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The Journal of Immunology, 1999, 162: 4781-4789.
Copyright © 1999 by The American Association of Immunologists

Enhancement of the Listeria monocytogenes p60-Specific CD4 and CD8 T Cell Memory by Nonpathogenic Listeria innocua1

Gernot Geginat2,*, Thomas Nichterlein*, Marianne Kretschmar*, Simone Schenk*, Herbert Hof*, Mio Lalic-Mülthaler{dagger}, Werner Goebel{dagger} and Andreas Bubert4,{dagger}

* Institut für Medizinische Mikrobiologie und Hygiene, Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Mannheim, Germany; and {dagger} Lehrstuhl für Mikrobiologie, Theodor-Boveri-Institut für Biowissenschaften, Am Hubland, Würzburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The contact of T cells to cross-reactive antigenic determinants expressed by nonpathogenic environmental micro-organisms may contribute to the induction or maintenance of T cell memory. This hypothesis was evaluated in the model of murine Listeria monocytogenes infection. The influence of nonpathogenic L. innocua on the L. monocytogenes p60-specific T cell response was analyzed. We show that some CD4 T cell clones raised against purified p60 from L. monocytogenes cross-react with p60 purified from L. innocua. The L. monocytogenes p60-specific CD4 T cell clone 1A recognized the corresponding L. innocua p60 peptide QAAKPAPAPSTN, which differs only in the first amino acid residue. In vitro experiments revealed that after L. monocytogenes infection of APCs, MHC class I-restricted presentation of p60 occurs, while MHC class II-restricted p60 presentation is inhibited. L. innocua-infected cells presented p60 more weakly but equally well in the context of both MHC class I and MHC class II. In contrast to these in vitro experiments the infection of mice with L. monocytogenes induced a strong p60-specific CD4 and CD8 T cell response, while L. innocua infection failed to induce p60-specific T cells. L. innocua booster infection, however, expanded p60-specific memory T cells induced by previous L. monocytogenes infection. In conclusion, these findings suggest that infection with a frequently occurring environmental bacterium such as L. innocua, which is nonpathogenic and not adapted to intracellular replication, can contribute to the maintenance of memory T cells specific for a related intracellular pathogen.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Listeria monocytogenes is a facultative intracellular bacterium that can cause severe systemic infections in human groups at risk, i.e., in immunosuppressed and elderly people, as well as in neonates and pregnant women (1). It represents the best studied member of the genus Listeria, which includes five additional species, L. ivanovii, L. innocua, L. seeligeri, L. welshimeri, and L. grayi. In addition to L. monocytogenes the only other potentially pathogenic species of the genus is L. ivanovii (2). All other members are considered harmless environmental bacteria. L. innocua shows the closest phylogenetic relationship to L. monocytogenes (3) and is the most frequent listerial isolate from human feces (4) and various food sources (5, 6, 7, 8). Pathogenic and nonpathogenic Listeria species differ in multiple pathogenicity-associated gene loci. The gene products of the PrfA-dependent gene cluster, which are crucial for the release from the host cell phagosome, intracellular replication, intracellular movement, and the cell-to-cell spread of pathogenic Listeria species (9), are lacking in L. innocua. In addition, genes coding for internalins, some of which have been shown to play a role in the bacterial uptake by various types of mammalian cells, do not seem to be present in nonpathogenic Listeria species (10).

The established model of the murine L. monocytogenes infection is characterized by the development of a long-lasting T cell-dependent immunity (11). The murine L. monocytogenes infection induces an initial induction and activation of Ag-specific effector T cells followed by subsequent contraction by apoptosis and the establishment of a T cell memory compartment (12, 13). It remains uncertain whether the prolonged life span of memory T cells is an intrinsic property of these cells or whether it reflects their intermittent stimulation by residual deposits of specific Ag, through cross-reactive contact with environmental Ags or through bystander stimulation (14, 15, 16). The investigation of listerial target Ags recognized by CD4 and CD8 T cells identified several secreted proteins of L. monocytogenes as potent T cell Ags. The listeriolysin O (LLO)3 and the p60 protein from L. monocytogenes are targets for both CD4 and CD8 T cells (17, 18, 19, 20). It has been shown that LLO-specific CD8 T cell clones and p60-specific CD4 or CD8 T cell clones mediate protective immunity against L. monocytogenes in vivo (20, 21, 22). In contrast to the PrfA-regulated virulence factors of L. monocytogenes, the PrfA-independent p60 protein is essential for cell viability, and it acts basically as a murein hydrolase required in a late step of cell division (23). It is the major extracellular protein from pathogenic L. monocytogenes and contributes to the uptake of this pathogen into some mammalian cell types such as fibroblasts and macrophages (23, 24, 25, 26). Because of the essential function of p60 for bacterial cell division, it is not surprising that a p60-related protein is produced by all members of the genus Listeria (25). The amino acid sequence comparison of the whole p60 protein family revealed that the amino- and carboxyl-terminal portions of p60 are highly conserved in all Listeria species, while the inner portions of p60 differ in a species-specific way (25).

The remarkable conservation of the p60 protein, which is a strong target Ag for protective L. monocytogenes-specific CD4 and CD8 T cells, provides an excellent model to study the influence of a related nonpathogenic species on the T cell-mediated immunity against a pathogenic bacterium. We show that infection with an environmental nonpathogenic bacterium such as L. innocua, which by itself is unable to induce a primary p60-specific T cell response, mediates the expansion of p60-specific memory T cells induced by previous L. monocytogenes infection. These findings suggest that contact with a frequently occurring harmless bacterium that is not adapted to an intracellular life can contribute to the maintenance of memory T cells specific for an antigenically related intracellular pathogen.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains

L. monocytogenes serovar 1/2a EGD, L. innocua sv6b, and the actA deletion mutant L. monocytogenes {Delta}actA were taken from the strain collection of the Institute of Microbiology at the University of Würzburg (Würzburg, Germany). The listeriolysin deletion mutant L. monocytogenes {Delta}hly was obtained from D. Portnoy, University of Pennsylvania (Philadelphia, PA), and Bacillus subtilis DB104 was provided by R. Doi, University of California (Davis, CA). All bacteria were grown in brain-heart infusion broth (Difco, Augsburg, Germany). The bacterial concentration was estimated by determination of the OD600 and was confirmed by colony counts on sheep blood agar plates.

Mice

Female BALB/cOlaHsd (H-2d) mice were purchased (Harlan-Winkelmann, Borchen, Germany), kept under conventional conditions, and used at 6–8 wk of age. For long term experiments mice were kept under specific pathogen-free conditions in a laminar air flow container.

Infection of mice and in vivo protection assay

Mice were infected by i.v. injection of Listeria in 0.2 ml of PBS. Primary L. monocytogenes infection was performed with 1 x 103 CFU of L. monocytogenes. Secondary L. monocytogenes infection was performed with 1 x 106 CFU of L. monocytogenes. Priming with low dose L. monocytogenes was performed with a dose of 1 x 102 CFU. Priming with high dose L. monocytogenes was performed with 5 x 103 CFU. Primary and secondary L. innocua infections were performed with 2 x 107 CFU. L. innocua booster infection of L. monocytogenes-primed mice was performed with 1 x 106 CFU. For the in vivo protection assay immunized mice were challenged by i.v. injection of 1 x 104 CFU of L. monocytogenes. Spleen and liver were removed 72 h after infection and were homogenized in Tenbroeck tissue grinders (Wheaton, Millville, NY) with 10 ml of sterile H2O. Homogenates were diluted serially, and aliquots of relevant dilutions were plated on tryptose agar. Colonies were counted after 48 h of incubation. Colony counts were corrected for dilution and averaged to yield CFU per organ. The level of protection was calculated as the log10 difference of the bacterial count from immunized mice and naive control mice. Data are presented as the average of individual experiments with five mice per group. Each experiment was performed at least twice with similar results. The statistical significance of results was tested as described below.

Purification of p60

The purification of p60 from the p60-overproducing strain L. monocytogenes MR1 pGB363–1p60 (LM-p60) was described previously (20). Purification of p60 from L. innocua (LI-p60) was performed similarly. In brief, supernatants from L. innocua cultures were harvested at early stationary growth phase, and the proteins were precipitated with TCA, washed with acetone, dissolved in sample buffer, heated at 95°C for 20 min, and separated by SDS-PAGE. After staining, the 60-kDa band representing the p60 protein was excised and transferred into a BIOTRAP BT 1000-chamber (Schleicher & Schuell, Dassel, Germany) for overnight elution. Purified proteins were used after dialysis against Tris-HCl buffer. To confirm purity, aliquots of the protein samples were reseparated by SDS-PAGE and either stained with Coomassie blue or transferred onto nitrocellulose for immunoblot analysis with a rabbit anti-p60 antiserum (27).

Peptide libraries and synthetic peptides

For mapping of the LI-p60 epitope recognized by the cross-reactive CD4 T cell clone 1A, soluble libraries of overlapping deca- and dodecapeptides (offset = one amino acid) covering the previously identified antigenic region of the p60 molecule from L. innocua were synthesized. The synthesis of a soluble peptide library representing the LM-p60 was described previously (20). The individual peptide spots were excised and distributed into different 1.5-ml tubes for cleavage due to diketopiperazine formation at neutral pH. The biological activity of individual peptides diluted 1/20 was performed with a T cell activation assay as described below. Definitive amounts of peptides capable of inducing T cell proliferation were synthesized on a Zinsser (Frankfurt, Germany) Analytic SMPS 350 A peptide synthesizer.

Accessory cells

Spleen cells, B7-transfected P815 cells, or P388D1 cells were used as accessory cells. Mitomycin C-inactivated spleen cells were prepared as described previously (20) and were used as accessory cells in T cell proliferation assays and T cell activation assays. MC201A5, P815 transfected with the human B7.1 gene (P815/B7) (28), were obtained from M. Reddehase (University Mainz, Mainz, Germany) with the permission of the DNAX Research Institute (Palo Alto, CA). Mitomycin C-inactivated P815/B7 were used as accessory cells for the restimulation of CD8 T cell lines. P815/B7 cells were cultured in RPMI supplemented with 5% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 x 10-5 M 2-ME, 2 mM glutamine, and 167 µg/ml G418. For in vitro infection experiments P388D1 cells were used as APCs.

T cell clones and lines

The generation and culture of the p60-specific CD4 T cell clones 1A, 3D, 5F, and 6G were described previously (20). CD8 T cell lines specific for p60217–225 and p60449–457 were derived from L. monocytogenes-infected BALB/c mice. Mice were primed with 5 x 103 CFU of L. monocytogenes i.p. and boosted with 1 x 106 CFU 14 days later. Spleens were removed 10 days later, and bulk cultures were set in 24-well flat-bottom plates with an initial concentration of 15 x 106 spleen cells/well in 1.5 ml of cell culture medium ({alpha} modification of Eagle’s medium; PAA, Wien, Austria) supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 x 10-5 M 2-ME, 2 mM glutamine, and 0.5 µg/ml amphothericin B ({alpha}MEM) and 10-8 M synthetic HPLC-purified p60217–225 or p60449–457 peptide, respectively. After an initial 5-day culture period T cells were grown in {alpha}MEM supplemented with 20 U of rmIL-2/ml (R&D Systems, Wiesbaden-Nordenstadt, Germany). CD8 T cell lines were restimulated every 3–4 wk with mitomycin C-inactivated P815/B7 cells as accessory cells. Per well of a 24-well plate, 0.4 x 106 T cells were seeded with 0.8 x 106 P815/B7 in {alpha}MEM containing 20 U of rmIL-2/ml and supplemented with 10-9 M p60217–225 or 10-9 M p60449–457 peptide. Cytotoxic CD8 T cell lines generated in the presence of P815/B7 showed the same characteristics as CD8 T cell lines generated in the presence of inactivated spleen cells.

T cell activation assays

T cell activation in the presence of inactivated spleen accessory cells and Ag was measured by [3H]thymidine incorporation into proliferating T cells or by detection of IL-2 in culture supernatants as previously described (20). In brief, 5 x 104 washed T cells were cultured with 2 x 105 inactivated accessory cells and the appropriate Ag in 150 µl of {alpha}MEM/well in round-bottom 96-well microtiter plates. Culture supernatants were harvested 18–24 h after initiation of cultures. IL-2 activity in supernatants was detected in a bioassay using IL-2-dependent HT-2 cells. Proliferation of T cells and HT-2 cells was measured by [3H]thymidine incorporation.

T cell activation by P388D1 macrophage-like cells was measured by the detection of IFN-{gamma} in culture supernatants. P388D1 cells were precultured for 48 h without antibiotics and in the presence of 20 U of rmIFN-{gamma}/ml (R&D Systems) to increase MHC class II expression and were plated in a final concentration of 1 x 105 cells in flat-bottom 96-well microtiter plates. After the removal of IFN-{gamma}-supplemented medium cells were either infected or loaded with synthetic peptides or purified p60. Bacterial infection was performed with centrifugal enhancement (10 min at 200 x g). After 30 min at 37°C infected cells were washed once with {alpha}MEM supplemented with 50 µg/ml gentamicin, and culture medium supplemented with 10 µg/ml gentamicin was added. After 6-h incubation at 37°C cells were fixed for 10 min with 1% paraformaldehyde in PBS (29). After washing with culture medium without FCS (wash medium) paraformaldehyde was inactivated by addition of a 1/1 mixture of lysine buffer (36.54 g of lysine in 500 ml of H2O) with wash medium for 20 min. Finally after four additional washing steps 5 x 104 T cells were added to each well in {alpha}MEM supplemented with 10 µg/ml gentamicin. Culture supernatants were harvested after 12–18 h at 37°C, and the IFN-{gamma} concentration was subsequently determined with an IFN-{gamma}-specific sandwich ELISA kit (R&D Systems), which binds and detects IFN-{gamma} with a pair of specific mAb. The ELISA assay was performed as suggested by the manufacturer. IFN-{gamma} production in individual samples was calculated from the titration of a supplied rmIFN-{gamma} standard. Results were corrected for the sample dilution to yield the sample concentration in picograms per milliliter.

Enzyme-linked immunospot (ELISPOT) assay

The frequency of p60-specific T lymphocytes was determined with the ELISPOT assay (12, 30). ELISPOT assays were performed in polyvinylidene fluoride-backed 96-well microtiter plates (Millipore, Eschborn, Germany) using a modified standard protocol provided by the manufacturer. Wells were coated overnight with bicarbonate coating buffer, pH 9.6, supplemented with 10 µg/ml of rat anti-mouse IFN-{gamma} mAb (RMMG-1; BioSource, Camarilla, CA) or rat anti-mouse IL-4 (clone BVD4-1D11; PharMingen, San Diego, CA) for the detection of IFN-{gamma}- or IL-4-producing cells, respectively. Before the addition of cells all wells were washed four times with H2O and subsequently blocked with {alpha}MEM for 30 min at 37°C. To determine the frequency of p60-specific cells in infected mice, 4 x 105 splenocytes were preincubated for 18 h in round-bottom 96-well microtiter plates in a final volume of 150 µl of {alpha}MEM in the presence of 5 µg/ml LM-p60, 5 µg/ml LI-p60, 10-8 M p60217–225, 10-8 M p60301–312, or 10-8 M p60449–457. Controls were performed without Ag or in the presence of 10-8 M LLO91–99. This preincubation step in round-bottom wells was required for the optimal activation of T cells. From these primary cultures 1 x 105 or 1 x 104 cells were transferred to anti-IFN-{gamma}- or anti-IL-4-coated wells. After 12–16 h at 37°C wells were washed 10 times with 200 µl PBS/0.25% Tween 20 (wash buffer). For the detection of bound IFN-{gamma} or IL-4, wells were incubated for 2 h at 37°C with 1 µg/ml biotin-labeled rat anti-mouse IFN-{gamma} mAb (clone XMG1.2, PharMingen) or rat anti-mouse IL-4 mAb (clone BVD6-24G2; PharMingen), respectively. Subsequently, after washing five times with wash buffer 50 µl of horseradish peroxidase-streptavidin conjugate (Dianova, Hamburg, Germany) diluted to a concentration of 4 µg/ml in wash buffer was added. After 2-h incubation at room temperature wells were washed four times with wash buffer and developed with 50 µl/well aminoethylcarbazole solution. A 20-mg tablet of 3-amino-9-ethylcarbazole (Sigma, Deisenhofen, Germany) was dissolved in 2.5 ml n,n-dimethylformamide (Sigma). After addition of 47.5 ml of sodium acetate buffer, pH 5.0, and 25 µl of H2O2 the solution was filtered through a 0.2-µm syringe filter and used immediately. After 20–30 min at room temperature the wells were washed three times with H2O and air-dried. The frequency of Ag-specific cells was calculated as the number of spots per number of splenocytes seeded. The specificity and sensitivity of the ELISPOT assay were controlled with IFN-{gamma}-secreting CD4 T cell clones (20) and an IL-4-transfected cell line provided by A. Limmer (Zentrum für Molekulare Biologie, Heidelberg, Germany). The plating efficiency was ~80% for both cell types.

Statistical analysis

Statistical analysis of the results of in vitro experiments was performed with the Newman-Keuls multiple comparison test at the 0.05 significance level. The statistical significance of the results of in vivo experiments was checked using the nonparametric Tukey multiple comparison test at the 0.05 significance level. All tests were performed using the WINKS statistical analysis software (Texasoft, Cedar Hill, TX).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Some CD4 T cell clones specific for L. monocytogenes p60 cross-react with L. innocua p60

L. monocytogenes infection induces a potent p60-specific Th1 immune response. We have shown previously that p60-specific Th1 clones mediate significant protection against L. monocytogenes infection (20). Because LM-p60 from pathogenic L. monocytogenes and LI-p60 from nonpathogenic L. innocua display about 90% homology (25), it was tested whether LM-p60-specific CD4 T cells cross-react with LI-p60. LI-p60 from culture supernatants of L. innocua was purified by preparative SDS-PAGE and subsequent gel elution. LM-p60 was purified from the p60-overproducing strain L. monocytogenes EGD MR1 pGB363-1p60 (23). SDS-PAGE analyses of TCA-precipitated supernatants revealed that p60 is a major secreted protein from L. innocua (Fig. 1GoA,lane 3). After purification, the LI-p60 preparation revealed a homogeneous molecular mass of 60 kDa without any visible contamination (Fig. 1GoA, lane 4). The identity of the 60-kDa protein was confirmed by Western blot analysis with an LM-p60-specific antiserum that cross-reacts with LI-p60 (25) (Fig. 1GoB).



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  		FIGURE 1. Purification of the p60 protein from L. innocua. Purification of p60 from culture supernatants was performed by TCA precipitation followed by preparative SDS-gel chromatography. Aliquots of p60 preparations were analyzed by SDS-PAGE. Proteins were visualized directly with Coomassie blue (A) or were blotted on nitrocellulose and analyzed with an anti-p60 antiserum (B). Lane M, The m.w. standard; lane 1, TCA-precipitated supernatant from the pGB363–1p60-transformed, LM-p60-overproducing strain L. monocytogenes EGD MR1; lane 2, purified LM-p60 protein from L. monocytogenes; lane 3, TCA-precipitated supernatant from L. innocua; lane 4, purified LI-p60 protein from L. innocua.

 
Several LM-p60-specific CD4 T cell clones were described previously (20). These LM-p60-specific T cell clones were now tested for cross-reactivity with LI-p60. T cells were cocultivated with inactivated spleen cells in the presence of graded amounts of either purified LM-p60 or LI-p60. T cell activation was measured by [3H]thymidine incorporation and expressed as the stimulation index. All clones revealed a dose-dependent LM-p60-specific proliferation (Fig. 2Go, open bars). In the presence of LI-p60 (Fig. 2Go, solid bars) only clones 1A and 3D showed Ag-specific proliferation, while clones 5F and 6G were strictly LM-p60 specific. The sensitivity of clone 1A was almost equal for both p60 preparations (Fig. 2Go, upper left panel). Clone 3D was ~5-fold more sensitive for LM-p60 than LI-p60 (Fig. 2Go, upper right panel).



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  		FIGURE 2. Th1 clones 1A and 3D raised against LM-p60 cross-react with LI-p60. T cell clones 1A, 3D, 5F, and 6G specific for LM-p60 were incubated with graded amounts of either purified LM-p60 protein (open bars) or LI-p60 (filled bars) in the presence of inactivated syngenic spleen cells as accessory cells. The proliferation of T cell clones was measured by incorporation of [3H]thymidine. Results are expressed as the mean stimulation index (SI) of triplicate determinations.

 
We have previously shown that the LM-p60-specific T cell clone 1A is specific for the 12-mer peptide LM-p60301–312 (EAAKPAPAPSTN) (20). The comparison of the known sequences of LM-p60 and LI-p60 reveals that this antigenic region is highly conserved between LM-p60 and LI-p60 (25). The LM-p60-specific CD4 T cell clone 1A was screened against a decamer and dodecamer peptide library with one residue offset covering the corresponding LI-p60 region LI-p60289–318. All fractions were tested with clone 1A in the presence of syngenic inactivated spleen cells, and the IL-2 activity in supernatants was screened with HT-2 cells. Only the peptide QAAKPAPAPSTN (LI-p60299–310) of the dodecamer library with one residue offset between subsequent peptides was found to be biologically active (Fig. 3GoA). No activity was obtained with any peptide of the decamer peptide library with one residue offset (data not shown). The antigenicity of the LI-p60 epitope 299–310 was confirmed with newly synthesized, HPLC-purified LI-p60299–310 peptide in a proliferation assay with clone 1A. Remarkably, the recognition of LI-p60299–310 by clone 1A was 5- to 10-fold less sensitive than the recognition of LM-p60301–312 (Fig. 3GoB), although recognition of the whole purified p60 proteins of both species was equally sensitive (Fig. 2Go, upper left panel). Thus, these results indicate that some LM-p60-specific CD4 T cell clones have the potential to cross-react with the LI-p60 derived from nonpathogenic L. innocua. Particularly, the epitope recognized by clone 1A is functionally conserved in the p60 proteins from L. monocytogenes and L. innocua.



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  		FIGURE 3. Characterization of the LI-p60 epitope recognized by the cross-reactive CD4 T cell clone 1A. A, Mapping of the cross-reactive antigenic peptide recognized by clone 1A with a set of dodecamer peptides with one-residue offset that maps the relevant region of LI-p60 (LI-p60289–318). Recognition of synthetic peptide by clone 1A was measured as IL-2 activity in culture supernatants. B, The results of the peptide mapping analysis were confirmed with synthetic HPLC-purified p60301–312 peptide. Ag-specific proliferation of clone 1A was measured in the presence of graded concentrations of synthetic LI-p60299–310 (circles) or LM-p60301–312 (squares). Results are expressed as the mean stimulation idex (SI) of triplicate cultures.

 
Differences in p60 Ag presentation by macrophages infected with L. monocytogenes and L. innocua

Induction of either Th cells or cytolytic T cells depends on the access of the Ag to the appropriate Ag presentation pathway (31). L. monocytogenes escapes into the cytosol and gains access to the cytosolic MHC class I presentation pathway mainly by the activity of LLO (32). To compare p60 Ag presentation by L. monocytogenes and L. innocua-infected cells, the recognition of Listeria-infected macrophage-like P388D1 cells by LM-p60301–312-specific CD4 (20) and LM-p60217–225 or LM-p60449–457-specific CD8 T cells (33) was analyzed. The sequences of both p60 CD8 T cell epitopes are completely conserved in LM-p60 and LI-p60 (25), and we have shown that LI-p60 is recognized by the CD4 T cell clone 1A (Fig. 2Go). T cell activation was measured by IFN-{gamma} secretion into the culture supernatant. P388D1 cells loaded with p60217–225 or with p60449–457 were recognized in a dose-dependent way by the corresponding peptide-specific CD8 T cells but not at all by p60-specific CD4 T cells (Fig. 4GoA). APC loaded either with LM-p60 or LI-p60 were recognized by the p60301–312-specific CD4 T clone with similar sensitivity but were not recognized by p60-specific CD8 T cells (Fig. 4GoB), indicating that the MHC class I-presented p60 peptides were not generated from exogenous p60 protein.



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  		FIGURE 4. Ag presentation of p60 by P388D1 cells infected with L. monocytogenes or L. innocua. Cloned p60301–312-specific CD4 T cells (filled diamonds), p60217–225-specific CD8 T cells (open circles), or p60449–457-specific CD8 T cells (open squares) were incubated with P388D1 cells. APC were loaded with p60217–225 or p60449–457 (A), loaded with LM-p60 or LI-p60 (B), or infected with L. monocytogenes wild-type (LM wt), L. innocua (LI), or the deletion mutants L. monocytogenes {Delta}actA (LM {Delta}actA) and L. monocytogenes {Delta}hly (LM {Delta}hly; C). Ag processing by APC was limited to a period of 6 h postinfection by fixation of APC with 1% paraformaldehyde before addition of T cells. Activation of T cells was measured as the IFN-{gamma} concentration in culture supernatants. Results are expressed as the IFN-{gamma} concentration in picograms per milliliter, and the SD of triplicate cultures is indicated. The dotted line at 20 pg/ml indicates the detection limit of the IFN-{gamma} ELISA.

 
The presentation of p60 by Listeria-infected cells was analyzed under conditions that limit Ag processing to a defined period. T cells were added after APC were fixed 6 h postinfection. L. monocytogenes-infected P388D1 APC were recognized preferentially by p60-specific CD8 T cells. These cells were recognized poorly by p60-specific CD4 T cells, which showed only weak IFN-{gamma} production (Fig. 4GoC, upper left panel). In contrast to L. monocytogenes-infected cells, which were recognized by p60-specific CD8 T cells even after infection with 10-2-10-3 CFU/cell, CD8 T cell recognition of L. innocua-infected cells required infection with at least 10 bacteria/cell. Remarkably, L. innocua-infected cells presented p60 equally in the MHC class I and the MHC class II context, as indicated by the activation of p60-specific CD4 and CD8 T cells (Fig. 4GoC, lower right panel). To study the influence of a possible cell-to-cell spread of L. monocytogenes on Ag presentation of p60 (29), APC were infected with the L. monocytogenes {Delta}actA deletion mutant, which lacks actA expression and is therefore unable to infect secondary cells by cell-to-cell spread (34). Under the experimental conditions used, recognition of L. monocytogenes {Delta}actA-infected cells by p60-specific CD4 and CD8 T cells (Fig. 4GoC, upper right panel) was similarly efficient as recognition of L. monocytogenes wild-type-infected cells, indicating that no active cell-to-cell spread occurred during the first 6 h postinfection. The relevance of active LLO secretion for the inhibition of the MHC class II-restricted p60 presentation by L. monocytogenes-infected cells was analyzed with the LLO deletion mutant L. monocytogenes {Delta}hly. APC infected with this mutant, which is unable to escape from the host cell phagosome (9), were recognized by p60-specific CD8 and CD4 T cells with similar efficacy, indicating that LLO secretion is responsible for the different p60 Ag presentation preferences of APC infected with L. monocytogenes or L. innocua, respectively (Fig. 4GoC, lower left panel). Thus, p60 Ag presentation by L. monocytogenes and L. innocua-infected macrophages is significantly different. Whereas the strong p60 presentation by L. monocytogenes-infected macrophage-like cells is restricted predominantly by MHC class I molecules, the p60 presentation by L. innocua-infected cells is weaker and occurs in the context of both MHC class I and MHC class II molecules.

Failure of L. innocua infection to induce a p60-specific T cell response in vivo

Because p60 Ag presentation by L. monocytogenes- and L. innocua-infected cells differed, we wondered to what extent this distinct preference of Ag presentation in vitro is mirrored by the frequency of p60-specific CD4 and CD8 T cells in Listeria-infected mice. ELISPOT assays were used to determine the frequency of p60-specific T cells in vivo (12). The frequency of p60217–225-specific or p60449–457-specific CD8 T cells was calculated as the number of IFN-{gamma} spots generated per 1 x 105 spleen cells in the presence of the corresponding synthetic peptide. The frequency of p60-specific CD4 T cells was recorded as the number of spots generated by stimulation with purified LM-p60 or LI-p60. Additionally, the frequency of p60301–312-specific CD4 T cells was determined. All assays were performed in parallel with plates coated with anti-IFN-{gamma} or with anti-IL4 to differentiate between p60-specific IFN-{gamma}-producing Th1 cells and IL-4-producing Th2 cells, respectively (35). Control experiments have shown that APC loaded with purified p60 protein are efficiently recognized by p60-specific CD4 T cells but not by CD8 T cell lines specific for p60217–225 or p60449–457 (Fig. 4GoB). Mice were primed with L. monocytogenes or with L. innocua and boosted on day 14 postinfection. Control mice were not infected. Spleens were removed 10 days after the booster infection and used for the ELISPOT test. L. monocytogenes-infected mice showed a significant (p < 0.05) increase in the number of IFN-{gamma}-producing cells reactive with p60217–225 (115 x 10-5), p60449–457 (24 x 10-5), LM-p60 (101 x 10-5), LI-p60 (93 x 10-5), or p60301–312 (28 x 10-5) compared with uninfected control mice (Fig. 5Go, upper panel). The background activity was ~10 x 10-5. No significant increase in the frequency of IL-4-producing T cells was found after L. monocytogenes infection, indicating that the p60-specific Th cell response was predominantly of the Th1 type. In contrast, infection with L. innocua did not induce a significant increase in the number of p60-reactive T cells compared with that in uninfected mice (Fig. 5Go, middle panel). The frequency of IFN-{gamma}-producing p60217–225-, p60449–457-, LM-p60-, LI-p60-, or p60301–312-reactive cells was not significantly (p < 0.05) different from that in controls without addition of Ag. The frequency of IL-4-producing cells in the presence of p60 proteins or p60 peptides varied only insignificantly, indicating that no prominent p60-specific Th2 cell response was induced by L. innocua infection. No significant p60-dependent increase in the frequency of IFN-{gamma}- or IL-4-producing cells was observed in uninfected mice (Fig. 5Go, lower panel). In summary, these experiments demonstrate that L. innocua infection is insufficient to induce a p60-specific CD4 or CD8 T cell response, while L. monocytogenes infection induces a strong p60-specific CD4 and CD8 T cell response.



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  		FIGURE 5. Frequency of Ag-specific T cells in the spleens of L. monocytogenes and L. innocua-infected mice. The frequency of Ag-specific T cells was determined with the ELISPOT assay as described in Materials and Methods. The frequency of cells reactive with p60449–457, p60217–225, p60301–312, LM-p60, or LI-p60 or without Ag is shown as the number of reactive cells per 1 x 105 splenocytes. The dotted line at 0.2 x 10-5 indicates the detection limit of the ELISPOT assay. The frequency of IFN-{gamma}-secreting (filled bars) or IL-4-secreting (open bars) T cells was determined in spleens of BALB/c mice infected and boosted with L. monocytogenes or L. innocua or of mice that remained uninfected as indicated. The SD of triplicate cultures is indicated. Asterisks indicate values significantly (p < 0.05) above background activity without Ag.

 
L. innocua infection enhances the p60-specific T cell memory established by previous L. monocytogenes infection

Although LI-p60 was presented by L. innocua-infected macrophages in vitro, L. innocua infection did not induce a primary p60-specific immune response. Some experimental evidence exists that primary induction of naive T cells requires a stimulus different from that required for the activation of experienced T cells (36). Therefore, it was tested whether L. innocua infection is able to boost p60-specific memory T cells established by prior L. monocytogenes infection. For these long term experiments specific pathogen-free mice were used; they were kept in a laminar air flow compartment to prevent contact with environmental micro-organisms. The primary infection was performed i.v. with a dose of either 1 x 102 CFU (low dose) or 5 x 103 CFU (high dose) of L. monocytogenes. In the booster groups mice were subsequently (1, 2, and 3 mo after L. monocytogenes infection) boosted with 1 x 106 CFU of L. innocua. In the control groups booster infections were not performed. The frequency of Ag-specific memory cells was determined 4 mo after primary L. monocytogenes infection or 1 mo after the last booster infection to obtain the frequency of p60-specific T cells in the early memory phase because from the study of L. monocytogenes infection it is known that after initial expansion of the T cell pool upon Ag contact most T cells die within 3 wk (12, 13). The frequency of Ag-specific, IFN-{gamma}-producing CD4 and CD8 T cells was determined with the IFN-{gamma} ELISPOT assay in the presence of LM-p60, LI-p60, p60301–312, p60217–225, or p60449–457 or without Ag (Fig. 6Go). In all groups the background frequency of spontaneously IFN-{gamma}-producing cells measured in the absence of Ag was <0.2 x 10-5 cells. Remarkably, repeated booster infections with L. innocua significantly (p < 0.05) increased the frequency of p60-specific T cells. In the group immunized with high dose L. monocytogenes the frequency of LM-p60 and LI-p60-specific Th1 cells increased 9- and 14-fold, respectively, and the frequency of p60217–225-specific cells increased 8-fold (Fig. 6GoA). In the group primed with low dose L. monocytogenes (Fig. 6GoB) the increases in the frequency of LM-p60- and LI-p60-specific Th1 cells were 13- and 15-fold, respectively, and the increase in the frequency of p60217–225-specific cells was 11-fold. In both groups the frequency of LLO91–99-specific cells was not significantly altered after L. innocua booster infection, indicating that the expansion of p60-specific T cells obtained by L. innocua infection was Ag specific. If the primary induction of p60-specific T cells by primary L. monocytogenes infection was omitted, and primary immunization was instead performed by L. innocua infection a p60- or LLO-specific T cell response was not observed (data not shown). Thus, although L. innocua infection is insufficient to induce a primary p60-specific T cell response in vivo, this nonpathogenic Listeria species is able to expand p60-specific CD4 and CD8 memory T cells induced by previous L. monocytogenes infection.



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  		FIGURE 6. L. innocua infection boosts a previously established LM-p60-specific T cell response. The frequency of Ag-specific IFN-{gamma}-producing cells was determined by ELISPOT either 4 mo after primary infection with L. monocytogenes only (filled bars) or 1 mo after primary infection with L. monocytogenes and boosting with L. innocua 1, 2, and 3 mo after primary infection (open bars). A, Primary infection with a high dose (5 x 103 CFU) of L. monocytogenes. B, Primary infection with a low dose (1 x 102 CFU) of L. monocytogenes. The frequency of cells reactive with LLO91–99, p60217–225, LM-p60, or LI-p60 or without Ag is shown as the number of reactive cells per 1 x 105 splenocytes. The SD of triplicate cultures is indicated. The dotted line at 0.2 x 10-5 indicates the detection limit of the ELISPOT assay. Asterisks indicate a significant (p < 0.05) difference between the booster group and the control group with primary infection only.

 
Limited protective efficacy of L. innocua infection

L. innocua is an environmental bacterium that is frequently isolated from food (5, 6, 7, 8). Frequent encounters with L. innocua could stimulate L. monocytogenes cross-reactive memory T cells and play a potential role in the maintenance of protective antilisterial immunity. To test whether infection with L. innocua improves the protective immune response against L. monocytogenes infection, mice were immunized by primary low dose or high dose L. monocytogenes infection and were subsequently boosted repeatedly with L. innocua as described in the previous section. Mice were challenged with L. monocytogenes 4 mo after primary infection. In the L. innocua booster groups this was 1 mo after the last booster infection. After primary immunization by high dose L. monocytogenes infection excellent protection was observed in the spleen (protection, >6) and in the liver (protection, >6) upon L. monocytogenes challenge (Fig. 7Go). This protection was not further improved by L. innocua booster infections. After primary immunization by low dose L. monocytogenes infection the protection upon L. monocytogenes challenge 4 mo postimmunization was less complete in the spleen (protection, 3.8) and liver (protection, 2.9). L. innocua booster infections significantly (p < 0.05) improved the protection in the liver (protection, 3.9), while protection in the spleen (protection, 4.0) was not significantly influenced. No increased protection was observed if mice were boosted with B. subtilis instead of L. innocua, indicating that the increased protection observed in the liver after L. innocua booster infection was not the result of an unspecific stimulation (Fig. 7Go). In contrast, primary L. innocua infection boosted by subsequent L. innocua infection after 1, 2, and 3 mo did not confer significant protection against challenge with L. monocytogenes. Together, these results demonstrate that repeated L. innocua infections fail to induce protective immunity against challenge with L. monocytogenes. However, L. innocua booster infections induced additional protection in the liver upon challenge with L. monocytogenes in the situation of suboptimal antilisterial immunity.



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  		FIGURE 7. Limited protective efficacy of L. innocua infection. Mice were immunized by primary infection with a low dose (LM-low dose) or a high dose (LM-high dose) inoculum of L. monocytogenes or by L. innocua (LI) infection. Subsequently, booster groups were repeatedly boosted, as indicated, by L. innocua or B. subtilis infection 1, 2, and 3 mo after primary infection. One month after the last booster infection mice were challenged with L. monocytogenes. The bacterial loads of spleen and liver were determined 72 h postinfection. Results are expressed as the mean log10 CFU ± SD of groups of five mice. A significant increase in protection after booster infection is indicated by an asterisk. Primary infection with L. monocytogenes and subsequent booster infection with L. innocua are indicated as LM-high dose/LI-boost if the primary inoculum was high dose L. monocytogenes and as LM-low dose/LI-boost if the primary inoculum was low dose L. monocytogenes. Primary infection with low dose L. monocytogenes and booster infection with B. subtilis is indicated as LM-low dose/BS-boost. Primary and booster infection with L. innocua is indicated as LI/LI-boost. Control mice were not infected.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study investigated the influence of L. innocua, a nonpathogenic environmental Listeria species, on the induction and maintenance of an L. monocytogenes-specific immune response. We show that although L. innocua infection itself fails to induce a primary L. monocytogenes-specific T cell response, infection with this nonpathogenic Listeria species has the potential to enhance a previously established L. monocytogenes-specific T cell memory. This is the first experimental support for the hypothesis that cross-reactive nonpathogenic micro-organisms influence the maintenance of T cell memory (14, 15, 37).

T cell memory is a hallmark of the immune system, and ever since its recognition there has been considerable interest in understanding how it is maintained. The idea that maintenance of long-lived T cell memory requires chronic exposure to Ag came from reports that memory T cells survive poorly on adoptive transfer unless accompanied by specific Ag (38, 39, 40). This observation has been challenged by the finding that after adoptive transfer of memory CD8 T cells these cells persist in the complete absence of specific Ag (41, 42, 43, 44). Several models were proposed to explain the maintenance of T cell memory in the absence of specific Ag. It was proposed that chronic stimulation of memory T cells could reflect repeated contact with cross-reactive environmental Ags (37). This speculation was supported by the idea that memory T cells are hyper-reactive to Ag (43, 45). In one study memory CD8 T cells responded to a peptide at a 10- to 50-fold lower concentration than that required for the stimulation of naive CD8 T cells (45). Alternative models, which are supported by experimental data mostly from viral infection systems, suggest that without specific TCR-mediated antigenic stimulation, memory T cells might participate as bystander cells from cytokines produced during the T cell response directed against an unrelated micro-organism (46, 47). In our experimental system the expansion of memory CD8 T cells was exclusively Ag specific. The frequency of CD8 T cells specific for the unrelated T cell Ag LLO was not altered by L. innocua booster infection. Thus, it has to be assumed that p60-specific memory T cells were specifically activated by contact with a related cross-reactive Ag. This observation corroborates the recent reports from viral infection models that most T cells that are activated during infection are strictly Ag specific and are not expanded during infection with a heterologous virus (48, 49).

The virulence-associated proteins of L. monocytogenes, such as LLO and the metalloprotease, which are targets for L. monocytogenes-specific T cells (17, 21, 50), are not shared with nonpathogenic Listeria (9). Because p60 is essential for cell viability, it is not surprising that a p60 homologue is also produced by nonpathogenic Listeria species, including L. innocua (23). The LM-p60 from pathogenic L. monocytogenes and the LI-p60 from nonpathogenic L. innocua display about 90% homology (25). In addition to the MHC class II epitope, both known MHC class I epitopes from LM-p60, p60217–225 and p60449–457, are conserved in both species (25) and are presented by L. innocua-infected macrophage-like P388D1 cells. The MHC class II epitope p60301–312 recognized by the CD4 T cell clone 1A is conserved in L. innocua, with the exception of position 1, which is a glutamine residue in the LI-p60 and a glutamic acid residue in LM-p60. The MHC class I-restricted Ag presentation of bacterial proteins by cells infected with L. innocua (51) or with LLO-negative L. monocytogenes strains (52) has been reported previously. However, no direct comparison of the relative strength of MHC class I and MHC class II-restricted presentation was performed. Although our experiments demonstrated MHC class I-restricted p60 presentation independent of LLO, the comparison with LLO-secreting L. monocytogenes clearly corroborates the importance of LLO for efficient MHC class I-restricted Ag presentation. This observation strengthens the results of a detailed study of the Ag presentation of LLO with LLO-specific MHC class I and MHC class II-restricted T cell hybridomas (29), which showed that MHC class I-restricted presentation of LLO depends on the hemolytic activity of LLO. In contrast to this study we investigated the influence of secreted LLO on the Ag presentation of an independent bacterial protein. The comparison of p60 presentation by cells infected with LLO-secreting L. monocytogenes with that by cells infected with LLO-negative L. monocytogenes or by L. innocua provided clear evidence that bacterial secretion of LLO strongly enhances the MHC class I-restricted presentation and inhibits the MHC class II-restricted presentation of a listerial protein in infected cells.

Although strong inhibition of MHC class II-restricted p60 Ag presentation occurred in L. monocytogenes-infected cells in vitro, a high frequency of p60-specific CD4 T cells was observed in vivo. The frequency of cells specific for the CD4 T cell epitope p60301–312 was similar to the frequency of cells specific for the CD8 T cell epitope p60449–457, which is subdominant relative to the p60217–225 epitope (12). The response to p60301–312 represented <10% of the total p60-specific CD4 T cell response. The frequency of T cells specific for the whole p60 was similar to the frequency of T cells specific for the immunodominant epitope p60217–225. A possible explanation for the efficient MHC class II-restricted in vivo presentation of p60 is the MHC class II-restricted presentation after secondary uptake of bacterial protein or killed bacteria released from dead cells by neighboring noninfected cells. This mechanism enables the MHC class II-restricted presentation of LLO in cultures of infected macrophages (29). In contrast to the strong p60-specific T cell response after L. monocytogenes infection, no p60-specific CD4 or CD8 T cell response was observed after primary L. innocua infection, although we found that p60 is presented by L. innocua-infected macrophages. One plausible explanation for this failure might be the rapid clearance of L. innocua from spleen and liver after infection (53, 54, 55). Similarly, it has been reported that antibiotic abridgement of L. monocytogenes infection by ampicillin treatment started as late as 5 days postinfection causes a significant reduction of protection against a secondary challenge infection (56). However, the activation of experienced p60-specific CD4 and CD8 memory T cells by L. innocua showed that in vivo the p60 of this species was indeed presented by MHC class I and MHC class II molecules, as predicted from the in vitro Ag presentation experiments. Generally, this observation suggests that different stimuli are required for the induction of a primary vs a recall T cell response similar to the situation observed with some nonimmunogenic tumor cell lines, which are only able to induce a recall T cell response in previously immunized mice (36). The reason for the different immunogenicities of L. monocytogenes and L. innocua is unknown. Recently, it was reported that dead bacteria suppress IL-12 production in contrast to live bacteria, which induce IL-12 in vivo (57). Because IL-12 plays an important role in the induction of a cellular immune response (58), the suppressive effect of killed L. innocua could prevent the induction of a T cell immune response.

Human infection by L. monocytogenes is a food-borne infection (5, 6, 7, 8). Using the ligated ileal loop model of the rat it was recently shown that L. monocytogenes is taken up in vivo by epithelium rather nonspecifically, i.e., no specific virulence factors of L. monocytogenes seem to be required for this step, and even nonpathogenic L. innocua are translocated at a similar rate as the L. monocytogenes wild-type strain (59). Remarkably, the screening of sera from healthy volunteers for the occurrence of p60-specific Abs revealed a high prevalence of exclusively LI-p60-specific Abs, indicating that nonpathogenic L. innocua from food are frequently exposed to the immune system of normal hosts (M. Lalic and A. Bubert, unpublished observations). In contrast to this natural mode of infection we studied L. innocua in a systemic infection model, because this is an established model for the study of the antilisterial T cell response. In our protection assays it was difficult to obtain additional protection after booster immunization with nonpathogenic Listeria, because even primary immunization with low doses of L. monocytogenes induced strong protection against reinfection. Upon challenge with L. monocytogenes 4 mo postimmunization, protection in the spleen was at least 10-fold better than that in the liver. Thus, it is not unexpected that L. innocua-mediated enhancement of protection against L. monocytogenes was limited to the situation of weak antilisterial immunity, as observed in the liver after low dose primary immunization. In summary, we conclude that after L. innocua booster infection weak Ag presentation of a cross-reactive protein is sufficient for the stimulation of L. monocytogenes-specific CD4 and CD8 memory T cells. A general model of the role of environmental micro-organisms for the maintenance of T cell memory would suggest that specific T cells established by primary infection with a pathogenic micro-organism can be boosted by contact with any antigenically related nonpathogenic species.


   Acknowledgments
 
We thank R. Holtappels (University of Mainz, Mainz, Germany) and J. Daniels (University of Würzburg, Würzburg, Germany) for critical review of the manuscript, and D. Palm (University of Würzburg) for his support with generation of the peptide library.


   Footnotes
 
1 This work was supported by the Forschungsfond der Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Projekt 13/1998. M.L.-M. received a stipend from the Graduiertenkolleg Infektiologie funded by the Deutsche Forschungsgemeinschaft. Back

2 Address correspondence and reprint requests to Dr. G. Geginat, Institut für Medizinische Mikrobiologie und Hygiene, Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany. E-mail address: Back

3 Abbreviations used in this paper: LLO, listeriolysin O; LM-p60, p60 produced by Listeria monocytogenes; LI-p60, p60 produced by Listeria innocua; rmIFN, recombinant murine IFN; ELISPOT, enzyme-linked immunospot. Back

4 Current address: Microbiological Analytics, Merck KGaA, Darmstadt, Germany. Back

Received for publication October 27, 1998. Accepted for publication January 14, 1999.


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 Discussion
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