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The Journal of Immunology, 2003, 171: 4689-4699.
Copyright © 2003 by The American Association of Immunologists

CD4+ T Cells Mediate IFN-{gamma}-Independent Control of Mycobacterium tuberculosis Infection Both In Vitro and In Vivo

Siobhán C. Cowley1 and Karen L. Elkins1

Laboratory of Mycobacterial Diseases and Cellular Immunology, Division of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation and Research/Food and Drug Administration, Rockville, MD 20852


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although IFN-{gamma} is necessary for survival of Mycobacterium tuberculosis infection in people and animal models, it may not be sufficient to clear the infection, and IFN-{gamma} is not a reliable correlate of protection. To determine whether IFN-{gamma}-independent mechanisms of immunity exist, we developed a murine ex vivo culture system that directly evaluates the ability of splenic or lung lymphocytes to control the growth of M. tuberculosis within infected macrophages, and that models in vivo immunity to tuberculosis. Surprisingly, CD4+ T cells controlled >90% of intracellular M. tuberculosis growth in the complete absence of IFN-{gamma} stimulation of macrophages, via a NO-dependent mechanism. Furthermore, bacillus Calmette-Guerin-vaccinated IFN-{gamma}-deficient mice exhibited significant protection against M. tuberculosis challenge that was lost upon depletion of CD4+ T cells. These findings demonstrate that CD4+ T cells possess IFN-{gamma}-independent mechanisms that can limit the growth of an intracellular pathogen and are dominant in secondary responses to M. tuberculosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tuberculosis is a significant global health problem, as one-third of the world’s population is estimated to be infected with Mycobacterium tuberculosis, and 8 million new active cases occur annually. The only current vaccine, live Mycobacterium bovis bacillus Calmette-Guerin (BCG),2 is of variable efficacy in different geographic regions, ranging from as little as 0% to as much as 80% protection (1). However, development of improved vaccine candidates is limited by our incomplete understanding of the cellular and molecular immune mechanisms required for the elimination of a M. tuberculosis infection (2).

M. tuberculosis is a facultative intracellular pathogen that survives and grows primarily within macrophages in the host. In both mice and humans, the immune response has difficulty eliminating a M. tuberculosis infection, resulting in the establishment of a latent infection in humans and a chronic disease state in mice. Similar to other intracellular pathogens, immunity to M. tuberculosis is cell mediated. Murine studies using in vivo Ab depletion, gene-disrupted mice, or adoptive transfer indicate that CD4+ T cells are key to the control of infection (3, 4, 5, 6, 7). Mice with disruptions in the genes for {beta}2-microglobulin, TAP, or CD8 are more susceptible to M. tuberculosis than wild-type (WT) mice, also implicating a role for CD8+ T cells and the MHC class I pathway (8, 9, 10). Both CD4+ and CD8+ T cells have been shown to produce IFN-{gamma} during an M. tuberculosis infection (2). In experimental models, IL-12 has a central role in regulating Th1 T cell production of IFN-{gamma}, and IFN-{gamma} is clearly key in the activation of macrophages (11). These findings, along with the extreme susceptibility of people (12) and mice (13, 14) with disruptions in the IFN-{gamma} or p40 gene to M. tuberculosis infection, has led to proposed use of IFN-{gamma} as a correlate of protection for new vaccines against tuberculosis (15).

However, numerous examples in the literature indicate that the levels of IFN-{gamma} produced by a mouse in response to a candidate vaccine do not always correlate with the effectiveness of that vaccine during M. tuberculosis challenge (16). Similarly, a recent investigation evaluated the efficacy of human BCG vaccination using several assays and found that mycobacterial growth inhibition did not correlate with IFN-{gamma} responses (17). Furthermore, mycobacterial infection of human and mouse macrophages disrupts a pivotal part of IFN-{gamma} intracellular signaling, resulting in inhibition of IFN-{gamma}-induced gene expression (18, 19). It also remains unclear whether the activity of IFN-{gamma} in vivo is primarily bactericidal or only bacteriostatic. Thus, although IFN-{gamma} is essential for the development of an immune response that prolongs the life span of an infected animal, it is not sufficient to eliminate an M. tuberculosis infection, as mice infected with M. tuberculosis develop a long-term chronic infection.

We are interested in investigating the other important immune mechanisms that contribute to the control of M. tuberculosis intracellular growth, particularly because understanding these mechanisms may provide improved correlates of protection for vaccine research. To more readily discover IFN-{gamma}-independent mechanisms, we have developed a murine ex vivo culture system that directly evaluates the ability of splenic or lung lymphocytes to control the growth of M. tuberculosis within infected macrophages, and that reflects the in vivo components of the immune system known to be involved in controlling an M. tuberculosis infection. Using this system, we demonstrate that a very substantial proportion of the ability of CD4+ T cells to control M. tuberculosis growth occurs in the absence of IFN-{gamma} stimulation of the macrophage. Furthermore, we show that IFN-{gamma}-deficient mice vaccinated with BCG exhibit limited but readily demonstrable protection against a subsequent virulent M. tuberculosis aerosol challenge that is dependent on CD4+ T cells. Thus, CD4+ T cells have IFN-{gamma}-independent mechanisms that can affect control of M. tuberculosis growth during secondary challenge.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Six- to twelve-week-old male specific pathogen-free C57BL6/J, 129S1/SvImJ, or IFN-{gamma}R-deficient mice on a C57BL6/J background were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were housed in sterile microisolator cages in a barrier environment at the Center for Biologics Evaluation and Research. Mice were fed autoclaved food and water ad libitum. All experiments were performed under protocols approved by the Center for Biologics Evaluation and Research/Food and Drug Administration Institutional Animal Care and Use Committee.

Culture and infection of bone marrow-derived macrophage (BMM{phi}) with bacteria

Bone marrow macrophages were used as the target cells for the in vitro system. BMM{phi} were cultured as previously described (20). Briefly, bone marrow was flushed from femurs of healthy C57BL6/J or 129S1/SvImJ mice with DMEM (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated FCS (HyClone, Logan, UT), 10% L-929-conditioned medium, 0.2 mM L-glutamine (Life Technologies), 10 mM HEPES buffer (Life Technologies), and 0.1 mM nonessential amino acids (Life Technologies) (complete DMEM (cDMEM)). Cells were plated at 2 x 106 in 24-well plates (Costar, Corning, NY), or at 1 x 106 in 48-well plates, in cDMEM supplemented with 50 µg/ml gentamicin (Life Technologies), and incubated at 37°C in 5% CO2. After 1 day of incubation, the medium was replaced with antibiotic-free cDMEM. The cells were incubated for an additional 5–7 days, with medium replaced every 2 days.

Following the 7-day culture period, the BMM{phi} formed a confluent monolayer, and the concentration of BMM{phi} was estimated to be 107 cells/well in the 24-well plates and 5 x 106 cells/well in the 48-well plates. BMM{phi} were then infected with M. tuberculosis Erdman, according to the following protocol: bacteria were diluted from frozen stocks in cDMEM and added at a multiplicity of infection (MOI) of 1:100 (bacterium-to-BMM{phi} ratio). A series of initial experiments studied the growth of M. tuberculosis in BMM{phi} at various MOIs; an MOI of 1:100 was selected to approximate a low dose in vivo infection, and because it permitted a controlled infection of the macrophage monolayer that allowed measurable growth of the bacteria to occur over a period of several days. M. tuberculosis was coincubated with BMM{phi} at 37°C in 5% CO2 for 2 h and then washed vigorously five times with PBS (BioWhittaker, Gaithersburg, MD) to eliminate extracellular bacteria. Less than 50 bacteria/well were detected in the supernatants from the final cell washes, indicating the elimination of the extracellular bacteria. Following the last wash, PBS was replaced with 1 or 2 ml/well cDMEM, and the cells were incubated at 37°C in 5% CO2 for the remainder of the experiment. To determine bacterial uptake, some BMM{phi} were lysed by adding sterile distilled water containing 0.1% saponin for 3 min. Culture lysates were serially diluted in PBS containing 0.05% Tween 80 and plated on 7H11 plates (supplemented with 10% OADC; BD Microbiology Systems, Sparks, MD); plates were incubated for ~3 wk; and colonies were counted. BMM{phi} uptake was routinely ~104 bacteria/ml (~10% of the number of bacteria added). Growth of M. tuberculosis in BMM{phi} was monitored by lysing cultures at various time points, serial dilution and plating of the lysates, and counting M. tuberculosis colonies, as described above.

Infection of mice with Mycobacteria or Listeria

Low dose aerosol infections with M. tuberculosis Erdman or KY/TN 95-031151 (CDC 1551; F. Collins, Center for Biologics Evaluation and Research) were performed as previously described (21). Briefly, mice were placed in an Inhalation Exposure System (A4212; Glas-Col, Terre Haute, IN). The nebulizer compartment was filled with a 10-ml suspension of the Mycobacterium strain of interest at appropriate concentrations (either 105 or 106 bacteria/ml, depending on the individual machine) diluted in sterile saline containing 0.05% Tween 80. These concentrations were demonstrated separately to reproducibly deliver 50–100 bacilli into the lungs over a 30-min exposure period. For M. bovis BCG Pasteur infections, mice were inoculated intradermally with 106 BCG bacteria to mimic the human vaccine inoculation route. This concentration was determined to establish a protective infection in C57BL6/J mice. For the IFN-{gamma}-deficient mouse ({gamma} knockout (GKO)) studies, mice (both GKO and WT controls) were inoculated intradermally with 106 BCG bacteria, and the infection was allowed to progress for 4–5 wk, at which point mice were subjected to chemotherapy (85 mg/L isoniazid and 50 mg/L rifampin in their drinking water ad libitum) for 2–3 mo to eliminate the BCG infection (22). This reduced the BCG numbers in the spleens and lungs to undetectable levels, as determined 1 wk postremoval of the antibiotic treatment; however, at later time points (~30 days), regrowth of BCG was evident in the spleens of the GKO animals. To generate a population of Listeria-specific T cells, mice were sublethally infected with 104 L. monocytogenes strain EGD intradermally, and spleens were harvested 4–6 wk later.

Depletion of T cell subsets in vivo

In vivo depletion of CD4+ cells in BCG-vaccinated mice was performed by i.p. administration of 500 µg of the anti-CD4 Ab GK1.5 twice weekly for the duration of the experiment. Successful in vivo depletion of CD4 cells was confirmed (<0.2% positive cells remaining) in the spleens of depleted animals using flow cytometry (see below).

Harvesting and enrichment of splenocytes

Spleens were aseptically removed from selected mice and disrupted with a 3-ml syringe plunger. A single-cell suspension was prepared, and erythrocytes were lysed with ammonium chloride. Cells were washed and viability was assessed by exclusion of trypan blue. In most experiments, macrophages were depleted from the starting splenic populations by incubation of the whole splenocyte preparation on plastic dishes for 1 h at 4°C, followed by recovery of the nonadherent population. Nonadherent cells were resuspended in Dulbecco’s PBS-2% FCS to the appropriate concentration, and splenocytes were added to BMM{phi} cultures at various concentrations, as indicated. Unless otherwise stated, 5 x 106 splenocytes were added to each well in the 24-well plate cultures, and 2.5 x 106 splenocytes were added to the 48-well cultures (~1 splenocyte to 2 BMM{phi}). In Fig. 4A, the numbers of B cells, CD4+ T cells, and CD8+ T cells added to the BMM{phi} were proportional to their percentages (as determined by flow cytometry) in infected C57BL/6J spleens. For example, the CD8+ T cell population was estimated to be ~10% of the splenocytes; thus, 5 x 105 purified CD8+ T cells were added per well in a 24-well plate. The same was done for the CD4+ T cells and B220+ cells, assuming these populations are ~25 and 45% of total splenocytes, respectively. B cells, CD4+ T cells, and CD8+ T cell populations were enriched using the appropriate beads and enrichment columns (MACS magnetic cell sorting system; Miltenyi Biotec, Auburn, CA), according to the manufacturer’s instructions. Cell populations were routinely greater than 90% of the desired population using this method of enrichment. In all cases, starting and enriched splenocyte populations were analyzed by flow cytometry using a flow cytometer (LSR; BD Biosciences, San Diego, CA), as described below.



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  		FIGURE 4. Control of M. tuberculosis growth by spleen cell subpopulations. Mice primed with a low dose aerosol infection of M. tuberculosis 4–6 wk previously served as a source of immune splenocytes. Splenocyte subpopulations were enriched from spleens of primed mice, as described in Materials and Methods; efficacy of enrichment was determined by flow cytometry. A, Lymphocyte subpopulations were added at concentrations similar to their relative percentage of abundance in the whole primed spleen (whole primed spleen, 100% or 5 x 106 cells/well; primed CD4+ cells, 25% of whole primed spleen or 1.25 x 106 cells/well; primed CD8+ cells, 10% or 5 x 105 cells/well; or primed B220+ cells, 45% or 2.25 x 106 cells/well), as described in Materials and Methods. Cells were added immediately following infection of BMMØ. Eight days after infection, cultures were assessed for intracellular bacteria, as described in the legend to Fig. 2. B, Immune CD4+ or CD8+ splenocytes were added to M. tuberculosis-infected BMMØs at the indicated concentrations, and intracellular bacteria numbers were assessed 8 days later, as described in the legend to Fig. 2. Asterisks (*) indicate p values <0.05 as compared with cocultures containing naive spleen cells. These results are representative of three experiments similar in design.

 
Due to the inability of mice to clear a M. tuberculosis infection, in some experiments M. tuberculosis-infected mice were subjected to chemotherapy, as described above. In other experiments, mice were not subjected to chemotherapy, and the bacterial loads in the spleen and lung cell preparations of these mice were enumerated by serial dilution and plating before their addition to the in vitro system.

In some experiments, cytokines were neutralized in the culture supernatants by addition of azide-free, low-endotoxin mouse anti-cytokine-neutralizing Abs at a concentration of 10–20 µg/ml. Abs were added to the cocultures at the same time as the splenocytes. Anti-cytokine Abs added to neutralize IFN-{gamma} were clones XMG1.2 and R4-6A2; for TNF {alpha}, clone MP6-XT3; for IL-12p40, clone C17.8; for IL-4, clone 11B11; and for IL-10, clone JES5-2A5 (all from BD PharMingen, San Diego, CA).

Harvesting of lung lymphocytes

Lungs were perfused with a solution of PBS-2% FCS, then aseptically removed and minced using sterile scalpels. The minced material was subjected to collagenase treatment (150 U/ml) for 1 h at 37°C in 5% CO2. The digested material was passed through a cell strainer and then washed in PBS-2% FCS; erythrocytes were lysed; and cells were counted as above before addition to BMM{phi} cultures at the concentration indicated.

Quantitation of cytokines and NO in BMM{phi} culture supernatants

Culture supernatants were assayed for IFN-{gamma}, IL-12p40, TNF {alpha}, IL-4, and IL-10 by standard sandwich ELISAs. All Ab pairs and standards were purchased from BD PharMingen, and ELISAs were performed in accordance with the manufacturer’s instructions. Samples were read at 405 nm on a VersaMax tunable microplate reader with a reference wavelength of 630 nm (Molecular Devices, Sunnyvale, CA). Cytokines were quantified by comparison with recombinant standards (all purchased from BD PharMingen) using four-parameter fit regression in the SoftMax Pro ELISA analysis software (Molecular Devices).

NO was detected in culture supernatants by the Griess reaction (23). Briefly, aliquots of culture supernatants were incubated with an equal volume of commercial Griess reagent (Sigma-Aldrich, St. Louis, MO) for 5 min at room temperature, and the absorbance of each sample at 490 nm was measured. Nitrite (NO2) was quantified by comparison with serially diluted NaNO2 as a standard using four-parameter fit regression as above.

Analysis of splenocyte populations using flow cytometry

Cells were prepared, as described above, and stained for lymphocyte cell surface markers, as previously described (20). Briefly, single-cell suspensions were blocked with anti-CD16 (Fc Block; BD PharMingen) and stained with either FITC-conjugated anti-CD45/B220 (RA3-6B2), PE-conjugated anti-CD4 (RM4-4), PE-conjugated anti-CD8{alpha} (53-6.7), PE-conjugated anti-CD11b (M1/70), or PE-conjugated anti-DX5 (DX5) mAbs (all from BD PharMingen) before fixation in 0.5% paraformaldehye. Appropriate isotype-matched controls were used as the point of comparison for analyses using a BD Biosciences LSR flow cytometer, with gates set to viable lymphocytes and monocytes according to forward and side scatter profiles. Among the total population of splenocytes, 129S1/SvlmJ and C57BL/6 mice had ~25–30% CD4+ cells, 10–15% CD8+ cells, 45% B220+ cells, 15% CD11b+ cells, and 2% DX5+ cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Control of growth of M. tuberculosis Erdman in murine BMM{phi} by M. tuberculosis-primed splenic and lung lymphocytes

Similar to previous studies demonstrating the ability of immune lymphocytes to control the intracellular growth of Francisella tularensis LVS in vitro (20), we tested the ability of M. tuberculosis-primed splenocytes to control the growth of M. tuberculosis in BMM{phi} in vitro. Because M. tuberculosis-infected mice develop a chronic infection, WT C57BL6/J mice were primed for 4 wk with an aerogenic infection of M. tuberculosis and then subjected to 8 wk of chemotherapy to clear the bacterial infection. At the end of the chemotherapy treatment, no bacteria were detected in lungs or spleens (limit of detection <50 CFU). Splenocytes from the infected and antibiotic-treated mice were added to M. tuberculosis-infected BMM{phi} cultures on the day that the BMM{phi} were infected, and growth of M. tuberculosis in the BMM{phi} monolayer was monitored for the next 10 days (Fig. 1). M. tuberculosis grew logarithmically in the BMM{phi} cultures, with a peak in the number of bacteria occurring 8 days after infection. Addition of M. tuberculosis-primed splenocytes to these cultures resulted in a significant reduction of M. tuberculosis growth (Fig. 1). Control of growth was evident as early as 4 days, and was maximal by 8 days after the addition of the primed splenocytes. The inhibition of growth in the BMM{phi} monolayers cocultured with immune splenocytes was found to be highly significant (p = 0.0072) at the day 8 time point, and in this example was equivalent to a 95% reduction in the absolute number of bacteria as compared with the M. tuberculosis-infected BMM{phi} monolayer alone. Subsequent experiments compared M. tuberculosis growth using the day 8 time point, when the control of M. tuberculosis growth was maximal.



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  		FIGURE 1. Control of M. tuberculosis Erdman growth by M. tuberculosis-immune splenocytes in BMM{phi}. BMM{phi} from WT C57BL/6 mice were infected at an MOI of 1:100 with M. tuberculosis Erdman. Infected BMM{phi} were either cultured alone (•), or with a 1:2 ratio of M. tuberculosis-immune splenocytes (splenocyte to BMM{phi} ratio; {circ}). At the indicated time points after infection, BMM{phi} were washed, lysed, and plated. Values shown are mean log CFU/ml of triplicate samples ± SEM. These results are representative of three experiments similar in design.

 
We next compared the ability of splenocytes from antibiotic-treated mice and actively infected mice to control M. tuberculosis growth (Fig. 2A). Using actively infected splenocytes added ~1 x 102 to 1 x 103 bacilli to the cultures. Despite the additional bacteria, cultures with splenocytes from actively infected mice exhibited the same net numbers of bacteria as those from antibiotic-treated mice, and thus comparable control of intracellular growth. Because of the ease of obtaining splenocytes from the actively infected mice, future experiments were performed using actively infected splenocytes, unless otherwise stated. In contrast to M. tuberculosis-primed splenocytes, splenocytes from naive mice reduced M. tuberculosis growth to a much smaller extent. In repeated experiments, the effect of naive splenocytes on M. tuberculosis growth in the BMM{phi} monolayer was found to be consistently small, but was usually significant (p < 0.05) as compared with growth of M. tuberculosis in BMM{phi} alone.



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  		FIGURE 2. Determination of the optimal conditions and specificity of control of M. tuberculosis growth in BMM{phi} by immune splenocytes. A, BMM{phi} were infected with M. tuberculosis and then cocultured with either naive splenocytes, splenocytes obtained from mice that were actively infected with M. tuberculosis 4 wk previously, or from mice that were infected with M. tuberculosis for 4 wk, followed by an 8-wk course of chemotherapy. Eight days after infection of the BMM{phi}, cultures were assessed for intracellular bacteria, as described in the legend to Fig. 2. B, BMM{phi} were infected with M. tuberculosis and then cocultured with either naive splenocytes (1:2 ratio), or M. tuberculosis-immune splenocytes from an actively infected mouse at the range of ratios shown. Eight days after infection, cultures were assessed for intracellular bacteria, as described in the legend to Fig. 2. C, BMM{phi} were infected with M. tuberculosis Erdman and then cocultured with splenocytes obtained from a mice sublethally infected with a variety of different intracellular pathogens 4–6 wk previously, as shown. All splenocyte populations were added at a 1:2 ratio (splenocyte:BMM{phi}). Eight days after infection, cultures were assessed for intracellular bacteria, as described in the legend to Fig. 2. Asterisks (*) indicate p values <0.05 as compared with cocultures containing naive spleen cells. These results are representative of three experiments similar in design.

 
We further tested the ability of different concentrations of M. tuberculosis-primed splenocytes to control M. tuberculosis growth (Fig. 2B). Maximal control of M. tuberculosis growth occurred using splenocytes at a 1:2 ratio (splenocytes:BMM{phi}). Higher concentrations of splenocytes were found either to be detrimental to the culture system or to provide no further control of growth. Thus, a 1:2 ratio was used for all subsequent experiments.

To determine whether the control of M. tuberculosis growth observed in this culture system was specific for Mycobacterium-primed spleen cells, we tested the ability of splenocytes from mice primed with a variety of bacteria to control M. tuberculosis Erdman growth. Groups of mice were primed with M. tuberculosis Erdman, M. tuberculosis CDC1551, M. bovis BCG, or a completely unrelated intracellular pathogen, Listeria monocytogenes EGD. Splenocytes from Erdman, CDC1551, and BCG-primed mice were found to control intracellular M. tuberculosis Erdman growth to a similar extent (Fig. 2C; p < 0.0002, as compared with naive spleen cells); in contrast, Listeria-primed splenocytes and naive splenocytes had only a small effect on M. tuberculosis Erdman growth (see above) and were not significantly different from each other (p > 0.3). Thus, the in vitro growth control of M. tuberculosis was specific to immune lymphocytes primed by exposure to Mycobacterium species.

Because the primary site of M. tuberculosis infection is the lung, we investigated the ability of Erdman-primed lung lymphocytes to control the growth of Erdman in BMM{phi} in vitro. Due to the difficulty in obtaining large numbers of lung cells, the ratio of immune lung cells to BMM{phi} was decreased to 1:4 in these cocultures. It was not practical to obtain a similar large number of naive lung lymphocytes, and therefore immune lung cell cocultures were compared with cultures containing the same numbers of naive spleen cells. In other experiments, control of intracellular M. tuberculosis growth by low numbers of naive lung lymphocytes was very similar to the control observed using the same number of naive splenocytes (data not shown). As shown in Fig. 3, lung cells from tuberculosis (TB)-immune mice significantly reduced Erdman growth by 79% (p = 0.032) as compared with control naive splenocytes. The lung lymphocytes were not significantly different from primed splenocytes (p = 0.914) in their ability to control M. tuberculosis growth in the BMM{phi}, even though lower numbers of immune lung cells were used. Thus, immune lung lymphocytes as well as immune splenic lymphocytes controlled M. tuberculosis intracellular growth.



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  		FIGURE 3. Control of M. tuberculosis growth by spleen and lung cell populations. BMM{phi} were infected with M. tuberculosis and then cocultured with either naive splenocytes, splenocytes obtained from mice given a low dose aerosol M. tuberculosis infection 4–6 wk previously, or lung lymphocytes obtained from the same infected mice. Eight days after infection, cultures were assessed for intracellular bacteria, as described in the legend to Fig. 2. Asterisks (*) indicate p values <0.05 as compared with cocultures containing naive spleen cells. These results are representative of three experiments similar in design.

 
Contribution of lymphocyte subpopulations to the control of M. tuberculosis infection in vitro

To examine the cell populations responsible for the inhibition of M. tuberculosis growth in vitro, we compared the ability of CD4+, CD8+, and B220+ spleen cell subpopulations from immune mice to control M. tuberculosis growth. CD4+-, CD8+-, and B220+-enriched splenocyte subpopulations from immune mice were added to the BMM{phi} cultures in numbers intended to mimic their relative proportions in the whole immune splenocyte cultures (see Materials and Methods). Whole splenocytes reduced M. tuberculosis growth by 95%, whereas there was a 89% reduction by the CD4+ T cells, 82% by the CD8+ T cells, and 64% by B220+ cells, as compared with cultures containing M. tuberculosis-infected macrophages alone (Fig. 4A). Similarly, there was a 73% reduction by the CD4+ T cells, 55% by the CD8+ T cells, and 8% by B220+ cells, as compared with cultures containing naive spleen cells (data not shown). The reduction of M. tuberculosis growth in cultures containing whole immune splenocytes and CD4+ T cells was significant as compared with either cultures containing M. tuberculosis-infected macrophages alone (both p < 0.0001) or cultures containing naive splenocytes (p = 0.0187 and 0.0325, respectively). In contrast, the control of growth mediated by these numbers of CD8+ and B220+ cells was significant as compared with cultures containing M. tuberculosis-infected macrophages alone, but not found to be significantly different from that of the naive spleen cell cultures (p = 0.162 and 0.494, respectively; data not shown).

To determine the relative effectiveness of T cell subpopulations in controlling M. tuberculosis growth, CD4+ and CD8+ T cells were added to infected BMM{phi} cultures at a range of concentrations. Both exhibited a dose-dependent inhibition of M. tuberculosis growth (Fig. 4B). However, on a cell-by-cell basis, CD4+ cells were more effective than CD8+ cells at all concentrations. Cocultures containing CD4+ T cells at all concentrations were found to produce a significant (p < 0.05) reduction of M. tuberculosis growth as compared with either cultures containing M. tuberculosis-infected macrophages alone or cocultures containing naive splenocytes, whereas CD8+ T cells only resulted in a significant reduction (p < 0.05) in M. tuberculosis growth at a concentration of 1.25 x 106 cells/well and higher.

To further characterize the quality of the T cell subpopulation responses, the levels of cytokines and nitrite (as an indicator of NO) were measured in the supernatants of these cultures. The concentrations of IFN-{gamma}, TNF-{alpha}, IL-12 p40, and NO were determined for cocultures containing the various cell subpopulations at the optimum number of 5 x 106 cells/culture (Fig. 4B). As shown in Fig. 5A, IFN {gamma} was undetectable in cocultures containing naive splenocytes or infected BMM{phi} alone, but was produced in abundance in cocultures containing either whole immune splenocytes or purified CD4+ T cells. In contrast, cocultures containing the same number of CD8+ T cells had ~10-fold lower levels of IFN-{gamma} in their supernatants. On the other hand, the cocultures containing purified CD8+ T cells were found to contain the most abundant levels of IL-12 p40 in their supernatants as compared with cocultures containing purified CD4+ T cells or whole immune splenocytes (Fig. 5B). Of note, although infected macrophages alone had undetectable levels of IL-12 p40 in their supernatants, cocultures containing naive splenocytes had readily detectable levels of IL-12 p40. Low levels of TNF-{alpha} were detected in cultures containing BMM{phi} alone, naive splenocytes, and CD8+ T cells, while higher levels were found in cocultures containing whole immune splenocytes and purified CD4+ T cells (Fig. 5C). NO was undetectable in cocultures containing naive splenocytes or infected BMM{phi} alone, but produced in large amount in cocultures containing either whole immune splenocytes or purified CD4+ T cells, and somewhat lower amounts in cocultures containing CD8+ T cells (Fig. 5D). Neither IL-10 nor IL-4 was detected in supernatants from any cultures (limit of detection 150 and 50 pg/ml, respectively).



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  		FIGURE 5. Secretion of cytokines and NO into culture supernatants following coculture of M. tuberculosis-infected BMM{phi} and immune splenocytes. Splenocytes from either unprimed mice or mice primed 4–6 wk previously with a low dose aerosol M. tuberculosis infection were cocultured with M. tuberculosis-infected macrophages at a 1:2 ratio (splenocytes to BMM{phi}). Similarly, CD4+ and CD8+ splenocyte subpopulations were added to infected BMM{phi} cultures at a 1:2 ratio. Culture supernatants were collected 8 days later and tested for IFN-{gamma} (A), IL-12 (B), TNF-{alpha} (C), and NO (D). Values shown are mean pg/ml of triplicate samples ± SEM. These results are representative of three experiments similar in design.

 
Contribution of cytokines to the control of M. tuberculosis infection in vitro

Given that IFN-{gamma}, TNF-{alpha}, and IL-12 are important for the development of a primary immune response in M. tuberculosis-infected mice, we tested the effect of neutralization of these and other cytokines on the ability of M. tuberculosis-immune splenocytes to control M. tuberculosis growth in BMM{phi}. As shown in Fig. 6, Ab neutralization of a variety of cytokines had only minimal effects on the ability of the macrophages to control intracellular growth of M. tuberculosis. Of the five cytokines tested, only the addition of neutralizing Abs to IFN-{gamma} and TNF-{alpha} reproducibly had small but significant effects on the ability of immune splenocytes to control M. tuberculosis growth compared with cocultures containing immune splenocytes (p = 0.048 and 0.042, respectively). In all cases, Ab neutralization was evaluated by ELISA and was found to reduce cytokine levels below the limits of detection of the assay (data not shown).



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  		FIGURE 6. Effect of cytokine neutralization on splenocyte inhibition of M. tuberculosis intracellular growth. BMM{phi} were infected with M. tuberculosis and cocultured with immune splenocytes. At the time of addition of the immune splenocytes, the indicated neutralizing anti-cytokine Abs (20 µg/ml) were added to the cocultures. Eight days after infection, cultures were assessed for intracellular bacteria, as described in the legend to Fig. 2. Asterisks (*) indicate p values <0.05 as compared with cocultures containing primed spleen cells. These results are representative of three experiments similar in design.

 
Contribution of T cell subpopulations and NO to the control of M. tuberculosis infection in vitro, in the presence or absence of responsiveness to IFN-{gamma}

We next investigated the ability of M. tuberculosis-immune splenocytes and purified T cell subpopulations to control M. tuberculosis growth in IFN-{gamma} receptor-deficient BMM{phi} (IFN-{gamma}R KO). This approach examines the ability of lymphocytes to control M. tuberculosis intracellular growth without IFN-{gamma} stimulation of macrophages, despite normal in vivo priming and unimpeded production of cytokines in the cocultures. Growth of M. tuberculosis in the WT BMM{phi} cocultured with M. tuberculosis-immune splenocytes was significantly reduced 97% as compared with growth in WT BMM{phi} alone (Fig. 7A; p = 0.004) and 89% as compared with coculture with naive splenocytes (data not shown). Remarkably, growth of M. tuberculosis in the IFN-{gamma}R KO BMM{phi} cocultured with WT splenocytes was also significantly reduced 83% as compared with growth in BMM{phi} alone (Fig. 7A; p = 0.002) and 76% as compared with coculture with naive splenocytes (data not shown). Thus, a large proportion of the ability of splenocytes to inhibit intracellular growth of M. tuberculosis in the BMM{phi} monolayer was independent of IFN-{gamma} stimulation of macrophages.



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  		FIGURE 7. Growth inhibition of M. tuberculosis by WT immune splenocytes or T cell subpopulations in BMM{phi} that lack the IFN-{gamma}R. A, WT (filled bars) and IFN-{gamma}R KO (gray bars) BMM{phi} were infected with M. tuberculosis and cocultured with immune splenocytes obtained from WT mice primed 4–6 wk earlier with a low dose M. tuberculosis aerosol infection. Eight days after infection, cultures were assessed for intracellular bacteria, as described in the legend to Fig. 2. B, WT (filled bars) and IFN-{gamma}R KO (gray bars) BMM{phi} were infected with M. tuberculosis and cocultured with immune T cell subpopulations obtained from WT mice primed 4–6 wk earlier with a low dose M. tuberculosis aerosol infection. Eight days after infection, cultures were assessed for intracellular bacteria, as described in the legend to Fig. 2. Asterisks (*) indicate p values <0.05 as compared with cocultures containing naive spleen cells. These results are representative of three experiments similar in design.

 
We further compared the ability of purified CD4+ and CD8+ T cells from immune mice to control M. tuberculosis growth in cocultures containing either type of M. tuberculosis-infected macrophages. In this study, 5 x 106 purified CD4+ or CD8+ T cells were added to cocultures, to reveal maximum effects of each cell type as well as to overcome any low cell density effects (Figs. 1 and 3). As seen in Fig. 7B (and earlier, Fig. 4), both CD4+ and CD8+ T cells had significant effects on M. tuberculosis intracellular growth in WT BMM{phi} compared with the growth in M. tuberculosis-infected macrophages alone (p = 0.002 and 0.007, respectively). The ability of whole immune splenocytes and CD4+ T cells to control growth in the IFN-{gamma}R KO BMM{phi} monolayer was significant as compared with cocultures containing M. tuberculosis-infected IFN-{gamma}R KO macrophages (p = 0.0004 and 0.0001, respectively; Fig. 7B), as well as compared with cultures containing naive spleen cells (p = 0.011 and 0.027, respectively; data not shown). In contrast, the ability of CD8+ T cells to control M. tuberculosis growth in the IFN-{gamma}R KO macrophages was only found to be significantly different as compared with cultures containing M. tuberculosis-infected IFN-{gamma}R KO macrophages alone (p = 0.01; Fig. 7B); it was not found to be significantly different from cocultures containing the same number of naive spleen cells (p = 0.320; data not shown). In multiple repeat experiments, the effect of CD8+ T cells on M. tuberculosis growth in the IFN-{gamma}R KO BMM{phi} monolayer was found to be consistently small and frequently insignificant as compared with growth of M. tuberculosis in IFN-{gamma}R KO BMM{phi} alone. Thus, only CD4+ cells, but not CD8+ cells, were consistently capable of inhibiting M. tuberculosis growth in macrophages that cannot respond to IFN-{gamma}, although both cell types controlled M. tuberculosis growth in WT macrophages that can respond to IFN-{gamma}.

To begin examination of the mechanism(s) responsible for CD4+ T cell IFN-independent control of intracellular M. tuberculosis growth, we investigated the levels of NO present in coculture supernatants. Cocultures containing WT M. tuberculosis-immune cells, or purified CD4+ T cells, with WT infected macrophages consistently produced more NO than those containing IFN-{gamma}R KO macrophages (Fig. 8A). In particular, cocultures containing CD4+ T cells with IFN-{gamma}R KO macrophages produced barely detectable amounts of NO, and the amounts detected were consistently below the reliable limit of quantitation (~6 µmol/ml). To determine whether these low levels provided biological activity, we examined the ability of M. tuberculosis-immune splenocytes and purified CD4+ T cells to control M. tuberculosis growth in BMM{phi} lacking the capacity to produce NO (iNOS KO). In contrast to the strong control of intracellular M. tuberculosis growth by either immune splenocytes or purified CD4+ T cells in cocultures containing WT macrophages, no significant control by either population was found in cocultures containing infected iNOS KO macrophages (Fig. 8B; p > 0.05 compared with cocultures containing naive splenocytes). Thus, the majority of the control of intracellular M. tuberculosis growth by murine CD4+ T cells, whether through IFN-{gamma} production or independently of IFN-{gamma}, depends on production of NO.



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  		FIGURE 8. Low levels of NO contribute to control of intracellular M. tuberculosis growth by WT immune splenocytes or CD4+ T cell subpopulations. A, Levels of nitrate, reflecting production of NO, were measured by Griess reaction in supernatants from cocultures containing the indicated cell populations and either WT (filled bars) or IFN-{gamma}R KO (gray bars) BMM{phi}. Immune splenocytes were obtained from WT mice primed 4–6 wk earlier with a low dose M. tuberculosis aerosol infection. B, WT (filled bars) and iNOS KO (gray bars) BMM{phi} were infected with M. tuberculosis and cocultured with the indicated cell populations, obtained from WT mice primed 4–6 wk earlier with a low dose M. tuberculosis aerosol infection. Eight days after infection, cultures were assessed for intracellular bacteria, as described in the legend to Fig. 2. These results are representative of three (A) or four (B) experiments similar in design. ND, Indicates cultures in which NO levels were not detected.

 
CD4+ T cells can mediate control of M. tuberculosis in vivo growth in BCG-vaccinated IFN-{gamma}-deficient mice following M. tuberculosis challenge

We next sought to determine whether the observed in vitro growth inhibition of M. tuberculosis in the absence of IFN-{gamma} responsiveness was operative in vivo. To this end, IFN-{gamma}-deficient mice (GKO) and WT mice were primed with an intradermal infection of M. bovis BCG for 4–5 wk, and then subjected to 8 wk of chemotherapy to clear the bacterial infection. At the end of the chemotherapy treatment, no bacteria were detected in lungs or spleens (limit of detection <50 CFU). One week after ending chemotherapy, groups of the BCG-vaccinated and naive mice were given a 102 CFU aerogenic challenge of M. tuberculosis Erdman. Organ burdens in the lungs and survival of these mice were then followed over time. As shown in Fig. 9A, a significant difference in lung burdens between the naive and vaccinated GKOs was evident at all time points examined, up until day 30 (p < 0.016), at which time the naive GKOs began to succumb to the infection. The mean survival time for the naive GKO mice was 32 ± 2 days, as compared with the BCG-vaccinated GKOs, which had a significantly increased mean survival time of 44.8 ± 2.9 days (p = 0.0003). All WT mice, both naive and vaccinated, survived for over 6 mo, at which point observation was terminated. Thus, secondary M. tuberculosis challenge of GKO mice results in decreased bacilli numbers in the lungs and significantly increases their survival time as compared with naive GKO mice, although ultimately these mice succumb to infection.



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  		FIGURE 9. Course of M. tuberculosis primary and secondary in vivo infections in the lungs of vaccinated GKO and WT mice in the presence and absence of CD4+ T cells. WT and GKO mice were given a 106 BCG intradermal vaccination that was allowed to progress for 4–5 wk, followed by an 8-wk chemotherapy treatment. Drug-cured vaccinated mice were then given a 102 CFU M. tuberculosis aerosol challenge, along with naive unvaccinated control WT and GKO mice. In this experiment (in which all groups were exposed to challenge simultaneously), day 0 lung burdens were determined for one mouse from each group, and were as follows: naive WT mice, 90 CFU; naive GKO mice, 150 CFU; BCG-vaccinated WT mice, 85 CFU; and BCG-vaccinated GKO mice, 120 CFU. A, At the indicated times after aerosol challenge, organs were harvested for determination of organ burdens. Values shown are mean log CFU/lung from four to five mice ± SEM. Signficant differences in CFU were found on day 10 (p = 0.011, vaccinated GKO vs naive WT; and p = 0.001, vaccinated GKO vs naive GKO), day 20 (p = 0.032, vaccinated GKO vs naive WT; and p = 0.0016, vaccinated GKO vs naive GKO), and day 30 (p = 0.016, vaccinated GKO vs naive WT; and p = 0.016 vaccinated GKO vs naive GKO). These results are representative of three experiments similar in design. B, Naive GKO mice, as well as GKO mice previously vaccinated with BCG and antibiotic treated as above, were given a 102 CFU M. tuberculosis aerosol challenge. Survival was followed over time. CD4+-depleted mice were given 0.5 mg GK1.5 Abs i.p. twice before the M. tuberculosis aerosol infection, and twice weekly thereafter. These results are representative of two experiments similar in design.

 
Given the results from the in vitro system, we sought to determine whether the observed increase in survival time of BCG-vaccinated GKO mice following M. tuberculosis challenge was mediated by CD4+ T cells. To this end, BCG-vaccinated GKO mice were given an aerogenic M. tuberculosis challenge and simultaneously in vivo depleted of CD4+ T cells (at the time of secondary challenge); survival and lung pathology of these mice were then compared with naive and vaccinated GKO controls. The mean survival times of the CD4+-depleted BCG-vaccinated GKO mice were significantly reduced compared with intact BCG-vaccinated GKO mice (Fig. 9B; 36.3 ± 1.3 days vs 53.3 ± 3.7 days, respectively, p = 0.0001) and were not significantly different from naive GKO mice (36.3 ± 1.3 days vs 39.3 ± 2.8 days, respectively, p = 0.095).

Histopathological examination of the lungs of these mice was performed at day 14 after M. tuberculosis aerosol challenge, at a time when the bacterial burden of the vaccinated GKO mice is similar to that of vaccinated WT mice. At this early time point, lungs of WT mice exhibited minimal pathology, but with some differences depending on CD4 depletion (Fig. 10, A, C, and E). Lung sections from naive mice showed minimal evidence of inflammation, while lung sections from BCG-vaccinated mice contained granulomas with some minor inflammation. Interestingly, lung sections from CD4-depleted, BCG-vaccinated WT mice had a poor granulomatous response and very minor inflammation, suggesting that CD4 depletion reduces granuloma formation even in WT mice. Striking differences are evident in the lung pathology of the different groups of GKO mice (Fig. 10, B, D, and F, respectively). As seen previously (13, 24), significant pathology with inflammation around airways was already evident at this time point in infected naive GKO mice; both activated macrophages and lymphocytes were evident in inflamed areas, but no organized granulomas were seen. In contrast, lung tissue from BCG-vaccinated GKO mice had less pathological damage; several large diffuse granulomas were apparent; and ~50% of the lung tissue appeared relatively unaffected. BCG-vaccinated GKO mice that were depleted of CD4+ T cells exhibited extensive lung tissue damage as compared with nondepleted mice, with virtually no intact tissue present, consolidation of inflamed areas, and extensive infiltration of macrophages and lymphocytes. This is consistent with the rapid death of these animals (Fig. 9B), which is most likely from respiratory insufficiency. Taken together, these data indicate that in vivo depletion of CD4+ T cells abrogates the IFN-{gamma}-independent protective immunity demonstrated by the BCG-vaccinated GKO mice during secondary M. tuberculosis challenge.



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  		FIGURE 10. Representative histology of lung tissues 14 days after M. tuberculosis aerogenic infection in: A, naive WT (C57BL/6J) mice; B, naive GKO mice; C, BCG-vaccinated WT mice; D, BCG-vaccinated GKO mice; E, BCG-vaccinated WT mice depleted of CD4+ T cells; and F, BCG-vaccinated GKO mice depleted of CD4+ cells. Tissues were Formalin fixed, embedded in paraffin, sectioned, and stained with H&E. Photomicrographs taken at x100 magnification using light microscopy are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Development of new vaccines for M. tuberculosis is currently limited by our incomplete understanding of the immune mechanisms that contribute to the control of M. tuberculosis infection in the host. Furthermore, although many studies have demonstrated the importance of several components of the immune system during M. tuberculosis infection, a reliable correlate of protection remains to be defined. Because it is difficult to study multiple components and their interactions in vivo, we wished to develop an in vitro system that models the in vivo immune response, permits direct study of various components of the immune system, and targets an important endpoint: the control of M. tuberculosis growth within its primary host cell, the macrophage. In this study, we present such a murine ex vivo model system of M. tuberculosis infection that was productively used to investigate lymphocyte control of M. tuberculosis growth within macrophages (Figs. 1–5). Of note, and consistent with the chronic nature of murine M. tuberculosis infection, control of intracellular M. tuberculosis growth was bacteriostatic, but not bactericidal. Remarkably, a large proportion of the in vitro intracellular growth of M. tuberculosis was affected by immune CD4+ T cells in the absence of macrophage responsiveness to IFN-{gamma}, but dependent on NO (Figs. 4, 6, 7, and 9). Correspondingly, CD4+ T cells mediated IFN-{gamma}-independent control of M. tuberculosis infection during a secondary challenge in vivo (Figs. 9 and 10).

The results obtained from our in vitro system are clearly consistent with murine in vivo studies (2). Similar to the in vivo protective capacity of BCG, lymphocytes from mice vaccinated in vivo with BCG controlled the intracellular growth of virulent M. tuberculosis; control was specific, in that lymphocytes from mice infected with Listeria did not control the intracellular growth of M. tuberculosis (Fig. 3C). CD4+ splenocytes were more potent effectors than CD8+ cells when compared on a cell-by-cell basis (Fig. 4, A and B). Similarly, in vivo studies have suggested that CD8+ T cells respond, but have a weaker capacity than CD4+ T cells to control M. tuberculosis infection. For example, in an adoptive transfer approach, CD4+ T cells were more effective than CD8+ T cells in reducing the in vivo growth of M. tuberculosis (5, 25, 26). Similarly, CD8+-deficient mice survived infection longer than CD4+-deficient mice, although neither survived as long as WT mice (3, 4, 6, 7).

Murine in vivo studies have shown that activated CD4+ and CD8+ T cells are quickly recruited to the lung. Although both are capable of producing IFN-{gamma}, CD4+ T cells appeared to be the predominant producers (27), and IFN-{gamma} production appears to be one of their primary effector functions. In contrast, lung CD8+ T cells during M. tuberculosis infection had other activities, including expression of perforin and lysis of infected macrophages. However, the effect of these activities in vivo and on M. tuberculosis viability remains to be determined (28). Interestingly, although our in vitro experiments indicate that CD8+ T cells produced relatively little IFN-{gamma} in the cocultures (Fig. 5A), coculture of M. tuberculosis-immune CD8+ T cells with infected IFN-{gamma}R KO BMM{phi} resulted in essentially complete loss of the ability of the CD8+ T cells to control M. tuberculosis growth. These results are consistent with in vivo protection studies using athymic mice, which showed that partial protection to M. tuberculosis infection could be provided by transfer of CD8+ T cells from WT mice, but that this protection was lost when these cells were transferred from IFN-{gamma}-deficient mice (29). Collectively, these studies suggest that the protective function of CD8+ T cells, at least in the absence of CD4+ T cells, is largely dependent upon their ability to produce IFN-{gamma} and activate macrophages.

In contrast, CD4+ T cells exerted a large amount of control of M. tuberculosis growth without IFN-{gamma} stimulation of the macrophage. Thus, CD4+ T cells have an IFN-{gamma}-independent mechanism for inducing macrophage growth inhibition; as shown in this study (Fig. 8), this mechanism depends largely on NO, at least in mice. The importance of IFN-{gamma} in generating a protective immune response against an M. tuberculosis infection is indisputable; IFN-{gamma} KO (GKO) mice, and people with IFN-{gamma} deficiencies, are exquisitely sensitive to a primary M. tuberculosis infection, and knockout mice may have disregulated T cell homeostasis (12, 13, 24, 30, 31). However, a high level of IFN-{gamma} production by an infected or vaccinated animal has not in itself proved to be a reliable indicator of protection against the disease, particularly with respect to the function of CD4+ T cells. For example, mice with a chronic M. tuberculosis infection that were depleted of CD4+ T cells lost the ability to control the disease, despite levels of IFN-{gamma} gene expression and iNOS protein expression in their lungs that were similar to control animals (4). Similarly, mice deficient in CD4+ cells were found to be unable to control an M. tuberculosis infection, despite having only transiently diminished levels of IFN-{gamma} in their lungs early in infection (3). Remarkably, in this study, we show that GKO mice primed with BCG developed CD4-dependent immunity that provided some protection against a virulent M. tuberculosis aerosol challenge (Figs. 9 and 10); organ burdens were lowered and survival times increased in BCG-vaccinated animals as compared with naive GKO mice, although they did not survive as long as WT mice. To our knowledge, these data are the first demonstration of in vivo protection against aerosol M. tuberculosis challenged in the complete absence of IFN-{gamma} during both priming and challenge. Taken together, therefore, these results strongly suggest that CD4+ cells provide both IFN {gamma}-dependent and IFN-{gamma}-independent activities during secondary M. tuberculosis infection.

The data presented in this study suggest that the mechanism CD4+ IFN-{gamma}-independent control of M. tuberculosis intracellular growth relies heavily upon NO (Fig. 8). Although NO quantities were near the limit of detection in the IFN-{gamma}R KO BMM{phi} cultures, CD4+ cells cocultured with iNOS KO BMM{phi} lost most of their ability to control M. tuberculosis intracellular growth; thus, it appears likely that the low NO levels in the IFN-{gamma}R KO BMM{phi} cultures contribute significantly to the observed control of growth. However, this does not rule out the possibility of other contributing mechanisms. Furthermore, there are several molecules that could activate NO-dependent and NO-independent macrophage antimycobacterial activities. Although the role of CD40 in mycobacterial infections remains to be fully defined, macrophages activated via the CD40-CD40 ligand interaction produce NO, IL-12, and possibly other effector mechanisms (32). Other candidate mediators of IFN-{gamma}-independent growth inhibition include other members of the TNF superfamily. CD4+ T cells produce TNF-{alpha}, lymphotoxin-{alpha}, and lymphotoxin-{beta}, including membrane-associated forms of these mediators (33), and in the M. tuberculosis infection model, mice deficient in TNF-{alpha}, lymphotoxin-{alpha}, and lymphotoxin-{beta} are more susceptible to infection than WT mice (34, 35, 36). TNF-{alpha} in particular may be a promising candidate. Blockade of TNF-{alpha} significantly reversed control of intracellular M. tuberculosis growth (Fig. 6), and TNF-{alpha} inhibits BCG growth in macrophages by NO-dependent and NO-independent mechanisms (37). We are currently investigating the contribution of these mechanisms to CD4+ cell-mediated IFN-{gamma}-independent growth inhibition. Whether these (or other) mechanisms are induced as part of the normal immune response to M. tuberculosis infection, or whether they only emerge as compensating mechanisms due to the absence of IFN-{gamma} activity in the KO models, remains to be determined. In this study, it is formally possible that compensatory activities contribute to the in vivo experiments using GKO mice (Figs. 9 and 10). It seems unlikely that compensatory activities explain the mechanisms reflected in the in vitro experiments (Figs. 1–8), because the effector cells were obtained from primed WT mice and developed in a normal environment.

In our cocultures, we found that addition of neutralizing Abs to IFN-{gamma} or TNF-{alpha} had significant effects on the ability of M. tuberculosis-immune whole spleen cells to control M. tuberculosis intracellular growth; this was not the case for anti-IL-12 p40, anti-IL-10, or anti-IL-4 (Fig. 6). Even the impact of anti-IFN or anti-TNF was relatively minor, although combinations of neutralizing anti-cytokine Abs remain to be tested. In repeated experiments, the impact of addition of neutralizing anti-IFN-{gamma} Abs (Fig. 6) was less than the use of IFN-{gamma}R KO macrophages (Fig. 7B). This may be due to the inherent difficulty in blocking all activity successfully by exogenous addition of blocking reagents. The results further emphasize the importance of using multiple technical approaches to examine complex questions.

In vitro models of human immunity to M. tuberculosis infection have previously been developed by others (38, 39), but surprisingly, analogous models have not been explored in animal studies, including mice. Silver et al. (38) cocultured M. tuberculosis-infected human mononuclear cells with nonadherent peripheral blood cells (PBLs) from either purified protein derivative (PPD)-positive or PPD-negative subjects, and assessed intracellular M. tuberculosis viability. Similar to results described in this work, growth inhibition mediated by PPD-positive cells was specific, relied primarily upon CD4+ cells, and was virtually unaffected by neutralizing anti-IFN-{gamma} Abs (38); lysis of M. tuberculosis-infected cells by immune CD4+ and CD8+ T cells did not involve Fas-Fas ligand interactions or perforin (40). The murine in vitro system described in this work intentionally uses immune lymphocytes that are primed in vivo and obtained from relevant organs in which M. tuberculosis replicates during infection, which is obviously more feasible in animal than in human models. Lymphocytes from different organs may also be studied to determine whether different mechanisms are used in the different organs to mediate M. tuberculosis growth inhibition. A further advantage of the murine model is the availability of mouse strains that are genetically deficient in a wide variety of different components of the immune system.

The discovery that a significant proportion of macrophage antimycobacterial activity in our in vitro system does not involve IFN-{gamma} stimulation is especially important given the ability of Mycobacteria species to interfere with host macrophage IFN-{gamma} intracellular signaling (41, 42); in particular, M. tuberculosis infection disrupted the association of STAT1 with the transcriptional coactivators CREB-binding protein and p300 (18). Thus, the ability of IFN-{gamma} to activate an M. tuberculosis-infected macrophage may be suboptimal, suggesting that much of the antimycobacterial macrophage activity observed in vivo and in vitro may necessarily use mechanisms that do not involve IFN-{gamma}.

The role of IFN-{gamma} during a secondary immune response has received little direct study. In vivo Ab neutralization studies indicate that neutralization of IFN-{gamma} during an M. tuberculosis secondary infection resulted in increased bacterial burdens in the spleen (43). However, the temporal importance of IFN-{gamma}, and the effect of IFN-{gamma} neutralization on M. tuberculosis growth in other organs, was not investigated. This is particularly significant given that the immune response during a secondary TB challenge is more relevant to vaccination and protection against subsequent exposure to TB. The in vitro culture system described in this work provides a new approach to examining the qualitative differences between T cell subpopulations obtained at different times after priming. The mechanisms used over time by M. tuberculosis-immune CD4+ cells, both IFN-{gamma} dependent and IFN-{gamma} independent, to inhibit M. tuberculosis intracellular growth will therefore be an important subject of our future studies. Further investigation of various immune cell types, cytokines, and other immune modulators capable of controlling M. tuberculosis intracellular growth will surely facilitate the identification of new correlates of protection and treatment strategies.


   Acknowledgments
 
We are grateful to our Center for Biologics Evaluation and Research colleagues, Dr. Sheldon Morris and Dr. Michael Brennan, for their thorough and thoughtful reviews of the manuscript, and to Elizabeth Hamilton for excellent technical assistance.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Siobhán Cowley or Dr. Karen Elkins, LMDCI/DBPAP/CBER/FDA, 1401 Rockville Pike, HFM 431, Rockville, MD 20852. E-mail address: cowley{at}cber.fda.gov or elkins{at}cber.fda.gov Back

2 Abbreviations used in this paper: BCG, bacillus Calmette-Guerin; BMM{phi}, bone marrow-derived macrophage; cDMEM, complete DMEM; GKO, {gamma} KO; KO, knockout; MOI, multiplicity of infection; PPD, purified protein derivative; TB, tuberculosis; WT, wild type. Back

Received for publication May 6, 2003. Accepted for publication August 21, 2003.


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 Introduction
 Materials and Methods
 Results
 Discussion
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