Processing and Major Histocompatibility Complex Class II Presentation of Legionella pneumophila Antigens by Infected Macrophages
http://www.100md.com
感染与免疫杂志 2005年第4期
Section of Microbial Pathogenesis, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut
ABSTRACT
To better understand interactions between the intracellular pathogen Legionella pneumophila and macrophages (Ms), host and bacterial determinants important for presentation of antigens on major histocompatibility complex class II molecules (MHC-II) were investigated. It was determined that immune CD4 T-cell responses to murine bone marrow-derived Ms (BMs) infected with wild-type L. pneumophila were higher than the responses to avirulent dotA mutant bacteria. Although this enhanced response by immune T cells required modulation of vacuole transport mediated by the Dot/Icm system, it did not require intracellular replication of L. pneumophila. Intracellular cytokine staining identified a population of immune CD4 T cells that produced gamma interferon upon incubation with BMs infected with wild-type L. pneumophila that did not respond to M infection with dotA mutant bacteria. Endocytic processing was required for presentation of L. pneumophila antigens on MHC-II as determined by a defect in CD4 T-cell responses when the pH of BM endosomes was neutralized with chloroquine. Investigation of MHC-II presentation of antigens by BMs infected with L. pneumophila icmR, icmW, and icmS mutants indicated that these mutants have an intermediate presentation phenotype relative to those of wild-type and dotA mutant bacteria. In addition, it was found that antigens from dot and icm mutants are presented earlier than antigens from wild-type L. pneumophila. Although immune CD4 T-cell responses to proteins secreted by the L. pneumophila Lsp system were not detected, it was found that the Lsp system is important for priming L. pneumophila-specific T cells in vivo. These data indicate that optimal antigen processing and MHC-II presentation to immune CD4 T cells involves synthesis of L. pneumophila proteins in an endoplasmic reticulum-derived compartment followed by transport to lysosomes.
INTRODUCTION
Naive T cells expressing a unique T-cell receptor become antigen-specific effector cells upon encountering a professional antigen-presenting cell that has a cognate peptide major histocompatibility complex (MHC) displayed on its surface (2). Effector T cells play an important role in immunity to pathogens by producing factors that either upregulate cellular antimicrobial functions or that kill infected target cells that display a cognate peptide MHC (11, 20). T cells producing the CD4 protein typically respond to peptides presented on MHC class II molecules (MHC-II), whereas a peptide-loaded MHC-I will stimulate T cells producing CD8. Most antigens loaded onto MHC-I are derived from proteins that have gained access to the cell cytosol, such as viral antigens or antigens released by bacterial pathogens that escape membrane-bound compartments and replicate in the cytosol of their host (21, 28). In contrast, peptides loaded onto MHC-II are derived from antigens that are transported to lysosomes, such as those produced by organisms that remain in vacuoles that undergo endocytic maturation after internalization by an antigen-presenting cell (8, 11).
There are many examples of intracellular pathogens that replicate in vacuoles that resist fusion with lysosomes (34). Paradoxically, the adaptive immune response mediated by CD4 T cells usually plays an important role in controlling infections by these pathogens (1, 29). How immunoreactive antigens from these pathogens intersect the MHC-II presentation pathway for presentation to CD4 T cells is not known. Questions related to how these antigens are released, how they move through the cell, and how they are processed for presentation on MHC-II are of fundamental importance to understanding host immunity to vacuolar pathogens and in the eventual development of effective vaccines that will help prevent diseases resulting from infections by these organisms.
We have been using Legionella pneumophila as a model system to understand how adaptive immune responses are generated against pathogens that replicate in specialized vacuoles. L. pneumophila infections can result in a severe pneumonia in humans known as Legionnaires' disease (12). The ability to multiply inside macrophages (Ms) is an important L. pneumophila virulence trait (17, 41). To replicate intracellularly, L. pneumophila alters transport of the vacuoles in which the bacteria reside to evade delivery to lysosomes and transform their compartment into an organelle that resembles the endoplasmic reticulum (ER) (6, 9, 18, 19). It is within this ER-derived organelle that bacterial multiplication occurs (15). The ability of L. pneumophila to redirect vacuole transport within bone marrow-derived Ms (BMs) is dependent on the delivery of bacterial proteins into the host cell by a type IVb secretion apparatus (5, 7, 22, 25), which is encoded by the dot and icm genes (33, 39). A subset of the proteins translocated by the L. pneumophila Dot/Icm system subvert host cell proteins involved in vesicle transport in the secretory pathway (9, 18, 25), leading to the creation of a replicative organelle. Strains of L. pneumophila that lack the Dot/Icm secretion system due to mutations in the dot or icm gene fail to remodel their vacuole, resulting in transport to lysosomes (3, 32, 40). At late stages of infection, L. pneumophila has been observed residing in vacuoles that have lysosomal properties, suggesting that additional maturation events may occur after establishment of an ER-derived vacuole (35). There is also evidence that the Dot/Icm system is involved in the process of egress of L. pneumophila from spent host cells (5, 24).
Previous studies on the presentation of L. pneumophila antigens by dendritic cells (DCs) revealed that CD4 T cells from immunized mice respond better to DCs infected with wild-type L. pneumophila than to DCs infected with L. pneumophila dotA mutant (27). Thus, the ability of L. pneumophila to evade lysosomal delivery after uptake was essential for the optimal presentation of bacterial antigens on MHC-II in DCs. To explain these results, it was hypothesized that there is a T-cell subset primed during infection that has the unique abilities to recognize and respond to antigens synthesized after L. pneumophila is internalized by DCs (27). This hypothesis predicts that T cells produced during infection with wild-type L. pneumophila should also have the ability to recognize Ms infected with wild-type L. pneumophila and respond by producing cytokines, such as gamma interferon (IFN-), that can lead to M activation. In this study, we examined CD4 T cells produced during L. pneumophila infection and investigated their ability to respond to infected BMs. These studies reveal host and bacterial factors that are important for the presentation of L. pneumophila peptides on MHC-II after BM infection.
MATERIALS AND METHODS
Bacterial cultures. The L. pneumophila serogroup 1 strain, Lp01 (3), and the isogenic dotA (42), icmW (42), icmS and icmR (6) mutant strains, as well as mspA (26), mspA dotA (26), mspA lspE, and mspA dotA lspE (this study) mutant strains, were cultured on charcoal-yeast extract (CYE) agar (10) for 2 days prior to use in experiments. The thyA mutant strain used was Lp02, which is a thymidine auxotroph derived from Lp01 (3). Allelic exchange was used to delete the entire dotA gene from Lp02 to generate the thyA dotA mutant. These strains were grown on CYE agar with thymidine added to a final concentration of 10 μg ml–1. Where indicated, tissue culture medium was also supplemented with thymidine at 10 μg ml–1. Heat-killed bacteria were obtained by incubating a 1-ml suspension of 109 L. pneumophila at 80°C for 45 min.
Construction of L. pneumophila lspE mutants. An in-frame deletion of the lspE gene was introduced into the L. pneumophila chromosome by allelic exchange as described previously (23). To construct the lspE deletion, two DNA fragments were generated by PCR. Primer E1 (5'-GCGAGCTCAAATTACTCGCGGACAAGG-3') and primer E2 (5'-TATGGGTAATTTATTTGGTCATTTTTGATG-3') were used to generate a DNA fragment homologous to the 5' region of the lspE gene, and primer E3 (5'-AATGACCAAATAAATTACCCATACTTTAGC-3') and primer E4 (5'-GCTCTAGATGAATTGCCAGGAAGTAAAG-3') were used to generate a region of 3' homology. These two DNA fragments were combined by recombinant PCR using primers E1 and E4. The lspE deletion allele was ligated into the gene replacement vector pSR47S (23) and was introduced into L. pneumophila mspA and mspA dotA strains to make the mspA lspE and mspA dotA lspE mutants, respectively.
Cell cultures. BMs were derived from A/J mice (Harlan Sprague Dawley). Cultures of BMs were prepared as described previously (4).
Intracellular growth and L. pneumophila uptake assays. Growth of L. pneumophila in BMs was measured as described previously (42). Assays to determine uptake of bacteria at different multiplicities of infection (MOIs) were performed similarly. Briefly, L. pneumophila was added to BM cultures at the MOI indicated, and plates were centrifuged for 5 min at 300 x g and warmed in a 37°C water bath for 15 min. Wells were then washed vigorously with ice-cold phosphate-buffered saline (PBS) to remove extracellular bacteria. For intracellular growth assays, fresh medium was added to the wells, and plates were returned to the incubator. Lysates from individual wells were prepared at the times indicated after infection and plated on CYE to determine the number of bacterial CFU.
Assays to measure presentation of L. pneumophila antigens. A/J mice were immunized with L. pneumophila to generate immune CD4 T cells as described previously (27). Briefly, mice were immunized with 106 L. pneumophila intraperitoneally and given similar booster doses 2 weeks later. Mice were sacrificed 1 week after the booster dose, and CD4 T cells were positively selected from the spleen using Miltenyi Biotech CD4 magnetic beads. Pooled CD4 T cells from three immunized mice were used in each experiment. Experiments shown in each figure were internally controlled, using the same pool of CD4 T cells. Independently derived pools of CD4 T cells could vary in the magnitude of their response to BMs infected with wild-type L. pneumophila, but the fold difference in their response compared to BMs infected with L. pneumophila dotA mutant was consistent.
To measure MHC-II presentation by BMs, 105 BMs were added to each well in 96-well flat-bottom tissue culture plates. BMs were infected with L. pneumophila at an MOI of 20, unless otherwise indicated. After the addition of L. pneumophila, plates were centrifuged at 300 x g for 5 min to enhance contact of the bacteria with the BMs. The plates were then warmed in a 37°C water bath for 15 min, before extracellular bacteria were removed by washing the wells three times with PBS. T cells (4 x 105) were added to each well in T-cell medium (RPMI 1640 containing 10% fetal bovine serum, 1% minimal essential medium nonessential amino acids, 1% minimal essential medium essential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 50 μM -mercaptoethanol, 10 mM HEPES [pH 7.55]). Tissue culture medium, supplements, and fetal bovine serum were obtained from Gibco. After 48 h of incubation, T-cell responses were measured by determining the amount of IFN- present in these cultures using an enzyme-linked immunosorbent assay (Pharmingen). Data are the average IFN- concentrations ± standard deviations (error bars) for three independent wells. Where indicated, bacterial protein synthesis was inhibited by the addition of chloramphenicol to a final concentration of 6.25 μg ml–1, and chloroquine was added to the medium to a final concentration of 40 μM to neutralize the pH of endocytic compartments.
Isolation of L. pneumophila supernatant proteins. L. pneumophila bacteria were grown in 100 ml of ACES-yeast extract (AYE) broth (10) in 1-liter flasks at 37°C with aeration until they reached stationary phase. Cultures were centrifuged twice at 10,000 x g for 20 min to remove bacterial cells. Proteins in the culture supernatant were precipitated with ammonium sulfate, using an initial cut at 20% saturation and a final cut at 80% saturation. Precipitated protein was dissolved and dialyzed extensively against PBS. Protein concentrations were determined by using a Bradford assay kit (Bio-Rad). For antigen presentation assays, protein concentrations were normalized, and equal volumes were added to give a final concentration of 10 μg ml–1 in the BM tissue culture medium.
Intracellular cytokine staining. Intracellular cytokine staining was performed using Pharmingen reagents (Cytofix/Cytoperm [catalog no. 554722], Perm/Wash [catalog no. 554723], phycoerythrin-conjugated anti-IFN- antibody [catalog no. 554412], phycoerythrin-conjugated isotype control [catalog no. 559318], Golgi-Plug [catalog no. 555029]), per the manufacturer's instructions. Briefly, T cells were incubated with either uninfected BMs or BMs infected with wild-type or dotA mutant strains of L. pneumophila. After the cells were incubated overnight, cytokine secretion was blocked by the addition of Golgi-Plug. Cells were incubated for an additional 5 h in the presence of Golgi-Plug. Cells were stained for the CD4 surface marker using a fluorescein isothiocyanate-conjugated anti-CD4 antibody (Pharmingen catalog no. 557307). Cells were then permeabilized in Cytofix/Cytoperm and stained for intracellular IFN-. Cell staining levels were determined by flow cytometry, and data were analyzed using Cell-Quest software on an Apple Macintosh G4 computer.
Statistical analysis. Values were analyzed by a Student's two-tailed t test for statistical significance.
RESULTS AND DISCUSSION
An optimal response by immune CD4 T cells requires establishment of an ER-derived vacuole but not replication of L. pneumophila in BMs. L. pneumophila-specific CD4 T cells isolated from immunized mice have previously been shown to produce more IFN- when they were incubated with BMs infected with wild-type L. pneumophila that establish an ER-derived organelle than when they were incubated with BMs infected with a dotA mutant strain that moves directly to lysosomes (27). It is possible that this difference in the response of CD4 T cells to BMs is due in part to an increased antigen load in the infected cells resulting from multiplication of wild-type L. pneumophila. To determine whether L. pneumophila replication within BMs influences the magnitude of this T-cell response, isogenic strains derived from a thymidine auxotroph were used. L. pneumophila thyA mutants create an ER-derived vacuole after host cell uptake but are unable to replicate proficiently unless exogenous thymidine is added to the tissue culture medium (3).
It was determined that the number of L. pneumophila internalized by BMs increased proportionally as the MOI was increased from 0.1 to 100 (Fig. 1A). Even though the uptake curve for the thyA dotA strain of L. pneumophila, which lacks a functional Dot/Icm system, was comparable to the curve for the isogenic thyA strain with a functional Dot/Icm system, IFN- production by L. pneumophila-specific T cells showed a dose-dependent increase only for BMs infected with L. pneumophila producing a functional Dot/Icm system (Fig. 1B). These data demonstrate that the response of immune CD4 T cells to a L. pneumophila dotA mutant that moves to lysosomes cannot be enhanced significantly by simply increasing the antigen load.
The dose-dependent increase in the response of CD4 T cells to BMs infected with the L. pneumophila thyA mutant could have resulted either from an increase in antigen load or from an increase in the number of BMs infected. If antigen load were the primary reason for this increase, then restoring the ability of the thyA mutant to replicate proficiently within infected cells should also increase the L. pneumophila-specific CD4 T-cell response. When intracellular growth to the thyA mutant was restored through the addition of thymidine to the tissue culture medium, the magnitude of the L. pneumophila-specific T-cell response did not change significantly (Fig. 1C). These data indicate that the dose-dependent increase in the CD4 T-cell response observed for this strain is due to an increase in the percentage of BMs infected, not a higher bacterial load in each infected cell.
BMs infected with wild-type L. pneumophila stimulate a higher percentage of immune CD4 T cells than BMs infected with dotA mutants. The increase in IFN- production observed for immune CD4 T cells that were incubated with BMs infected with wild-type L. pneumophila could result from stimulation of a subset of T cells that do not respond to antigens presented by BMs infected with dotA mutants. Alternatively, this enhanced response could result from a single subset of T cells producing more IFN-. To distinguish between these two possibilities, the number of responding T cells was determined by flow cytometry after incubation of CD4 T cells with BMs infected with either wild-type L. pneumophila or the L. pneumophila dotA mutant strain (Fig. 2). These data show that as determined by intracellular IFN- staining, the number of T cells responding was greater after incubation with BMs infected with wild-type L. pneumophila than after incubation with BMs infected with dotA mutants. There was roughly a threefold increase in the number of T cells that stain positive for IFN- when BMs infected with wild-type L. pneumophila were compared to BMs infected with dotA mutants. Thus, nearly two-thirds of the L. pneumophila-specific T cells in this population of immune effectors fail to respond to BMs infected with L. pneumophila dotA mutants. These data demonstrate that there is a subset of immune CD4 T cells that respond only to L. pneumophila with the capacity to evade fusion with lysosomes and establish an ER-derived vacuole.
L. pneumophila antigens presented on MHC-II require processing in acidified endocytic organelles. MHC-II are translocated into the ER during synthesis and are then transported sequentially through the secretory and endocytic pathways to lysosomes. Nonself peptides presented on MHC-II are typically derived from exogenous antigens processed in acidified endocytic organelles (14). Given that L. pneumophila resides in a nonendocytic ER-derived organelle, two possibilities exist for how MHC-II acquire L. pneumophila antigens. One possible mechanism for presentation involves the loading of MHC-II with L. pneumophila antigens directly in the ER, bypassing the need for an acidified endocytic intermediate. The other possibility is that L. pneumophila antigens are delivered to acidified endocytic organelles for processing and MHC-II presentation. To address the mechanism of L. pneumophila antigen processing, chloroquine was used to neutralize the pH of endocytic compartments to prevent the processing of antigens in these organelles.
These data show that the CD4 T-cell response to BMs infected with wild-type L. pneumophila was severely impaired upon the addition of chloroquine to the tissue culture medium (Fig. 3A). Chloroquine and the bacterial protein synthesis inhibitor chloramphenicol both interfered with MHC-II presentation of antigens after BM infection by wild-type L. pneumophila. The concentration of chloroquine used to inhibit antigen processing in endocytic compartments did not affect the multiplication of L. pneumophila within BMs (Fig. 3B), which indicates that chloroquine is not directly interfering with trafficking or the formation of the ER-derived organelle in which L. pneumophila replicates. Chloroquine interfered with CD4 T-cell responses when it was added to the culture medium at the time of L. pneumophila infection, but it did not affect the response when added 18 h postinfection (Fig. 3C) when T cells were added to the assay. These data indicate that chloroquine is preventing the processing and presentation of L. pneumophila antigens on MHC-II and not interfering with IFN- secretion by immune T cells.
From these data, we conclude that acidified endocytic organelles play an important role in the processing of L. pneumophila antigens that are produced intracellularly by bacteria that create an ER-derived vacuole. The pathway antigens travel for delivery from an ER-derived vacuole to an acidified lysosome remains unknown. One possibility is that vacuoles containing replicating L. pneumophila eventually fuse with lysosomes. Consistent with this hypothesis, a previous study using murine BMs found that during the late stages of infection, a high percentage of vacuoles containing replicating L. pneumophila stain positive for lysosomal markers (35). Direct fusion of a replicative vacuole with lysosomes would result in the processing and MHC-II presentation of both secreted and somatic proteins produced intracellularly by L. pneumophila. A second possibility is that vesicles containing proteins shed from or secreted by L. pneumophila may exit the ER-derived compartment occupied by L. pneumophila and intersect the endocytic pathway for MHC-II processing and presentation. This would restrict the presentation of somatic antigens and enhance presentation of an antigen subset that would be displayed preferentially by BMs infected with wild-type L. pneumophila. These two pathways would not be mutually exclusive and may both be contributing antigen for presentation on MHC-II.
Optimal stimulation of immune CD4 T cells requires that the L. pneumophila-containing vacuole evade fusion with lysosomes after uptake and be converted into an ER-derived organelle. Immune CD4 T-cell responses were measured after infection of BMs with dot and icm mutants with distinct phenotypes to further investigate how Dot/Icm-dependent signaling processes might influence presentation of L. pneumophila antigens on MHC-II. Similar to dotA mutants, L. pneumophila icmW and icmS mutants reside in vacuoles that fuse rapidly with lysosomes (6, 42). These mutants are unable to replicate in BMs, but the Dot/Icm system is only partially defective in these mutants. L. pneumophila icmS and icmW mutants retain a Dot/Icm-dependent activity that forms pores in the host cell membrane and have the ability to recruit ER-derived vesicles to their vacuole membrane (6, 42). When examined for antigen presentation, the magnitude of the immune CD4 T-cell response to BMs infected with either the icmS or icmW mutant was greater than the response to BMs infected with the dotA mutant but was not as robust as the response detected to BMs infected with wild-type L. pneumophila (Fig. 4A). This intermediate response was insensitive to chloramphenicol treatment but was inhibited by chloroquine. These data suggest that either the recruitment of ER-derived vesicles or the pore-forming activity retained by the icmS and icmW mutants enhances presentation of L. pneumophila antigens to immune CD4 T cells after the rapid delivery of these mutants to lysosomes.
Although icmR mutants of L. pneumophila are also defective for growth in BMs, the kinetics of endocytic maturation for the vacuoles in which they reside is slower than for the vacuoles in which dotA or icmS or icmW mutants reside (6). Previous studies indicate that a small subset of the vacuoles containing icmR mutants will permit limited L. pneumophila replication in BMs prior to their fusion with lysosomes (6). Unlike icmS or icmW mutants, the icmR mutants are unable to form pores in the host plasma membrane, and they appear to be unable to recruit ER-derived vesicles to their vacuole membrane (6, 37). Similar to what was observed using icmS and icmW mutants, the magnitude of the immune CD4 T-cell response to BMs infected with icmR mutants was at an intermediate level compared to responses detected to BMs infected with wild-type L. pneumophila and dotA mutant L. pneumophila (Fig. 4A). The immune CD4 T-cell response to BMs infected with icmR mutants was sensitive to chloroquine treatment and dropped slightly when bacterial protein synthesis was inhibited with chloramphenicol. These data are consistent with limited de novo synthesis of proteins intracellularly by icmR mutant bacteria leading to a slight enhancement of MHC-II antigen presentation after fusion of their vacuoles with lysosomes.
We next examined the kinetics of antigen presentation to determine whether MHC-II processing was more rapid after BM infection with mutant L. pneumophila that are unable to avoid fusion with lysosomes after uptake. For these studies, BMs were infected, and antigen processing was interrupted at 4-h intervals by the addition of chloroquine to the culture medium (Fig. 4B). These data show that antigen presentation had peaked for dot and icm mutants within 8 h of infection. After infection with wild-type bacteria, L. pneumophila-specific T-cell responses were slightly lower at 4 h than the responses to the icm mutants, but they were higher than the responses observed for the dot and icm mutants at later time points.
Previous studies using immunofluorescence microscopy to analyze L. pneumophila trafficking in BMs indicate that most vacuoles containing dot or icm mutants have undergone fusion with lysosomes either within a few minutes or within 2 to 4 h for the icmR mutants (6, 32, 40), which would be consistent with the peak in MHC-II presentation we observe for these mutants at 8 h postinfection. In contrast, the majority of vacuoles containing wild-type L. pneumophila are devoid of lysosomal markers during the first 10 to 12 h of infection (16). Interestingly, it was reported previously that a high percentage of replicative vacuoles stain positive for lysosomal markers in BMs infected for more than 12 h with wild-type L. pneumophila (35), which is consistent with our observation that peak MHC-II presentation after infection of BMs with wild-type L. pneumophila occurs at later time points (>8 h). Thus, these data indicate that there is a correlation between the kinetics of lysosome fusion observed for vacuoles containing L. pneumophila and the kinetics of antigen presentation.
Protein secretion by the L. pneumophila Lsp system is important for priming of CD4 T cells in vivo but not for immune CD4 T-cell responses ex vivo. The L. pneumophila lsp genes encode a type II protein secretion system that can deliver bacterial proteins across the bacterial outer membrane (13). Unlike type IV systems, type II secretion systems are not involved in the direct transfer of bacterial proteins across a host membrane. Proteins secreted by the Lsp system might move out of the lumen of the L. pneumophila-containing vacuole and be delivered to lysosomes where they would become available for presentation on MHC-II. Thus, proteins secreted by the Lsp system could constitute a subset of antigens that are important for stimulation of immune CD4 T cells after infection of BMs with wild-type L. pneumophila. To determine the contribution of the Lsp system on MHC-II presentation of L. pneumophila antigens, an lspE mutant was characterized for its abilities to prime T-cell responses in vivo and MHC-II presentation ex vivo.
To determine whether the Lsp system is important for the priming and response of CD4 T cells, mice were immunized with wild-type L. pneumophila, an isogenic mspA lspE mutant, or heat-killed wild-type L. pneumophila. Responses from these immune CD4 T cells were then measured after infection of BMs with wild-type L. pneumophila, a mspA mutant, a mspA lspE mutant, and a mspA lspE dotA mutant (Fig. 5A). The mspA gene encodes an L. pneumophila protease that is secreted by the Lsp secretion system (13). Because MspA is responsible for the degradation of L. pneumophila proteins secreted into broth culture (26, 36), mspA mutants were used to diminish the possibility that other substrates secreted by the Lsp system would be rapidly degraded. CD4 T cells isolated from mice immunized with wild-type L. pneumophila gave responses to BMs infected with either a mspA mutant or a mspA lspE mutant that were similar to the responses observed for BMs infected with wild-type L. pneumophila. These data indicate that protein secretion by the Lsp system is not required for stimulation of immune CD4 T cells.
Interestingly, compared to the CD4 T cells from mice immunized with wild-type L. pneumophila, it was found that the CD4 T cells from mice immunized with the mspA lspE mutant were slightly less responsive to BMs infected with wild-type L. pneumophila, the mspA mutant, or the mspA lspE mutant, though this difference did not reach statistical significance (P value of 0.065). These data indicate that the Lsp system may be important for the priming of CD4 T cells in vivo. Because lsp mutants are less pathogenic than wild-type L. pneumophila (31), this priming defect might simply be due to reduced virulence. Alternatively, proteins secreted by the Lsp system may have a direct role in enhancing MHC-II presentation in vivo.
Although substrate proteins of the Lsp system are not secreted by an lspE mutant (30), they are still produced by this mutant and would become available for MHC-II presentation if intact L. pneumophila were degraded in lysosomes. To directly address whether Lsp substrates are major antigens recognized by immune CD4 T cells, secreted proteins were isolated from supernatants of wild-type L. pneumophila or dotA, mspA lspE, and mspA lspE dotA mutant strains grown in broth. These supernatant proteins were then added to BM cultures, and the responses of CD4 T cells isolated from mice immunized with wild-type L. pneumophila were measured (Fig. 5B). Proteins added directly to cultures of Ms are internalized by fluid-phase endocytosis, transported along the endocytic pathway to lysosomal compartments, processed, and loaded onto MHC-II (38). The CD4-mediated T-cell response did not diminish in response to L. pneumophila supernatants when proteins secreted by the Lsp system were eliminated. These data demonstrate that proteins secreted by the Lsp system were not required for maximal stimulation of immune CD4 T cells, consistent with the observation than CD4 T cells respond equally well to Ms infected with the mspA mutant strain and the mspA lspE mutant strain. From these data, we conclude that Lsp substrate proteins are not the major antigenic determinants of the CD4-mediated immune response generated during L. pneumophila infection, although their role in the process of priming of the T-cell response in vivo has yet to be determined.
In summary, these studies have defined events that are important for the presentation of L. pneumophila antigens on MHC-II after M infection. Establishment of the unique ER-derived vacuole that provides an initial safe haven for L. pneumophila within the M is clearly important for optimal stimulation of immune CD4 T cells. Flow cytometry data indicate that a subset of CD4 T cells primed during infection recognize antigens presented preferentially by L. pneumophila that reside within this specialized organelle. The finding that acidified endocytic organelles are required for MHC-II presentation of L. pneumophila antigens indicates that lysosomal degradation plays an important role in processing of the antigens that are presented by infected Ms. How antigens are delivered from the ER-derived vacuole to lysosomes remains unknown. It does not appear as if antigens secreted into the vacuole lumen by the Lsp system constitute the primary determinants of the adaptive CD4-mediated immune response against L. pneumophila. It is possible that a subset of immune CD4 T cells recognize unique L. pneumophila determinant that are produce intracellularly, but additional studies are required to identify antigens recognized by L. pneumophila-specific immune T cells. Determining the regulation, subcellular localization, and genetic requirements for presentation of these antigens should provide important details on the process by which L. pneumophila proteins become available for presentation on MHC-II.
ACKNOWLEDGMENTS
This work was supported in part by NIH grant AI48770 (C.R.R.).
We thank Jonathan Kagan for helpful discussions and critical review of the manuscript.
REFERENCES
1. Andersen, P. 1997. Host responses and antigens involved in protective immunity to Mycobacterium tuberculosis. Scand. J. Immunol. 45:115-131.
2. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392:245-252.
3. Berger, K. H., and R. R. Isberg. 1993. Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol. Microbiol. 7:7-19.
4. Celada, A., P. W. Gray, E. Rinderknecht, and R. D. Schreiber. 1984. Evidence for a gamma-interferon receptor that regulates macrophage tumoricidal activity. J. Exp. Med. 160:55-74.
5. Chen, J., K. S. de Felipe, M. Clarke, H. Lu, O. R. Anderson, G. Segal, and H. A. Shuman. 2004. Legionella effectors that promote nonlytic release from protozoa. Science 303:1358-1361.
6. Coers, J., J. C. Kagan, M. Matthews, H. Nagai, D. M. Zuckman, and C. R. Roy. 2000. Identification of Icm protein complexes that play distinct roles in the biogenesis of an organelle permissive for Legionella pneumophila intracellular growth. Mol. Microbiol. 38:719-736.
7. Conover, G. M., I. Derre, J. P. Vogel, and R. R. Isberg. 2003. The Legionella pneumophila LidA protein: a translocated substrate of the Dot/Icm system associated with maintenance of bacterial integrity. Mol. Microbiol. 48:305-321.
8. Cresswell, P. 1994. Assembly, transport, and function of MHC class II molecules. Annu. Rev. Immunol. 12:259-293.
9. Derre, I., and R. R. Isberg. 2004. Legionella pneumophila replication vacuole formation involves rapid recruitment of proteins of the early secretory system. Infect. Immun. 72:3048-3053.
10. Feeley, J. C., R. J. Gibson, G. W. Gorman, N. C. Langford, J. K. Rasheed, D. C. Mackel, and W. B. Baine. 1979. Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. J. Clin. Microbiol. 10:437-441.
11. Flynn, J. L., and J. Chan. 2001. Immunology of tuberculosis. Annu. Rev. Immunol. 19:93-129.
12. Fraser, D. W., T. R. Tsai, W. Orenstein, W. E. Parkin, H. J. Beecham, R. G. Sharrar, J. Harris, G. F. Mallison, S. M. Martin, J. E. McDade, C. C. Shepard, and P. S. Brachman. 1977. Legionnaires' disease: description of an epidemic of pneumonia. N. Engl. J. Med. 297:1189-1197.
13. Hales, L. M., and H. A. Shuman. 1999. Legionella pneumophila contains a type II general secretion pathway required for growth in amoebae as well as for secretion of the Msp protease. Infect. Immun. 67:3662-3666.
14. Harding, C. V., and H. J. Geuze. 1993. Immunogenic peptides bind to class II MHC molecules in an early lysosomal compartment. J. Immunol. 151:3988-3998.
15. Horwitz, M. A. 1983. Formation of a novel phagosome by the Legionnaires' disease bacterium (Legionella pneumophila) in human monocytes. J. Exp. Med. 158:1319-1331.
16. Horwitz, M. A. 1983. The Legionnaires' disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J. Exp. Med. 158:2108-2126.
17. Horwitz, M. A., and S. C. Silverstein. 1980. Legionnaires' disease bacterium (Legionella pneumophila) multiplies intracellularly in human monocytes. J. Clin. Investig. 66:441-450.
18. Kagan, J. C., and C. R. Roy. 2002. Legionella phagosomes intercept vesicular traffic from endoplasmic reticulum exit sites. Nat. Cell Biol. 4:945-954.
19. Kagan, J. C., M. P. Stein, M. Pypaert, and C. R. Roy. 2004. Legionella subvert the functions of rab1 and sec22b to create a replicative organelle. J. Exp. Med. 199:1201-1211.
20. Kagi, D., B. Ledermann, K. Burki, R. M. Zinkernagel, and H. Hengartner. 1996. Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo. Annu. Rev. Immunol. 14:207-232.
21. Lehner, P. J., and P. Cresswell. 1996. Processing and delivery of peptides presented by MHC class I molecules. Curr. Opin. Immunol. 8:59-67.
22. Luo, Z. Q., and R. R. Isberg. 2004. Multiple substrates of the Legionella pneumophila Dot/Icm system identified by interbacterial protein transfer. Proc. Natl. Acad. Sci. USA 101:841-846.
23. Merriam, J. J., R. Mathur, R. Maxfield-Boumil, and R. R. Isberg. 1997. Analysis of the Legionella pneumophila fliI gene: intracellular growth of a defined mutant defective for flagellum biosynthesis. Infect. Immun. 65:2497-2501.
24. Molmeret, M., O. A. Alli, S. Zink, A. Flieger, N. P. Cianciotto, and Y. A. Kwaik. 2002. icmT is essential for pore formation-mediated egress of Legionella pneumophila from mammalian and protozoan cells. Infect. Immun. 70:69-78.
25. Nagai, H., J. C. Kagan, X. Zhu, R. A. Kahn, and C. R. Roy. 2002. A bacterial guanine nucleotide exchange factor activates ARF on Legionella phagosomes. Science 295:679-682.
26. Nagai, H., and C. R. Roy. 2001. The DotA protein from Legionella pneumophila is secreted by a novel process that requires the Dot/Icm transporter. EMBO J. 20:5962-5970.
27. Neild, A. L., and C. R. Roy. 2003. Legionella reveal dendritic cell functions that facilitate selection of antigens for MHC class II presentation. Immunity 18:813-823.
28. Pamer, E. G., A. J. Sijts, M. S. Villanueva, D. H. Busch, and S. Vijh. 1997. MHC class I antigen processing of Listeria monocytogenes proteins: implications for dominant and subdominant CTL responses. Immunol. Rev. 158:129-136.
29. Perry, L. L., K. Feilzer, and H. D. Caldwell. 1997. Immunity to Chlamydia trachomatis is mediated by T helper 1 cells through IFN-gamma-dependent and -independent pathways. J. Immunol. 158:3344-3352.
30. Rossier, O., and N. P. Cianciotto. 2001. Type II protein secretion is a subset of the PilD-dependent processes that facilitate intracellular infection by Legionella pneumophila. Infect. Immun. 69:2092-2098.
31. Rossier, O., S. R. Starkenburg, and N. P. Cianciotto. 2004. Legionella pneumophila type II protein secretion promotes virulence in the A/J mouse model of Legionnaires' disease pneumonia. Infect. Immun. 72:310-321.
32. Roy, C. R., K. H. Berger, and R. R. Isberg. 1998. Legionella pneumophila DotA protein is required for early phagosome trafficking decisions that occur within minutes of bacterial uptake. Mol. Microbiol. 28:663-674.
33. Segal, G., and H. A. Shuman. 1998. How is the intracellular fate of the Legionella pneumophila phagosome determined Trends Microbiol. 6:253-255.
34. Sinai, A. P., and K. A. Joiner. 1997. Safe haven: the cell biology of nonfusogenic pathogen vacuoles. Annu. Rev. Microbiol. 51:415-462.
35. Sturgill-Koszycki, S., and M. S. Swanson. 2000. Legionella pneumophila replication vacuoles mature into acidic, endocytic organelles. J. Exp. Med. 192:1261-1272.
36. Szeto, L., and H. A. Shuman. 1990. The Legionella pneumophila major secretory protein, a protease, is not required for intracellular growth or cell killing. Infect. Immun. 58:2585-2592.
37. Tilney, L. G., O. S. Harb, P. S. Connelly, C. G. Robinson, and C. R. Roy. 2001. How the parasitic bacterium Legionella pneumophila modifies its phagosome and transforms it into rough ER: implications for conversion of plasma membrane to the ER membrane. J. Cell Sci. 114:4637-4650.
38. Unanue, E. R., and P. M. Allen. 1986. Biochemistry and biology of antigen presentation by macrophages. Cell. Immunol. 99:3-6.
39. Vogel, J. P., H. L. Andrews, S. K. Wong, and R. R. Isberg. 1998. Conjugative transfer by the virulence system of Legionella pneumophila. Science 279:873-876.
40. Wiater, L. A., K. Dunn, F. R. Maxfield, and H. A. Shuman. 1998. Early events in phagosome establishment are required for intracellular survival of Legionella pneumophila. Infect. Immun. 66:4450-4460.
41. Yamamoto, Y., T. W. Klein, C. A. Newton, R. Widen, and H. Friedman. 1988. Growth of Legionella pneumophila in thioglycolate-elicited peritoneal macrophages from A/J mice. Infect. Immun. 56:370-375.
42. Zuckman, D. M., J. B. Hung, and C. R. Roy. 1999. Pore-forming activity is not sufficient for Legionella pneumophila phagosome trafficking and intracellular growth. Mol. Microbiol. 32:990-1001.(Annie Neild, Takahiro Mur)
ABSTRACT
To better understand interactions between the intracellular pathogen Legionella pneumophila and macrophages (Ms), host and bacterial determinants important for presentation of antigens on major histocompatibility complex class II molecules (MHC-II) were investigated. It was determined that immune CD4 T-cell responses to murine bone marrow-derived Ms (BMs) infected with wild-type L. pneumophila were higher than the responses to avirulent dotA mutant bacteria. Although this enhanced response by immune T cells required modulation of vacuole transport mediated by the Dot/Icm system, it did not require intracellular replication of L. pneumophila. Intracellular cytokine staining identified a population of immune CD4 T cells that produced gamma interferon upon incubation with BMs infected with wild-type L. pneumophila that did not respond to M infection with dotA mutant bacteria. Endocytic processing was required for presentation of L. pneumophila antigens on MHC-II as determined by a defect in CD4 T-cell responses when the pH of BM endosomes was neutralized with chloroquine. Investigation of MHC-II presentation of antigens by BMs infected with L. pneumophila icmR, icmW, and icmS mutants indicated that these mutants have an intermediate presentation phenotype relative to those of wild-type and dotA mutant bacteria. In addition, it was found that antigens from dot and icm mutants are presented earlier than antigens from wild-type L. pneumophila. Although immune CD4 T-cell responses to proteins secreted by the L. pneumophila Lsp system were not detected, it was found that the Lsp system is important for priming L. pneumophila-specific T cells in vivo. These data indicate that optimal antigen processing and MHC-II presentation to immune CD4 T cells involves synthesis of L. pneumophila proteins in an endoplasmic reticulum-derived compartment followed by transport to lysosomes.
INTRODUCTION
Naive T cells expressing a unique T-cell receptor become antigen-specific effector cells upon encountering a professional antigen-presenting cell that has a cognate peptide major histocompatibility complex (MHC) displayed on its surface (2). Effector T cells play an important role in immunity to pathogens by producing factors that either upregulate cellular antimicrobial functions or that kill infected target cells that display a cognate peptide MHC (11, 20). T cells producing the CD4 protein typically respond to peptides presented on MHC class II molecules (MHC-II), whereas a peptide-loaded MHC-I will stimulate T cells producing CD8. Most antigens loaded onto MHC-I are derived from proteins that have gained access to the cell cytosol, such as viral antigens or antigens released by bacterial pathogens that escape membrane-bound compartments and replicate in the cytosol of their host (21, 28). In contrast, peptides loaded onto MHC-II are derived from antigens that are transported to lysosomes, such as those produced by organisms that remain in vacuoles that undergo endocytic maturation after internalization by an antigen-presenting cell (8, 11).
There are many examples of intracellular pathogens that replicate in vacuoles that resist fusion with lysosomes (34). Paradoxically, the adaptive immune response mediated by CD4 T cells usually plays an important role in controlling infections by these pathogens (1, 29). How immunoreactive antigens from these pathogens intersect the MHC-II presentation pathway for presentation to CD4 T cells is not known. Questions related to how these antigens are released, how they move through the cell, and how they are processed for presentation on MHC-II are of fundamental importance to understanding host immunity to vacuolar pathogens and in the eventual development of effective vaccines that will help prevent diseases resulting from infections by these organisms.
We have been using Legionella pneumophila as a model system to understand how adaptive immune responses are generated against pathogens that replicate in specialized vacuoles. L. pneumophila infections can result in a severe pneumonia in humans known as Legionnaires' disease (12). The ability to multiply inside macrophages (Ms) is an important L. pneumophila virulence trait (17, 41). To replicate intracellularly, L. pneumophila alters transport of the vacuoles in which the bacteria reside to evade delivery to lysosomes and transform their compartment into an organelle that resembles the endoplasmic reticulum (ER) (6, 9, 18, 19). It is within this ER-derived organelle that bacterial multiplication occurs (15). The ability of L. pneumophila to redirect vacuole transport within bone marrow-derived Ms (BMs) is dependent on the delivery of bacterial proteins into the host cell by a type IVb secretion apparatus (5, 7, 22, 25), which is encoded by the dot and icm genes (33, 39). A subset of the proteins translocated by the L. pneumophila Dot/Icm system subvert host cell proteins involved in vesicle transport in the secretory pathway (9, 18, 25), leading to the creation of a replicative organelle. Strains of L. pneumophila that lack the Dot/Icm secretion system due to mutations in the dot or icm gene fail to remodel their vacuole, resulting in transport to lysosomes (3, 32, 40). At late stages of infection, L. pneumophila has been observed residing in vacuoles that have lysosomal properties, suggesting that additional maturation events may occur after establishment of an ER-derived vacuole (35). There is also evidence that the Dot/Icm system is involved in the process of egress of L. pneumophila from spent host cells (5, 24).
Previous studies on the presentation of L. pneumophila antigens by dendritic cells (DCs) revealed that CD4 T cells from immunized mice respond better to DCs infected with wild-type L. pneumophila than to DCs infected with L. pneumophila dotA mutant (27). Thus, the ability of L. pneumophila to evade lysosomal delivery after uptake was essential for the optimal presentation of bacterial antigens on MHC-II in DCs. To explain these results, it was hypothesized that there is a T-cell subset primed during infection that has the unique abilities to recognize and respond to antigens synthesized after L. pneumophila is internalized by DCs (27). This hypothesis predicts that T cells produced during infection with wild-type L. pneumophila should also have the ability to recognize Ms infected with wild-type L. pneumophila and respond by producing cytokines, such as gamma interferon (IFN-), that can lead to M activation. In this study, we examined CD4 T cells produced during L. pneumophila infection and investigated their ability to respond to infected BMs. These studies reveal host and bacterial factors that are important for the presentation of L. pneumophila peptides on MHC-II after BM infection.
MATERIALS AND METHODS
Bacterial cultures. The L. pneumophila serogroup 1 strain, Lp01 (3), and the isogenic dotA (42), icmW (42), icmS and icmR (6) mutant strains, as well as mspA (26), mspA dotA (26), mspA lspE, and mspA dotA lspE (this study) mutant strains, were cultured on charcoal-yeast extract (CYE) agar (10) for 2 days prior to use in experiments. The thyA mutant strain used was Lp02, which is a thymidine auxotroph derived from Lp01 (3). Allelic exchange was used to delete the entire dotA gene from Lp02 to generate the thyA dotA mutant. These strains were grown on CYE agar with thymidine added to a final concentration of 10 μg ml–1. Where indicated, tissue culture medium was also supplemented with thymidine at 10 μg ml–1. Heat-killed bacteria were obtained by incubating a 1-ml suspension of 109 L. pneumophila at 80°C for 45 min.
Construction of L. pneumophila lspE mutants. An in-frame deletion of the lspE gene was introduced into the L. pneumophila chromosome by allelic exchange as described previously (23). To construct the lspE deletion, two DNA fragments were generated by PCR. Primer E1 (5'-GCGAGCTCAAATTACTCGCGGACAAGG-3') and primer E2 (5'-TATGGGTAATTTATTTGGTCATTTTTGATG-3') were used to generate a DNA fragment homologous to the 5' region of the lspE gene, and primer E3 (5'-AATGACCAAATAAATTACCCATACTTTAGC-3') and primer E4 (5'-GCTCTAGATGAATTGCCAGGAAGTAAAG-3') were used to generate a region of 3' homology. These two DNA fragments were combined by recombinant PCR using primers E1 and E4. The lspE deletion allele was ligated into the gene replacement vector pSR47S (23) and was introduced into L. pneumophila mspA and mspA dotA strains to make the mspA lspE and mspA dotA lspE mutants, respectively.
Cell cultures. BMs were derived from A/J mice (Harlan Sprague Dawley). Cultures of BMs were prepared as described previously (4).
Intracellular growth and L. pneumophila uptake assays. Growth of L. pneumophila in BMs was measured as described previously (42). Assays to determine uptake of bacteria at different multiplicities of infection (MOIs) were performed similarly. Briefly, L. pneumophila was added to BM cultures at the MOI indicated, and plates were centrifuged for 5 min at 300 x g and warmed in a 37°C water bath for 15 min. Wells were then washed vigorously with ice-cold phosphate-buffered saline (PBS) to remove extracellular bacteria. For intracellular growth assays, fresh medium was added to the wells, and plates were returned to the incubator. Lysates from individual wells were prepared at the times indicated after infection and plated on CYE to determine the number of bacterial CFU.
Assays to measure presentation of L. pneumophila antigens. A/J mice were immunized with L. pneumophila to generate immune CD4 T cells as described previously (27). Briefly, mice were immunized with 106 L. pneumophila intraperitoneally and given similar booster doses 2 weeks later. Mice were sacrificed 1 week after the booster dose, and CD4 T cells were positively selected from the spleen using Miltenyi Biotech CD4 magnetic beads. Pooled CD4 T cells from three immunized mice were used in each experiment. Experiments shown in each figure were internally controlled, using the same pool of CD4 T cells. Independently derived pools of CD4 T cells could vary in the magnitude of their response to BMs infected with wild-type L. pneumophila, but the fold difference in their response compared to BMs infected with L. pneumophila dotA mutant was consistent.
To measure MHC-II presentation by BMs, 105 BMs were added to each well in 96-well flat-bottom tissue culture plates. BMs were infected with L. pneumophila at an MOI of 20, unless otherwise indicated. After the addition of L. pneumophila, plates were centrifuged at 300 x g for 5 min to enhance contact of the bacteria with the BMs. The plates were then warmed in a 37°C water bath for 15 min, before extracellular bacteria were removed by washing the wells three times with PBS. T cells (4 x 105) were added to each well in T-cell medium (RPMI 1640 containing 10% fetal bovine serum, 1% minimal essential medium nonessential amino acids, 1% minimal essential medium essential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 50 μM -mercaptoethanol, 10 mM HEPES [pH 7.55]). Tissue culture medium, supplements, and fetal bovine serum were obtained from Gibco. After 48 h of incubation, T-cell responses were measured by determining the amount of IFN- present in these cultures using an enzyme-linked immunosorbent assay (Pharmingen). Data are the average IFN- concentrations ± standard deviations (error bars) for three independent wells. Where indicated, bacterial protein synthesis was inhibited by the addition of chloramphenicol to a final concentration of 6.25 μg ml–1, and chloroquine was added to the medium to a final concentration of 40 μM to neutralize the pH of endocytic compartments.
Isolation of L. pneumophila supernatant proteins. L. pneumophila bacteria were grown in 100 ml of ACES-yeast extract (AYE) broth (10) in 1-liter flasks at 37°C with aeration until they reached stationary phase. Cultures were centrifuged twice at 10,000 x g for 20 min to remove bacterial cells. Proteins in the culture supernatant were precipitated with ammonium sulfate, using an initial cut at 20% saturation and a final cut at 80% saturation. Precipitated protein was dissolved and dialyzed extensively against PBS. Protein concentrations were determined by using a Bradford assay kit (Bio-Rad). For antigen presentation assays, protein concentrations were normalized, and equal volumes were added to give a final concentration of 10 μg ml–1 in the BM tissue culture medium.
Intracellular cytokine staining. Intracellular cytokine staining was performed using Pharmingen reagents (Cytofix/Cytoperm [catalog no. 554722], Perm/Wash [catalog no. 554723], phycoerythrin-conjugated anti-IFN- antibody [catalog no. 554412], phycoerythrin-conjugated isotype control [catalog no. 559318], Golgi-Plug [catalog no. 555029]), per the manufacturer's instructions. Briefly, T cells were incubated with either uninfected BMs or BMs infected with wild-type or dotA mutant strains of L. pneumophila. After the cells were incubated overnight, cytokine secretion was blocked by the addition of Golgi-Plug. Cells were incubated for an additional 5 h in the presence of Golgi-Plug. Cells were stained for the CD4 surface marker using a fluorescein isothiocyanate-conjugated anti-CD4 antibody (Pharmingen catalog no. 557307). Cells were then permeabilized in Cytofix/Cytoperm and stained for intracellular IFN-. Cell staining levels were determined by flow cytometry, and data were analyzed using Cell-Quest software on an Apple Macintosh G4 computer.
Statistical analysis. Values were analyzed by a Student's two-tailed t test for statistical significance.
RESULTS AND DISCUSSION
An optimal response by immune CD4 T cells requires establishment of an ER-derived vacuole but not replication of L. pneumophila in BMs. L. pneumophila-specific CD4 T cells isolated from immunized mice have previously been shown to produce more IFN- when they were incubated with BMs infected with wild-type L. pneumophila that establish an ER-derived organelle than when they were incubated with BMs infected with a dotA mutant strain that moves directly to lysosomes (27). It is possible that this difference in the response of CD4 T cells to BMs is due in part to an increased antigen load in the infected cells resulting from multiplication of wild-type L. pneumophila. To determine whether L. pneumophila replication within BMs influences the magnitude of this T-cell response, isogenic strains derived from a thymidine auxotroph were used. L. pneumophila thyA mutants create an ER-derived vacuole after host cell uptake but are unable to replicate proficiently unless exogenous thymidine is added to the tissue culture medium (3).
It was determined that the number of L. pneumophila internalized by BMs increased proportionally as the MOI was increased from 0.1 to 100 (Fig. 1A). Even though the uptake curve for the thyA dotA strain of L. pneumophila, which lacks a functional Dot/Icm system, was comparable to the curve for the isogenic thyA strain with a functional Dot/Icm system, IFN- production by L. pneumophila-specific T cells showed a dose-dependent increase only for BMs infected with L. pneumophila producing a functional Dot/Icm system (Fig. 1B). These data demonstrate that the response of immune CD4 T cells to a L. pneumophila dotA mutant that moves to lysosomes cannot be enhanced significantly by simply increasing the antigen load.
The dose-dependent increase in the response of CD4 T cells to BMs infected with the L. pneumophila thyA mutant could have resulted either from an increase in antigen load or from an increase in the number of BMs infected. If antigen load were the primary reason for this increase, then restoring the ability of the thyA mutant to replicate proficiently within infected cells should also increase the L. pneumophila-specific CD4 T-cell response. When intracellular growth to the thyA mutant was restored through the addition of thymidine to the tissue culture medium, the magnitude of the L. pneumophila-specific T-cell response did not change significantly (Fig. 1C). These data indicate that the dose-dependent increase in the CD4 T-cell response observed for this strain is due to an increase in the percentage of BMs infected, not a higher bacterial load in each infected cell.
BMs infected with wild-type L. pneumophila stimulate a higher percentage of immune CD4 T cells than BMs infected with dotA mutants. The increase in IFN- production observed for immune CD4 T cells that were incubated with BMs infected with wild-type L. pneumophila could result from stimulation of a subset of T cells that do not respond to antigens presented by BMs infected with dotA mutants. Alternatively, this enhanced response could result from a single subset of T cells producing more IFN-. To distinguish between these two possibilities, the number of responding T cells was determined by flow cytometry after incubation of CD4 T cells with BMs infected with either wild-type L. pneumophila or the L. pneumophila dotA mutant strain (Fig. 2). These data show that as determined by intracellular IFN- staining, the number of T cells responding was greater after incubation with BMs infected with wild-type L. pneumophila than after incubation with BMs infected with dotA mutants. There was roughly a threefold increase in the number of T cells that stain positive for IFN- when BMs infected with wild-type L. pneumophila were compared to BMs infected with dotA mutants. Thus, nearly two-thirds of the L. pneumophila-specific T cells in this population of immune effectors fail to respond to BMs infected with L. pneumophila dotA mutants. These data demonstrate that there is a subset of immune CD4 T cells that respond only to L. pneumophila with the capacity to evade fusion with lysosomes and establish an ER-derived vacuole.
L. pneumophila antigens presented on MHC-II require processing in acidified endocytic organelles. MHC-II are translocated into the ER during synthesis and are then transported sequentially through the secretory and endocytic pathways to lysosomes. Nonself peptides presented on MHC-II are typically derived from exogenous antigens processed in acidified endocytic organelles (14). Given that L. pneumophila resides in a nonendocytic ER-derived organelle, two possibilities exist for how MHC-II acquire L. pneumophila antigens. One possible mechanism for presentation involves the loading of MHC-II with L. pneumophila antigens directly in the ER, bypassing the need for an acidified endocytic intermediate. The other possibility is that L. pneumophila antigens are delivered to acidified endocytic organelles for processing and MHC-II presentation. To address the mechanism of L. pneumophila antigen processing, chloroquine was used to neutralize the pH of endocytic compartments to prevent the processing of antigens in these organelles.
These data show that the CD4 T-cell response to BMs infected with wild-type L. pneumophila was severely impaired upon the addition of chloroquine to the tissue culture medium (Fig. 3A). Chloroquine and the bacterial protein synthesis inhibitor chloramphenicol both interfered with MHC-II presentation of antigens after BM infection by wild-type L. pneumophila. The concentration of chloroquine used to inhibit antigen processing in endocytic compartments did not affect the multiplication of L. pneumophila within BMs (Fig. 3B), which indicates that chloroquine is not directly interfering with trafficking or the formation of the ER-derived organelle in which L. pneumophila replicates. Chloroquine interfered with CD4 T-cell responses when it was added to the culture medium at the time of L. pneumophila infection, but it did not affect the response when added 18 h postinfection (Fig. 3C) when T cells were added to the assay. These data indicate that chloroquine is preventing the processing and presentation of L. pneumophila antigens on MHC-II and not interfering with IFN- secretion by immune T cells.
From these data, we conclude that acidified endocytic organelles play an important role in the processing of L. pneumophila antigens that are produced intracellularly by bacteria that create an ER-derived vacuole. The pathway antigens travel for delivery from an ER-derived vacuole to an acidified lysosome remains unknown. One possibility is that vacuoles containing replicating L. pneumophila eventually fuse with lysosomes. Consistent with this hypothesis, a previous study using murine BMs found that during the late stages of infection, a high percentage of vacuoles containing replicating L. pneumophila stain positive for lysosomal markers (35). Direct fusion of a replicative vacuole with lysosomes would result in the processing and MHC-II presentation of both secreted and somatic proteins produced intracellularly by L. pneumophila. A second possibility is that vesicles containing proteins shed from or secreted by L. pneumophila may exit the ER-derived compartment occupied by L. pneumophila and intersect the endocytic pathway for MHC-II processing and presentation. This would restrict the presentation of somatic antigens and enhance presentation of an antigen subset that would be displayed preferentially by BMs infected with wild-type L. pneumophila. These two pathways would not be mutually exclusive and may both be contributing antigen for presentation on MHC-II.
Optimal stimulation of immune CD4 T cells requires that the L. pneumophila-containing vacuole evade fusion with lysosomes after uptake and be converted into an ER-derived organelle. Immune CD4 T-cell responses were measured after infection of BMs with dot and icm mutants with distinct phenotypes to further investigate how Dot/Icm-dependent signaling processes might influence presentation of L. pneumophila antigens on MHC-II. Similar to dotA mutants, L. pneumophila icmW and icmS mutants reside in vacuoles that fuse rapidly with lysosomes (6, 42). These mutants are unable to replicate in BMs, but the Dot/Icm system is only partially defective in these mutants. L. pneumophila icmS and icmW mutants retain a Dot/Icm-dependent activity that forms pores in the host cell membrane and have the ability to recruit ER-derived vesicles to their vacuole membrane (6, 42). When examined for antigen presentation, the magnitude of the immune CD4 T-cell response to BMs infected with either the icmS or icmW mutant was greater than the response to BMs infected with the dotA mutant but was not as robust as the response detected to BMs infected with wild-type L. pneumophila (Fig. 4A). This intermediate response was insensitive to chloramphenicol treatment but was inhibited by chloroquine. These data suggest that either the recruitment of ER-derived vesicles or the pore-forming activity retained by the icmS and icmW mutants enhances presentation of L. pneumophila antigens to immune CD4 T cells after the rapid delivery of these mutants to lysosomes.
Although icmR mutants of L. pneumophila are also defective for growth in BMs, the kinetics of endocytic maturation for the vacuoles in which they reside is slower than for the vacuoles in which dotA or icmS or icmW mutants reside (6). Previous studies indicate that a small subset of the vacuoles containing icmR mutants will permit limited L. pneumophila replication in BMs prior to their fusion with lysosomes (6). Unlike icmS or icmW mutants, the icmR mutants are unable to form pores in the host plasma membrane, and they appear to be unable to recruit ER-derived vesicles to their vacuole membrane (6, 37). Similar to what was observed using icmS and icmW mutants, the magnitude of the immune CD4 T-cell response to BMs infected with icmR mutants was at an intermediate level compared to responses detected to BMs infected with wild-type L. pneumophila and dotA mutant L. pneumophila (Fig. 4A). The immune CD4 T-cell response to BMs infected with icmR mutants was sensitive to chloroquine treatment and dropped slightly when bacterial protein synthesis was inhibited with chloramphenicol. These data are consistent with limited de novo synthesis of proteins intracellularly by icmR mutant bacteria leading to a slight enhancement of MHC-II antigen presentation after fusion of their vacuoles with lysosomes.
We next examined the kinetics of antigen presentation to determine whether MHC-II processing was more rapid after BM infection with mutant L. pneumophila that are unable to avoid fusion with lysosomes after uptake. For these studies, BMs were infected, and antigen processing was interrupted at 4-h intervals by the addition of chloroquine to the culture medium (Fig. 4B). These data show that antigen presentation had peaked for dot and icm mutants within 8 h of infection. After infection with wild-type bacteria, L. pneumophila-specific T-cell responses were slightly lower at 4 h than the responses to the icm mutants, but they were higher than the responses observed for the dot and icm mutants at later time points.
Previous studies using immunofluorescence microscopy to analyze L. pneumophila trafficking in BMs indicate that most vacuoles containing dot or icm mutants have undergone fusion with lysosomes either within a few minutes or within 2 to 4 h for the icmR mutants (6, 32, 40), which would be consistent with the peak in MHC-II presentation we observe for these mutants at 8 h postinfection. In contrast, the majority of vacuoles containing wild-type L. pneumophila are devoid of lysosomal markers during the first 10 to 12 h of infection (16). Interestingly, it was reported previously that a high percentage of replicative vacuoles stain positive for lysosomal markers in BMs infected for more than 12 h with wild-type L. pneumophila (35), which is consistent with our observation that peak MHC-II presentation after infection of BMs with wild-type L. pneumophila occurs at later time points (>8 h). Thus, these data indicate that there is a correlation between the kinetics of lysosome fusion observed for vacuoles containing L. pneumophila and the kinetics of antigen presentation.
Protein secretion by the L. pneumophila Lsp system is important for priming of CD4 T cells in vivo but not for immune CD4 T-cell responses ex vivo. The L. pneumophila lsp genes encode a type II protein secretion system that can deliver bacterial proteins across the bacterial outer membrane (13). Unlike type IV systems, type II secretion systems are not involved in the direct transfer of bacterial proteins across a host membrane. Proteins secreted by the Lsp system might move out of the lumen of the L. pneumophila-containing vacuole and be delivered to lysosomes where they would become available for presentation on MHC-II. Thus, proteins secreted by the Lsp system could constitute a subset of antigens that are important for stimulation of immune CD4 T cells after infection of BMs with wild-type L. pneumophila. To determine the contribution of the Lsp system on MHC-II presentation of L. pneumophila antigens, an lspE mutant was characterized for its abilities to prime T-cell responses in vivo and MHC-II presentation ex vivo.
To determine whether the Lsp system is important for the priming and response of CD4 T cells, mice were immunized with wild-type L. pneumophila, an isogenic mspA lspE mutant, or heat-killed wild-type L. pneumophila. Responses from these immune CD4 T cells were then measured after infection of BMs with wild-type L. pneumophila, a mspA mutant, a mspA lspE mutant, and a mspA lspE dotA mutant (Fig. 5A). The mspA gene encodes an L. pneumophila protease that is secreted by the Lsp secretion system (13). Because MspA is responsible for the degradation of L. pneumophila proteins secreted into broth culture (26, 36), mspA mutants were used to diminish the possibility that other substrates secreted by the Lsp system would be rapidly degraded. CD4 T cells isolated from mice immunized with wild-type L. pneumophila gave responses to BMs infected with either a mspA mutant or a mspA lspE mutant that were similar to the responses observed for BMs infected with wild-type L. pneumophila. These data indicate that protein secretion by the Lsp system is not required for stimulation of immune CD4 T cells.
Interestingly, compared to the CD4 T cells from mice immunized with wild-type L. pneumophila, it was found that the CD4 T cells from mice immunized with the mspA lspE mutant were slightly less responsive to BMs infected with wild-type L. pneumophila, the mspA mutant, or the mspA lspE mutant, though this difference did not reach statistical significance (P value of 0.065). These data indicate that the Lsp system may be important for the priming of CD4 T cells in vivo. Because lsp mutants are less pathogenic than wild-type L. pneumophila (31), this priming defect might simply be due to reduced virulence. Alternatively, proteins secreted by the Lsp system may have a direct role in enhancing MHC-II presentation in vivo.
Although substrate proteins of the Lsp system are not secreted by an lspE mutant (30), they are still produced by this mutant and would become available for MHC-II presentation if intact L. pneumophila were degraded in lysosomes. To directly address whether Lsp substrates are major antigens recognized by immune CD4 T cells, secreted proteins were isolated from supernatants of wild-type L. pneumophila or dotA, mspA lspE, and mspA lspE dotA mutant strains grown in broth. These supernatant proteins were then added to BM cultures, and the responses of CD4 T cells isolated from mice immunized with wild-type L. pneumophila were measured (Fig. 5B). Proteins added directly to cultures of Ms are internalized by fluid-phase endocytosis, transported along the endocytic pathway to lysosomal compartments, processed, and loaded onto MHC-II (38). The CD4-mediated T-cell response did not diminish in response to L. pneumophila supernatants when proteins secreted by the Lsp system were eliminated. These data demonstrate that proteins secreted by the Lsp system were not required for maximal stimulation of immune CD4 T cells, consistent with the observation than CD4 T cells respond equally well to Ms infected with the mspA mutant strain and the mspA lspE mutant strain. From these data, we conclude that Lsp substrate proteins are not the major antigenic determinants of the CD4-mediated immune response generated during L. pneumophila infection, although their role in the process of priming of the T-cell response in vivo has yet to be determined.
In summary, these studies have defined events that are important for the presentation of L. pneumophila antigens on MHC-II after M infection. Establishment of the unique ER-derived vacuole that provides an initial safe haven for L. pneumophila within the M is clearly important for optimal stimulation of immune CD4 T cells. Flow cytometry data indicate that a subset of CD4 T cells primed during infection recognize antigens presented preferentially by L. pneumophila that reside within this specialized organelle. The finding that acidified endocytic organelles are required for MHC-II presentation of L. pneumophila antigens indicates that lysosomal degradation plays an important role in processing of the antigens that are presented by infected Ms. How antigens are delivered from the ER-derived vacuole to lysosomes remains unknown. It does not appear as if antigens secreted into the vacuole lumen by the Lsp system constitute the primary determinants of the adaptive CD4-mediated immune response against L. pneumophila. It is possible that a subset of immune CD4 T cells recognize unique L. pneumophila determinant that are produce intracellularly, but additional studies are required to identify antigens recognized by L. pneumophila-specific immune T cells. Determining the regulation, subcellular localization, and genetic requirements for presentation of these antigens should provide important details on the process by which L. pneumophila proteins become available for presentation on MHC-II.
ACKNOWLEDGMENTS
This work was supported in part by NIH grant AI48770 (C.R.R.).
We thank Jonathan Kagan for helpful discussions and critical review of the manuscript.
REFERENCES
1. Andersen, P. 1997. Host responses and antigens involved in protective immunity to Mycobacterium tuberculosis. Scand. J. Immunol. 45:115-131.
2. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392:245-252.
3. Berger, K. H., and R. R. Isberg. 1993. Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol. Microbiol. 7:7-19.
4. Celada, A., P. W. Gray, E. Rinderknecht, and R. D. Schreiber. 1984. Evidence for a gamma-interferon receptor that regulates macrophage tumoricidal activity. J. Exp. Med. 160:55-74.
5. Chen, J., K. S. de Felipe, M. Clarke, H. Lu, O. R. Anderson, G. Segal, and H. A. Shuman. 2004. Legionella effectors that promote nonlytic release from protozoa. Science 303:1358-1361.
6. Coers, J., J. C. Kagan, M. Matthews, H. Nagai, D. M. Zuckman, and C. R. Roy. 2000. Identification of Icm protein complexes that play distinct roles in the biogenesis of an organelle permissive for Legionella pneumophila intracellular growth. Mol. Microbiol. 38:719-736.
7. Conover, G. M., I. Derre, J. P. Vogel, and R. R. Isberg. 2003. The Legionella pneumophila LidA protein: a translocated substrate of the Dot/Icm system associated with maintenance of bacterial integrity. Mol. Microbiol. 48:305-321.
8. Cresswell, P. 1994. Assembly, transport, and function of MHC class II molecules. Annu. Rev. Immunol. 12:259-293.
9. Derre, I., and R. R. Isberg. 2004. Legionella pneumophila replication vacuole formation involves rapid recruitment of proteins of the early secretory system. Infect. Immun. 72:3048-3053.
10. Feeley, J. C., R. J. Gibson, G. W. Gorman, N. C. Langford, J. K. Rasheed, D. C. Mackel, and W. B. Baine. 1979. Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. J. Clin. Microbiol. 10:437-441.
11. Flynn, J. L., and J. Chan. 2001. Immunology of tuberculosis. Annu. Rev. Immunol. 19:93-129.
12. Fraser, D. W., T. R. Tsai, W. Orenstein, W. E. Parkin, H. J. Beecham, R. G. Sharrar, J. Harris, G. F. Mallison, S. M. Martin, J. E. McDade, C. C. Shepard, and P. S. Brachman. 1977. Legionnaires' disease: description of an epidemic of pneumonia. N. Engl. J. Med. 297:1189-1197.
13. Hales, L. M., and H. A. Shuman. 1999. Legionella pneumophila contains a type II general secretion pathway required for growth in amoebae as well as for secretion of the Msp protease. Infect. Immun. 67:3662-3666.
14. Harding, C. V., and H. J. Geuze. 1993. Immunogenic peptides bind to class II MHC molecules in an early lysosomal compartment. J. Immunol. 151:3988-3998.
15. Horwitz, M. A. 1983. Formation of a novel phagosome by the Legionnaires' disease bacterium (Legionella pneumophila) in human monocytes. J. Exp. Med. 158:1319-1331.
16. Horwitz, M. A. 1983. The Legionnaires' disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J. Exp. Med. 158:2108-2126.
17. Horwitz, M. A., and S. C. Silverstein. 1980. Legionnaires' disease bacterium (Legionella pneumophila) multiplies intracellularly in human monocytes. J. Clin. Investig. 66:441-450.
18. Kagan, J. C., and C. R. Roy. 2002. Legionella phagosomes intercept vesicular traffic from endoplasmic reticulum exit sites. Nat. Cell Biol. 4:945-954.
19. Kagan, J. C., M. P. Stein, M. Pypaert, and C. R. Roy. 2004. Legionella subvert the functions of rab1 and sec22b to create a replicative organelle. J. Exp. Med. 199:1201-1211.
20. Kagi, D., B. Ledermann, K. Burki, R. M. Zinkernagel, and H. Hengartner. 1996. Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo. Annu. Rev. Immunol. 14:207-232.
21. Lehner, P. J., and P. Cresswell. 1996. Processing and delivery of peptides presented by MHC class I molecules. Curr. Opin. Immunol. 8:59-67.
22. Luo, Z. Q., and R. R. Isberg. 2004. Multiple substrates of the Legionella pneumophila Dot/Icm system identified by interbacterial protein transfer. Proc. Natl. Acad. Sci. USA 101:841-846.
23. Merriam, J. J., R. Mathur, R. Maxfield-Boumil, and R. R. Isberg. 1997. Analysis of the Legionella pneumophila fliI gene: intracellular growth of a defined mutant defective for flagellum biosynthesis. Infect. Immun. 65:2497-2501.
24. Molmeret, M., O. A. Alli, S. Zink, A. Flieger, N. P. Cianciotto, and Y. A. Kwaik. 2002. icmT is essential for pore formation-mediated egress of Legionella pneumophila from mammalian and protozoan cells. Infect. Immun. 70:69-78.
25. Nagai, H., J. C. Kagan, X. Zhu, R. A. Kahn, and C. R. Roy. 2002. A bacterial guanine nucleotide exchange factor activates ARF on Legionella phagosomes. Science 295:679-682.
26. Nagai, H., and C. R. Roy. 2001. The DotA protein from Legionella pneumophila is secreted by a novel process that requires the Dot/Icm transporter. EMBO J. 20:5962-5970.
27. Neild, A. L., and C. R. Roy. 2003. Legionella reveal dendritic cell functions that facilitate selection of antigens for MHC class II presentation. Immunity 18:813-823.
28. Pamer, E. G., A. J. Sijts, M. S. Villanueva, D. H. Busch, and S. Vijh. 1997. MHC class I antigen processing of Listeria monocytogenes proteins: implications for dominant and subdominant CTL responses. Immunol. Rev. 158:129-136.
29. Perry, L. L., K. Feilzer, and H. D. Caldwell. 1997. Immunity to Chlamydia trachomatis is mediated by T helper 1 cells through IFN-gamma-dependent and -independent pathways. J. Immunol. 158:3344-3352.
30. Rossier, O., and N. P. Cianciotto. 2001. Type II protein secretion is a subset of the PilD-dependent processes that facilitate intracellular infection by Legionella pneumophila. Infect. Immun. 69:2092-2098.
31. Rossier, O., S. R. Starkenburg, and N. P. Cianciotto. 2004. Legionella pneumophila type II protein secretion promotes virulence in the A/J mouse model of Legionnaires' disease pneumonia. Infect. Immun. 72:310-321.
32. Roy, C. R., K. H. Berger, and R. R. Isberg. 1998. Legionella pneumophila DotA protein is required for early phagosome trafficking decisions that occur within minutes of bacterial uptake. Mol. Microbiol. 28:663-674.
33. Segal, G., and H. A. Shuman. 1998. How is the intracellular fate of the Legionella pneumophila phagosome determined Trends Microbiol. 6:253-255.
34. Sinai, A. P., and K. A. Joiner. 1997. Safe haven: the cell biology of nonfusogenic pathogen vacuoles. Annu. Rev. Microbiol. 51:415-462.
35. Sturgill-Koszycki, S., and M. S. Swanson. 2000. Legionella pneumophila replication vacuoles mature into acidic, endocytic organelles. J. Exp. Med. 192:1261-1272.
36. Szeto, L., and H. A. Shuman. 1990. The Legionella pneumophila major secretory protein, a protease, is not required for intracellular growth or cell killing. Infect. Immun. 58:2585-2592.
37. Tilney, L. G., O. S. Harb, P. S. Connelly, C. G. Robinson, and C. R. Roy. 2001. How the parasitic bacterium Legionella pneumophila modifies its phagosome and transforms it into rough ER: implications for conversion of plasma membrane to the ER membrane. J. Cell Sci. 114:4637-4650.
38. Unanue, E. R., and P. M. Allen. 1986. Biochemistry and biology of antigen presentation by macrophages. Cell. Immunol. 99:3-6.
39. Vogel, J. P., H. L. Andrews, S. K. Wong, and R. R. Isberg. 1998. Conjugative transfer by the virulence system of Legionella pneumophila. Science 279:873-876.
40. Wiater, L. A., K. Dunn, F. R. Maxfield, and H. A. Shuman. 1998. Early events in phagosome establishment are required for intracellular survival of Legionella pneumophila. Infect. Immun. 66:4450-4460.
41. Yamamoto, Y., T. W. Klein, C. A. Newton, R. Widen, and H. Friedman. 1988. Growth of Legionella pneumophila in thioglycolate-elicited peritoneal macrophages from A/J mice. Infect. Immun. 56:370-375.
42. Zuckman, D. M., J. B. Hung, and C. R. Roy. 1999. Pore-forming activity is not sufficient for Legionella pneumophila phagosome trafficking and intracellular growth. Mol. Microbiol. 32:990-1001.(Annie Neild, Takahiro Mur)