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The Journal of Immunology, 1999, 162: 6341-6350.
Copyright © 1999 by The American Association of Immunologists

Antigen Secreted from Noncytosolic Listeria monocytogenes Is Processed by the Classical MHC Class I Processing Pathway1

Heather L. Zwickey* and Terry A. Potter2,*

* Division of Basic Immunology, Department of Medicine, National Jewish Medical and Research Center, Denver, CO 80206; and Department of Immunology and the Cancer Center, University of Colorado Health Sciences Center, Denver, CO 80262


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Intracellular bacteria can reside in a vacuolar compartment, or they can escape the vacuole and become free living in the cytoplasm. The presentation of Ag by class I MHC molecules has been defined primarily for Ag present in the cytoplasm. It was therefore thought that Ags from bacteria that remain in a vacuole would not be presented by MHC class I molecules. Although some studies have provided data to support this idea, it is not necessarily true for all intracellular bacteria. For example, we have previously demonstrated that an epitope from the p60 protein secreted by LLO- Listeria monocytogenes, which does not reside in the cytoplasm, can be presented by MHC class I molecules to a T cell clone specific for the epitope, p60217–225. We have further examined the route by which Ag secreted by LLO- L. monocytogenes is presented by MHC class I molecules. Using pharmacological inhibitors, we demonstrate that MHC class I presentation of the p60 epitope derived from by LLO- L. monocytogenes requires phagolysosome fusion and processing by the proteasome. Lysosomal cathepsins, however, are not required for processing of the p60 epitope. Similarly, processing of the AttM epitope, secreted by LLO- L. monocytogenes and presented by H2-M3, also requires phagolysosome fusion and cleavage by the proteasome. Thus, p60 and AttM secreted by LLO- L. monocytogenes are processed via the classical class I pathway for presentation by MHC class I molecules.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The cell-mediated arm of the immune response is generally required to fight cellular pathogens such as viruses and intracellular bacteria. A key effector cell of this response is the CD8+ CTL, which recognizes peptide Ags presented by MHC class I molecules on the infected cell. The Ag processing pathway leading to presentation of antigenic peptides in association with MHC class I molecules involves several steps, including the digestion of ubiquitinated proteins by the proteasome into peptide fragments (reviewed in 1). Binding of the digested peptides to the TAP transporters located in the membrane of the endoplasmic reticulum (ER),3 enables them to be transported into the ER, where they may subsequently bind nascent MHC class I molecules. The peptide/MHC class I complexes are transported through the Golgi apparatus to the surface of the cell. The steps involved in this classical pathway of Ag processing have been defined using pharmacologic inhibitors and mutant cell lines.

Transcription and translation of viral encoded proteins are performed by the machinery of the host cell, and therefore epitopes from these proteins are readily presented by MHC class I molecules. In contrast, bacteria and intracellular pathogens other than viruses use their own machinery for transcription and translation. Furthermore, while some organisms become free living in the cytosol of the host cell, other organisms remain in the phagocytic vesicle, where they are thought to be sequestered from pathways leading to Ag presentation. Historic evidence suggested that epitopes from bacterial pathogens that were free living in the cytosol were readily presented by MHC class I molecules, whereas Ags from bacteria that remain in a vacuole were not presented (2, 3). There are several published reports in which we and others have demonstrated that at least some Ags from organisms retained within a phagocytic vesicle can be presented by MHC class I molecules (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). One hypothesis to explain how proteins from bacteria remaining in a vacuole can gain access to the MHC class I processing pathway is that the protein is actively transported out of the phagosome into the cytoplasm. An alternative hypothesis is that the phagosome ruptures, releasing phagocytosed material into the cytosol. The cytosolic Ag could then be processed by the classical class I pathway. In addition to the classical pathway of class I presentation, alternative mechanisms such as peptide regurgitation have been described (9). In this regurgitation pathway, Ag phagocytosed by the APC is digested in the phagosome and peptide fragments are excreted into the extracellular milieu, where they bind surface MHC class I molecules. Peptide regurgitation therefore bypasses the classical class I processing pathway, and it cannot be inhibited by the drugs that block classical class I processing.

The bacterium, Listeria monocytogenes, is an intracellular pathogen that can infect macrophages, hepatocytes, and fibroblasts. L. monocytogenes secretes a protein, listeriolysin O (LLO), that lyses the phagosome, allowing the escape of the bacterium from the phagosome into the cytoplasm of the cell. L. monocytogenes can replicate in the cytosol and can subsequently invade other cells. Proteins secreted by L. monocytogenes living in the cytosol are processed in the classical MHC class I processing pathway (15, 16, 17). These proteins include p60 and LLO, which contain epitopes presented by the classical MHC class I molecules, as well as other proteins or peptides, such as Lem A and AttM, which contain epitopes presented by the nonclassical MHC molecule, H2-M3. Mutant strains of L. monocytogenes that are unable to produce functional LLO (referred to herein as LLO- L. monocytogenes) do not escape the phagolysosome. It has been shown that these mutant bacteria do not enter the cytosol and are incapable of presenting some MHC class I-restricted epitopes to T cells (3). In contrast, epitopes from p60 secreted by noncytosolic L. monocytogenes can be presented in association with MHC class I molecules on the surface of APCs (18, 19). Since epitopes derived from LLO- L. monocytogenes can be presented by MHC class I molecules, other intracellular bacteria that remain in a phagocytic vesicle may also produce peptide epitopes that are presented by MHC class I molecules. In this paper we have examined whether Ag secreted by LLO- L. monocytogenes is processed by the classical MHC class I pathway. We have used pharmacological inhibitors of different processes of the classical MHC class I pathway to determine whether presentation of two different epitopes was affected. Our studies demonstrate that these epitopes produced by noncytosolic L. monocytogenes are presented by MHC class I molecules via the classical processing pathway. Furthermore, the classical route of Ag presentation occurs regardless of whether bacteria can divide in the cells.


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

BALB/c mice (H-2d) were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 mice (H-2b) were purchased from Taconic Farms (Germantown, NY).

Bacterial strains

Wild-type (LLO+) and mutant (LLO-PIPLC-PCPLC- (phosphatidylinositol phospholipase C negative and phosphatidylcholine phospholipase C negative)) strains of L. monocytogenes were obtained from Dr. Helene Marquis (Department of Microbiology, University of Colorado Health Sciences Center, Denver, CO). The LLO-PIPLC-PCPLC- strain was derived by eliminating the PrfA operon (20, 21). These bacteria do not produce LLO, PIPLC, or PCPLC. For experiments involving bacterial infections, overnight cultures of these strains of L. monocytogenes were diluted 1/10 with fresh TPB and grown for 3 h at 37°C. Bacteria were then harvested and washed twice in PBS before they were used to infect APCs.

APCs

The cell line J774A.1 (macrophage-like) was obtained from American Type Culture Collection (Manassas, VA) Cells were maintained in antibiotic-free MEM supplemented with 7.5% FCS. Bone marrow macrophages were derived from bone marrow harvested from either BALB/c or C57BL/6 mice. Cells were differentiated in antibiotic-free MEM supplemented with 7.5% FCS and GM-CSF.

Enriched peritoneal macrophage populations were harvested following i.p. injection of thioglycolate or protease peptone. Cells were harvested from the peritoneal cavity 4 or 3 days, respectively, postinjection and were cultured in antibiotic-free MEM supplemented with 7.5% FCS.

T cells reacting with epitopes derived from L. monocytogenes

The CTL clone referred to as 10Bp60 was generated by immunizing two 8- to 12-wk-old BALB/c mice i.p. with 5 x 102 CFU of wild-type L. monocytogenes. After 2 mo, the immunized mice were challenged i.v. with 2 x 103 CFU of wild-type L. monocytogenes. At 4 days postchallenge, the spleen and lymph node cells were harvested. RBCs were lysed with Tris-buffered ammonium chloride. Spleen and lymph node cells were cultured in MEM supplemented with nonessential amino acids, vitamins, 1 mM pyruvate, 50 µg/ml penicillin, 50 µg/ml streptomycin, 1 x 10-5 M 2-ME, and 10% FCS hereafter referred to as CTM) supplemented with 4% Con A Sup in 24-well plates at a concentration of 4 x 106/well. Irradiated J774 cells (1 x 105) infected with wild-type L. monocytogenes were added to each well as APCs for restimulation. In addition, irradiated spleen cells from a BALB/c mouse were added as feeder cells for each restimulation. The CTLs were restimulated every week with either L. monocytogenes-infected J774 cells or with J774 cells pulsed with the peptide KYGVSVQDI (an epitope of the p60 protein that binds to H-2Kd) on an alternating schedule. After 4 wk the CTLs were cloned by limiting dilution and screened for activity. After being cloned, 10Bp60 was maintained in culture by restimulation with J774A.1 cells infected with L. monocytogenes every 7 days.

The C10.4 cells were obtained from the lymph nodes and spleen of a TCR transgenic mouse (provided by Dr. Uwe Staerz, National Jewish Medical and Research Center). These cells recognize the peptide f-MIVTL in the context of the H2-M3 nonclassical MHC class I molecule (22). C10.4 was maintained in culture by restimulation with BMC 2.3 macrophages infected with wild-type L. monocytogenes.

Infection of APCs

Cells (1 x 105) in antibiotic-free MEM/10% FCS were infected with 100 µl of washed L. monocytogenes at various dilutions. After 35 min gentamicin was added at a final concentration of 5 µg/ml to kill remaining extracellular bacteria. Cells were infected for 7 h, at which point the medium was replaced with CTM. T lymphocytes were added at a final E:T cell ratio of 10:1 and were incubated with infected cells overnight (14 h).

Measurement of IFN-{gamma}

Supernatants from the overnight coculture of T cells and APCs infected with L. monocytogenes were assayed for the presence of IFN-{gamma} by ELISA. The capture Ab (XMG) and the detecting Ab (biotinylated R46A2) were used at 1 µg/ml. The assay was monitored with streptavidin conjugated with HRP and was developed colorimetrically. A control curve for titrated amounts of IFN-{gamma} (20 to 0.15 ng/ml) was run in parallel so that the amount of IFN-{gamma} could be quantitated.

Measurement of IL-12

Protease peptone- or thioglycolate macrophage-enriched populations, or bone marrow-derived macrophages were infected in antibiotic-free medium as described above. Gentamicin was added at 35 min to kill the remaining extracellular bacteria. After 7 h, the medium was removed and replaced with CTM. The cells were then incubated overnight at 37°C when 100 µl of supernatant was collected and assayed for the presence of IL-12 by ELISA.

The DuoSet Kit (Genzyme, Cambridge, MA) was used to measure IL-12 by ELISA. The IL-12-coating Ab was used at 3.0 µg/ml, and capture Ab was used at 1 µg/ml. The streptavidin-HRP conjugate was used in concert with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) peroxidase for detection. The IL-12 standard was diluted from 16.2 to 0.1 ng/ml, which is the lower level of detection for this system.

Use of pharmacological reagents to inhibit Ag processing

Bafilomycin, which inhibits a vacuolar ATPase, was dissolved in DMSO and added to APCs at a final concentration of 1 µM 30 min before infection with L. monocytogenes.

Lactacystin, an inhibitor of the proteasome, was obtained from E. J. Corey Laboratories (Harvard University, Boston, MA) and was added to APCs at a final concentration of 1 nM for 30 min before infection. Leupeptin, N-acetyl-Leu-Leu-norleucinal (LLnL), and acetyl-Leu-Leu Methional (LLM), which are protease inhibitors, were used at 0.75 M, 50 µM, and 100 µM, respectively, and were added to cells 30 min before infection. Ammonium chloride, which inhibits vacuolar pH, was added to APCs at the time of infection at a final concentration of 20 mM.

Assay for peptide regurgitation

C57BL/6 (H-2b) bone marrow macrophages were infected as described above. After infection, the cells were washed extensively and mixed with an equal number BALB/c (H-2d) bone marrow macrophages that had been fixed with 1% paraformaldehyde. 10Bp60 (H-2 Kd/p60 reactive) T cells were added, and 14 h later the supernatants were harvested and assayed for the presence of IFN-{gamma}. To measure regurgitation of the AttM epitope, B10.CAS fibroblasts (H2-M3a) were infected with L. monocytogenes for 7 h and then mixed with fixed C57BL/6 bone marrow macrophages and C10.4 (H2-M3b/AttM-reactive) T cells. To ensure that B10.CAS fibroblasts were infected with L. monocytogenes, 1 x 106 B10.CAS cells were infected for 7 h, then extensively washed and lysed in double-distilled H2O. Dilutions of these lysates were plated on Luria Bertoni agar, and after an overnight incubation the number of bacterial colonies was counted.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We have previously assayed the expression of antigenic epitopes of L. monocytogenes using CTL assays. A problem inherent to using CTL populations to monitor the expression of antigenic epitopes is that the uptake of L. monocytogenes by different cells in the macrophage populations is heterogeneous and not all cells are infected. Thus, the percent specific release of 51Cr from a population of cells infected with L. monocytogenes upon interaction with CTL can be quite low. We therefore analyzed the release of IFN-{gamma} from CD8+ T cell clones reactive with L. monocytogenes epitopes as a measure of epitope expression by the population of infected cells. An initial concern with this approach was that the macrophages may produce IL-12 upon infection with L. monocytogenes, and this IL-12 may elicit IFN-{gamma} production by the T cell clones in an Ag-independent manner. We therefore analyzed whether different populations of macrophages differed in their abilities to produce IL-12 in response to infection with wild-type or LLO- L. monocytogenes.

Macrophages elicited with thioglycolate from either BALB/c or C57BL/6 mice secrete IL-12 upon infection with L. monocytogenes. Although protease peptone-elicited macrophages from C57BL/6 mice produce IL-12 in response to infection, protease peptone-elicited macrophages from BALB/c mice do not produce measurable amounts of IL-12. In contrast, macrophages derived from the bone marrow from either BALB/c or C57BL/6 mice do not produce detectable IL-12 upon infection with L. monocytogenes. The production of IL-12 after infection of thioglycolate-elicited or protease peptone-elicited populations of macrophages with L. monocytogenes stimulates the secretion of IFN-{gamma} in an Ag-independent manner (data not shown). Therefore, bone marrow-derived macrophages that do not produce IL-12 in response to infection with L. monocytogenes were used in the experiments described herein.

Antigenic peptides from L. monocytogenes are not regurgitated from infected cells

One alternative pathway by which peptides can be loaded onto class I molecules is by the binding of peptides to MHC class I molecules expressed on the cell surface. This extracellular loading can occur following regurgitation of peptide from cells into the extracellular milieu (9). Peptide regurgitation allows peptide to bind to MHC molecules expressed on either the infected cell or uninfected bystander cells. To examine whether peptides from LLO- L. monocytogenes were being loaded onto cells following regurgitation of peptides, macrophages derived from C57BL/6 (H-2b) mice were infected with wild-type or LLO- L. monocytogenes. The p60 peptide, which stimulates 10Bp60 when bound to H-2Kd, does not stimulate these T cells in the presence of H-2b APCs only. Therefore, to stimulate the 10Bp60 cells, peptide must be released from the C57BL/6 cells and bind to H-2Kd on the surface of the fixed (and uninfected) BALB/c macrophages. Secretion of IFN-{gamma} by 10Bp60 cells was used as a readout for Ag presentation. As shown in Fig. 1GoA, the addition of synthetic peptide to the fixed cells activates 10Bp60 to produce IFN-{gamma}. Incubation of fixed BALB/c macrophages and infected C57BL/6 macrophages did not elicit IFN-{gamma} production by 10Bp60, suggesting that peptide regurgitation does significantly contribute to the Ag presentation of p60.



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  		FIGURE 1. Cells infected with LLO+ or LLO- L. monocytogenes do not regurgitate either the p60 (A) or the AttM (B) epitope onto fixed bystander macrophages. C57BL/6 macrophages (A) were infected at a MOI of 50. Fibroblasts from B10.CAS mice (B) were infected with L. monocytogenes for 3 h before addition of gentamicin; the infection was continued for a total of 7 h. The cultures also contained BALB/c bone marrow-derived macrophages that had been fixed by treatment with 1% paraformaldehyde. After 7 h the cells were washed, and either 10Bp60 (A) or C10.4 (B) T cells were added at a ratio of 10:1 and cultured overnight. Peptide cultures received 10 ng/ml of the synthetic Ag peptide. Control cultures received no peptide. Supernatants were collected and analyzed for the presence of IFN-{gamma} by ELISA.

 
A similar experiment was performed using C10.4 T cells that recognize the AttM epitope bound to the H2-M3 nonclassical MHC class I molecule. Since both C57BL/6 and BALB/c macrophages express H2-M3, fibroblasts obtained from the B10.CAS mouse, which has a mutation in H2-M3 that prevents presentation to C10.4, were infected with L. monocytogenes. The infected B10.CAS cells were mixed with fixed C57BL/6 macrophages and incubated with C10.4 T cells. The coculture of B10.CAS fibroblasts infected with L. monocytogenes and fixed C57BL/6 macrophages did not elicit IFN-{gamma} production by C10.4 T cells (Fig. 1GoB). This suggests that peptide regurgitation from infected cells is insufficient to activate C10.4. We conclude that release of peptide into the extracellular milieu is not the primary mechanism by which cells infected with LLO- L. monocytogenes present antigenic epitopes from p60 or AttM.

Phagolysosome fusion is required for Ag presentation of p60 and AttM epitopes

We used pharmacological inhibitors to determine which cellular organelles were required for presentation of Ags derived from LLO- L. monocytogenes. The inhibitors used in these experiments did not have a direct effect on the growth of L. monocytogenes (data not shown) and were added to bone marrow macrophages before the infection with L. monocytogenes. If the activity of the inhibitor was irreversible, it was removed by washing the cells before infection; however, if the action of the inhibitor was transient, the inhibitor was maintained in the culture throughout the entire experiment. Secretion of IFN-{gamma} by p60- or AttM-specific CTLs was used to monitor Ag presentation.

Following phagocytosis of L. monocytogenes by macrophages, the bacteria resides in a phagosome that fuses with lysosomes to form an acidic phagolysosome. Bacteria such as LLO- L. monocytogenes that are unable to escape the phagolysosome are maintained within this compartment. In contrast, wild-type L. monocytogenes produce LLO that, under conditions of acidic pH, permeabilizes the phagolysosome, allowing the bacteria to escape into the cytoplasm. Bafilomycin has multiple effects on cells infected with L. monocytogenes. Importantly, bafilomycin inhibits a vacuolar ATPase and thereby prevents acidification of vacuoles, phagosomes, and lysosomes (23, 24, 25). In the presence of bafilomycin, both LLO+ and LLO- L. monocytogenes remain within the phagosome, since LLO cannot function at a neutral pH (26). Furthermore, bafilomycin inhibits the fusion of the phagosome with the lysosome (45). We used bafilomycin to test whether acidification of the phagosome or fusion of the phagosome and lysosomes is required for presentation of LLO- L. monocytogenes. As demonstrated in Fig. 2GoA, bafilomycin completely inhibits presentation of the p60 epitope recognized by 10Bp60 in cells infected with either wild-type or LLO- bacteria. Bafilomycin also blocks presentation of the AttM epitope recognized by C10.4 (Fig. 2GoC). Bafilomycin does not affect the presentation of exogenously added peptides (Fig. 2Go, B and D). In control experiments, bafilomycin does not affect the class I presentation of peptide derived from cytosolic protein (data not shown). This suggests that an acidic pH in the phagosome or fusion with lysosomes is required for processing and/or presentation of the epitopes from p60 and AttM.



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  		FIGURE 2. Phagolysosome fusion is required for the processing of both the p60 (A and B) and the AttM (C and D) epitopes. BALB/c (A) or C57BL/6 (C) bone marrow-derived macrophages were cultured in the presence (open symbols) or the absence (filled symbols) of bafilomycin for 30 min. These cells were then infected with LLO+ (squares) or LLO- (circles) L. monocytogenes at several MOIs for 7 h. Bafilomycin-treated (diamonds) or untreated (triangles) BALB/c (B) or C57BL/6 (D) bone marrow-derived macrophages were pulsed with the antigenic peptide. After 7 h cells were washed, and 10Bp60 (A and B) or C10.4 (C and D) T cells were added at a ratio of 10:1. Supernatants from the overnight cocultures were analyzed for the presence of IFN-{gamma} by ELISA.

 
Processing by calpain, cathepsin S, cathepsin B, or cathepsin L is not required for presentation of p60 or AttM from LLO- L. monocytogenes

When the phagosome fuses with the lysosome, lysosomal proteases such as cathepsins have access to phagocytosed proteins. Cathepsin S, cathepsin L (a cysteine-type protease), and cathepsin B (a serine protease) are involved in the processing of endocytosed Ags presented by MHC class II molecules. Similarly, proteins secreted into the phagocytic vesicle by LLO- L. monocytogenes may be digested by lysosomal cathepsins. The protease inhibitorLLM blocks the proteolytic activity of calpain and the lysosomal cathepsins B and L (27, 28). It was unlikely that LLM would affect processing of p60 secreted by wild-type L. monocytogenes because wild-type L. monocytogenes resides in the cytoplasm. We examined whether LLM had an effect on processing of p60 secreted by LLO- L. monocytogenes. As shown in Fig. 3Go LLM does not inhibit processing and presentation of the p60 epitope to 10Bp60 in cells infected with either wild-type or LLO- L. monocytogenes. This suggests that cathepsins B and L are not required for processing of p60 secreted by L. monocytogenes even if the bacteria reside in the phagolysosome. To confirm that the concentration of LLM used in these experiments was able to inhibit the activity of cathepsin B and L, we demonstrated that it could inhibit the processing of the MHC class II invariant chain (data not shown).



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  		FIGURE 3. Presentation of the p60 (A–C) and the AttM (D–F) epitopes does not require cathepsin B, cathepsin L, or calpain. BALB/c (A and B) or C57BL/6 (D and E) bone marrow-derived macrophages were cultured in the presence (open symbols) or the absence (filled symbols) of LLM for 30 min. These cells were then infected with LLO+ (squares) or LLO- (circles) L. monocytogenes at several MOIs for 7 h. LLM-treated (diamonds) or untreated (triangles) BALB/c (C) or C57BL/6 (F) bone marrow-derived macrophages were pulsed with the antigenic peptide. After 7 h, cells were washed, and 10Bp60 (A–C) or C10.4 (D–F) T cells were added at a ratio of 10:1. Supernatants from the overnight cocultures were analyzed for the presence of IFN-{gamma} by ELISA.

 
Leupeptin is a protease inhibitor that blocks cathepsin S in addition to cathepsins B and L (29, 30, 31, 32). Cathepsin S is involved in processing Ag in the endocytic pathway destined for presentation by MHC class II molecules on professional APCs, such as macrophages (33). We tested the effects of leupeptin on the processing and presentation of the p60 epitope secreted by both wild-type and LLO- L. monocytogenes. As shown in Fig. 4Go leupeptin did not inhibit presentation of the p60 epitope to 10Bp60 T cells. In control experiments (not shown) processing of the invariant chain was inhibited by the addition of leupeptin. Therefore, processing of p60 secreted by LLO- L. monocytogenes does not require cathepsin S.



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  		FIGURE 4. Presentation of the p60 (A–C) and the AttM (D–F) epitopes does not require cathepsin S. BALB/c (A and B) or C57BL/6 (D and E) bone marrow-derived macrophages were cultured in the presence (open symbols) or the absence (filled symbols) of leupeptin for 30 min. These cells were then infected with LLO+ (squares) or LLO- (circles) L. monocytogenes at several MOIs for 7 h. Leupeptin-treated (diamonds) or untreated (triangles) BALB/c (C) or C57BL/6 (F) bone marrow-derived macrophages were pulsed with the antigenic peptide. After 7 h, cells were washed, and 10Bp60 (A–C) or C10.4 (D–F). T cells were added at a ratio of 10:1. Supernatants from the overnight cocultures were analyzed for the presence of IFN-{gamma} by ELISA.

 
As discussed previously, the AttM epitope recognized by C10.4 is a short, hydrophobic peptide that requires some processing before presentation. AttM secreted by L. monocytogenes must be processed by cellular proteases before presentation by H2-M3, because the AttM peptide is 23 aa long, and the maximum length for peptides binding to H2-M3 is around 9 aa. Furthermore, the ideal length of AttM for eliciting a response by C10.4 T cells is 7 aa (22). Presentation of the AttM epitope secreted by LLO- L. monocytogenes does not require processing by the lysosomal cathepsins B, L, or S (Figs. 3Go and 4Go). The inability of LLM and leupeptin to block processing epitopes from p60 or AttM secreted from LLO- L. monocytogenes demonstrates that neither calpains nor cathepsins B, S, and L are required for processing of epitopes from AttM or p60.

Processing of p60 secreted by LLO- L. monocytogenes requires the proteasome

Most cytosolic proteins require digestion by the proteasome to create epitopes for presentation by MHC class I molecules. The proteasome cleaves ubiquitinated proteins into peptide fragments that can be transported into the ER where they bind newly synthesized class I molecules. Therefore, the proteasome is one of the components of the classical MHC class I pathway. The generation of the p60 epitope from wild-type L. monocytogenes located in the cytosol requires processing by the proteasome. If the polypeptides p60 and AttM secreted by LLO- L. monocytogenes traffic to the cytoplasm, the proteasome could be involved in processing them to antigenic epitopes.

Pharmacologic inhibitors of the proteasome were used to determine whether the proteasome is involved in the processing of AttM and p60. The protease inhibitor LLnL transiently blocks lysosomal cathepsins and in addition prevents processing by the proteasome (28, 34). When APCs were incubated with LLnL before and during infection with either LLO+ or LLO- L. monocytogenes, there was no processing and presentation of p60 or AttM epitopes (Fig. 5Go, A and C). The presentation of synthetic peptide was slightly inhibited by LLnL (Fig. 5Go, B and D), and this was probably due to a reduced level of class I molecules on the surface of the cell. Since the proteasome inhibitor LLnL blocks presentation of both p60 and AttM in cells infected with either wild-type or LLO- L. monocytogenes, the proteasome is involved in the processing of these two epitopes.



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  		FIGURE 5. LLnL blocks processing of p60 and AttM. BALB/c (A) or C57BL/6 (C) bone marrow-derived macrophages were cultured in the presence (open symbols) or the absence (filled symbols) of LLnL for 30 min. These cells were then infected with LLO+ (squares) or LLO- (circles) L. monocytogenes at several MOIs for 7 h. LLnL-treated (diamonds) or untreated (triangles) BALB/c (B) or C57BL/6 (D) bone marrow-derived macrophages were pulsed with the antigenic peptide. After 7 h, cells were washed, and 10Bp60 (A and B) or C10.4 (C and D) T cells were added at a ratio of 10:1. Supernatants from the overnight cocultures were analyzed for the presence of IFN-{gamma} by ELISA.

 
We also investigated the effects of another proteasome inhibitor, lactacystin, on presentation of Ags from L. monocytogenes. Lactacystin inhibits the proteasome through binding irreversibly to the ß-threonine subunit (35, 36). This property allows cells to be preincubated with lactacystin and washed before infection. For this reason, there is no effect of lactacystin on T cells or L. monocytogenes. Also, unlike LLnL, which is a peptide analogue inhibitor and therefore affects both the proteasome and other cellular proteases, lactacystin has only been reported to affect the proteasome. Similar to LLnL, lactacystin inhibited processing of p60 (Fig. 6GoA) and AttM (Fig. 6GoC) secreted from both wild-type and LLO- L. monocytogenes. Lactacystin does not significantly reduce the level of expression of H-2Kd (data not shown.) Therefore, the observed effects of lactacystin are most likely due to its effects on the proteasome.



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  		FIGURE 6. The proteasome is required for processing of both the p60 (A and B) and the AttM (C and D) epitopes. BALB/c (A) or C57BL/6 (C) bone marrow-derived macrophages were cultured in the presence (open symbols) or the absence (filled symbols) of lactacystin for 30 min. These cells were then washed and infected with LLO+ (squares) or LLO- (circles) L. monocytogenes at several MOIs for 7 h. Lactacystin-treated (diamonds) or untreated (triangles) BALB/c (B) or C57BL/6 (D) bone marrow-derived macrophages were pulsed with the antigenic peptide. After 7 h, cells were washed again, and 10Bp60 (A and B) or C10.4 (C and D) T cells were added at a ratio of 10:1. Supernatants from the overnight cocultures were analyzed for the presence of IFN-{gamma} by ELISA.

 
The complete inhibition of processing of p60 and AttM by LLnL and lactacystin implicates the proteasome in the processing of both these Ags following secretion by LLO- L. monocytogenes. However, the observation that both p60 and AttM require processing by the proteasome also suggests that although the LLO- L. monocytogenes does not enter the cytoplasm of the cell, the p60 and AttM polypeptides are present in the cytosol.

Presentation of AttM requires TAP but does not require the invariant chain

In addition to pharmacological inhibitors, MHC processing and presentation can also be investigated using TAP knockout mice. These mice are on the H-2b genetic background and are therefore not appropriate for analyzing presentation of p60 epitopes, but can be used to analyze the processing of AttM and its presentation by H2-M3 molecules, which is identical in H-2b and H-2d haplotypes.

One potential mechanism of cross-talk leading to presentation by MHC class I molecules is a class II-like pathway in which class I molecules traffic with the class II invariant chain to a class II-like compartment (37). This trafficking would place the MHC class I molecule in the same compartment as the Ag, facilitating the direct binding of the peptide to the class I molecule. This pathway is disrupted in the invariant chain knockout mouse (38). Ag presentation by bone marrow macrophages derived from the invariant chain knockout mouse was analyzed to determine whether this pathway contributes to the presentation of AttM secreted by LLO- L. monocytogenes. Macrophages from invariant chain knockout mice infected with LLO- L. monocytogenes (Fig. 7GoC) were able to present AttM as efficiently as macrophages from C57BL/6 mice (Fig. 7GoA). This suggests that the invariant chain is not significantly involved in the presentation of AttM from L. monocytogenes within the phagolysosome.



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  		FIGURE 7. Processing of AttM requires TAP (E), but does not require the invariant chain (D). C57BL/6 (A and B), invariant chain-/- (C and D), or TAP-/- (E and F) bone marrow-derived macrophages were infected with LLO+ (filled squares) or LLO- (filled circles) L. monocytogenes at various MOIs for 7 h. In the right panels the antigenic peptide was used to pulse bone marrow macrophages. After 7 h the cells were washed, and C10.4 T cells were added at a ratio of 10:1. Supernatants from the overnight coculture were collected and analyzed for IFN-{gamma} secretion by ELISA.

 
The evidence obtained from the use of pharmacological inhibitors suggested that AttM secreted by LLO- L. monocytogenes uses the classical class I processing pathway, including the proteasome, for presentation by MHC class I molecules. Bone marrow macrophages from mice in which the proteasomal subunits, LMP2 and LMP7, have been deleted were not capable of presenting AttM to C10.4 T cells even when pulsed with 100 µg/ml of synthetic AttM peptide (data not shown.) It is likely that the inability of these macrophages to present exogenously added peptide to C10.4 T cells is due to low levels of H2-M3 expression in LMP knockout mice. As a consequence, the inability of macrophages from LMP knockout mice to present AttM upon infection with L. monocytogenes is difficult to interpret.

One component of the classical MHC class I processing pathway for which there is no pharmacological inhibitor is the TAP transporter. The TAP transporter knockout mouse has been derived and is unable to transport cytoplasmic peptides into the ER (39). Macrophages from TAP knockout mice were used to determine whether TAP transport is required for processing and presentation of AttM. As shown in Fig. 7GoE, macrophages derived from TAP-/- mice were unable to present AttM to C10.4 T cells. The level of expression of H2-M3 on the surface of macrophages derived from TAP-/- mice is likely to be very low, because pulsing TAP-/- macrophages with the AttM peptide elicits only low levels of IFN-{gamma} secretion by C10.4 T cells. Similarly, TAP-defective RMA/S cells are unable to present the AttM peptide to C10.4 T cells (data not shown). Therefore, the observation that macrophages from TAP-/- mice are unable to present Ag to T cells following infection with L. monocytogenes is consistent with involvement of the TAP transporter in presentation of this epitope; however, these experiments are also difficult to interpret.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Wild-type LLO+ L. monocytogenes escape from the phagosome and reside in the cytosol of the infected cell. It is therefore not surprising that proteins derived from LLO+ L. monocytogenes are processed to antigenic epitopes by the classical MHC class I processing pathway. Previous studies claimed that epitopes from proteins secreted by LLO- L. monocytogenes that are retained within the phagolysosome are not presented by MHC class I molecules (2, 3). In contrast, we recently demonstrated that T cells specific for the secreted protein, p60, were able to lyse cells infected with LLO- L. monocytogenes (10). In addition, cells infected with three different strains of LLO- L. monocytogenes were recognized by T cells isolated from the spleens of infected mice (13). Thus, it appears that at least some Ags produced by LLO- L. monocytogenes are indeed presented by MHC class I molecules. It is possible that the discrepancy in the ability to detect the expression of epitopes from LLO- L. monocytogenes may be due to the source of the epitopes recognized by the T cell. For example, in some experiments performed by Bouwer and co-workers, the bulk population of T cells was maintained in culture by restimulating with cells infected with heat-killed bacteria (3). Since heat-killed bacteria do not secrete Ag, only T cells specific for nonsecreted Ag are likely to be stimulated by this method. It may be that secreted proteins, such as p60, or short polypeptides, such as the AttM attenuator peptide, are much more likely to contribute to the epitopes recognized following infection with LLO- L. monocytogenes. In addition, epitopes of the secreted LLO protein are immunodominant, and therefore, many Listeria-specific T cells contained in a bulk population are specific for epitopes of LLO (17). These T cells would be unable to respond to cells infected with LLO- L. monocytogenes.

One mechanism by which epitopes from intracellular bacteria can be presented by MHC class I molecules is peptide regurgitation. Phagocytic cells infected with Escherichia coli containing the OVA minigene can release, or regurgitate, the OVA epitope, which could then bind to MHC molecules expressed on the surface of uninfected cells (9). We did not observe peptide regurgitation in the L. monocytogenes system; however, we used a different bacterium, and the Ags recognized by our T cells are secreted by the bacteria. In contrast, the experiments in which peptide regurgitation was observed targeted the Ag (OVA) to either the periplasm or the surface of the bacterium. Furthermore, the recombinant E. coli contained an OVA minigene construct that encoded only the epitope that binds to H-2Kb. In contrast, both epitopes that we examined (p60 and AttM) required further processing before presentation. Because peptide regurgitation did not significantly contribute to presentation of the p60 or AttM epitopes from LLO- L. monocytogenes, we examined other potential routes of processing for presentation by MHC class I molecules.

The effects of bafilomycin established that either phagolysosome fusion or vacuolar acidification was required for presentation of both LLO+ and LLO- L. monocytogenes. Bafilomycin inhibits a vacuolar ATPase, thereby blocking both acidification of the phagosome and phagosome lysosome fusion. An acidic pH in the phagosome is required for the lytic activity of LLO (26), and bafilomycin inhibits pore formation by LLO. We therefore expected that the effects of bafilomycin on the presentation of Ags from LLO+ and LLO- L. monocytogenes would be similar. Indeed, bafilomycin blocked presentation of p60 and AttM secreted by wild-type and LLO- L. monocytogenes. Phagosome lysosome fusion could be required for presentation of Ag from the phagolysosome for a variety of reasons. For example, there could be a protein in the lysosome required for transporting the antigenic protein into the cytoplasm. Another possibility is that heat shock proteins may be required for protecting antigenic epitopes. Although heat shock proteins are present in the lysosome, they have not been demonstrated in the phagosome.

Phagolysosome fusion could also be required because lysosomal proteases are responsible for digesting the proteins into peptides that can bind to MHC molecules. If this is the case, blocking the activity of lysosomal proteases could affect presentation of p60 or AttM epitopes. We used the inhibitors LLM, leupeptin, and LLnL to block the lysosomal cathepsins B, L, and S. LLM and leupeptin had no effect on presentation of the p60 or AttM epitopes, suggesting that cathepsins B, L, and S were not involved in the processing of Ags secreted by LLO- L. monocytogenes. LLnL is a peptide aldehyde inhibitor that blocks cleavage by the proteasome as well as by cathepsins. LLnL blocked processing of both p60 and AttM. We also used a second inhibitor of the proteasome, lactacystin, to determine whether activity of the proteasome was required for presentation of the p60 or AttM epitopes. Lactacystin inhibits the activity of the proteasome by a different mechanism than LLnL. Lactacystin completely blocked presentation of both the p60 and AttM epitopes, suggesting that the proteasome was required for processing and/or presentation of these epitopes. Overall, these results suggest that p60 and AttM are processed by the classical pathway for presentation by MHC class I molecules.

Brefeldin A is a pharmacological inhibitor that blocks transport from the Golgi to the surface of the cell and is often used to inhibit classical presentation of Ag by MHC class I molecules. Brefeldin A acts transiently, and upon removal of the drug, protein transport quickly (within 5 min) resumes. Therefore, experiments that use brefeldin A require that the drug be present throughout the course of the experiment. Unfortunately, we were unable to use brefeldin A in these experiments because the presence of brefeldin A in the medium prevents the secretion of IFN-{gamma} by the T cells.

Using OVA coupled to latex beads, other workers have demonstrated that exogenous Ags phagocytosed by macrophages can be processed for presentation by MHC class I molecules (reviewed in Refs. 40 and 41). Ag coupled to latex beads and LLO- L. monocytogenes are similar systems in that both are phagocytosed into intracellular compartments surrounded by an organelle membrane, yet epitopes from each are presented by MHC class I molecules. The ability of OVA coupled to latex beads to be processed and presented by MHC class I molecules is analogous to our observation that p60 and AttM secreted by LLO- L. monocytogenes are presented by MHC class I molecules to T cells. The processing of OVA coated onto latex beads is resistant to chloroquine, suggesting that an acidic pH in the endocytic compartments is not required. Furthermore, the inability of leupeptin to inhibit presentation of the OVA epitope indicates that lysosomal cathepsins are not involved in the processing of OVA. The requirement of the proteasome for the processing of exogenously added OVA suggests that the Ag is degraded in the cytoplasm of the cell. Therefore, the pathway by which exogenous OVA is processed for presentation by MHC class I molecules is similar to the pathway used by p60 and AttM secreted by LLO- L. monocytogenes. Using the TAP knockout mouse, it was further shown that presentation of exogenous OVA requires TAP transport into the endoplasmic reticulum. Bone marrow macrophages from TAP-/- mice are unable to present AttM to C10.4 T cells following infection with wild-type or LLO- L. monocytogenes, suggesting that processing of AttM requires TAP transport into the endoplasmic reticulum. Thus, a classical route of processing for presentation by MHC class I molecules is likely in both of these systems.

The isolation of CD8+ T lymphocytes that recognize Ags from other bacteria that remain within a vacuolar compartment is consistent with the idea that proteins from these bacteria are presented in association with MHC class I molecules. However, relatively few epitopes have been identified for these pathogens (42, 43). The expression of model Ags by recombinant bacteria such as Salmonella, E. coli, and Calmette-Guérin bacillus demonstrates that Ags from vacuolar bacteria are presented by MHC class I molecules (7, 9, 14, 44). Since the p60 and AttM epitopes were previously identified using wild-type L. monocytogenes, LLO- L. monocytogenes provides a good model to identify the pathway by which Ag secreted by noncytoplasmic bacteria can be presented by MHC class I molecules.


   Acknowledgments
 
We thank Drs. Mike Princiotta and Uwe Staerz (National Jewish Medical and Research Center) for providing the C10.4 T cells, and Dr. Helene Marquis (University of Colorado Health Sciences Center) for the generous gift of the mutant strains of L. monocytogenes and her technical advice.


   Footnotes
 
1 This work was supported by Grants AI37905 and AI38407 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Terry Potter, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206-2761. E-mail address: Back

3 Abbreviations used in this paper: ER, endoplasmic reticulum; LLO, listeriolysin O; PIPLC, phosphatidylinositol phospholipase C; PCPLC, phosphatidylcholine phospholipase C; CTM, MEM supplemented with nonessential amino acids, vitamins, 1 mM pyruvate, 50 µg/ml penicillin, 50 µg/ml streptomycin, 1 x 10-5 M 2-ME, and 10% FCS; MOI, multiplicity of infection; LLnL, N-acetyl-L-leucyl-L-norleucinal; LLM, acetyl-Leu-Leu Methional. Back

Received for publication October 26, 1998. Accepted for publication March 9, 1999.


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