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The Journal of Immunology, 2001, 167: 6480-6486.
Copyright © 2001 by The American Association of Immunologists

Immunization with gp96 from Listeria monocytogenes-Infected Mice Is Due to N-Formylated Listerial Peptides1

Anne-Marit Sponaas*, Ulrich Zuegel{ddagger}, Stephan Weber*, Robert Hurwitz*, Ralf Winter{dagger}, Stephanie Lamer{dagger}, Peter R. Jungblut{dagger} and Stefan H. E. Kaufmann*

* Department of Immunology, {dagger} Central Support Unit Biochemistry, Max-Planck Institute for Infection Biology, Berlin, Germany; and {ddagger} Department of Experimental Dermatology, Schering AG, Berlin, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
N-Formylated (N-f-met) peptides derived from proteins of the intracellular bacterium Listeria monocytogenes generate a protective, H2-M3-restricted CD8 T cell response in C57BL/6 mice. N-f-met peptide-specific CTL were generated in vitro when mice previously immunized with gp96 isolated from donor mice infected with L. monocytogenes were stimulated with these peptides. No significant peptide-specific CTL activity was observed in mice immunized with gp96 from uninfected animals. Masses corresponding to one N-f-met peptide were found by matrix-assisted laser desorption/ionization-mass spectrometry on gp96 isolated from C57BL/6 mice infected with L. monocytogenes, but not on gp96 from noninfected mice. Therefore, bacterial N-f-met peptides from intracellular bacteria can bind to gp96 in the infected host, and gp96 loaded with these peptides can generate N-f-met-peptide-specific CTL. We assume a unique role of gp96 in Ag processing through the H2-M3 pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Experimental listeriosis represents an elegant model that has significantly contributed to our understanding of the immune responses to microbial invaders (1). Investigations of this model have revealed that in addition to conventional CD8 T cells restricted by classical MHC class I molecules, CD8 T cells controlled by the nonclassical MHC class Ib molecules participate in protection (2, 3). In particular, the primary immune response against Listeria monocytogenes in C57BL/6 mice is dominated by such T cells (4, 5). The nonclassical H2-M3 class Ib molecules restrict CD8 T cells specific for Ags coded for by mitochondrial or bacterial DNA (6). Hydrophobic peptides that possess N-terminal formyl groups (N-f-met)3 bind to H2-M3 with high affinity (7). Thus far, three H2-M3-restricted T cell epitopes from L. monocytogenes have been identified. The fMIGWII peptide is derived from the LemA protein (8), fMIVIL was found in supernatants of bacterial culture (9), and fMIVTLF was derived from a leader peptide of a bacterial tRNA attenuator (10). Although it is not fully understood how H2-M3-restricted T cells contribute to protective immunity, adoptive transfer of a fMIGWII-specific T cell clone into mice conferred partial protection against L. monocytogenes (11).

Heat shock proteins (HSP) are highly conserved polypeptides performing various biological functions (12). HSP act as molecular chaperones that promote protein translocation and degradation, protein folding and unfolding, as well as assembly of multimeric protein complexes. HSP carry out these functions by binding to exposed hydrophobic regions of protein targets (reviewed in Ref. 13), suggesting that the substrates for the binding sites of HSP are hydrophobic. Indeed, it has been shown that the binding site of HSP70 prefers short peptides composed of clusters of hydrophobic amino acid residues flanked by basic residues (14). Although gp96 is predicted to contain a hydrophobic binding site (15), it appears to bind to a greater variety of peptides (16, 17) with a preference for noncharged amino acids at key positions 2 and 9 (18).

The ability of several HSP cognates to trigger the immune system is well appreciated. For example, mice injected with HSP derived from tumors generate a strong anti-tumor response that is mediated by T cells (19). The underlying mechanism is currently being elucidated. APCs express cell surface receptors for HSP (20, 21). After receptor-mediated endocytosis, HSP-associated peptides are transferred to MHC class I (22, 23). The receptor for the endoplasmic reticulum (ER) resident HSP gp96 has been identified as CD91 ({alpha}2-macroglobulin receptor; Ref. 24). HSP are able to activate dendritic cells and macrophages through CD14 signaling (25), and are able to induce maturation and cytokine secretion in these cells (26, 27). This in turn stimulates the specific activation of naive T cells. Such properties indicate that HSP function as "danger signals" when tissue damage is initiated by malignancy, infection, or inflammation.

In only a few instances have peptides been isolated from gp96 (28, 29, 30). This could be due to the physicochemical features of the peptides associated with HSP. During the course of our attempts to identify antigenic peptides in mice infected with L. monocytogenes, we have identified and isolated N-f-met peptides from gp96. These are hydrophobic and highly promiscuous peptides, characteristic not only for the prokaryotic, but also for the mitochondrial proteome (31, 32). Therefore, it is tempting to speculate that gp96 plays a unique role in the processing and presentation of bacterial and mitochondrial Ags through the H2-M3 pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice, cells, Abs, and materials

Female C57BL/6 mice were obtained from our breeding facilities at the Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin (Berlin, Germany) and were used between 6 and 8 wk of age. Breeding pairs of MHC class II-deficient mice (A{beta}-/- mice) (33) were obtained from Dr. D. Mathis (Strasbourg, France). All mice were bred under specific pathogen-free conditions. The RMA mouse cell line (H-2b) was cultured in RPMI 1640 supplemented with 10% FCS (Sigma-Aldrich, Deisenhofen, Germany), 1 mM L-glutamine, 10 mM HEPES, 5 x 10-5 M 2-ME, 100 µg/ml penicillin, and 100 U/ml streptomycin. All tissue culture reagents were purchased from Biochrom (Berlin, Germany). Ab to gp96 (SPA 850) was obtained from StressGen Biotechnologies (Victoria, British Columbia, Canada). Formylated peptides (fMIVIL, fMIGWII, and fMIVTLF) were synthesized by Jerini BioTools (Berlin, Germany).

Infection and immunization of mice and purification of gp96

Mice were infected i.v. in the tail vein with 5 x 103 CFU L. monocytogenes strain EGD. Spleens and livers were harvested 3 days later, and were homogenized in hypotonic buffer (30 mM sodium carbonate buffer, pH 7.1) containing protease inhibitors (2 mM Pefabloc and 1 µM leupeptin and pepstatin). After centrifugation (35,000 x g for 30 min at 4°C), supernatants were filter sterilized (0.2-µm pore size; Millipore, Bedford, MA), and precipitated with ammonium sulfate (30–70% saturation). The redissolved proteins were applied to a Con A-Sepharose column (CL-4B; Pharmacia Biotech, Uppsala, Sweden; 1 ml of Con A-Sepharose/50 mg of protein). After extensive washing with PBS (pH 7.2), Con A-bound material was eluted with PBS containing 10% {alpha}-methylmannoside. The eluate was further separated by ion-exchange chromatography using a MonoQ column (HR 5/5; Pharmacia Biotech) with a linear NaCl gradient from 0.15 to 1 M. Gp96 was eluted at high salt concentrations. Minor impurities were further removed by applying pooled fractions to a gel filtration column (Superdex 200HR 10/30; Pharmacia Biotech). The purity and identity of gp96 were judged by SDS-PAGE and Western blot using mAb specific for gp96 using 0.1–1 µg of protein per lane. Gp96 was isolated from noninfected mice by the same method. LPS content was tested for by Limulus Amebocyte Lysate Coatest (Charles River Endosafe, Charleston, SC). Mice were immunized s.c. with 30 µg of gp96 in 100–200 µl of PBS. As a control for the presence of contaminating LPS, mice were immunized with gp96 previously boiled at 95°C for 5 min.

Generation of formyl-35S-labeled MIGWII and peptide binding assay

Resin-bound IGWII was synthesized by Jerini BioTools and was coupled to F-moc-protected [35S]methionine (Amersham, Buckinghamshire, U.K.) by the o-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate method (34). The specific radioactivity of methionine was 1 Ci/mmol. The resin-bound peptide was deprotected by treatment with 20% piperidine and was formylated with an excess of formic acid in the presence of dicyclohexylcarbodiimide. The formylated peptide was cleaved from the resin by treatment with 95% trifluoroacetic acid (TFA). Purity and identity of the formylated peptide was confirmed by thin layer chromatography and mass spectrometry (MS). RMA cells pulsed with this peptide were also specifically lysed by a fMIGWII-specific T cell clone (11) (data not shown). Peptide binding assays were performed using PAGE on 8% polyacrylamide gels according to Laemmli. Protein (100 pmol) were incubated with 1 nmol f[35S]MIGWII (1 µCi) at 20°C for 30 min in 50 µl of 20 mM Tris-HCl, pH 7.5, and 1 mM MgCl2. The sample was mixed with 50 µl of 0.2% SDS and 10% glycerol without heating and was applied to the gel. Following electrophoresis, the gel was dried and the radioactive band was visualized and quantified with a phosphoimager (Ray test).

Generation of CTL

Mice were sacrificed 10–14 days after immunization with gp96, or 7 days after infection with L. monocytogenes. Spleens from two mice were prepared to a single cell suspension and were cultured at 4 x 106 cells/ml in 10 ml of IMDM supplemented with 10% FCS (Sigma-Aldrich), 1 mM L-glutamine, 10 mM HEPES, 5 x 10-5 M 2-ME, 100 µg/ml penicillin, and 100 U/ml streptomycin together with 3 x 106/ml (3000 rad) syngeneic stimulator cells. The stimulator cells had been pulsed for 1 h at 37°C in the presence of 10-6 M peptide and washed twice before addition to the responder cells. The cells were incubated in a 25-cm2 flask (Falcon, Heidelberg, Germany) at 37°C in a humidified atmosphere containing 7% CO2 for 7 days.

CTL assay

RMA target cells were incubated with 10-6 M peptide and 100 µCi 51Cr for 90 min. Targets were washed twice and were added at 5 x 103 targets per well to a round-bottom 96-well tissue culture plate (Falcon) together with responder T cells at different E:T ratios. After 4 h of incubation, 100 µl of supernatants were collected and 51Cr activity was determined. The percentage of specific lysis was determined as (experimental value - spontaneous release)/(maximum release - spontaneous release) x 100. Each value presents the mean of triplicate values and the standard deviation was always less than 2{varsigma}. Each experiment uses C57BL/6 mice infected with sublethal doses of L. monocytogenes as positive control and naive C57BL/6 mice as negative control. For Figs. 1aGond 2, the percentage of chromium release was determined from the linear part of the curve.



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  		FIGURE 1. Purity of gp96 preparations. gp96 preparations isolated from spleen and livers of C57BL/6 mice were analyzed by SDS-PAGE gel electrophoresis followed by Coomassie blue staining and Western blot analysis of the same samples. In Western blot analysis, the blot was stained with anti gp96 mAb. Lane 1, gp96 from L. monocytogenes-infected mice; Lane 2, gp96 from noninfected mice.

 
Peptide elution from gp96

One milligram of gp96 was desalted using a centrifugal filter with 30-kDa cut-off (Ultrafree; Millipore). The sample was precipitated with 2% TFA and 40% methanol at -20°C for 24 h, and was then centrifuged at 50,000 rpm at 4°C for 30 min. The nonprecipitated material, i.e., peptides, was removed. This sample was tested for presence of remaining protein (gp96) by Bradford and was then vacuum dried.

Matrix-assisted laser desorption/ionization (MALDI)-MS

The mass spectra measurements were obtained by a Voyager Elite MALDI-time of flight mass spectrometer (Applied Biosystems, Foster City, CA). All measurements were performed in the positive-ion reflector mode at an accelerating voltage of 20 kV, 70% grid voltage, 0.05% guide wire voltage, and a delay of 100 ns. Two hundred fifty-six scans were averaged per spectrum. The low-mass gate was set at 500 m/z. The matrix for MALDI-MS was a saturated solution of {alpha}-cyano-4-hydroxy cinnamic acid (20 mg/ml) in 50% acetonitrile and 0.3% TFA. Two microliters of the sample was mixed with 2 µl of matrix solution, and 2 µl was applied onto the sample plate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CTL specific for listerial N-f-met peptides are induced in C57BL/6 mice after immunization with gp96 from infected mice

Gp96 was isolated from infected organs of C57BL/6 mice and as control, from organs of normal, noninfected C57BL/6 mice. A standard protocol (see Materials and Methods) was used for purification of gp96 from organ lysates. The purified gp96 was Coomassie stained after SDS-PAGE, and Western Blot analysis confirmed the presence of gp96 (Fig. 1Go). We also checked for possible bacterial contaminants in gp96 samples by preparing tryptic digests of the 100-kDa band from a Coomassie gel. These digests were analyzed by MALDI-MS, and the mass spectrum analysis indicated that besides the expected gp96, no major bacterial contaminant was present (data not shown). A small amount of mouse binding Ig protein was detected.

C57BL/6 mice were immunized with gp96 from Listeria-infected mice. After 10 days, the spleen cells were cultured in the presence of either of the three known H2-M3-restricted N-f-met peptides, and the CTL activity was determined. T cells from mice immunized with gp96 from Listeria-infected organs recognized N-f-met peptides (Fig. 2Go, A–C). In contrast, mice immunized with gp96 from noninfected mice and cultured with N-f-met peptides generated minimal levels of N-f-met specific lysis (Fig. 2Go, D–F), excluding LPS contamination or other nonspecific adjuvant effects of gp96 (35) or activation of N-f-met-specific precursors in naive mice (8). Around 10 ng of LPS/mg protein was detected in the purified gp96 preparations (data not shown). Induction of CTL by contaminating LPS was ruled out because mice immunized with boiled gp96 failed to generate significant CTL against the N-f-met peptides (data not shown). However, we cannot formally exclude synergistic effects of LPS for gp96 from infected organs. When spleen cells from mice immunized with gp96 from infected organs were cultured with an irrelevant peptide, no fMIVIL-specific CTL were generated (Fig. 2GoG). The level of CTL activity induced by immunization with gp96 was comparable with that generated by i.v. infection with L. monocytogenes (Fig. 2GoH).



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  		FIGURE 2. gp96 from L. monocytogenes-infected organs generate specific CTL to N-f-met peptides. 51Cr release assay with responder T cells from C57BL/6 mice immunized s.c. with 30 µg of gp96 from L. monocytogenes-infected mice (A–C) or responder T cells from mice immunized with gp96 from noninfected mice (D–F). Spleen cells were removed 10 days after immunization and were cultured with irradiated syngeneic stimulator cells pulsed with 10-6 M fMIVIL (A and D), fMIGWII (B and E), fMIVTLF (C and F), and listeriolysin (LLO) 91-99 (G). After 7 days of culture, responder T cells were tested in a 4-h 51Cr release assay on peptide-pulsed RMA cells (•) and RMA cells without peptide ({circ}). H shows levels of 51Cr release at an E:T ratio of 25:1 for C57BL/6 mice immunized with gp96 from infected organs ({blacksquare}) and mice infected 7 days previously with 5 x 103 CFU L. monocytogenes ({square}). Splenocytes were cultured with N-f-met peptide-pulsed (H) stimulator cells and after 7 days were tested on peptide-pulsed RMA cells. The lysis of unpulsed RMA target cells is also indicated (). Splenocytes from naive C57BL/6 mice stimulated in vitro with N-f-met peptides failed to induce peptide-specific lysis (data not shown). Each point in the curves represents the mean of triplicates and has a standard deviation of less than 2{varsigma}. Mann Whitney tests were performed on the original cpm data and significant differences were found between peptide loaded vs peptide unloaded data set in A–C (p < 0.05). The data shown in Fig. 3Go are representative of five independent experiments.

 
The role of MHC class II-restricted T cells in the gp96-induced CTL response

The role of MHC class II-restricted T cell responses to HSP and their associated peptides is insufficiently understood. Therefore, we wanted to determine whether the immune responses to the N-f-met peptides could be induced in the absence of conventional CD4 T cells. To this end, we immunized A{beta}-/- mice with gp96 from L. monocytogenes-infected mice (Fig. 3GoA) or noninfected mice (Fig. 3GoB). Only spleen cells from mice immunized with gp96 from Listeria-infected organs and restimulated with fMIVIL generated a relatively weak specific CTL response. Stimulation with the two other peptides, fMIGWII and fMIVTLF, failed to induce measurable cytotoxicity. Fig. 3GoC shows the CTL activity C57BL/6 mice infected with L. monocytogenes.



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  		FIGURE 3. A CTL response to fMIVIL is generated in MHC class II-deficient mice immunized with gp96 from infected organs. A{beta}-/- mice were immunized with gp96 from infected mice (A) and noninfected mice (B), and after 10 days, spleen cells were cultured in the presence of 10-6 M fMIVIL ({circ} , •), fMIGWII ({square} , {blacksquare}), and fMIVTLF ({triangleup} , {blacktriangleup}). After 7 days of culture the cells were tested in a 51Cr release assay on peptide pulsed (• , {blacksquare} , {blacktriangleup}) or unpulsed ({circ} , {square} , {triangleup}) RMA target cells. As positive control, C57BL/6 mice were infected i.v. with 5000 L. monocytogenes and 7 days later, the spleen cells were cultured with 10-6 M fMIVIL-, fMIGWII-, and fMIVTLF-pulsed irradiated stimulator cells (C). Each point in the curves represents the mean of triplicates and has a standard deviation of less than 2{varsigma}. Mann Whitney tests were performed on the original cpm data and significant differences were found between the peptide loaded vs peptide unloaded data set in C (p < 0.05). Similar results were obtained in three separate experiments.

 
L. monocytogenes infection induced CTL activity comparable with that of gp96 immunization (Fig. 2GoH). However, the result cannot be explained by an inherent failure of the A{beta}-/- mice to generate CTL against N-f-met peptides, because A{beta}-/- mice infected with L. monocytogenes induced anti-N-f-met CTL activity comparable with that of C57BL/6 control mice (data not shown). Thus, MHC class II-restricted T cells are important for anti-fMIGWII and -fMIVTLF CTL responses generated by gp96. The CTL activity against fMIVIL in MHC class II-/- mice indicates that some Th-independent CTL responses developed against this peptide. This could be explained by higher affinity of fMIVIL compared with fMIGWII and fMIVTLF peptides to H2-M3 molecules. A similar mechanism has recently been described for a group of CD4-independent CD8 T cell responses, where Th-independent CD8 T cells were generated in response to MHC class I loaded with high-affinity peptides (36). The observation that the Th-independent response to fMIVIL was relatively weak could be best explained by the participation of both CD4 Th-dependent and -independent CD8 T cells.

gp96 from L. monocytogenes-infected organs induces CTL that recognize all three listerial N-f-met peptides

Although H2-M3 molecules are nonpolymorhic, they can present different N-f-met peptides, resulting in a protective, heterogeneous T cell response. This response could be the result of the ability of single N-f-met peptides to activate a multitude of H2-M3-restricted T cells with different peptide specificities. Such cross-reactivity of N-f-met-specific T cells has been described earlier (37). We determined whether the CTL response generated by gp96 immunization was similar to that induced by L. monocytogenes infection and investigated whether the CTL were cross-reactive or expressed exclusive specificity. Fig. 4GoA shows that CTL stimulated with fMIVIL also killed targets pulsed with fMIGWII and fMIVTLF. Conversely, T cells stimulated with fMIGWII killed targets pulsed with fMIVIL and fMIVTLF (Fig. 4GoB), and fMIVTLF-stimulated T cells recognized fMIVIL- and fMIGWII-pulsed targets (Fig. 4GoC). This cross-reaction developed not only after gp96 immunization, but also during natural L. monocytogenes infection in mice (Fig. 4Go, D–F). The N-f-met peptides stimulated a variety of distinct V{beta}-expressing T cells, and stimulation with each of the peptides lead to the same pattern of V{beta} usage (data not shown). Therefore, we consider it likely that a polyclonal immune response comprising a variety of T cell clones with overlapping specificity was induced.



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  		FIGURE 4. Infection with L. monocytogenes and immunization with gp96 from infected organs generate CTL cross-reactive with the three N-f-met peptides. Spleen cells were removed from C57BL/6 mice 10 days after immunization with 30 µg of gp96 from L. monocytogenes-infected mice and were cultured in the presence of syngeneic, irradiated stimulator cells pulsed with 10-6 M fMIVIL (A), fMIGWII (B), or fMIVTLF (C) for 7 days and then tested in 51Cr release assay on fMIVIL- ({blacksquare}), fMIGWII- (), or fMIVTLF- () labeled RMA cells and RMA cells without peptide ({square}). As control, C57BL/6 mice were infected i.v. with 5 x 103 L. monocytogenes, and after 7 days, the spleen cells were cultured in the presence of 10-6 M fMIVIL (D), fMIGWII (E), and fMIVTLF (F) as described above. After 7 days of culture, these cells were also tested in 51Cr release assay on RMA targets with and without peptide. The percentage of chromium release was determined from the linear part of the curves. Each point in the original curves represents the mean of triplicates and has a standard deviation of less than 2{varsigma}. Mann-Whitney U tests were performed on the original cpm data and significant differences were found between groups of peptide pulsed vs unpulsed target cells in A–F (p < 0.05). Similar results were obtained in three separate experiments.

 
N-f-met peptides bind to gp96

Next, we determined binding of a synthetic N-f-met peptide to gp96 in vitro. To radioactively label the N-f-met peptide, 35S-radiolabeled methionine was introduced into the fMIGWII peptide. Fig. 5Go reveals that fMIGWII bound more efficiently to gp96 because significantly more radioactive peptide was associated with gp96 than with BSA and HSP90. Under the given conditions, ~0.5% of the total peptide was found in the gp96 band after gel electrophoresis when added at 10-fold molar excess over gp96. The addition of cold fMIGWII, fMIVIL, or fMIVTLF at 10-fold molar excess reduced the binding of [35S]fMIGWII (Fig. 5Go). These data suggest that the binding site of gp96 can accommodate N-f-met peptides.



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  		FIGURE 5. FMIGWII binds to gp96. One hundred picomoles protein were incubated with 1 nmol f[35S]MIGWII (1 µCi) at 20°C for 30 min in 50 µl of 20 mM Tris-HCl (pH 7.5) and 1 mM MgCl2. The sample was mixed with 50 µl 0.2% SDS and 10% glycerol without heating and was applied to 8% SDS-PAGE. The radioactive bands were quantified with a phosphoimager. Binding of radiolabeled fMIGWII to gp96 was inhibited 5-fold in the presence of excess amounts of cold fMIGWII, fMIVIL, or fMIVTLF peptides. This experiment was performed three times with similar results.

 
Elution of fMIVIL from gp96 from L. monocytogenes-infected organs

Gp96 from L. monocytogenes-infected organs was precipitated with TFA and methanol, and the eluted peptides were directly analyzed by MALDI-MS. Two masses corresponding to fMIVIL were identified (638 as Na+ and 616 as H+ peaks) on gp96 from infected, but not from noninfected mice (Fig. 6Go, A and B). Masses corresponding to fMIGWII and fMIVTLF were not found on gp96 from infected organs. This was not due to a difference in sensitivity of detection of the three N-f-met peptides, because the masses of synthetic fMIVIL, fMIGWII, and fMIVTLF could all be detected above 10 pmol (data not shown). There could be several reasons for the failure to detect fMIGWII and fMIVTLF. First, the precursor polypeptides from which fMIGWII and fMIVTLF are derived could be in lower abundance in the ER than the polypeptide from which fMIVIL is generated. Second, by assuming higher peptide affinity or preferential binding of the fMIVIL peptide to gp96 compared with fMIGWII and fMIVTLF, higher amounts of fMIVIL than fMIGWII and fMIVTLF could associate with gp96. Alternatively, due to differential peptide binding affinity to gp96, it could be more difficult to elute fMIGWII and fMIVTLF from gp96 than fMIVIL. In addition, the in vivo priming may arise solely from recognition of fMIVIL and the responses to the other epitopes after in vitro restimulation could be due to the cross-reactivity demonstrated in Fig. 4Go.



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  		FIGURE 6. Comparative MS of eluates from gp96 from L. monocytogenes-infected and noninfected mice. MALDI-MS spectra of peptides eluted from gp96 after precipitation with 2% TFA and 40% methanol. The protein was removed by centrifugation. A, Peptides eluted from gp96 from L. monocytogenes-infected C57BL/6 mice. B, Peptides eluted from gp96 from noninfected C57BL/6 mice. C, 100 pmol synthetic fMIVIL peptide. This experiment was performed three times with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our finding is consistent with two major functions of HSP, namely their chaperone function in protein folding and unfolding, and their ability to activate T cells (14, 19). The role of HSP as chaperones would require a specific binding site capable of binding to hydrophobic parts of the target protein. N-f-met peptides are hydrophobic and are recognized by H2-M3-restricted CD8 T cells during listeriosis.

Several groups have succeeded in eluting peptides from HSP (28, 29, 30). The majority of these peptides have been class Ia binding peptides, but recently, a heptameric hepatitis B virus peptide has been isolated from gp96 derived from liver tissues from patients with hepatocellular carcinoma, indicating that peptides other than standard MHC class Ia binding peptides can be found associated with gp96 (38). In most instances, MHC class Ia binding peptides isolated from gp96 are identified functionally by prescreening HPLC fractions of eluted peptides with Ag-specific T cell clones. Mass spectrum analysis of the positive HPLC fractions revealed that these MHC I binding peptides only represented a fraction of the peptides eluted from gp96 (28, 29, 30). This opens the possibility that additional peptides could be present on gp96 and, therefore, we speculate that T cells generated by gp96 immunization recognize distinct peptides with unusual physiochemical features such as N-f-met peptides. Previous identification of such peptides on HSP could have been hampered by their hydrophobicity.

The protective, anti-listerial immune response in C57BL/6 mice is dominated by T cells specific for N-f- met peptides, and no classical, protective H-2b-restricted listerial epitopes for CD8 T cells have yet been identified (5). In contrast, BALB/c mice infected with sublethal doses of L. monocytogenes develop H2Kd-restricted, protective T cells specific for dominant LLO and p60 epitopes (reviewed in Ref. 39). Interestingly, no L. monocytogenes-encoded LLO or p60 peptides were identified on gp96 from Listeria-infected BALB/c mice (data not shown) despite the fact that these Ags are transported into and are abundant in the ER (40).

The gp96-induced T cell response to fMIVIL did not depend on MHC class II-restricted Th cells, but was augmented by the presence of both MHC class I and class II molecules. Depletion experiments have lead to similar conclusions in the tumor system (41). At present, we do not know whether this was due to MHC class II-restricted bacterial Ags present on gp96 or whether gp96 was processed and served as Ag for CD4 Th cells. After uptake, HSP ends up in early endosomes (21), and could therefore be processed and presented via the MHC class II pathway.

Modest but significant reduction of bacterial numbers was found in the spleens and livers of mice immunized with gp96 from L. monocyogenes compared with mice immunized with gp96 from noninfected animals (42). This was not unexpected because HSP-based tumor therapies appear to be most efficient in preventing metastasis rather than in reducing a large tumor mass (43). Therefore, it may be conceivable that to control the assault of large doses of bacteria, HSP-based therapies are more efficient if administered together with other vaccine candidates.

Efficacious vaccines against intracellular bacteria, in particular Mycobacterium tuberculosis, are not available (1). We set out to investigate the capacity of gp96 to bind immunogenic peptides and to express natural adjuvant activity against intracellular bacteria. Ideally, such a subunit vaccine would comprise the smallest immunogenic entity—that is, the epitope recognized by protective T cells. Unfortunately, the high polymorphism of MHC molecules strongly restricts the feasibility of peptide vaccines in out-breed populations. HSP involved in Ag processing are virtually nonpolymorphic and hence are well suited for the identification of promiscuous precursor peptides for T cell epitopes.

The isolation of N-f-met peptides from gp96 derived from Listeria-infected organs is consistent with the hydrophobic character of the gp96 binding site. Immune responses to intracellular bacteria such as M. tuberculosis are characterized by significant nonclassical immune T cell to hydrophobic proteins and lipids (1, 44). Therefore, our approach may be useful for the development of novel subunit vaccines.


   Acknowledgments
 
We thank Dr. Hans Willy Mittruecker for helpful discussions and Drs. Peter Seiler and Peter Aichele for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by a grant from the Deutsche Forschungsgemeinschaft (KA 573/4-1). Back

2 Address correspondence and reprint requests to Dr. Stefan H. E. Kaufmann, Department of Immunology, Max-Planck Institute for Infection Biology, Schumannstrasse 21-22, 10117 Berlin, Germany. E-mail address: kaufmann{at}mpiib-berlin.mpg.de Back

3 Abbreviations used in this paper: N-f-met, N-terminal formyl groups; HSP, heat shock proteins; LLO, listeriolysin; TFA, trifluoroacetic acid; ER, endoplasmic reticulum; MALDI-MS, matrix-assisted laser desorption ionization-mass spectrometry. Back

Received for publication June 18, 2001. Accepted for publication September 24, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kaufmann, S. H. E.. 2000. Is the development of a new tuberculosis vaccine possible?. Nat. Med. 6:955.[Medline]
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