 T cells (CD8+ CD44hi) (2, 7, 13), we sought to determine whether proliferation of CD8 T cells was impaired in the absence of IL-15 and whether CD8 T cells were more prone to apoptosis after M. tuberculosis infection in an IL-15-deficient environment.
Proliferation of CD8 T cells in IL-15–/– and wild-type mice was determined by flow cytometry. Sixteen hours prior to each experiment, mice were injected with BrdU intraperitoneally, and the percentage of BrdU+ cells within the CD8 gate was determined (Fig. 3A). In IL-15–/– mice, CD8 T cells did not show any defects in proliferation in response to M. tuberculosis infection. In fact, a significantly higher percentage of proliferating CD8 T cells in IL-15–/– mice was detected during acute and chronic infection, suggesting that even in the face of persistent exposure to M. tuberculosis antigens in IL-15-deficient mice, CD8 T cells did not lose their capacity to proliferate (Fig. 3A). There were no differences in proliferation of CD8 T cells from the lymph nodes of IL-15–/– and wild-type mice (data not shown).
Since CD8 T cells may be more sensitive to activation-induced cell death in the absence of IL-15, we measured the amount of apoptosis in the CD8 T-cell population of IL-15–/– and wild-type mice; apoptotic CD8 T cells were defined as being annexin V and 7-AAD positive. Similar percentages of lung CD8 T cells from IL-15–/– and wild-type mice underwent apoptosis following M. tuberculosis infection (Fig. 3B). The dynamic T-cell responses in the lungs over time, with waxing and waning of proliferation and apoptosis, have been observed previously (V. Lazarevic et al., submitted).
Collectively, these findings suggest that the remaining CD8 T cells in IL-15–/– mice were capable of proliferating and were not more susceptible to apoptosis than wild-type CD8 T cells following M. tuberculosis infection.
IFN- production by CD4 and CD8 T cells in IL-15–/– mice. The number of IFN- producing CD4 and CD8 T cells was determined after ex vivo stimulation of lung cells with M. tuberculosis-infected dendritic cells in the presence of blocking anti-MHC-I and anti-MHC-II antibodies, respectively. During acute infection, there were no differences in the numbers of IFN--producing CD4 and CD8 T cells in the lungs of IL-15–/– and wild-type mice (Fig. 3C and D). In chronic infection, the number of IFN--producing CD4 and CD8 T cells increased in IL-15–/– mice, which could be due in part to a slightly higher bacterial burden in the lungs of IL-15–/– mice. Although there were significantly fewer CD8 T cells overall in the IL-15–/– mice (50% fewer), the number of IFN--producing CD8 T cells was actually higher in IL-15–/– mice than in wild-type mice during chronic infection as a result of the increased frequency of IFN--producing T cells within the CD8 T-cell population (data not shown). These findings indicate that long-lasting effector CD8 T cells can develop in the absence of IL-15 during M. tuberculosis infection.
Cytotoxic activity of M. tuberculosis-specific CD8 T cells in the absence of IL-15. The cytotoxic potential of IL-15–/– and wild-type CD8 T cells was determined by LDA using isolated lung T cells as effector cells and M. tuberculosis-infected antigen-presenting cells as stimulators. The data are summarized as the mean number of CTLp per lung for each group of mice (Fig. 3E). Our results demonstrate that the cytotoxic CD8 T cells develop and function normally in an IL-15-deficient environment during acute infection. Both groups of mice followed the same kinetics of cytotoxic activity, which in the murine model of tuberculosis is characterized by high cytotoxic activity during acute infection and loss of cytolytic CD8 T cells during chronic infection (V. Lazarevic et al., submitted). Although there were significantly more CTLp in the lungs of IL-15–/– mice 9 weeks postinfection, the cytotoxicity was negligible in both groups of mice by 12 weeks postinfection (Fig. 3E).
Cytokine profile of IL-15–/– mice. Since effector T-cell responses developed normally under IL-15-deficient conditions, we next investigated whether another cytokine(s) could replace IL-15 in providing signals for survival, proliferation, and maintenance of CD4 and CD8 T-cell effector functions. We determined the relative expression levels of IL-2, IL-7, IL-10, and IL-12 cytokine genes in the lungs of IL-15–/– and wild-type mice by quantitative RT-PCR. IL-2, IL-7, and IL-15 belong to the same c family of cytokines. IL-10 and IL-12 cytokines were of interest due to their anti-inflammatory and proinflammatory effects, respectively. We postulated that increased frequency and numbers of IFN--producing CD4 and CD8 T cells in IL-15–/– mice during chronic M. tuberculosis infection (Fig. 3C and D) could be due to either decreased IL-10 or increased IL-12 gene expression. Our results show there was significantly less IL-2 mRNA in the lungs of IL-15–/– than in wild-type mice, while there were no detectable differences in the amounts of IL-7, IL-10, and IL-12 mRNA (Fig. 4).
Effects of exogenous IL-15 administration on the development of T-cell responses and control of M. tuberculosis infection. Initial studies in which the potential of IL-15 as an immunotherapeutic agent against M. tuberculosis was investigated suggested that overexpression of IL-15 can augment T-cell-mediated immune responses to M. tuberculosis (16, 26, 27). We administered recombinant IL-15 (1 μg/mouse) to wild-type (C57BL/6) mice daily from day 5 to day 26 after low-dose aerosol infection and examined the development of T-cell effector functions and protection against M. tuberculosis infection. Daily administration of IL-15 for 21 days had no significant effect on the control of bacterial growth in the lungs of treated mice (Fig. 5A). Both IL-15-treated and control wild-type mice had similar percentages and numbers of CD4 and CD8 T cells in the lungs (Fig. 5B and C and data not shown). No differences in survival or in the numbers of IFN--producing CD4 and CD8 T cells were detected in the lungs of IL-15- and PBS-treated wild-type mice (data not shown).
IL-15–/– mice successfully control secondary infection with M. tuberculosis. C57BL/6 wild-type mice control M. tuberculosis infection, but they are unable to eliminate the bacteria, even with a robust immune response. To study memory responses, previously infected mice were cleared of mycobacteria by treating IL-15–/– and wild-type mice with a combination of pyrazinamide and isoniazid beginning 4 weeks postinfection for 2 months. Prolonged treatment with this combination of antibiotics was previously shown to have sterilizing activity in wild-type mice (21). After completion of a 2-month antibiotic treatment, two IL-15–/– mice and one wild-type mouse were sacrificed and whole-lung homogenates were plated to determine whether antibiotic treatment cleared the bacteria. Surprisingly, IL-15–/– mice were unable to clear mycobacteria and harbored 6 and 63 CFU, respectively. In contrast, no bacteria were recovered from the lungs of wild-type mice. At this stage, the antibiotic regimen was changed to isoniazid (0.1 g/liter) and rifampin (0.15 g/liter). Three months after antibiotic treatment commenced, one mouse from each experimental group was sacrificed to check for the presence of viable bacteria. The IL-15–/– mouse harbored 24 CFU, while again no bacteria were detected in wild-type mice. It was not until mice underwent 5 months of antibiotic treatment that no bacteria were detected in the lungs of IL-15–/– mice. After 2 months of rest, mice were challenged with a low dose of M. tuberculosis via the aerosol route, and the ability of "immune" IL-15–/– and wild-type mice to control secondary M. tuberculosis infection was evaluated. Both "immune" IL-15–/– and wild-type mice were equally efficient at controlling bacterial burden after M. tuberculosis challenge (Fig. 1B).
Memory CD4 and CD8 T-cell responses in IL-15–/– mice. Previous reports have demonstrated that IL-15–/– or IL-15R–/– mice have reduced numbers of memory CD8 T cells (7, 13), mainly due to decreased proliferation and decreased homing of IL-15R–/– lymphocytes to peripheral lymph nodes (13). We investigated the importance of IL-15 in the development of M. tuberculosis-specific T-cell memory after challenging immune mice with a low dose of M. tuberculosis. Infiltration of immune cells into the lungs of IL-15–/– "immune" mice was delayed at 3 weeks postchallenge, but by 6 weeks postchallenge infiltrations of cells into the lungs of IL-15–/– and wild-type mice were similar (Fig. 6A). No apparent differences were observed in lymph node cell numbers between the two groups of mice throughout the secondary infection (Fig. 6A). Although there were similar numbers of CD4 T cells in the lymph nodes, infiltration of CD4 T cells into the lungs of IL-15–/– mice initially lagged behind wild-type mice (Fig. 6B). The absence of IL-15 resulted in a significantly lower percentage of activated CD69+ CD4 T cells after challenge (Fig. 6D). IL-15–/– mice had significantly fewer CD8 T cells in the lungs and lymph nodes initially after challenge. In the lungs, it was not until 9 weeks postinfection that CD8 T cells in IL-15–/– mice finally reached wild-type numbers (Fig. 6C). In the lymph nodes, it appeared that CD8 T cells never expanded in numbers after secondary infection (Fig. 6C). Similar percentages of activated CD8 T cells were observed in the lungs of IL-15–/– and wild-type mice after secondary M. tuberculosis infection (Fig. 6D).
Proliferation of memory CD4 and CD8 T-cell responses in IL-15–/– mice. In contrast to previous findings where numbers of virus-specific memory CD8 T cells slowly declined in IL-15-deficient mice (2, 22), the most dramatic difference in memory T-cell responses of IL-15–/– and wild-type mice was observed early after secondary M. tuberculosis infection. "Immune" IL-15–/– mice had significantly fewer CD4 and CD8 T cells until 6 weeks postchallenge (Fig. 6B and C). This difference in total numbers of CD4 and CD8 T cells during early recall response was not due to impaired proliferation, as similar percentages of proliferating CD4 and CD8 T cells were detected in the lungs of "immune" IL-15–/– and wild-type mice (Fig. 7A). These differences are most likely due to delayed infiltration of memory CD4 and CD8 T cells into the infected lungs following secondary M. tuberculosis infection. It is important to note that any effect of IL-15 deficiency could be masked by de novo priming of effector T cells later in infection. Therefore, similar numbers of lung cells at 6 weeks postchallenge could be attributed to the influx of primary effector T cells rather than memory T cells.
IFN- production by memory CD4 and CD8 T cells is not dependent on IL-15. We assessed the ability of memory CD4 and CD8 T cells from IL-15–/– and wild-type mice to produce IFN- in response to M. tuberculosis-infected dendritic cells. Equal numbers of IFN--producing CD4 and CD8 memory T cells were present in the lungs of "immune" IL-15–/– and wild-type mice (Fig. 7B), suggesting that IFN- production by memory T cells is not dependent on IL-15.
DISCUSSION
The main goal of this study was to investigate whether IL-15 is required for the generation and maintenance of effector and memory T-cell responses following M. tuberculosis infection. The results of this study indicate that IL-15–/– mice were not substantially impaired in their ability to control primary and secondary M. tuberculosis infection. Similar numbers of mycobacteria were detected in the lungs, lymph nodes, and spleens of IL-15–/– and wild-type mice. There was a tendency toward a slightly higher bacterial burden in the lungs of IL-15–/– mice during chronic M. tuberculosis infection, and IL-15–/– mice were less able to clear the infection in the presence of antibiotics. CD4 and CD8 T-cell effector functions were not affected by IL-15 deficiency after primary and secondary M. tuberculosis infections. In view of recent studies, the most relevant finding is that CD8 T cells were not more prone to apoptosis following M. tuberculosis infection, and there was no sign of reduced proliferation of effector CD8 T cells in IL-15-deficient mice. The most dramatic difference in "immune" IL-15–/– and wild-type mice was characterized by delayed infiltration of memory cells into the lungs of "immune" mice after secondary challenge. We did not find a beneficial effect of IL-15 treatment on the ability of wild-type mice to control M. tuberculosis infection. However, the route or dose of infection, the timing of administration, or the background of the mouse strains may be confounding factors.
In this study, we sought to determine whether IL-15 was required for the generation of primary and recall T-cell responses against M. tuberculosis. The baseline number of CD8 T cells in the lungs and lymph nodes in uninfected IL-15–/– mice was at least twofold lower than in uninfected wild-type mice. Initial studies with IL-15–/– and IL-15R–/– mice revealed that in the absence of IL-15 signaling there was a 30 to 50% reduction in peripheral CD8 T cells (7, 13). IL-15 signaling is important for the survival of peripheral nave CD8 T cells, which is most likely mediated by increased expression of antiapoptotic proteins, such as Bcl-2 (3, 28). Although the initial CD8 T-cell deficiency in IL-15–/– mice could be explained by reduced survival of nave T cells associated with IL-15-deficient mice, there was still a possibility that a reduction in the overall CD8 effector population after primary infection could be due to reduced proliferation or enhanced apoptosis of remaining CD8 T cells in the absence of IL-15. We tested these hypotheses, and our data indicate that a significantly higher percentage of effector CD8 T cells proliferated in IL-15–/– mice during acute and chronic M. tuberculosis infection. Therefore, the proliferative ability of CD8 T cells in the face of persistent stimulation with M. tuberculosis antigens was not undermined in the absence of IL-15. Since CD8 T cells may be more prone to undergo apoptosis after antigenic stimulation in the absence of IL-15, we determined the percentage of apoptotic CD8 T cells during acute and chronic M. tuberculosis infections. There were similar percentages of apoptotic CD8 T cells in the lungs of IL-15–/– and wild-type mice. Therefore, the low-magnitude CD8 T-cell responses during primary M. tuberculosis infection of IL-15–/– mice is not a result of reduced proliferation or enhanced apoptosis but more likely due to the reduced numbers of CD8 T cells inherent in IL-15–/– mice. Furthermore, the cytokine production and cytotoxic activity of the remaining CD8 T cells were normal or even higher in IL-15–/– mice, supporting the idea that IL-15 is not required for the priming or effector functions of M. tuberculosis-specific CD8 T cells.
In accordance with previous studies (7, 13), CD4 T-cell responses were not affected by the absence of IL-15 signaling. Similar percentages of proliferating and apoptotic CD4 T cells were detected in the lungs and lymph nodes of IL-15–/– and wild-type mice (data not shown). The effector function of CD4 T cells was normal in IL-15–/– mice, as evidenced by a high percentage of activated CD69+ CD4 T cells and potent IFN- CD4 T-cell responses.
Although there were significant differences in the absolute numbers of CD8 T cells between IL-15–/– and wild-type mice, the increased frequency of IFN--producing CD8 T cells in the lungs of IL-15–/– mice resulted in equivalent or even significantly higher numbers of IFN--producing CD8 T cells in IL-15–/– mice during chronic infection. We postulated that enhanced IFN- T-cell responses in IL-15–/– mice could be due to either decreased IL-10 or increased IL-12 gene expression. Since there were no significant differences in IL-10 and IL-12 mRNA in the lungs of IL-15–/– and wild-type mice, it is possible that this boosted IFN- response may be due to increased bacterial burden in the lungs of IL-15–/– mice during the chronic stage of infection. Notably, the number of IFN--producing CD4 and CD8 T cells did not decline in the absence of IL-15.
Using the aerosol route of infection, we have previously demonstrated that after primary M. tuberculosis infection a pool of memory T cells is generated in the lungs of immune mice (23). In the first 2 weeks after challenge with M. tuberculosis, more than 30% of both CD4 and CD8 T cells in the lungs of immune mice expressed activation marker CD69 and produced IFN- upon restimulation. In contrast, less than 5% of T cells in the lungs of nave mice were CD69+ and produced IFN- during the first 2 weeks postinfection (23). These results suggest that M. tuberculosis infection leads to the development of memory CD4 and CD8 T-cell populations capable of rapid activation and effector function in the lungs upon rechallenge (23). However, these memory T-cell responses do not protect mice from reinfection. In fact, protective immunity due to previous exposure to M. tuberculosis generally results in a 10-fold-lower bacterial burden after rechallenge compared to nave mice. The secondary infection is established and bacterial burden is stringently controlled at 105 to 106 CFU per lung over 6 months. Therefore, a log difference in bacterial numbers is a level of protection conferred by memory T-cell responses upon secondary infection of mice with M. tuberculosis.
The inability of memory T cells in the lungs to control initial M. tuberculosis replication and to prevent reinfection remains the largest obstacle that immunologists face, and better understanding of lung-specific immunity is required if we are to design more-effective vaccines against M. tuberculosis.
There are no phenotypic markers that reproducibly represent the functional characteristics of memory CD4 and CD8 T cells in a murine model of tuberculosis. A combination of CD44, Ly6C, and CD62L markers failed to clearly delineate memory cells from effector cells after M. tuberculosis infection of nave or memory mice (unpublished data). The only distinguishing feature of memory response in a murine model of tuberculosis is a higher percentage of activated CD69+ T cells in the lungs of "immune" mice compared with nave mice within the first 3 weeks postinfection (reference 23 and unpublished data). Our data indicate that protective immune responses were generated in the absence of IL-15, as evidenced by the similar control of bacterial loads in the lungs and spleens of challenged IL-15–/– and wild-type mice. Several groups reported that in the absence of IL-15 or IL-15R signaling, the homeostatic proliferation of memory CD8 T cells was significantly diminished, leading to a slow decline in virus-specific memory CD8 T cells (2, 22). There was no indication of reduced proliferation or a decline in memory CD4 and CD8 T-cell responses in IL-15–/– mice in our studies. The most dramatic difference between "immune" IL-15–/– and wild-type mice was observed early after secondary challenge with M. tuberculosis. There was a significant delay in the infiltration of CD4 and CD8 T cells into the lungs of IL-15–/– "immune" mice at 3 weeks postchallenge. By 6 weeks postchallenge, the kinetics and magnitude of memory T-cell responses were similar for IL-15–/– and wild-type mice. As M. tuberculosis is not cleared by secondary immune response, and reinfection is established, it is important to note that any effect of IL-15–/– deficiency on maintenance of memory T-cell responses could be masked by the influx of de novo primed effector T cells after 3 weeks postinfection.
Although development of primary T-cell responses occurred normally in IL-15–/– mice, these mice had difficulty clearing M. tuberculosis infection when treated with antibiotics. We observed previously that CD4–/– mice were unable to eliminate M. tuberculosis during antibiotic treatment (unpublished data). Since CD4 T cells play a pivotal role in controlling acute and persistent M. tuberculosis infection, the efficacy of antibiotics was undermined in the absence of this important T-cell subset. These findings suggest antibiotic treatment must be prolonged in cases of immunodeficiency, such as the absence of CD4 T cells. That IL-15–/– mice had a similar difficulty in eliminating mycobacteria during antibiotic treatment suggested that this cytokine may have other unidentified roles in modulating the "immune" system that may be overlooked using standard immunological techniques. Flow cytometric analysis revealed similar numbers of dendritic cells, neutrophils, and macrophages in the lungs of IL-15–/– and wild-type mice (data not shown), and cytokine profile analysis revealed no significant differences in IL-7, IL-10, and IL-12 mRNA expression in the lungs. Since no major differences in the functionality of T cells, composition of innate immune cells, and cytokine profile (except for IL-2 mRNA) were detected in IL-15–/– mice, it remains unresolved why IL-15–/– mice were unable to clear M. tuberculosis infection in the presence of a standard course of antibiotics and why chronic infection is less well controlled in these mice.
Collectively, our results indicate that IL-15 is not essential for the generation and maintenance of effector CD4 and CD8 T-cell responses. The magnitude of the recall response in "immune" IL-15–/– mice was significantly smaller than in "immune" wild-type mice, which could be due to delayed infiltration or impaired maintenance of the memory cells or a consequence of a smaller burst size of effector CD8 T-cell responses during primary infection of IL-15–/– mice. Development of tetramer staining reagents for M. tuberculosis antigens and better phenotypic definition of M. tuberculosis-specific memory T cells will enable a detailed investigation of development and maintenance of memory T-cell responses following secondary M. tuberculosis infection.
ACKNOWLEDGMENTS
We are grateful to Jacques Pechon at Immunex for providing IL-15–/– breeding pairs as well as recombinant IL-15. We acknowledge the technical assistance of Amy Myers and Carolyn Bigbee.
These studies were supported by the National Institutes of Health grants AI37859 and AI50732 (J.L.F.) and the American Lung Association (CL-016; J.L.F.).
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