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Immunology. Mar 2000; 99(3): 473–480.
PMCID: PMC2327172

Mycobacterium tuberculosis-activated dendritic cells induce protective immunityin mice

Abstract

Activated dendritic cells are critically important in the priming of T-cell responses. In this report we show that the infection of a conditionally immortalized dendritic cell line (tsDC) with Mycobacterium tuberculosis resulted in the up-regulation of B7-1 and B7-2 co-stimulatory molecules and the induction of several inflammatory cytokines, including tumour necrosis factor-α and interleukin-6, -1β and -12. In addition, we show that these activated dendritic cells were capable of eliciting antigen-specific T-cell responses and potent anti-mycobacterial protective immunity in a murine model of experimental tuberculosis infection.

Introduction

Tuberculosis remains one of the most important threats to human health and represents an enormous problem world-wide. Bacillus Calmette–Guérin (BCG) provides only limited protection against the infection1 and the cellular basis for the induction of protective CD4+ and CD8+ T-cell responses remains poorly understood.2,3 Interestingly, although BCG gives variable levels of protection in studies in human populations, it consistently gives good protection in small rodent models, and new generations of subunit or attenuated vaccines rarely provide the levels of protection seen with BCG in these models.

Dendritic cells (DC) in addition to macrophages are readily infected with Mycobacterium tuberculosis. 4 They are present in the airway epithelium and lung parenchyma5 and it is likely that they play a principal role in priming anti-mycobacterial T-cell responses. In peripheral tissues DC are present in a so-called ‘immature state’, unable to stimulate T cells; however, they are specialized in antigen-uptake and after interaction with pathogens or inflammatory products they acquire the ability to migrate to lymphoid tissues for priming specific T-cell responses.6,7

Immortalized DC lines are a good model for the study of the molecular mechanisms involved in the activation and presentation of bacterial antigens to naive T cells; in addition they could be used directly as cell-based vaccines for studying the induction of protective anti-bacterial immunity. In this report we used a conditionally immortalized DC line (tsDC) generated from the bone marrow of transgenic mice carrying a temperature-sensitive mutant of the simian virus 40 (SV40) large T antigen.8 These cells grow in a semi-adherent fashion at 37° in the absence of granulocyte–macrophage colony-stimulating factor (GM-CSF) and exhibit typical features of ‘immature’ DC; here we show that, in response to infection by M. tuberculosis these cells up-regulate several cytokine genes and co-stimulatory molecules indicating transition to a ‘mature’ phenotype. Furthermore we demonstrate that adoptively transferred tsDC infected with M. tuberculosis were effective inducers of M. tuberculosis-specific cellular and humoral responses and elicited a significant level of protective immunity against experimental infection, superior to that seen with BCG. These results demonstrate that, even in rodent models in which BCG is effective, it is possible to obtain improved levels of protective immunity, and reinforce the potential role of DC in priming anti-mycobacterial T-cell immunity.

Materials and methods

Mice and cells

Six- to eight-week-old CBA/ca or C57/BL6 female mice were kept in conventional animal facilities at the National Institute for Medical Research. The dendritic cell line tsDC (generously provided by A. Volkmann and B. Stockinger, The National Institute for Medical Research, UK) was cultured in flasks (Life Technologies, Paisley, UK) at 1 × 105 cells/ml in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Paisley, UK), supplemented with 10% fetal calf serum (FCS) and 10 mm glutamine, and kept at 36·5°. Medium for culturing the cells in the antigen presentation assay was Optimem (Life Technologies, Paisley, UK). Bone marrow-derived DC (BMDC) were purified from C57/BL6 mice as described previously;8 briefly, bone marrow cells were cultured at 37° in Petri dishes in 10 ml of Iscove’s modified Dulbecco’s medium supplemented with 5–10% FCS, 10 mm glutamine and 10% supernatant of Ag 8653 myeloma cells transfected with mouse GM-CSF cDNA (20–30 U/ml). On day 4 non-adherent cells were removed. Loosely adherent cells were used at days 6–8 as a source of DC. Their purity, as assessed by major histocompatibility complex (MHC) class II expression, was 70–90%.

Bacterial cultures

Mycobacterium tuberculosis H37Rv was grown in Dubos 7H9 broth for 14 days, aliquoted and stored in liquid nitrogen. Aliquots were thawed and diluted in phosphate-buffered saline (PBS) prior to infection of cells or mice.

Infection of tsDC, BMDC and vaccination protocol

Cells were incubated at 36·5° and infected with M. tuberculosis at a multiplicity of infection of five bacteria to one cell; after 12–18 hr extracellular bacteria were removed by washing and the percentage of cells infected was assessed by staining with the Ziehl–Neelsen method for acid-fast bacteria. For the vaccination experiments the cells were irradiated at 2·5 megaRads and 1 × 106−4 × 106 cells were injected intraperitoneally in 0·5 ml of incomplete Iscove’s modified Dulbecco medium (Flow Laboratories).

Flow cytometry and cell sorting

Uninfected and infected tsDC were washed twice with PBS and, after blocking Fc receptors using anti-mouse CD16/CD32 (Pharmingen, Oxford, UK) for 15 min, the cells were stained for 20 min on ice with directly conjugated antibodies. Two-colour flow cytometry was used using the following monoclonal antibodies: phycoerythrin (PE)-conjugated anti-B7-1 clone 1G10, PE-conjugated anti-B7-2 clone GL-1, PE-conjugated anti-intercellular adhesion molecule-1 (ICAM-1) clone YNI/1.7.4, PE-conjugated anti-heat stable antigen clone MI/69, fluorescein isothiocyanate (FITC)-conjugated streptavidin (all obtained from Pharmingen) and biotin-conjugated anti-MHC class II I-Eαk,d clone 14.4.4 ATCC HB32 (kindly supplied by Dr B. Stockinger). Acquisition was performed on a fluorescence-activated cell sorter (FACScan; Becton Dickinson, Mountain View, CA) using forward- and side-scatter characteristics to exclude dead cells. Data were analysed using winmdi 2·6 (The Scripps Research Institute, CA).

Detection of cytokine gene expression by reverse transcription polymerase chain reaction (RT-PCR)

Total RNA was isolated from 1 × 106 DC using RNAzol B (AMS Biotech, Abingdon, UK) in accordance with the manufacturer’s instructions. A DNA digestion step was included to avoid any genomic DNA contamination. PCR amplification was carried out using specific amplimer pairs for murine tumour necrosis factor-α (TNF-α) [giving a 750-base pair (bp) product], interleukin-6 (IL-6) (giving a 638-bp product), IL-1β (giving a 587-bp product) and IL-12p40 (giving a 394-bp product) (Clontech, Palo Alto, CA).The PCR mixture contained 5 µl of 10 × PCR buffer, 5 µl of 25 mm MgCl2, 1 µl of 10 mm dNTP, 0·5 µl of 20 µm primers, 0·5 µl of Taq polymerase (5 U/µl) and 5 µl of cDNA. The β-actin primers used as controls for the PCR reactions were (sense: 5′-ATG GAT GAC GAT ATC GCT-3′; anti-sense: 5′-ATG AGG TAG TCT GTC AGG T-3′ giving a 540-bp product). PCR products were visualized by electrophoresis through 1·0% or 1·2% agarose containing ethidium bromide.

Cytokine assay

Quantification of cytokines produced by cultured cells was carried out by enzyme-linked immunosorbent assay (ELISA) using commercially available kits (Genzyme, Cambridge, MA for the determination of total IL-12 or Amersham, Buckinghamshire, UK for IL-6, TNF-α and IL-1β). For the determination of these cytokines in culture fluid, supernatants were collected at different times after M. tuberculosis-infection and filtered through a 0·2-µm filter (Millipore, Hertfordshire, UK) before they were analysed according to the manufacturer’s protocol.

Electron microscopy

Infected or uninfected tsDC were washed with PBS and fixed with 2·5% glutaraldehyde/2% formaldehyde in 0·1 m sodium cacodylate buffer (pH 7·2). Cells were post-fixed with 1% osmium tetroxide and 1% aqueous uranyl acetate, dehydrated and embedded in Epon (Agar Scientific, Essex, UK). Thin sections (55 nm) were mounted on 200 carbon-coated grids and stained with ethanolic uranyl acetate and Reynold’s lead citrate. Observation was made with a Jeol Cx 100 transmission electron microscope.

M. tuberculosis viability assay

Infected tsDC or peritoneal macrophages were lysed at different time-points with 2% saponin at 37°. Supernatants were plated on 7H11 Middlebrook agar (Difco Laboratories, Surrey, UK) at 10-fold dilutions. The plates were incubated at 37° for 3 weeks before colony-forming units (CFU) were counted.

Peritoneal macrophages

Peritoneal macrophages were collected, and cultured in DMEM medium (Gibco BRL, UK) supplemented with 20% FCS and 2-mercaptoethanol. The cells were cultured at 1 × 106/well and after 2 days were infected as described above.

T-cell culture and antibody levels

T cells were purified from the spleens of three to five mice using T-cell separation columns (R & D Systems, Oxfordshire, UK) following the manufacturer’s instructions. Pooled T cells were then cultured in duplicate in 96-well plates (Nunc) at 3 × 105−5 × 105 cells/well in AIM V serum-free medium (Gibco BRL) supplemented with 2-mercaptoethanol and antibiotics. Infected or uninfected tsDC were used as antigen-presenting cells at 3 × 104−5 × 104 cells/well; cells were cultured at 37° for 24–48 hr. Supernatants were collected and filtered for cytokine assays. Interferon-γ (IFN-γ) and IL-2 were quantified using commercial ELISA kits (Amersham) according to the manufacturer’s instructions.

Serum antibody titres of M. tuberculosis-specific immunoglobulin G1 (IgG1) and IgG2a were assayed by ELISA using 0·5 µg/well of a M. tuberculosis-cell free extract (kindly donated by Patricia Brookes, National Institute for Medical Research). After blocking with 5% milk in PBS, sera dilutions were incubated in duplicate with the coated antigen for 1 hr at room temperature. After washing, biotinylated anti-mouse IgG1 or IgG2a (Pharmacia, Buckinghamshire, UK) was added at a 1 : 1000 dilution for 1 hr. Plates were then washed and incubated for 30 min with a 1 : 1000 dilution of a streptavidin–peroxidase-labelled antibody (Pharmacia). After washing, the enzyme activity was detected using tetramethylbenzidine (ICN, Hampshire, UK). The amount of reaction product was assessed using an ELISA plate reader (Biotek-Instruments, Hertfordshire, UK) at an optical density of 450 nm.

Induction of anti-mycobacterial protective immunity

To test the protective immune response following vaccination with antigen-pulsed tsDC, two experiments were carried out. In expt 1, groups of four or five CBA/ca mice received one or two intraperitoneal injections of 1 × 106−4 × 106M. tuberculosis-infected tsDC, uninfected tsDC, or 5 × 106 irradiated M. tuberculosis. Four weeks after the final injection the mice were challenged intravenously with 1 × 106 CFU of M. tuberculosis. As a positive control, one group of mice received a single intradermal vaccination with 1 × 106 CFU of BCG (Glaxo, Middlesex, UK) 6–9 weeks before the infectious challenge. Protection was evaluated by determining the numbers of CFU in the lungs 6 weeks after the challenge. In expt 2, groups of four or five CBA/ca mice received two intraperitoneal injections of 1 × 106−4 × 106M. tuberculosis-infected tsDC, uninfected tsDC, or tsDC which had been allowed to take up irradiated M. tuberculosis. As a positive control, one group of mice received a single intradermal vaccination with 1 × 106 CFU of BCG (Glaxo) 12 weeks before the infectious challenge. In the experiment using BMDCs (expt 3) mice received three injections of 1 × 106−4 × 106M. tuberculosis-infected BMDC, BMDC, or 5 × 106 irradiated M. tuberculosis at 3-week intervals; one group of mice received a single intradermal vaccination with 1 × 104 BCG (Glaxo) 12 weeks before the challenge. Differences between the groups were compared using Student’s t-test.

Results

Infection of tsDc with M. tuberculosis

We incubated confluent cultures of tsDC with M. tuberculosis as described in the Materials and Methods, and examined the cells at different times post-infection by electron microscopy. As shown in Fig. 1(a) 12–24 hr after the infection bacilli were observed within membrane-bound phagosomes and no free bacteria were observed in the cytoplasm. In some infected cells, multivesicular organelles containing cell membrane-damaged bacteria were observed and their occurrence was paralleled by the appearance of membrane-bound inclusions having pale homogeneous but granular contents (Fig. 1b). The bacteria were also frequently found in multilaminar and multivesicular vacuoles (Fig. 1c) reminiscent of MHC class II-enriched compartments (MIIC)9 previously described as the major site for antigen degradation and peptide loading onto MHC class II molecules in DC10 and B cells.11 By 48–96 hr, many of these vacuoles appeared largely devoid of their contents, and of intact bacteria (Fig. 1d).

Figure 1
Electron micrographs showing M. tuberculosis-infected tsDC. (a) × 30 000 magnification showing membrane-bound phagosomes containing M. tuberculosis 12–24 hr after infection. (b) × 48 150 magnification illustrating some membrane-damaged ...

To assess the progression of the bacterial infection, tsDC cultures were lysed at different intervals and viable counts of bacteria were carried out. Cultures of peritoneal macrophages were infected in parallel and used as controls to demonstrate the ability of the bacteria to replicate within permissive cells. As shown in Fig. 2, viable M. tuberculosis were still present in the tsDC at day 5 after the infection; however, the number of viable bacteria decreased significantly suggesting that these cells were able, to some extent, to control the growth of the tubercle bacilli; growth of the identical inoculum in peritoneal macrophages was confirmed.

Figure 2
Growth of M. tuberculosis in tsDC and peritoneal macrophages measured by the colony-forming unit assay at different times post-infection. Data represent the mean counts from three separate well monolayers.

Infection of tsDC with M. tuberculosis up-regulates cytokine expression

To analyse if infection with M. tuberculosis resulted in stimulation to produce inflammatory cytokines, tsDC were infected with M. tuberculosis and the expression of TNF-α, IL-1β, IL-6 and IL-12 were monitored by RT-PCR 24 hr after infection, and by ELISA at 12 and 48 hr after infection. As shown in Fig. 3(a), there was an up-regulation of expression of all four cytokines when tsDC were infected with M. tuberculosis; expression of these cytokines was not observed in uninfected cells. To confirm these results we investigated if these cytokines were secreted after M. tuberculosis infection. As can be seen in Fig. 3(b), we detected high levels of TNF-α and IL-6 and low levels of IL-1β and IL-12 in the supernatants of tsDC infected with M. tuberculosis; in contrast, very low levels of these cytokines were observed in supernatants obtained from uninfected tsDC used as controls. TNF-α and IL-6 were secreted at very high levels as early as 12 hr post infection.

Figure 3
Infection of tsDC with M. tuberculosis up-regulates cytokine expression. (a) RT-PCR analysis for the detection of IL-6-, TNF-α-, IL-1β-, IL-12- and β-actin-specific mRNAs after M. tuberculosis infection. RNA obtained from non-infected ...

Infection of tsDC with M. tuberculosis up-regulatesco-stimulatory molecules

Activated DC secrete inflammatory cytokines and express high levels of co-stimulatory molecules.6 To analyse if the infection with M. tuberculosis resulted in increased cell surface expression of co-stimulatory molecules, we infected tsDC with M. tuberculosis and analysed the expression of B7-1, B7-2, ICAM-1 and heat stable antigen (HSA) by FACS analysis. As shown in Fig. 4, the surface expression of B7-1 and B7-2 molecules was clearly up-regulated after the infection; however, no significant increase in the surface levels of ICAM-1 and HSA was observed. In addition, the up-regulation of co-stimulatory molecules in M. tuberculosis-infected tsDC was paralleled by concomitant up-regulation of MHC class II molecules. Based on these results we conclude that the infection with M. tuberculosis is sufficient to induce DC to mature into an ‘activated’ phenotype.

Figure 4
FACS analysis of tsDC or M. tuberculosis-infected tsDC for surface expression of the indicated antigens. Staining with isotype-matched control antibodies as negative controls is shown in hatched histograms; non-infected tsDC in filled histograms and ...

M. tuberculosis-activated tsDC induce specific immune responses

We next investigated whether infected tsDC were able to induce specific immune responses. In these experiments, we adoptively transferred uninfected tsDC, or tsDC that had been infected or pulsed with irradiated M. tuberculosis, into naive CBA/ca mice as described in the Materials and Methods. We tested the splenic T cells for antigen-specific cytokine production and the sera of vaccinated mice by ELISA for anti-M. tuberculosis IgG1 and IgG2a specific antibodies. As shown in Fig. 5(a), the transfer of M. tuberculosis-infected tsDC or M. tuberculosis-pulsed tsDC, into mice resulted in effective priming of T cells as demonstrated by the high levels of IL-2 and IFN-γ produced in response to antigen-specific stimulation. In addition, and consistent with the effective priming of T-cell responses, these mice generated high levels of anti-M. tuberculosis IgG2a and IgG1-specific antibodies (Fig. 5b); by contrast, the specific anti-M. tuberculosis cellular and humoral immune response in naive mice or mice to which uninfected tsDC had been transferred as controls was very low.

Figure 5
Specific cellular (a) or humoral (b) immune responses to M. tuberculosis antigens after injection of M. tuberculosis-infected tsDC, tsDC pulsed with irradiated M. tuberculosis, or tsDC. For analysing the specific T-cell responses, mice received one injection ...

Mice immunized with M. tuberculosis-infected tsDC or M. tuberculosis-infected BMDC are protected against M. tuberculosis infection

We next studied whether the injection of M. tuberculosis-infected tsDC was sufficient to confer protection against experimental infection with M. tuberculosis by challenging the immunized CBA/ca mice with viable M. tuberculosis 4 weeks after immunization. As shown in Fig. 6 (expt 1), mice receiving two injections of M. tuberculosis-infected tsDC were significantly better protected than mice receiving BCG (P < 0·05), although BCG itself, given in this case 6–9 weeks before infection, only had a small (though significant, P < 0·05) effect in this experiment. Interestingly, mice that received a single injection of M. tuberculosis-infected tsDc were not better protected than the BCG group. In a second experiment (Fig. 6, expt 2) mice immunized twice with M. tuberculosis-infected tsDC (DC/INF × 2), were significantly protected against the infection when compared to naı¨ve mice, or mice that received uninfected tsDC (DC Controls; P < 0·05) and the protection achieved in the lungs was again significantly better than BCG (P < 0·05); in this experiment BCG was given 12 weeks before infection and appeared to give much better protection than that seen in expt 1. Protection was also achieved in C57/BL6 mice that received injections of BMDC infected with M. tuberculosis (Fig. 6; expt 3) showing that the ability to prime protective responses was not a special feature of the immortalized tsDC. In this strain of mice the protection observed in the lungs was not significantly greater than the protection conferred by vaccination with BCG (P < 0·05). In the spleen, BCG gave better protection (P < 0·05).

Figure 6
Protection against M. tuberculosis infection after immunization with M. tuberculosis-infected tsDC or M. tuberculosis-infected BMDC. In expt 1, groups of four or five CBA/ca mice received either one or two intraperitoneal injections of M. tuberculosis ...

Discussion

In the present study we evaluated the ability of M. tuberculosis to infect the conditionally immortalized DC line tsDC; we find that these cells effectively phagocytose M. tuberculosis and are able to control the intracellular growth of the bacteria; the mechanism responsible for this effect is not clear but since it has been shown that DC can effectively up-regulate nitric oxide synthase in response to activation,12 it is possible that l-arginine-dependent generation of reactive nitrogen intermediates participates in mediating this anti-mycobacterial effect.

We also observed up-regulation of several cytokine genes in response to mycobacterial infection; recent work4 demonstrated that human DC generated from peripheral blood were capable of up-regulating co-stimulatory molecules and secreting elevated levels of TNF-α, IL-1 and IL-12 in response to M. tuberculosis infection. Our data, using murine cells confirm these results and show that the infection with M. tuberculosis is sufficient to activate DC to prime specific immune responses and to induce significant levels of protection against experimental infection with M. tuberculosis. Importantly, we confirmed the specific anti-M. tuberculosis protective responses in mice vaccinated with primary BMDC infected with M. tuberculosis, showing that the immunogenicity of these cells is not a special feature of the immortalized cell line tsDC.

DC are the most efficient antigen-presenting cells for priming T-cell responses, therefore it is likely that in our experiments specific anti-mycobacterial T-cell priming was accomplished directly by the infected DC; however, because the DC were irradiated prior to injection, it is possible that cross-presentation of mycobacterial antigens contributed to the protective effect in this model.

Previous work13 showed that murine DC infected with BCG were able to prime specific anti-mycobacterial T-cell responses; in our experiments, two injections of M. tuberculosis-infected DC cells were necessary to induce protective responses comparable or better than that achieved with BCG; our data are consistent with the interpretation that boosting immunizations are required to increase the frequency and/or differentiation of the protective T cells.

Our results demonstrate for the first time that M. tuberculosis-activated DC are able to induce a protective immune response that may be more effective than BCG vaccination in mice. This reinforces the importance of DC in generating protective immune responses against infectious agents. Other groups have shown, for example, that similar strategies can be used to protect against experimental infections with Chlamydia trachomatis14 and Toxoplasma gondii 15. Recently, BCG-infected DC were shown to protect mice against an aerosol challenge with M. tuberculosis;16 however, in these experiments the DC were given intratracheally a route which would favour local presentation to T cells but would not necessarily indicate systemic priming of naı¨ve T cells.

Our results support the development of anti-mycobacterial vaccine strategies able to target DC in vivo; in addition, because DC need to be activated in order to effectively prime protective T-cell responses, vaccine vectors capable of activating DC ‘in vivo’ should increase the protective efficacy of the immunization protocol. We and others have previously shown that polynucleotide vaccination effectively generates anti-mycobacterial protective responses in mice;17,18 it is likely that the efficacy obtained by these immunization strategies depends largely on the ability of bacterial DNA to target and activate DC in vivo.19

Acknowledgments

We thank the Praxis XXI Programme of the Portuguese Ministry of Science and Technology for funding Clara S. Soares

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