INTRODUCTION

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Tuberculosis (TB) remains the single most important bacterial infection worldwide, with a third of the world's population infected with Mycobacterium tuberculosis and eight million new cases of clinical TB reported to the World Health Organization each year (15). Meta-analysis of trials with the only currently available vaccine, Mycobacterium bovis BCG, has concluded that BCG confers only 50 and 80% protective efficacy against pulmonary and disseminated TB, respectively (8). Although beneficial to individuals, BCG vaccination appears to be insufficient to control the spread of TB, and new immunization strategies are urgently needed.

The heterodimeric cytokine interleukin 12 (IL-12) provides an important bridge between innate and adaptive immunities and is essential for protection against mycobacterial infections (11, 12, 18). IL-12 is required for sensitization of Th1-like CD4⁺ T cells, stimulates the production of gamma interferon (IFN-) by NK cells, and, upon restimulation, contributes to the expansion of IFN--producing CD4⁺ T cells (34). Therefore, vaccination strategies optimizing IL-12 production by antigen-presenting cells (APC) in response to BCG may have increased protective efficacy against M. tuberculosis infection.

Several lines of evidence have demonstrated that dendritic cells (DC) are the major APC for primary T-cell responses as well as the initial source of IL-12 in microbial infections (6, 28, 29). M. tuberculosis and BCG infection of human or murine myeloid DC induces a coordinate process of cell maturation and up-regulation of IL-12 production (13, 23, 33). Subsequent transfer of BCG-infected DC into mice led to rapid IFN- responses against mycobacterial antigens (13), and M. tuberculosis-infected DC induced potent immunity against experimental TB in mice (32). These data suggest that during mycobacterial infections, host DC located in the lung migrate to the T-cell areas of the draining lymph nodes, where they present antigens to T cells and promote the expansion of IFN--secreting CD4⁺ T cells. Stimulating the IL-12 response of DC to mycobacterial antigens may thus represent a way to elicit earlier and more potent protective responses.

Interaction between the CD40 receptor on APC and its ligand (CD40L) on activated T cells plays a critical role in immunity to intracellular pathogens by up-regulating the production of IL-12 (16, 20, 21). CD40- or CD40L-deficient mice have an increased susceptibility to leishmanial infection and show an impaired priming of Th1-type cells, correlating with a lack of activation of the macrophage effector functions required for parasite clearance (5, 25, 31). By contrast, CD40L knockout mice showed no difference in susceptibility to M. tuberculosis infection compared to wild-type mice (9), suggesting that cell-mediated immunity and protection against mycobacteria develop independently of CD40L. This would imply that mycobacterial components stimulate IL-12 production by DC and macrophages without the involvement of CD40. Therefore, it is possible that additional stimulation of the CD40 signaling pathway in mycobacterium-infected APC may further enhance IL-12 production and the resultant T-cell protective immunity. In this study, we have investigated whether mycobacterium-infected DC are responsive to CD40 signaling and whether ex vivo stimulation of these cells via CD40 enhanced their ability to confer protective immunity against M. tuberculosis infection.

	MATERIALS AND METHODS

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Mice. Six- to 8-week-old C57BL/6 mice were obtained from the Animal Resources Centre (Perth, Australia) and kept under specific-pathogen-free conditions at the Centenary Institute animal facility.

DC cultures. Bone marrow-derived DC were generated by a modification of a method previously described (24). Briefly, murine bone marrow cell suspensions were incubated with a mixture of M5/114 (anti-major histocompatibility complex [MHC] class II), RA3-6B2 (anti-B220), 53-6.7 (anti-CD8), GK1.5 (anti-CD4), and RB6-8C5 (anti-Ly-6G) monoclonal antibodies (MAbs), and stained cells were eliminated by negative selection using Dynabeads M-450 coated with sheep anti-rat immunoglobulin G (IgG). The remaining cells were cultured in complete medium consisting of RPMI 1640 containing 5% fetal calf serum, 50 µM 2-mercaptoethanol, and 2 mM glutamine supplemented with 2.5 ng of recombinant murine granulocyte/macrophage colony-stimulating factor/ml and 5 ng of recombinant murine IL-4/ml. To test the effect of CD40 ligation on DC, a rat anti-CD40 IgG (1C10; DNAX, Palo Alto, Calif.) or an irrelevant rat IgG (GL113; ATCC HB11679) was added to the complete medium. The cultures were fed by changing 75% of the medium every 2 days and resulted in DC of immature phenotype after 6 days. For BCG infection, day-4 DC cultures were incubated with live BCG (Tokyo strain; ATCC 35737) at a multiplicity of infection of 10:1. After 12 h, the free mycobacteria were removed by centrifugation. The cells were washed and cultured in fresh medium for an additional 48 h. For T-cell-priming assays, day-6 DC were -irradiated (2,500 rads), and serial dilutions in serum-free lymphocyte medium (AIM-V; Life Technologies, Grand Island, N.Y.) were plated in 96-well plates. Syngeneic splenocytes were then added at a density of 5 × 10⁵ well. Total cell proliferation was monitored after 48 h by [³H]thymidine incorporation, and the IFN- content of the culture supernatants was assessed as previously described (13).

Analysis of cytokine mRNA expression. Expression of cytokine mRNA was measured by a multiprobe RNase protection assay as previously described (13). Total mRNAs from cultured DC or lymph node cell suspensions were prepared using RNAzol (Tel-Test, Inc., Friendswood, Tex.), hybridized with template mRNA probes specific for a number of cytokines and chemokines (Pharmingen, San Diego, Calif.), and then digested with RNase. Protected probes were analyzed by migration on an acrylamide gel following the manufacturer's protocol. Templates for the housekeeping genes L32 and GADPH (glyceraldehyde-3-phosphate dehydrogenase) were used to normalize the total RNA content of the samples.

Cytokine production assays. IL-12 production by DC was measured with a bioassay. Briefly, IL-12 in test samples was captured by the rat anti-mouse IL-12 (p40) MAb C15.6 (Pharmingen) on a 96-well plate. After being washed, test samples were replaced with concanavalin A-activated mouse lymphoblasts (10⁵/well). Lymphoblast proliferation in response to captured IL-12 was then monitored by [³H] thymidine incorporation.

Flow cytometry. Day-6 DC were stained by incubation with anti-CD80, anti-CD86, or anti-MHC class II MAbs followed by fluorescein isothiocyanate-conjugated goat anti-rat IgGs. Staining of cells was performed in a 96-well round-bottom plate (BD Pharmingen, San Diego, Calif.) with 2 × 10⁵ cells per well. The cells were pelleted by centrifugation (480 × g; 4°C; 1 min), and the supernatant was aspirated. The cells were washed by centrifugation in 2% bovine serum albumin-0.1% NaN₃ phosphate-buffered saline (PBS), and diluted antibody combinations were then added (15 min; 4°C). After being washed, the samples were analyzed on a FACScan (Becton Dickinson, San Jose, Calif.).

Elispot for cytokine-producing cells. Four weeks after M. tuberculosis challenge, mediastinal lymph nodes (MLN) were harvested and single-cell suspension were prepared by sieving them through 200-µm-pore-size mesh and resuspending the cells in culture medium. Nitrocellulose wells of an Immobilon-P plate (Millipore, Bedford, Mass.) were coated with an anti-IFN- MAb (AN18), washed, and coated with PBS containing 2% fetal calf serum. The cell suspensions were then plated at 5 × 10⁵ per well with either medium alone, purified protein derivative of M. tuberculosis (Statens Seruminstitut, Copenhagen, Denmark), or concanavalin A (Sigma, St. Louis, Mo.). The plates were incubated at 37°C for 18 h and then extensively washed with PBS. Subsequently, biotinylated XMG1.2 MAb was added to the wells. After 2 h of incubation and washing, the plates were incubated with avidin-alkaline phosphatase (Sigma). The presence of IFN--producing cells was determined by using an alkaline phosphatase conjugate substrate kit (Bio-Rad Laboratories, Hercules, Calif.).

Immunization and M. tuberculosis challenge protocols. Groups of five mice were subjected to various vaccination protocols prior to M. tuberculosis infection. Unvaccinated animals, as well as mice immunized using a reference vaccination protocol corresponding to subcutaneous injection of 5 × 10⁴ BCG Tokyo 12 weeks prior to challenge, were included as controls. BCG-infected DC initiate specific T-cell responses in mice by 5 days after adoptive transfer (13). Further experiments showed that this response is generated within 48 h after DC transfer in the draining lymph nodes. Therefore, the other groups were immunized 2 days before aerosol M. tuberculosis infection by intratracheal instillation of 2 × 10⁵ DC. A Middlebrook airborne-infection apparatus (Glas-Col Inc., Terre-Haute, Ind.) was used to deliver 100 bacilli of M. tuberculosis H37Rv (ATCC 27294) into the lungs of exposed mice. The numbers of viable bacteria in the lungs were measured 4 weeks after infection by plating serial dilutions of whole-organ homogenates on supplemented Middlebrook 7H11 nutrient agar (Difco, Detroit, Mich.) and counting the bacterial colonies formed after incubation for 20 days at 37°C. The data are expressed as mean log₁₀ CFU and standard error of the mean and (SEM) per lung.

	RESULTS

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Stimulation of DC with an anti-CD40 MAb results in cell maturation. The MAb 1C10 recognizes the extracellular domain of murine CD40 and mimics the stimulatory activities of CD40L on B cells (22). To examine whether this agonistic antibody could simulate the effect of CD40 engagement on DC, bone marrow-derived DC cultures were generated and incubated with increasing concentrations of the 1C10 MAb. A dose-dependent maturation of DC, as shown by the up-regulation of the costimulatory molecules B7-1 (CD80) and B7-2 (CD86) and MHC class II expression, was observed (Fig. 1). Maximal stimulation was obtained with 10 µg of anti-CD40 MAb/ml. Equivalent amounts of an irrelevant antibody had no effect on the DC phenotype, confirming that the 1C10 MAb was able to activate DC selectively via CD40.

View larger version (34K): [in this window] [in a new window]   FIG. 1.   Stimulation of DC with an anti-CD40 MAb results in cell maturation. The expression of CD80, CD86, and MHC class II on DC in cultures supplemented with increasing doses of anti-CD40 MAb (CD40) or an irrelevant MAb (ctrl) were compared. The flow cytometry histograms are representative of two independent experiments.

Effect of CD40 stimulation on cytokine expression by BCG-infected DC. BCG infection of DC induces changes in the mRNA expression for a number of cytokines, including the p40-inducible chain of IL-12 (13). We examined whether CD40 stimulation modified the cytokine mRNA expression of BCG-infected DC in an RNase protection assay (Fig. 2). CD40 ligation and BCG infection induced similar patterns of cytokine expression in DC, with significant enhancement of mRNA for the proinflammatory cytokines IL-12 p40, IL-1 ( and  subunits), IL-6, and tumor necrosis factor (TNF). mRNA for the anti-inflammatory cytokine transforming growth factor  was down-regulated, and the IL-1 receptor antagonist (IL-1Ra) mRNA level remained unchanged. Expression of mRNA for IL-10 in CD40-stimulated cells was not detected. When BCG-infected DC were stimulated via CD40, this cytokine expression profile was maintained. Increased production of mRNA for IL-1, IL-6, transforming growth factor , and IL-10 was observed.

View larger version (62K): [in this window] [in a new window]   FIG. 2.   Expression of mRNAs for inflammatory cytokines by DC is induced by BCG infection and maintained under CD40 ligation. mRNA profiles are shown for uninfected and BCG-infected DC stimulated with 10 µg of anti-CD40 (CD40) or an irrelevant MAb (ctrl)/ml. The profiles are representative of two independent experiments.

CD40 ligation promotes the release of biologically active IL-12 by BCG-infected DC. To determine if CD40-derived stimulation of IL-12 p40 mRNA results in the effective release of functional IL-12 heterodimer, we compared the IL-12 production of CD40-stimulated DC to that of DC incubated with an irrelevant antibody in a biological assay (Fig. 3). Both CD40 ligation and BCG infection increased the production of bioactive IL-12 by DC, although CD40 stimulation was a less potent IL-12 inducer in our model. Importantly, when BCG-infected DC were stimulated with the anti-CD40 MAb, IL-12 production was significantly augmented.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   CD40 ligation promotes the release of biologically active IL-12 by BCG-infected DC. IL-12 production, as measured by a bioassay, is shown for uninfected and BCG-infected DC without antibody stimulation or in the presence of 10 µg of irrelevant (ctrl) or anti-CD40 (CD40) MAb/ml. Mean IL-12 levels (+SEM) for triplicate cultures are shown. Differences between groups were analyzed using analysis of variance (**, P < 0.01). unv, unvaccinated.

CD40-stimulation augments the ability of BCG-infected DC to generate IFN--producing T cells in vitro. Since CD40 stimulation of BCG-infected DC appeared to augment their IL-12 production capacity, we examined whether stimulated cells had an increased ability to prime IFN--secreting T cells against mycobacterial antigens. Naïve splenocytes were prepared and incubated with serial dilutions of BCG-infected DC prestimulated with anti-CD40 or an irrelevant antibody. After 48 h, total cell proliferation was significantly higher in the CD40-stimulated DC cultures (Fig. 4A). Since splenocytes were not proliferating in the absence of DC (not shown) and the DC were irradiated before incubation with splenocytes, T cells certainly represented the major splenocyte subset able to proliferate in response to DC. Moreover, larger amounts of IFN- were generated in these cultures (Fig. 4B), suggesting that CD40-stimulated BCG-infected DC induced stronger Th1-oriented T-cell responses in vitro.

View larger version (17K): [in this window] [in a new window]   FIG. 4.   CD40 stimulation augments the ability of BCG-infected DC to generate IFN--producing cells in vitro. Proliferation (A) of and production (B) of IFN- by naïve splenocytes incubated with serial dilutions of uninfected DC (), BCG-infected DC incubated with an irrelevant antibody (), and CD40-stimulated BCG-infected DC () are shown.

Increased expression of Th1 cytokines in mice primed with CD40-stimulated BCG-infected DC. To examine whether CD40 stimulation had an impact on the immunogenicity of BCG-infected DC in vivo, the ability of stimulated cells to induce Th1-oriented immune responses against M. tuberculosis infection was tested by aerosol challenge. Mice were immunized by intratracheal injection of CD40-stimulated or unstimulated BCG-infected DC and subsequently challenged with M. tuberculosis infection, as previously described (13). mRNA for inflammatory cytokines in pooled MLN cells at the peak of infection, which is 4 weeks postchallenge, were analyzed (Fig. 5). Expression of mRNAs for the proinflammatory cytokines IL-12 p40 and IL-18 was significantly augmented in the mice vaccinated with CD40-stimulated BCG-infected DC, suggesting that a more potent Th1 response had been initiated. In accordance with this, the IFN- mRNA signal was approximately five times greater in this group. mRNA expression for other cytokines, such as IL-1 ( and  subunits), IL-1Ra, and IL-10, was also increased.

View larger version (48K): [in this window] [in a new window]   FIG. 5.   CD40 stimulation enhances the ability of BCG-infected DC to generate IFN- response in vivo. Cytokine mRNA profiles are shown for unvaccinated mice (unv), mice immunized subcutaneously with BCG (bcg), and mice vaccinated by the intratracheal route with uninfected or BCG-infected DC treated with an irrelevant (ctrl) or anti-CD40 (CD40) antibody. The samples correspond to pools of RNAs from MLN cells of five mice sacrificed 4 weeks after M. tuberculosis aerosol challenge. Templates for the housekeeping genes L32 and the GADPH gene were used to normalize the total RNA content in the samples. The data are representative of two independent experiments. MIF, macrophage migration inhibitory factor.

Generation of IFN--producing T cells. To determine if this increased production of IFN- mRNA correlated with the generation of larger populations of Th1-type T cells, the number of IFN--producing cells in the MLN was measured by Elispot (Fig. 6). The mean number of IFN--producing cells was higher in mice immunized with CD40-stimulated BCG-infected DC than in unimmunized animals or in mice vaccinated with uninfected DC. However, there was no statistical difference between mice vaccinated with CD40-stimulated BCG-infected DC and the other BCG-immunized groups.

View larger version (21K): [in this window] [in a new window]   FIG. 6.   Generation of specific IFN--producing T cells following priming with CD40-stimulated DC. The numbers of purified protein derivative-specific IFN--producing cells in MLN of mice immunized intratracheally with uninfected or BCG-infected DC or treated with an irrelevant (ctrl) or anti-CD40 (CD40) antibody are shown. The control groups corresponded to unvaccinated animals (unv) or to mice vaccinated with subcutaneous BCG (bcg). The data are the mean numbers of IFN--producing cells (+SEM) measured by Elispot for groups of five animals and are representative of two independent experiments. Differences between animal groups were analyzed by analysis of variance (*, P < 0.05; NS, nonsignificant).

Protection against aerosol M. tuberculosis infection. The ability of CD40-stimulated BCG-infected DC to induce a protective immune response against aerosol M. tuberculosis infection was then examined. Analysis of the number of mycobacteria recovered from the lungs of vaccinated animals showed that all groups immunized with BCG, either alone or internalized in DC, had protection levels which were significantly different from those of the unvaccinated group (Fig. 7). There were, however, no statistical differences between the animals immunized with BCG-infected DC or CD40-stimulated BCG-infected DC under the experimental conditions tested.

View larger version (68K): [in this window] [in a new window]   FIG. 7.   CD40 stimulation of BCG-infected DC does not improve their ability to confer protection against aerosol M. tuberculosis infections. The data points represent the mean lung CFU (+SEM) from five mice 4 weeks after aerosol challenge: unvaccinated mice (unv) and mice immunized subcutaneously with BCG (bcg) or by the intratracheal route with uninfected DC or BCG-infected DC treated with an irrelevant (ctrl) or anti-CD40 (CD40) antibody. The Data are representative of two independent experiments. Differences in CFU between experimental groups were analyzed by analysis of variance (*, P < 0.05; NS, nonsignificant).

	DISCUSSION

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In order to investigate the impact of CD40 stimulation on DC, we have used an anti-CD40 antibody with proven agonistic activity on B cells (22). Incubation of bone marrow-derived DC of immature phenotype with this MAb led to a dose-dependent maturation of the DC phenotype and the up-regulation of a number of cytokines, including IL-1, IL-6, IL-12p40, and TNF. This pattern of activation reproduces the functional effects of DC binding to the CD40L present on activated T cells (35), confirming the activity of this anti-CD40 MAb on DC. The IL-12 production levels following CD40 ligation in DC cultures were consistent with those obtained from murine splenic DC stimulated under similar conditions (26). Stimulation of human blood-derived DC using CD40L-transfected cells triggered higher levels of IL-12 production (7), possibly reflecting the differential efficacy of CD40 stimulation with dimeric reagents, such as anti-CD40 antibodies, compared to trimeric CD40L.

Cytokines play a major role in protective immunity against M. tuberculosis infection, since they contribute to the development of appropriate T-cell-mediated immunity and the generation of inflammatory responses (27). The IL-12 production induced by mycobacterial infection potentiates the development of T cells producing IFN- and TNF, both potent activators of the killing of bacteria by infected macrophages (4, 10, 17). The generation of IL-1, IL-6, and TNF in the lung initiates the development of the inflammation process, which leads to granuloma formation and containment of bacterial dissemination (4). Infection of DC with BCG (13, 33) and M. tuberculosis (23) up-regulates the expression of these inflammatory cytokines in vitro. Here we show that CD40 stimulation further enhances the production of IL-1, IL-1, and IL-6 by BCG-infected DC, suggesting that stimulation of infected DC via CD40 may potentiate the development of inflammatory responses in vivo. The presence of significantly higher levels of mRNA for IL-1 in the draining MLN of mice vaccinated with CD40-stimulated DC and challenged with aerosol M. tuberculosis suggests that a stronger inflammatory response is also induced in draining lymphoid organs.

Incubation of BCG-infected DC with agonistic anti-CD40 antibody significantly augmented their ability to release biologically active IL-12 and to activate IFN--producing T cells in vitro. This finding suggests that BCG-induced maturation of DC does not saturate their capacity for cytokine production and that BCG-infected DC are responsive to additional costimulatory signals from T cells. Following transfer in vivo, CD40-stimulated BCG-infected DC demonstrated an increased ability to generate Th1 immune responses to M. tuberculosis challenge compared to unstimulated cells, as shown by enhanced mRNA production for IL-12 p40 and IFN- in the MLN. Interestingly, mRNA for IL-18, a potent IFN--inducing cytokine which acts in synergy with IL-12 to maximize IFN- production (3), was also up-regulated. This enhanced immune response, however, was not sufficient to generate increased protection against M. tuberculosis challenge. Several factors can account for this lack of efficiency. First, it is possible that the amplitude of the additional IFN- response generated by CD40-stimulated DC is not sufficient. Indeed, our results show that although mRNA expression for IFN- was increased in this group, the number of IFN--producing cells was not significantly augmented. This suggested that immunization with CD40-stimulated DC may have increased the expression of IFN- by activated T cells rather than the frequency of M. tuberculosis-specific T cells. Improved generation of protective T cells may be observed in a strain of mice with stronger response to exogenous IL-12 than C57BL/6, such as BALB/c (18). Alternatively, the immunization protocol used to deliver DC may not be optimal. We are currently examining whether longer delays between vaccination with stimulated DC and challenge, or other routes of immunization, leads to better protective efficacy.

Another possibility raised by Reis e Sousa et al. (30) may be that restimulation of lymphoid DC by microbial challenge is paralyzed by concurrent expression of modulatory factors suppressing IL-12 production by DC. We observed that mRNA for IL-10, a potent suppressor of DC activation and IL-12 secretion (14, 26), was up-regulated in animals immunized with CD40-stimulated DC. In accordance with this, stimulation of BCG-infected DC via CD40 resulted in increased expression of mRNA for IL-10 in vitro. CD40-stimulated BCG-infected DC show an increased ability to activate Th1 cells in vitro despite IL-10 release in the culture supernatant. Following transfer of CD40-activated DC in vivo, however, the release of this anti-inflammatory cytokine in the close environment of DC in lymphoid organs may result in a more potent regulation of their activation.

Given the major role of IL-12 in the generation of protective responses against M. tuberculosis infection, developing strategies for optimal delivery of this cytokine is of central importance. Exogenous IL-12 increased the protective efficacy of BCG against M. tuberculosis challenge (19). Coinjection of a plasmid encoding both chains of IL-12 increased the protective efficacy of DNA subunit vaccines against M. tuberculosis infection (U. Palendira et al., submitted for publication). This supports the concept that IL-12 may have an adjuvant role when delivered with the antigen. Since DC possess the dual ability to present antigens and to secrete IL-12, DC manipulation may be another attractive approach to deliver immunologically active IL-12. In support of this, DC pulsed with leishmanial antigens and engineered to produce IL-12 were found to be potent vaccines in a murine model of leishmanial infection (1, 2). We have shown that CD40 stimulation of BCG-infected DC not only promotes their ability to secrete IL-12 but increases their release of other inflammatory mediators which play a critical role in anti-mycobacterial immunity. Therefore, CD40 stimulation of DC is a relevant method for increasing Th1-like T-cell responses to mycobacteria. Although this approach was not sufficient by itself to increase protection against M. tuberculosis infection, CD40 stimulation of DC combined with additional strategies may contribute to this aim.