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The Journal of Immunology, 2007, 179, 1825 -1833
Copyright © 2007 by The American Association of Immunologists, Inc.
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Inhibition of Phagosome Maturation by Mycobacteria Does Not Interfere with Presentation of Mycobacterial Antigens by MHC Molecules1

Laleh Majlessi2,3,*,{dagger}, Benoit Combaluzier3,{ddagger}, Imke Albrecht{ddagger}, Jessica E. Garcia*,{dagger}, Clémence Nouze*,{dagger}, Jean Pieters{ddagger} and Claude Leclerc*,{dagger}

* Institut Pasteur, Unité de Régulation Immunitaire et Vaccinologie, Paris, France; {dagger} Institut National de la Santé et de la Recherche Médicale, Unité 883, Paris, France; and {ddagger} Biozentrum, University of Basel, Klingelbergstrasse 50, Basel, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Pathogenic mycobacteria escape host innate immune responses by surviving within phagosomes of host macrophages and blocking their delivery to lysosomes. Avoiding lysosomal delivery may also be involved in the capacity of living mycobacteria to modulate MHC class I- or II-dependent T cell responses, which may contribute to their pathogenicity in vivo. In this study, we show that the presentation of mycobacterial Ags is independent of the site of intracellular residence inside professional APCs. Infection of mouse macrophages or dendritic cells in vitro with mycobacterial mutants that are unable to escape lysosomal transfer resulted in an identical efficiency of Ag presentation compared with wild-type mycobacteria. Moreover, in vivo, such mutants induced CD4+ Th1 or CD8+ CTL responses in mice against various mycobacterial Ags that were comparable to those induced by their wild-type counterparts. These results suggest that the limiting factor for the generation of an adaptive immune response against mycobacteria is not the degree of lysosomal delivery. These findings are important in the rational design of improved vaccines to combat mycobacterial diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mycobacterium tuberculosis is one of the most successful pathogens known today, killing millions of individuals worldwide each year. Importantly, while about one-third of the global population is infected with M. tuberculosis, only a relatively small percentage of infected individuals develop disease. The reason for this lies within the robust host immunity that is generated within healthy individuals, resulting in the establishment of a latent infection that is kept under control by both innate and adaptive immunity. Host control of mycobacterial infections relies on the activation of CD4+ and CD8+ T cells against epitopes generated in professional APC (1).

In APC, proteolysis of Ags leads to the generation of peptides able to bind to recycled or newly synthesized MHC class II (MHC-II)4 molecules. Newly synthesized MHC-II molecules associated with invariant chain (Ii) are transported to endocytic vesicles, the so-called MHC-II compartment or MIIC, where the loading of the antigenic peptides occurs (2, 3, 4, 5). However, phagosomes containing particulate Ags possess MHC-II molecules and other components involved in MHC-II presentation machinery. Thus, phagosomes, like MIIC, are equipped with the machinery to generate appropriate peptide-MHC-II complexes (6). In addition, cross-presentation of peptides, derived from particulate Ags, can occur via a putative phagosome-to-cytosol mechanism and thereby the conventional MHC-I pathway (7). Alternatively, this can occur by fusion and fission of phagosomes with endoplasmic reticulum-derived vesicles containing newly synthesized MHC-I molecules (8, 9, 10).

Presentation of mycobacterial Ags to the immune system involves the internalization, intracellular transport, and proteolytic processing of mycobacteria in macrophages and dendritic cells (1). Mycobacterial internalization occurs through phagocytosis mediated via different cell surface receptor molecules. However, the hallmark of pathogenic mycobacteria is that following phagocytosis, they resist lysosomal delivery, instead residing within mycobacterial phagosomes that do not fuse with lysosomes. Retention within the nonlysosomal mycobacterial phagosome allows the mycobacteria to replicate (11, 12). Mycobacterial phagosomes acquire markers of early endosomes but display limited acquisition of the late endosomal/lysosomal markers rab 7 and lysosome-associated membrane proteins (LAMP) (4) (12, 13, 14, 15) and exclude vacuolar ATP-dependent proton pumps (vH+-ATPases), explaining their limited acidification (16). Moreover, these phagosomes retain coronin 1 (also known as P57 or TACO, for tryptophan-aspartate-containing coat protein) (17). In parallel to these events, processing and presentation of mycobacterial Ags by MHC-II is hampered in the case of infection with live mycobacteria compared to their heat-killed counterparts (18). Because the latter are unable to inhibit phagosome maturation (19), it is important to determine whether phagosome maturation is required for efficient Ag presentation.

The eukaryotic-like serine/threonine protein kinase G (PknG) is an important mycobacterial virulence factor, because it is responsible for blocking the fusion of the mycobacterial phagosome with lysosomes, allowing the bacteria to survive intracellularly (20). In this study, using Mycobacterium bovis bacillus Calmette-Guérin (BCG) strain lacking the pkng gene (M. bovis BCG {Delta}pkng) (20, 21), we investigated the contribution of PknG to the inhibition of phagosome-lysosome fusion in murine dendritic cells. In addition, we addressed the possible influence of PknG on the efficiency of in vitro processing and presentation of mycobacterial Ags by these APC and on the in vivo induction of CD4+ and CD8+ T cell responses specific to diverse mycobacterial Ags.


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

BALB/c (H-2d) or C57BL/6 (H-2b) mice were purchased from Charles River Breeding Laboratories. Animal studies were approved by the Institut Pasteur safety committee in accordance with French and European guidelines.

Wild-type M. bovis BCG and M. bovis BCG {Delta}pkng (Montreal strain) expressing GFP (20) were propagated in 7H9 mycobacterial or Dubos medium (Difco and BD Biosciences) supplemented with 10% OADC Middlebrook supplement. Hygromycin (50 µg/ml) was included to select {Delta}pkng-resistant bacteria. Mycobacteria were harvested at mid-log phase (OD600 nm {approx} 0.5), resuspended in PBS, aliquoted, and then frozen at –80°C. A defrosted aliquot was used to quantify the CFU before Ag presentation assays or immunizations.

Preparation of APC

Bone marrow (BM)-derived macrophages or BM-dendritic cells were generated, in antibiotic-free conditions from BALB/c or C57BL/6 mouse femur marrow cell cultures in complete RPMI 1640 supplemented with 2 ng/ml M-CSF or GM-CSF, respectively (20, 22, 23). The source of these differentiation factors was supernatant from L929 or Ag8653 cells, respectively, transfected with cDNA coding for murine M-CSF or GM-CSF. The adherent cells from BM-macrophage cultures or total cells from BM-dendritic cell cultures were recovered at day 5 or 7, respectively, for use in cell fractionation, confocal microscopy, and Ag presentation assays. At day 7, BM-dendritic cell cultures were composed of 93.6% CD11c+CD11b+ cells, 8.3% CD11c+Gr1+, and only 1.5% CD11c–CD11b+.

Cell fractionation

BM-macrophages or BM-dendritic cells were infected for 3 h with mycobacteria, homogenized, and then subjected to organelle electrophoresis (3, 17). In brief, cells were washed twice, pelleted at 450 x g for 7 min, resuspended in 1 ml of buffer, and homogenized using a cell cracker. The postnuclear supernatant (15 min, 240 x g) was then trypsinized (25 µg/mg protein; Calbiochem) for 5 min at 37°C, and the reaction was stopped by adding soybean trypsin inhibitor (625 µg/mg protein; Calbiochem). Membranes were sedimented (60 min at 100,000 x g), resuspended in 0.5 ml of 6% Ficoll-70 (Pharmacia) in homogenization buffer, and loaded in the middle of a Ficoll gradient (10–0%). Electrophoresis was conducted for 80 min at 10.4 mA. Fractions were collected and assayed for protein concentration (Bradford method) and beta-hexosaminidase activity. To analyze the presence of bacteria, fractions were pelleted on glass slides, fixed with paraformaldehyde, and acid fast stained (BD Biosciences). Slides were analyzed using fluorescent microscopy to determine the number of bacteria in the fractions.

Confocal microscopy

BM-dendritic cells, at day 7 of culture, were incubated with GFP-expressing wild-type M. bovis BCG or M. bovis BCG {Delta}pkng (OD600 nm = 0.1) in the cell culture dish for 3 h at 37°C. For subsequent staining, BM-dendritic cells were harvested, resuspended in PBS, and transferred onto poly-L-lysine-coated glass slides. Following an incubation for 20 min on ice to allow the cells to adhere to the slides, cells were fixed in methanol (4 min at –20°C) and stained for LAMP-1 (ID4B; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) using a mouse anti-rat IgG (H + L)-Alexa 568 (Molecular Probes) as secondary Ab. Infection of BM-macrophages with GFP-expressing wild-type M. bovis BCG or M. bovis BCG {Delta}pkng was performed directly onto the glass slide. After a 3 h incubation, cells were washed, fixed with methanol, and stained for LAMP-1. Slides were analyzed by confocal microscopy confocal laser scanning module LSM510 Meta (Zeiss), connected to an Axiovert 200M (Zeiss).

Peptides

TB10.3/4:20–28 (24), TB10.3:74-88 (our unpublished data), TB10.4:74–88 (25), Ag85A:101–120, Ag85A:241–260 (26), Ag85A:144–152 (27) and MalE:100–140 (28) peptides were synthesized by NeoMPS.

In vitro Ag presentation assays

BM-macrophages or BM-dendritic cells from C57BL/6 (H-2b) or BALB/c (H-2d) mice were plated (1 x 105 cells/well) in 96-well flat-bottom plates and infected with various concentrations of wild-type M. bovis BCG or M. bovis BCG {Delta}pkng for 2, 4, or 24 h. Cells were then washed three times with RPMI 1640 and fixed with glutaraldehyde (0.025% final concentration) for 3 min at 37°C. The fixation was stopped by addition of lysine (0.1 M final concentration) at 4°C. The infected and fixed APC were then incubated overnight with 1 x 105 appropriate T cell hybridoma. DE10 T cell hybridoma is specific to Ag85A:241–260 and is restricted by I-Ab, whereas the CG11 T cell hybridoma is specific to Ag85A:101–120 and is restricted by I-Ed (22). The presence of IL-2 in the supernatants of cocultures was monitored by a standard IL-2-specific ELISA as described elsewhere (22).

T cell assays

Eight—10-wk-old female BALB/c mice received s.c. 1 x 107 CFU/mouse of wild type M. bovis BCG or M. bovis BCG {Delta}pkng. Three weeks later, to measure T cell proliferative responses to mycobacterial Ags, splenocytes were cultured (1 x 106 cells/well) in 96-well flat-bottom plates in synthetic HL-1 medium (BioWhittaker), in the presence of various concentrations of bovine tuberculin purified protein derivative (PPD) (Veterinary Laboratories Agency) or of appropriate synthetic peptides, as previously described (22). Cultures were pulsed 72 h later with 1 µCi of [methyl-3H]thymidine/well for 16 h and incorporated cpm were counted in a Wallac Microbeta counter.

IFN-{gamma} production was measured in the supernatants of splenocyte cultures after 72 h of stimulation with 5 µg/ml PPD, 10 µg/ml TB10.3:74–88, Ag85A:101–120 or MalE:100–140 peptides, or 2 µg/ml TB10.4:74–88 peptide by ELISA using R4-6A2 mAb for coating and biotin-conjugated XMG1.2 mAb for detection (BD Pharmingen).

CTL responses were measured at 3 wk postimmunization after a 5-day in vitro stimulation with 10 µg/ml TB10.3/4:20–28 (24) or Ag85A:144–152 (27) peptides in RPMI 1640 complemented with 10% FCS. The CTL activity was determined in a 4-h in vitro 51Cr release assay using P815 (H-2d) mastocytoma loaded with 50 µM of the homologous peptide, at diverse E:T ratios, as detailed elsewhere (24).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Influence of protein kinase G on phagosome maturation and intracellular trafficking of mycobacteria in professional APC

Pathogenic mycobacteria survive within macrophages by expressing the eukaryotic-like serine/threonine PknG. PknG is secreted within the cytosol of macrophages and blocks the fusion of mycobacterial phagosomes with lysosomes, preventing intracellular degradation of the mycobacteria (20). To analyze whether PknG also blocks phagosome-lysosome fusion in dendritic cells, BM-macrophages and BM-dendritic cells were incubated with either wild-type M. bovis BCG or M. bovis BCG {Delta}pkng-expressing GFP for 3 h. Following fixation and permeabilization, the cells were stained for the lysosomal marker protein LAMP-1 and analyzed by confocal laser scanning microscopy. As shown in Fig. 1, wild-type M. bovis BCG remained largely in nonlysosomal phagosomes both in BM-macrophages and in BM-dendritic cells. In contrast, in the absence of PknG, the majority of the bacilli were delivered to lysosomes, as shown by the colocalization with LAMP-1 (Fig. 1).


Figure 1
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  		FIGURE 1. Influence of mycobacterial PknG on the phagosome maturation and intracellular localization of mycobacteria in BM-macrophages or BM-dendritic cells. A, BM-dendritic cells (lower panels) were incubated with wild-type M. bovis BCG (left) or M. bovis BCG {Delta}pkng (right) expressing the GFP (OD600 = 0.1) for 3 h at 37°C. Subsequently, adherent and nonadherent dendritic cells were removed from the cell culture dish, pelleted by centrifugation, resuspended in PBS, and transferred to poly-L-lysine-coated glass slides. After 20 min of incubation on ice to allow the cells to adhere, the cells were fixed with methanol and stained for LAMP-1 followed by Alexa Fluor-568-labeled secondary Ab. Incubation of BM-macrophages (upper panels) with wild-type M. bovis BCG or M. bovis BCG {Delta}pkng-expressing GFP (OD600 = 0.1) was performed directly onto glass slides. After 3 h of infection, cells were washed and stained for LAMP-1. Bar, 2 µm. B, Quantification of lysosomal delivery of internalized wild-type M. bovis BCG or M. bovis BCG {Delta}pkng in BM-dendritic cells and BM-macrophages. Shown are the values ± SD (n = 50) of one representative experiment.

 
To independently analyze the intracellular transport of wild-type M. bovis BCG or M. bovis BCG {Delta}pkng in macrophages vs dendritic cells, subcellular fractionation by organelle electrophoresis was used (3, 17, 29). To that end, BM-macrophages and BM-dendritic cells were infected with wild-type M. bovis BCG or M. bovis BCG {Delta}pkng for 3 h, followed by homogenization and organelle separation by electrophoresis (Fig. 2). During organelle electrophoresis, acidic organelles such as lysosomes migrate toward the anode, while nonacidified organelles, including mycobacterial phagosomes remain deflected toward the cathode. After electrophoresis, the different fractions can be analyzed for organelle-specific markers as well as for the presence of mycobacteria. In both BM-macrophages as well as BM-dendritic cells, as expected, lysosomes were shifted toward the anode following electrophoresis as analyzed using the lysosomal marker beta-hexosaminidase (30, 31). Importantly, while in both BM-macrophages as well as BM-dendritic cells, wild-type M. bovis BCG was recovered in nonshifted fractions, mycobacteria lacking PknG were largely transferred to fractions positive for the lysosomal marker beta-hexosaminidase (17, 18, 19, 20, 21). Together, these data suggest that in both macrophages and dendritic cells, PknG is essential to avoid lysosomal delivery.


Figure 2
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  		FIGURE 2. Influence of mycobacterial PknG on the intracellular trafficking of mycobacteria in BM-macrophages or BM-dendritic cells. BM-macrophages or BM-dendritic cells were incubated for 3 h with wild-type M. bovis BCG (A) or M. bovis BCG {Delta}pkng (B), homogenized, and subjected to organelle electrophoresis. The distribution of organelle-specific markers and the amount of bacteria per fraction were determined.

 
Presentation of mycobacterial Ag upon infection of APC with wild-type M. bovis BCG or M. bovis BCG {Delta}pkng

The finding that mycobacteria lacking PknG are readily delivered to lysosomes prompted us to compare the Ag processing and presentation of wild-type and PknG-deficient mycobacteria after uptake by macrophages or dendritic cells. To do so, wild-type M. bovis BCG or M. bovis BCG {Delta}pkng were used to infect APC derived from C57BL/6 (H-2b) mice. To analyze Ag processing and presentation, the well-defined Ag85A was chosen as a reporter mycobacterial Ag (32) and the DE10 T cell hybridoma, specific to Ag85A:241–260 peptide and restricted by I-Ab (22), was used to detect the presentation by the MHC-II molecules of the corresponding immunodominant epitope. At different time points after infection with various concentrations of wild-type M. bovis BCG or M. bovis BCG {Delta}pkng, APC from C57BL/6 (H-2b) were fixed to arrest Ag processing and presentation and were cocultured with the DE10 T cell hybridoma to assess the amount of surface MHC-II-peptide complexes. In this model, the efficiency of the presentation of Ag85A by MHC-II molecules of BM-macrophages infected either with wild-type M. bovis BCG or M. bovis BCG {Delta}pkng strain was comparable after 2 h of infection and increased similarly after 4 or 24 h of infection (Fig. 3, A–C). The presentation of Ag85A by MHC-II molecules of BM-dendritic cells was also comparable upon infection by wild-type M. bovis BCG or M. bovis BCG {Delta}pkng strain after 2 h and increased in a similar manner after 4 or 24 h of infection (Fig. 3, D–F). Important to note and as expected, the efficiency of Ag presentation by BM-dendritic cells was much higher as compared with BM-macrophages (see different scales of IL-2 production used for BM-macrophages (Fig. 3, A–C) and for BM-dendritic cells (Fig. 3, D–F)). However, BM-macrophages and BM-dendritic cells showed the same efficiency in the presentation of the synthetic Ag85A:241–260 peptide (Fig. 3G), indicating that the difference in Ag presentation between BM-dendritic cells and BM-macrophages was not due to any other culture parameters than the intrinsic capacity of these two cell types in Ag processing and presentation. Importantly, comparable data were obtained by use of the CG11 T cell hybridoma specific to another Ag85A epitope, i.e., Ag85A:101–120 and restricted by I-Ed, following the presentation of Ag85A by BM-macrophages or BM-dendritic cells generated from BALB/c (H-2d) mice (data not shown). These data demonstrate that the in vitro presentation of Ag85A through the MHC-II-dependent processing and presentation pathway of either macrophages or dendritic cells is not influenced by the marked differential intracellular localization of mycobacteria due to the PknG function.


Figure 3
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  		FIGURE 3. In vitro presentation of mycobacterial Ag85A by C57BL/6 (H-2b) BM-macrophages or BM-dendritic cells infected with wild-type M. bovis BCG or M. bovis BCG {Delta}pkng. BM-macrophages (A–C) or BM-dendritic cells (D–F) from C57BL/6 (H-2b) mice were infected with various concentrations of wild-type M. bovis BCG or M. bovis BCG {Delta}pkng for 2, 4, or 24 h. G, BM-macrophages or BM-dendritic cells were incubated with various concentrations of Ag85A:241–260 or a negative control peptide for 24 h. Cells were then washed, fixed, and cocultured overnight with the I-Ab-restricted, Ag85A:241–260-specific, DE10 T cell hybridoma. The IL-2 production detected by ELISA was used as the readout of hybridoma activation. Results are mean ± SD and are representative of two independent experiments.

 
Induction of CD4+ Th1 or CD8+ CTL responses upon immunization of mice with wild-type M. bovis BCG or M. bovis BCG {Delta}pkng

We further sought to determine in vivo the possible consequence of different intracellular trafficking of mycobacteria on the presentation of a panel of various mycobacterial Ags in both MHC-II and MHC-I pathways. To that end, we investigated the efficiency of induction of CD4+ or CD8+ T cell responses against several mycobacterial Ags upon immunization of mice with wild-type M. bovis BCG or M. bovis BCG {Delta}pkng. BALB/c (H-2d) mice were chosen for these experiments due to the availability of not only MHC-II-, but also well-defined MHC-I-restricted mycobacterial T cell epitopes in the H-2d haplotype (24, 27).

CD4+ T cell responses were studied against PPD and a panel of well-defined BCG Ags, i.e., Ag85A and immunogens from the early secreted antigenic target-6-kDa (ESAT-6) family (33), namely, TB10.3 and TB10.4. CD4+ T cell responses were evaluated at 3 wk postimmunization with wild-type M. bovis BCG or M. bovis BCG {Delta}pkng. Splenocytes were in vitro stimulated with PPD, Ag85A:101–120 (26), TB10.3:77–84 (our unpublished data), TB10.4:77–84 (25), or the unrelated MalE:100–114 peptide (28). All of these peptides have been previously shown to contain only MHC-II-restricted T cell epitopes. Comparable intensities and sensitivities of proliferative (Fig. 4) as well as IFN-{gamma} (Fig. 5) responses were observed in individual BALB/c mice (n = 3) immunized with wild-type M. bovis BCG or M. bovis BCG {Delta}pkng.


Figure 4
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  		FIGURE 4. Proliferative CD4+ T cell response to a panel of mycobacterial Ags induced upon immunization of BALB/c mice with wild-type M. bovis BCG or M. bovis BCG {Delta}pkng. BALB/c (H-2d) mice (n = 3) were injected s.c. with 1 x 107 CFU/mouse of wild-type M. bovis BCG (left panels) or M. bovis BCG {Delta}pkng (right panels). Three weeks after immunization, splenocytes of individual mice were stimulated in vitro with various concentrations of PPD or of peptides containing immunodominant epitopes of TB10.3, TB10.4, or Ag85A Ags or with MalE:100–114 as negative control peptide. Proliferation was measured as described in Materials and Methods. Results are mean ± SD from each individual mouse per group (n = 3) and are representative of three independent experiments.

 

Figure 5
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  		FIGURE 5. IFN-{gamma} CD4+ T cell response to a panel of mycobacterial Ags induced upon immunization of BALB/c mice with wild-type M. bovis BCG or M. bovis BCG {Delta}pkng. BALB/c (H-2d) mice were injected s.c. with 1 x 107 CFU/mouse of wild-type M. bovis BCG (left panels) or M. bovis BCG {Delta}pkng (right panels). At 3 wk after injection, splenocytes of individual mice were stimulated in vitro with different Ags or peptides (see legend to Fig. 4). IFN-{gamma} production was measured as described in Materials and Methods. Results are mean ± SD from each individual mouse per group (n = 3) and are representative of three independent experiments.

 
We then compared the CD8+ CTL responses induced in BALB/c mice immunized with wild-type M. bovis BCG or M. bovis BCG {Delta}pkng. Three weeks after immunization, splenocytes of immunized mice were investigated for their CTL activity against Kd-restricted Ag85A:144–152 (27) or TB10.3/4:20–28 peptides, the latter being shared by TB10.3 and TB10.4 immunogens (24). Comparable lytic activities against Ag85A:144–152 or TB10.3/4:20–28 epitopes were detected with splenocytes of mice immunized with either wild-type M. bovis BCG or M. bovis BCG {Delta}pkng (Fig. 6).


Figure 6
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  		FIGURE 6. CD8+ CTL response to mycobacterial Ags induced upon immunization of BALB/c mice with wild-type M. bovis BCG or M. bovis BCG {Delta}pkng. BALB/c (H-2d) mice (n = 3) were injected s.c. with 1 x 107 CFU/mouse of wild-type M. bovis BCG or M. bovis BCG {Delta}pkng. At 3 wk after injection, pooled splenocytes of mice were stimulated in vitro with 10 µg/ml Kd-restricted TB10.3/4:20–28 or Ag85A:144–152 epitope. The CTL response was measured in a conventional 51Cr release assay, with P815 mastocytoma unloaded or loaded with the homologous peptides as targets. Results correspond to the percentage of specific lysis and are representative of two independent experiments.

 
Taken together, our results show that the marked differential trafficking of mycobacteria within professional APC has no consequence for the efficiency of induction of MHC-II-restricted CD4+ Th1 or MHC-I-restricted CD8+ CTL responses in mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Inhibition of phagosome-lysosome fusion has been long proposed to represent a mechanism by which pathogenic mycobacteria bias the efficiency of Ag presentation by host MHC molecules to evade immune surveillance (34, 35, 36). In addition to the fact that a low phagosomal pH can be directly toxic to microorganisms, the terminal degradation of proteins into peptides, i.e., unfolding of Ags by thiol reductases and acidic endopeptidases such as the cathepsins, occurs at low pH. Moreover, the removal of the Ii chaperone from MHC-II{alpha}beta heterodimers and interaction of the latter with H-2M for replacement of class II-associated Ii-derived peptide by antigenic peptides into the groove of mature MHC-II molecules, all take place in acidified endosomes (5, 37, 38, 39). In the present study, however, we demonstrate that the presentation of mycobacterial Ags is identical whether or not mycobacteria are transferred to lysosomes or remain sequestered in nonlysosomal phagosomes.

In vivo processing and presentation of mycobacterial Ags occur by both macrophages as well as dendritic cells (1). However, the relative contribution of each of these types of professional APC in the generation of CD4+ and CD8+ T cell responses in vivo are not clear. We thus investigated both of these APC types in this study. We first established that mycobacterial PknG was able to block lysosomal fusion in dendritic cells as occurs in macrophages (20). Subsequently, we compared the efficiency of mycobacterial Ag presentation by host macrophages or dendritic cells infected with wild-type or PknG-deficient BCG. As evaluated in vitro by use of anti-Ag85A T cell hybridomas (22), BM-macrophages or BM-dendritic cells infected with wild-type M. bovis BCG or M. bovis BCG {Delta}pkng presented this mycobacterial reporter Ag with similar efficiency, independent of the multiplicity of infection and following short (2 or 4 h) or long (24 h) time periods subsequent to infection. Moreover, following infection, the presentation of Ag85A was similar in the context of two distinct restricting elements (I-Ab and I-Ed) and in two different mouse genetic backgrounds (C57BL/6 (H-2b) and BALB/c (H-2d)) whether wild-type or PknG-deficient mycobacteria were used. Also, in vivo immunization with either wild-type or PknG-deficient BCG strains induced similar proliferative and IFN-{gamma} CD4+ T cell responses to the PPD Ag mixture as well as to diverse well-defined mycobacterial immunogens.

Exogenous protein Ags may display different intracellular trafficking as well as different sensitivity to proteolysis and can give rise to epitopes with various affinity for the restricting elements (5). To understand the capacity of distinct mycobacterial Ags to trigger CD4+ T cell responses upon immunization with wild-type or PknG-deficient mycobacteria, we selected a subset of mycobacterial Ags, i.e., the 32-kDa Ag85A expressed in complex with Ag85B and C (32), and 10-kDa small proteins from the ESAT-6 family of proteins (TB10.3 and TB10.4), which most probably are secreted by an ESAT-6–1-like secretion machinery (40, 41, 42). Regardless of the nature of the Ag, these were presented in a similar fashion whether the mycobacteria were retained in phagosomes (wild-type M. bovis BCG) or were transported to lysosomes (M. bovis BCG {Delta}pknG).

It should also be noted that the precise organelles of the endocytic pathway where the processing from phagocytosed Ags and peptide loading to MHC-II molecules occurs is still unclear (43). However, the results reported here suggest that the lack of delivery of mycobacteria to lysosomes does not limit the ability of the APC to stimulate T cell responses. Indeed, wild-type mycobacteria that resist lysosomal delivery can give rise to identical MHC-I- and -II-restricted T cell responses as compared with PknG-deficient bacilli that are immediately shuttled to lysosomes.

Although we cannot exclude that also in vivo, a small proportion of mycobacteria are routed to lysosomes, our data show that the capacity of mycobacteria to resist lysosomal delivery does not prevent the generation of an immune response. In fact, when all of the infecting mycobacteria are directly transferred to lysosomes, as in the case of PknG-deficient mycobacteria, an immune response identical to the one generated by wild-type mycobacteria was found. This is surprising in light of previous work showing that the route of Ag uptake and the final subcellular localization defines the outcome of an immune response (30, 44, 45, 46). For example, for both macrophages and dendritic cells, it has been established that lysosomal transfer is important for the processing and presentation of Ags (30, 46). The results presented here show that in the case of an infection with mycobacteria, the degree of lysosomal transfer of mycobacteria does not influence the extent to which responses are generated against mycobacterial Ags. These results are not only important for the understanding of the immune escape mechanisms of pathogenic mycobacteria but are also relevant for strategies aimed at improving existing vaccines.

The effective formation of MHC-II-mycobacterial peptide complexes has been detected in the immature mycobacterial phagosomes (18). The phagosomes containing live mycobacteria are {approx}1 pH unit more acidic than the host cell cytosol, even they are {approx}1 pH unit less acidic than the phagosomes containing dead mycobacteria or particulate Ags (47). In contrast, proteases of the endocytic vesicles present all along the endosomal/lysosomal pathway can possess broad optimal pH for their enzymatic activities. For instance, one of the predominant endocytic proteases, i.e., cathepsin S, involved in both Ag proteolysis (48) and maturation of MHC-II molecules (49), maintains its enzymatic function even at neutral pH (50). Moreover, MHC-II-Ii complexes and cathepsin S have been detected not only in the acidic lysosomes, but also in early endosomes of relatively high pH (51). Therefore, existence of a lysosome-independent mechanism of mycobacterial Ag presentation involving such mechanisms has to also be taken into account.

It has been recently shown that the generation of peptide-MHC-II complexes in a phagosome-autonomous manner is controlled by TLRs, possibly due to a TLR-mediated regulation of the proteolysis of Ii to class II-associated Ii-derived peptide (52). Therefore, it is possible that the numerous mycobacterial TLR ligands acting in mycobacterial phagosomes can provide optimal conditions for efficient formation of peptide-MHC-II complexes in phagosomes for further epitope presentation at the cell surface.

Our results also show that immunization of mice with wild-type or PknG-deficient BCG induces comparable CD8+ T cell responses to Ag85A and TB10.3/TB10.4 mycobacterial immunogens. Therefore, the distinct trafficking of mycobacteria within the host APC and the marked phagosome maturation due to the loss-of-function of PknG do not affect the presentation of mycobacterial Ags within the MHC-I presentation pathway. Little is known about the mechanisms of presentation of mycobacterial Ags by MHC-I molecules. It has been shown that phagosomes, via interaction with endoplasmic reticulum-derived vesicles, can acquire the MHC-I presentation machinery (8, 53). Maturation of phagosomes into phagolysosomes, as occurs upon infection with PknG-deficient mycobacteria, would therefore impair epitope editing and loading to MHC-I molecules, the latter being unstable and binding peptides inefficiently in acidic conditions (54). However, the comparable CD8+ T cell induction following immunization with wild-type or PknG-deficient mycobacteria suggests that MHC-I loading does not occur in phagolysosomes.

Cross-presentation of mycobacterial Ags may also occur via the cytosolic pathway by proteasome- and TAP-dependent mechanisms (55), most probably following phagosome-to-cytosol translocation of mycobacterial Ags. According to this scheme, degradation of Ags depends on the proteasome and not on the endosome/lysosome. Thus, maturation of phagosomes, for instance upon infection with PknG-deficient mycobacteria, would have no consequence on mycobacterial Ag presentation by MHC-I molecules. Recently, the presentation of mycobacterial Ags by MHC-I and -II molecules has been shown to involve uptake by bystander dendritic cells of apoptotic vesicles generated from primary infected macrophages (56, 57). Mycobacterial Ags then enter the MHC-I or -II pathway of dendritic cells. Therefore, in this model, the cells that present mycobacterial Ags in vivo are not those initially infected with mycobacteria. Maturation of phagosomes, as induced by PknG-deficient BCG in primarily infected macrophages, would not have an appreciable consequence on the Ag presentation by dendritic cells which capture subsequently the apoptotic vesicles that are generated.

In conclusion, the present investigation demonstrates that the inhibition of phagosome-lysosome fusion by mycobacteria and the sequestration of the bacilli in APC (34) does not affect mycobacterial Ag presentation by MHC-I or -II molecules and that these phenomena cannot explain how this successful intracellular pathogen can escape from immune surveillance.


   Acknowledgment
 
We gratefully acknowledge Priscille Brodin for precious help and discussion.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Institut Pasteur (Programme Transversal de Recherche 110, to L.M.), the European Community (Cellprom, to C.L.), the Swiss National Science Foundation the Swiss Life Jubileum Fund, and the World Health Organization (to J.P.). Back

2 Address correspondence and reprint requests to Dr. Laleh Majlessi, Régulation Immunitaire et Vaccinologie, Institut National de la Santé et de la Recherche Médicale, Unité 883, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, Cedex 15, France. E-mail address: lmajless{at}pasteur.fr Back

3 L.M. and B.C. contributed equally to this work. Back

4 Abbreviations used in this paper: MHC-II,I. MHC class II/I; BCG, bacillus Calmette-Guérin; LAMP, lysosome-associated membrane protein; PknG, protein kinase G; Ii, invariant chain; BM, bone marrow; PPD, purified protein derivative; ESAT-6, early secreted antigenic target-6-kDa. Back

Received for publication September 9, 2006. Accepted for publication May 25, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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