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The Journal of Immunology, 2001, 167: 2209-2218.
Copyright © 2001 by The American Association of Immunologists

Dynamic Antigen Presentation Patterns of Listeria monocytogenes-Derived CD8 T Cell Epitopes In Vivo1

Mojca Skoberne*, Rafaela Holtappels{dagger}, Herbert Hof* and Gernot Geginat2,*

* Institut für Medizinische Mikrobiologie und Hygiene, Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Mannheim, Germany; and {dagger} Institut für Virologie, Universität Mainz, Mainz, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Little information exists regarding the presentation of antigenic peptides in infected tissues. In this study the in vivo presentation of four different CD8 T cell epitopes of Listeria monocytogenes was monitored. Peptide presentation was measured by a new, highly sensitive, ex vivo Ag presentation assay that was based on the testing of freshly isolated cells from infected spleens with peptide-specific CD8 T cell lines in an IFN-{gamma}-specific ELISPOT assay. Remarkably, the peptide presentation pattern of splenocytes and that of macrophages purified from spleens of L. monocytogenes-infected mice were different from those of in vitro infected macrophage-like cell lines. The in vivo Ag presentation pattern of splenocytes also exhibited dynamic changes during the first 48 h of infection. In vivo peptide presentation at later time points postinfection was biased toward immunodominant CD8 T cell epitopes, while at an early time point, 6 h postinfection, subdominant and dominant CD8 T cell epitopes were presented with similar strength. In summary, our studies show that Ag presentation during an infection is a highly dynamic process that only can be fully appreciated by the study of cells infected in their physiological environment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The host response against infection with a complex micro-organism comprises T cells specific for a multitude of different antigenic peptides. Generally, the magnitude of the T cell response against different antigenic peptides exhibits a remarkably constant hierarchy, and the majority of the responding T cells are directed against few immunodominant T cell epitopes (1). Numerous studies in different infectious disease model systems revealed that the strength of the CD8 T cell response against a peptide is the result of the complex interplay of three major factors: the quantity and stability of peptide MHC class I/complexes expressed on APC, the TCR repertoire of the responding T cell population, and the suppression of T cells specific for subdominant epitopes by T cells specific for immunodominant epitopes (reviewed in Ref. 2). Ag presentation is certainly required for the induction and expansion of CD8 T cells. However, only a few studies exist about the processing and presentation of antigenic peptides in vivo. In principle, the extraction and quantification of naturally processed antigenic peptides allow the direct analysis of Ag processing in tissues (3). Due to the relatively large number of infected cells required for this method the extraction of antigenic peptides from organs was only successful in a few model infections (4, 5). More indirectly, bacteria and viruses that express genetically engineered proteins were used to analyze the effects of variations in Ag presentation on the strength of the CD8 T cell response in vivo. Examples are the enhanced immunogenicity of preprocessed Ags that are directly targeted into the endoplasmic reticulum (6) or the modulation of the immunogenicity of T cell Ag by variations in the sequences flanking a T cell epitope (7, 8).

The murine infection with Listeria monocytogenes is one of the infection models where the mechanisms governing CD8 T cell induction and expansion were studied in detail. Mice infected with L. monocytogenes mount MHC class I Kd-restricted CD8 T cell responses against peptides encompassing aa 91–99 of listeriolysin O (LLO)3 (4); aa 217–225 (9), 449–457 (10), and 476–484 (11) of the p60 protein; and aa 84–92 of the listerial metalloprotease (12), respectively. In vivo the majority of CD8 T cells are specific for the immunodominant epitopes LLO91–99 and p60217–225, while relatively few T cells are directed against the subdominant epitopes p60449–457 and Mpl84–92 (13). The frequency of p60476–484-specific CD8 T cells is intermediate between the frequency of p60217–225 and that of p60449–457-specific T cells (11). Remarkably, among these four L. monocytogenes-derived peptides the immunodominant LLO91–99 is the least abundant endogenously processed peptide in infected cell lines, while the subdominant p60449–457 is the most abundant antigenic peptide in infected cell lines (14). Thus, a paradoxical inverse correlation exists between the abundance of naturally processed antigenic peptides in infected cells and the frequency of peptide-specific CD8 T cells in vivo.

It must be kept in mind that the quantitative analysis of peptide processing is based on in vitro infected cells and that it is not known to what extent this in vitro model represents the in vivo situation. Therefore, in the current study the presentation of L. monocytogenes-derived antigenic peptides was monitored in vivo. We used a novel approach for the direct measurement of Ag presentation in tissues that is based on the testing of in vivo-infected cells with peptide-specific CD8 T cell lines in a sensitive ELISPOT assay. Remarkably, the peptide presentation pattern of splenocytes infected with L. monocytogenes in vivo exhibited dynamic changes during the first 48 h of infection. In light of this new finding the possible correlation between peptide presentation and in vivo CD8 T cell expansion and function was reevaluated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and infection of mice

Female BALB/cOlaHsd (H-2d) mice were purchased (Harlan-Winkelmann, Borchen, Germany), kept under conventional conditions and used at 8–10 wk of age. Mice were infected with L. monocytogenes serovar 1/2a EGD in 0.2 ml PBS either i.v. or i.p. as indicated. Infectious doses were 1 x 106 and 1 x 103 CFU i.v. for ex vivo peptide presentation experiments and T cell induction studies, respectively. Bacteria used for infection were in the logarithmic growth phase. The bacterial concentration was estimated from the OD at 600 nm.

APC, in vitro infection of APC, and CD8 T cell lines

P815 mastocytoma cells were used as targets in the cell-mediated cytotoxicity assay. For in vitro infection experiments macrophage-like J774A1 (J774) and P388D1 (P388) cells were used as APC. Approximately 1 x 108 P388 or J774 cells were infected with L. monocytogenes at a multiplicity of infection of 10. After 1 h at 37°C, cells were washed once, and the medium was exchanged with medium containing 5 µg/ml gentamicin. After an additional 5-h incubation at 37°C, cells were either detached by trypsin treatment and tested in the ELISPOT-based Ag presentation assay or, alternatively for peptide extraction experiments, were harvested with a cell scraper without addition of trypsin.

CD8 T cell lines specific for p60217–225, p60449–457, p60476–484, and LLO91–99 were derived from the spleens of L. monocytogenes-infected BALB/c mice. CD8 T cell lines were propagated by repeated restimulation with P815 cells transfected with the human B7.1 gene (P815/B7) (15) in the presence of the appropriate synthetic peptide in medium supplemented with IL-2 as described previously (16). Synthetic peptides were purchased (Jerini Biotools, Berlin, Germany). The peptide concentration used for restimulation was 10-10 M for all CD8 T cell lines. The detection limit of the CD8 T cell lines used was between 10-11 and 10-12 M peptide as measured using a standard chromium release assay.

Immunomagnetic isolation of macrophages from infected mice

Macrophages from spleens of L. monocytogenes-infected mice were isolated by immunomagnetic cell sorting using paramagnetic microbeads conjugated to monoclonal hamster anti-mouse CD11b (clone M1/70.15.11.5) Abs (Miltenyi, Bergisch Gladbach, Germany). Spleens were removed 48 h after i.v. infection of mice with 1 x 105 CFU L. monocytogenes. Spleens were injected with 500 µl of a 1 mg/ml solution of collagenase D (Roche Diagnostics, Mannheim, Germany) in HBSS. Subsequently, spleens were cut in small pieces and incubated for 30 min at 37°C in 5% CO2 in the collagenase D buffer. Cells were collected by centrifugation and subsequently separated twice on MS+ selection columns (Miltenyi) following the standard positive selection protocol provided by the manufacturer. For each experiment spleens from three mice were pooled. At the end of the positive selection procedure, between 0.5 x 106 and 1 x 106 cells were obtained per 1 x 108 spleen cells. Aliquots of the selected cells were stained with FITC-labeled rat anti-mouse F4/80 IgG2b (clone CI:A3-1; Serotec, Eching, Germany), rat anti-mouse CD11b IgG2b mAb (clone, 5C6; Roche Diagnostics), rat Ig2b isotype control mAb (Serotec), and hamster anti-mouse CD11c IgG mAb (clone HL3; PharMingen, San Diego, CA), respectively, and subjected to FACS analysis. The remaining cells were tested in the ELISPOT-based Ag presentation assay described below.

Isolation of endogenously processed peptides

Peptide extraction from infected cells and organs was performed as described previously with minor modifications (5). Spleens were removed 48 h after i.v. infection with 1 x 106 CFU L. monocytogenes. Organs from five mice were pooled and passed through a steel mesh. After removal of an aliquot for plating of bacteria, trifluoroacetic acid (TFA) was added to achieve a pH of 2.0. The lysis solution was supplemented with COMPLETE proteinase inhibitor (final concentration, 1 tablet/50 ml lysis buffer) and 1 µg/ml pepstatin (both from Roche Diagnostics). After homogenization extracts were sonicated, left for 30 min on ice, and centrifuged for 1 h at 50,000 x g. Supernatants were removed and passed through a Sephadex G-25 (Amersham Pharmacia, Freiburg, Germany) column with an isocratic flow of 1 ml 0.1% TFA/min. Low m.w. fractions were collected and passed through a Sep-Pak C18 reverse phase, solid phase extraction unit (Waters, Eschborn, Germany). After washing with 5 ml 10% acetonitrile (AcN), bound material was eluted with 1.5 ml 50% AcN and 1.5 ml 100% AcN, pooled, concentrated to a final volume of 0.5 ml by vacuum centrifugation, and further fractionated by HPLC on a reverse phase C18 column ({Delta} Pak C18–300A, 3.9 x 300 mm; Waters): 1 ml peptide extract was loaded and eluted with a flow rate of 1 ml/min on a linear AcN gradient. Solution A was 0.1% TFA; solution B was 70% AcN and 0.09% TFA. The gradient was 0–5 min of 0% B, 5–55 min linear increase to 50% B, 55–63 min linear increase to 100% B, 63–66 min of 100% B, and 66–74 min linear decrease to 0% B. One-minute fractions were collected and stored at -70°C.

Isolation of naturally processed peptides from infected cell lines was performed similarly. Six hours postinfection (p.i.) ~1 x 108 adherent L. monocytogenes-infected P388 or J774 cells were washed twice with ice-cold PBS and harvested with a cell scraper. Cell pellets were disrupted, sonicated, and lysed for 30 min in 2 ml 0.5% TFA supplemented with proteinase inhibitors as described above. Subsequently, cell lysates were centrifuged 30 min at 20,000 x g, and supernatants were further purified by ultrafiltration using MICROSEP (Pall-Gelman, Dreieich, Germany) ultrafiltration units with a 10-kDa cutoff. The low Mr fraction was further fractionated by HPLC as described above.

Quantification of endogenously processed peptides

For the quantification of endogenously processed antigenic peptides HPLC fractions were dried by vacuum concentration and resolved in 1 ml cell culture medium. The precise amount of antigenic peptides in HPLC fractions was determined as described previously (5). Fractions were tested in a standard chromium release assay with 51Cr-labeled P815 cells as APC. The peptide concentration of the fractions was calculated by linear interpolation from lysis data obtained with a synthetic peptide standard. The recoveries of p60217–225, p60449–457, p60476–484, and LLO91–99 were determined in a preliminary experiment after admixture of synthetic peptides to mock extracts of cell lines or spleens. Recovery from spleen extracts was ~20% for p60217–225 and p60476–484, 10% for p60449–457, and 60% for LLO91–99. Recovery from cell extracts was ~20% for all three p60 peptides and 70% for LLO91–99. In the calculation of the total number of peptides per organ or per cell the different recovery rates were counted.

ELISPOT-based Ag presentation assay

Ag presentation by in vivo-infected splenocytes was assessed with an ELISPOT-based Ag presentation assay. This assay applies the basic principle of the ELISPOT assay for the detection of Ag presentation by target cells infected in vitro or in vivo. Spleens were removed between 6 and 48 h after infection of mice. Splenocytes were used as APC after passing through nylon gaze (80 mesh) and RBC lysis. Alternatively, in some experiments macrophages isolated from infected spleens or in vitro infected P388 and J774 cells were also used as APC. P388 and J774 cells were infected as described above. The setting of the assay was similar for these different APC types. All APC were tested in the presence of 10 µg/ml gentamicin and 20 µg/ml tetracycline. In round-bottom 96-well microtiter plates, 3 x 104 peptide-specific CD8 T cells/well were added to graded numbers of APC in a final volume of 150 µl. Plates were subsequently incubated for 5 h at 37°C in 5% CO2. This preincubation step in round-bottom plates was required for optimal contact of APC and responder CD8 T cells and resulted in a significantly better T cell activation compared with tests that were performed directly in flat-bottom ELISPOT plates (data not shown). After the preincubation cells were resuspended, and 100 µl cell suspension were transferred to rat anti-mouse IFN-{gamma} mAb-coated (RMMG-1; BioSource International, Camarillo, CA) nylon membrane-backed 96-well microtiter plates (Nunc, Wiesbaden, Germany) and incubated overnight. During the primary incubation step <20% of infected P388 and J774 cells attached; thus, the majority of APC were transferred to the ELISPOT plates together with CD8 T cells. ELISPOT plates were developed with biotin-labeled rat anti-mouse IFN-{gamma} mAb (clone XMG1.2; BD Biosciences, Heidelberg, Germany), HRP-streptavidin conjugate (Dianova, Hamburg, Germany), and aminoethylcarbazole dye solution.

Ex vivo enumeration of peptide-specific CD8 T cells

The frequency of peptide-specific CD8 T lymphocytes was determined in an IFN-{gamma}-specific ELISPOT assay 10 days after i.v. infection of mice with 1 x 103 CFU L. monocytogenes as described previously (11). Unseparated splenocytes (6 x 105/well) were stimulated for 6 h in round-bottom 96-well microtiter plates in the presence of 10-7 M peptide. Subsequently, activated cells (4 x 105 or 4 x 104/well) were transferred to anti IFN-{gamma}-coated ELISPOT plates that were developed as described above. The frequency of Ag-specific cells was calculated as the number of spots per splenocytes seeded. The specificity and sensitivity of the ELISPOT assay were controlled with IFN-{gamma}-secreting CD8 T cell lines specific for p60217–225, p60449–457, p60476–484, and LLO91–99. The recovery of seeded CD8 T cells was >90% for all T cell lines.

Adoptive transfer

Mice were infected i.v. with 1 x 103 CFU L. monocytogenes and subsequently received 5 x 106 peptide-specific CD8 T cells i.v. in PBS. The number of CFU in the organ homogenates was determined 72 h later as described previously (16). The statistical significance of results was checked with the Newman-Keuls multiple comparison test at the 0.05 significance level. All tests were performed using WINKS statistical analysis software (Texasoft, Cedar Hill, TX). All experiments were repeated at least twice, with similar results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Applying the ELISPOT assay for measuring Ag presentation ex vivo

Ag presentation studies are generally performed with permanent cell lines that are cultured and infected in vitro. Ex vivo analysis of antigenic peptides from infected organs was only reported in a few instances (4, 5, 17). Acidic extraction of naturally processed peptides from tissues is a time-consuming task that involves multiple separation steps and generally requires a relatively large input of material. Another disadvantage of the quantitative peptide extraction approach is that peptides presented on the cell surface cannot be measured selectively. To overcome these restrictions we applied the principle of the ELISPOT assay for analysis of in vivo-infected APC. The ELISPOT assay is generally used for the detection of cells reactive against a defined Ag (13). For the ELISPOT-based Ag presentation assay the test principle was reversed, and the assay was used to detect the activation of peptide-specific CD8 T cells by infected APC.

To detect Ag presentation in vivo, mice were infected i.p. with a high dose (1 x 107 or 1 x 106 CFU) of L. monocytogenes. Spleens were removed 48 h p.i., and splenocytes were tested in the ELISPOT-based Ag presentation assay (Fig. 1GoA). To quantify the strength of Ag presentation the number of spots per well was counted (Fig. 1GoB). In vivo infected splenocytes showed a distinct reaction pattern with CD8 T cells specific for p60217–225, p60449–457, p60476–484, and LLO91–99. The strongest response was obtained with CD8 T cells specific for LLO91–99 and p60217–225. CD8 T cells specific for p60476–484 yielded an intermediate response, and p60449–457-specific CD8 T cells yielded the weakest response. The reactivity of the CD8 T cell lines was dependent on the dose used for infection of mice (Fig. 1GoB). Spleens of mice infected with 1 x 107 CFU compared with mice infected with 1 x 106 CFU revealed an ~5-fold increased load with L. monocytogenes (56 x 106 and 11 x 106 CFU/spleen, respectively). Although the absolute strength of the CD8 T cell response against infected splenocytes varied in both groups of mice, the principal recognition pattern remained unchanged. The signal strength was also dependent on the number of APC added per well. A 5-fold reduction of the number of APC added per well (1 x 105 vs 5 x 105/well) also reduced the absolute number of spots per well, but did not alter the principal recognition pattern obtained with the panel of CD8 T cell lines tested (Fig. 1GoB). If CD8 T cell lines were cocultivated with noninfected spleen cells or if spleen cells were cultivated in the absence of CD8 T cells, a background activity between one and four spots per well was observed (data not shown).



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  		FIGURE 1. Applying the ELISPOT assay for measuring Ag presentation ex vivo. Mice were infected i.p. with the indicated dose of L. monocytogenes. Ag presentation by splenocytes was tested 48 h p.i. with a panel of CD8 T cell lines in the ELISPOT-based Ag presentation assay as described in Materials and Methods. CD8 T cells specific for p60217–225, p60449–457, p60476–484, or LLO91–99 were added to either 5 x 105 or 1 x 105 splenocytes as indicated. Scans of individual wells (A) and the total number of spots per 2 x 104 CD8 T cells (B) are shown. The peptide sensitivity of the used CD8 T cells was tested in an ELISPOT assay with splenocytes as APC in the presence of the indicated peptide concentrations (C). Shown is the total number of spots per 2 x 104 CD8 T cells of the indicated specificity.

 
To enable quantitative comparison of the presentation of different peptides, CD8 T cell lines were selected that showed comparable peptide sensitivities (Fig. 1GoC). The peptide sensitivity of CD8 T cell lines was measured in the ELISPOT-based Ag presentation assay. Noninfected splenocytes were incubated with graded doses of synthetic p60217–225, p60449–457, p60476–484, and LLO91–99 peptide, respectively, and were tested with the CD8 T cell line of corresponding specificity. As shown in Fig. 1GoC all CD8 T cell lines required a similar minimal peptide concentration for a significant response.

Naturally processed antigenic peptides were detected after infection with a high dose of L. monocytogenes. When spleens were removed 48 h p.i., a significant bacterial load was present in the organ. Thus, the possibility exists that further replication of bacteria occurs in the in vitro phase of the assay. To address the possible effect of ongoing intracellular bacterial replication on the Ag presentation assay control experiments were performed. Graded numbers of splenocytes were tested in the ELISPOT-based Ag presentation assay either in the absence of antibiotics (Fig. 2Go, upper panel) or in the presence of gentamicin and tetracycline (Fig. 2Go, lower panel). As shown in Fig. 2Go the presence of antibiotics did not influence the principle peptide presentation pattern of L. monocytogenes-infected splenocytes. Thus, the peptide presentation pattern of L. monocytogenes-infected splenocytes was not influenced by the inhibition of bacterial protein synthesis. To exclude any possible interference of viable bacteria in the Ag presentation assay, all tests were performed in the presence of gentamicin and tetracycline.



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  		FIGURE 2. Ex vivo Ag presentation analysis is independent of bacterial replication. To test the influence of ongoing bacterial replication and protein secretion on the ex vivo peptide presentation pattern the ELISPOT-based Ag presentation assay was performed either in the absence of antibiotics (upper panel) or in the presence of 10 µg/ml gentamicin and 20 µg/ml tetracycline (lower panel). Graded numbers of splenocytes were tested with peptide-specific CD8 T cell lines. Shown is the mean total number of spots per 2 x 104 CD8 T cells of the indicated specificity. Error bars indicate the SD of triplicate determinations.

 
Taken together, these results demonstrate that the ELISPOT-based Ag presentation assay allows the ex vivo detection of antigenic peptides presented on the surface of L. monocytogenes-infected splenocytes.

L. monocytogenes-infected cells exhibit distinct Ag presentation patterns in vivo and in vitro

Long term in vitro propagated cell lines poorly represent the characteristics of natural APC, which, e.g., are always exposed to a specific local cytokine environment. Therefore, we used the ELISPOT-based Ag presentation assay to compare in vitro and in vivo Ag presentation. Graded numbers of APC infected in vivo or in vitro were added to CD8 T cells specific for p60217–225, p60449–457, p60476–484, or LLO91–99. Fig. 3GoA shows the average number of spots/well obtained in the presence of 2 x 104 splenocytes, 103 J774, or 103 P388 cells/well, respectively. The reactivity pattern of CD8 T cell lines with in vivo-infected splenocytes (Fig. 3GoA, upper panel) differed consistently from the reaction pattern obtained with in vitro infected P388 (Fig. 3GoA, middle panel) or J774 (Fig. 3GoA, lower panel) cell lines. Remarkably, in comparison to L. monocytogenes-infected P388 or J774 cells, LLO91–99 was clearly presented stronger by infected splenocytes. In contrast, the peptide presented strongest by P388 and J774 cells was p60449–457, which was barely detectable on infected splenocytes. As it is not known whether the APC type principally influences peptide recognition by CD8 T cells control experiments were performed with peptide-loaded APC. Splenocytes, P388, or J774 cells were loaded for 2 h in the presence of 10-9 M synthetic peptides, washed, and tested with peptide-specific CD8 T cells of the corresponding specificity. Fig. 3GoB shows the number of spots per 2 x 104 responder CD8 T cells. To directly compare the Ag presentation efficacy of different cell types, the number of APC was adjusted to 1000 APC/well. As shown in Fig. 3GoB splenocytes were weaker APC than P388 or J774 cells. However, different APC types did not exert a selective influence on the recognition of any of the four antigenic peptides tested. In summary, these results show that ex vivo isolated L. monocytogenes-infected splenocytes and in vitro infected cell lines exhibit different Ag presentation patterns.



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  		FIGURE 3. L. monocytogenes-infected cells exhibit different Ag presentation patterns in vivo and in vitro. Ag presentation by in vivo-infected splenocytes (upper panel) and in vitro infected P388 (middle panel) and J774 (lower panel) cells was compared (A). The peptide presentation was tested in the ELISPOT-based Ag presentation assay with CD8 T cell lines of the indicated specificity. Shown is the mean total number of spots per 2 x 104 CD8 T cells ± SD of triplicate determinations. The possible cell line dependency of the peptide recognition pattern was analyzed with peptide-loaded APC (B). J774, P388, and splenocytes were loaded 2 h with 10-9 M peptide and were tested with CD8 T cells of the corresponding specificity as indicated. Shown is the number of spots per 2 x 104 CD8 T cells and 1000 peptide-loaded APC per well.

 
Ag presentation pattern of in vivo-infected macrophages

Splenocytes are not a homogenous cell population. To test whether the disparate Ag presentation patterns of whole spleen cells and in vitro infected macrophage-like cell lines are the result of a mixture of different APC types in the spleen, the Ag presentation pattern of in vivo-infected macrophages was analyzed. CD11b+ cells, which are mostly macrophages, were separated from spleens 48 h after i.v. infection of mice with 1 x 105 CFU L. monocytogenes. The immunomagnetic selection of CD11b+ cells yielded a highly enriched macrophage population. Approximately 90% of cells stained positively for F4/80, and all cells were negative for the dendritic cell marker CD11c (Fig. 4GoA). These CD11b+ cells were tested with peptide-specific CD8 T cell lines in the ELISPOT-based Ag presentation assay (Fig. 4GoB). The Ag presentation pattern of isolated macrophages repeated the principle peptide presentation pattern of whole spleen cells, which is characterized by strong p60217–225 and LLO91–99 presentation. However, the direct comparison of the T cell activation by isolated macrophages and unseparated spleen cells showed that the CD11b-selected cells are much stronger Ag presenters (Fig. 4GoB).



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  		FIGURE 4. Ag presentation pattern of in vivo-infected macrophages. Macrophages were isolated by immunomagnetic separation from spleens 48 h after i.v. infection with 1 x 105 CFU L. monocytogenes. A, Cells were stained as indicated with anti-F4/80- and anti-CD11c-mAb or with an rat IgG2b isotype control. The cytofluorometric analysis of ex vivo isolated macrophages is shown. B, Isolated macrophages or unseparated splenocytes were tested in the ELISPOT-based Ag presentation assay. The number of spots per 2 x 104 CD8 T cells and the indicated number of APC per well is shown.

 
Thus, isolated, in vivo-infected macrophages demonstrate the same Ag presentation pattern as unseparated spleen cells, which is clearly different form the peptide presentation pattern of in vitro infected macrophage-like cell lines.

Acidic extraction of naturally processed antigenic peptides from infected organs

The ELISPOT-mediated Ag presentation assay measures selectively cell surface presentation of peptides. To determine whether the Ag presentation pattern reflects the total peptide composition of infected cells, peptides were also quantified in whole cell extracts. Mice were infected i.v. with 1 x 106 CFU L. monocytogenes. Spleens were removed 48 h p.i., and peptides were extracted from a pool of five organs. Naturally processed antigenic peptides were also extracted from L. monocytogenes-infected P388 cells. After HPLC separation, fractions containing antigenic activity were identified with peptide-specific CD8 T cells in a 51Cr release assay. Fig. 5GoA shows the results of a representative CTL test of HPLC fractions from a peptide extract of infected spleens. The total peptide content of positive fractions was finally determined by linear interpolation from a synthetic peptide standard (data not shown). In L. monocytogenes-infected spleens only the peptides LLO91–99 and p60217–225 were detectable (Fig. 5GoB, upper panel). Both peptides were similarly abundant, in the range between 3 x 109 and 5 x 109 peptides/spleen. The peptides p60476–484 and p60449–457 both ranged below the detection limit of the assay, which was ~5 x 108 peptides/spleen for both peptides. In contrast to infected splenocytes, the most abundant peptides in P388 cells were p60449–457 and p60217–225, while LLO91–99 was significantly less abundant than these p60-derived peptides (Fig. 5GoB, lower panel). The p60476–484 peptide was not detected in cell extracts, indicating that less than five p60476–484 peptides were presented per cell. Thus, if the peptide presentation patterns of P388 cells and spleens are compared, it is clear that p60449–457 was significantly less abundant than p60217–225 in infected spleens. As in infected spleens the amount of p60217–225 peptides was ~10-fold greater than the detection limit of p60449–457-specific CD8 cells, it is unlikely that the presence of an equal amount of p60449–457 was just overlooked. Taken together, the quantitative peptide extraction procedure confirmed the results obtained with the ELISPOT-based Ag presentation assay (Fig. 3GoA). Therefore, we conclude that the Ag presentation pattern displayed by infected splenocytes and P388 cells mirrors the total peptide composition of the infected cells.



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  		FIGURE 5. Acidic extraction of naturally processed antigenic peptides from L. monocytogenes-infected cell lines and organs. Naturally processed antigenic peptides were extracted and quantified from the spleen of L. monocytogenes-infected mice and from infected P388 cells. After HPLC separation, fractions containing antigenic activity were identified with peptide-specific CD8 T cell lines in a 51Cr release assay. Peptides p60217–225, p60449–457, p60476–484, and LLO91–99 were detected with CD8 T cells of corresponding specificity as indicated. A representative CTL test of HPLC fractions prepared from a peptide extract of infected spleens 48 h p.i. is shown (A). Arrows indicate the HPLC fraction where the indicated peptides elute. The total peptide content of positive fractions was determined by linear interpolation from a synthetic peptide standard (B). The total number of peptides per L. monocytogenes-infected spleen is shown (upper panel). For infected P388 cells the number of peptides per cell is indicated (lower panel). Dotted lines indicate the detection limit of the assay.

 
Kinetics of Ag presentation in vivo

After sublethal L. monocytogenes infection of mice the bacterial load in the spleen peaks around day 3 p.i., and specific CD8 T cells can be detected 5 days p.i. and peak around day 7 p.i. (18). To study the kinetics of Ag presentation after L. monocytogenes infection the peptide presentation pattern of splenocytes was tested at different time points postinfection. Two different experimental approaches were chosen. To achieve a similar bacterial load in the spleen mice were infected with different doses of L. monocytogenes. Spleens were tested 6, 24, and 48 h after i.v. infection with 1 x 108, 1 x 107, and 1 x 106 CFU/mouse, respectively (Fig. 6GoA). Alternatively, all mice were infected with the same dose of 1 x 105 CFU L. monocytogenes i.v. and tested 6, 24, and 48 after infection (Fig. 6GoB). The number of CFU per spleen of the different experimental groups is shown in Fig. 6GoC. Later time points p.i. were not included due to the increasing background activity of ex vivo isolated splenocytes. Spleen cells of all mice were tested in the ELISPOT-based Ag presentation assay with a set of CD8 T cell lines. Remarkably, the relative abundance of naturally processed antigenic peptides in vivo changed dramatically during the first 48 h of L. monocytogenes infection. Around 6 h p.i., p60217–225, p60449–457, p60476–484, and LLO91–99 were all presented with comparable strength. Later, between 24 and 48 h p.i., this relation changed. At 48 h p.i. LLO91–99 and p60217–225 were presented much more strongly than p60449–457 and p60476–484 in both experimental groups.



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  		FIGURE 6. Kinetics of Ag presentation in vivo. The ex vivo Ag presentation pattern was determined 6, 24, and 48 h after L. monocytogenes infection of mice. Freshly isolated splenocytes from infected mice were tested in the ELISPOT-based Ag presentation assay using peptide-specific CD8 T cell lines. The strength of Ag presentation is shown as the mean total number of spots per 2 x 104 CD8 T cells of the indicated specificity. Error bars indicate the SD of triplicate determinations. A, Mice were tested 6 h after infection with 1 x 108 CFU, 24 h after infection with 107 CFU, and 48 h after infection with 106 CFU L. monocytogenes, respectively. B, Mice were tested 6, 24, and 48 h after infection with 105 CFU L. monocytogenes. The different time points of each of the two experimental groups (A and B) were tested in the same experiment. C, Shown are the bacterial loads of spleens tested in A and B.

 
In summary, these data show that the Ag presentation pattern of L. monocytogenes-infected cells in vivo is highly dynamic, with rapid peptide-specific changes during the first 48 h p.i.

Frequency and protective potential of peptide-specific CD8 T cells in vivo

In previous reports a paradoxical inverse correlation between the abundance of naturally processed antigenic peptides extracted from in vitro infected cells and the frequency of peptide-specific CD8 T cells in vivo was noted (13, 14). We found that the Ag presentation pattern of infected cell lines differed from the pattern displayed by ex vivo isolated splenocytes. Furthermore, we found that in vivo the peptide presentation pattern of L. monocytogenes-infected cells changed during the course of infection. As Ag presentation is principally required for CD8 T cell induction and also for the recognition of infected target cells, we analyzed the possible correlation between in vivo peptide presentation and the frequency and effector function of peptide-specific CD8 T cells. As shown in Fig. 7GoA the frequency of peptide-specific CD8 T cells in primarily infected BALB/c mice on day 10 p.i. exhibited a distinct hierarchy. This hierarchy of L. monocytogenes-specific CD8 T cells (LLO91–99 > p60217–225 > p60476–484 > p60449–457) corroborates results published previously by Sijts et al. (13). The recently described p60476–484 epitope exhibited an intermediate strength among the three known CD8 T cell epitopes of p60, confirming previous results obtained after secondary L. monocytogenes infection (11). Remarkably, the immunodominant peptides LLO91–99 and p60217–115 were also the peptides that revealed the strongest Ag presentation 48 h p.i. (Fig. 6Go, A and B).



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  		FIGURE 7. Frequency and protective potential of peptide-specific CD8 T cells in vivo. The frequency of peptide-specific CD8 T cells in vivo 10 days p.i. was determined in an ELISPOT assay in the presence of 10-7 M peptide (A). The mean number of IFN-{gamma}-positive cells per 4 x 105 splenocytes is shown. Error bars indicate the SD of three individual mice. The protective potential of peptide-specific CD8 T cells was tested in adoptive transfer experiments (B and C). Mice were injected i.v. with 1 x 103 CFU L. monocytogenes and subsequently received 5 x 106 peptide-specific CD8 T cells of the indicated specificity. Results show the number of CFU per spleen ± SD of groups of five mice. Statistical analysis of results was performed with the Newman-Keuls multiple comparison test (p < 0.05). T cell lines were ranked according to the protection mediated in the spleen. The graphical representation of the Newman-Keuls test (C) indicates the rank and group of each T cell line. The means of any two lines belonging to the same group (underscored by the same line) are not significantly different.

 
To test the possible correlation of the protective potential of peptide-specific CD8 T cells to the peptide presentation pattern in vivo, adoptive transfer experiments were performed. The CD8 T cell lines were matched for comparable peptide sensitivity and lytic potential in vitro. Mice received 5 x 106 CD8 T cells immediately after L. monocytogenes infection, and the bacterial load of the spleen was determined 3 days later (Fig. 7GoB). All CD8 T cell lines conferred significant (p < 0.05) protection against L. monocytogenes. Generally, LLO91–99-specific CD8 T cells conferred the strongest protection, but this difference was not statistically significant (Fig. 7GoC). Corroborating earlier results by Dunn and North (19) CD8 T cells transferred 24 h p.i. conferred no significant protection against L. monocytogenes infection (data not shown). Compared with the quantitative hierarchy of CD8 T cells, the protection assay revealed a different hierarchy of the protective potential of transferred CD8 T cells. Remarkably, the protection mediated by CD8 T cells did not correlate with the Ag presentation pattern at 48 h p.i., when only poor presentation of p60449–457 and p60476–484 was detected. However, the protection pattern of CD8 T cells correlated well with the Ag presentation pattern 6 h p.i., when all peptides were presented with similar strength (Fig. 6Go, A and B).

In summary, these data show that the protective capacity of adoptively transferred L. monocytogenes-specific CD8 T cells does not correlate to the immunodominance of these CD8 T cells in vivo. Further it is shown that the expansion and the effector function of CD8 T cells correlate with the in vivo Ag presentation pattern at different time points p.i.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We used a novel approach for the ex vivo measurement of Ag presentation that is based on the testing of in vivo-infected APC with peptide-specific CD8 T cells in a sensitive ELISPOT assay. The high sensitivity of this assay enabled the measurement of peptide presentation without the technically difficult classical peptide extraction method that is generally used for the quantification of naturally processed peptides in tissues (3, 4, 5, 17). Remarkably, the comparison of in vivo-infected splenic macrophages and in vitro-infected macrophage-like cell lines revealed distinct peptide presentation patterns for in vivo- and in vitro-infected APC. As different APC types and also APC harvested at different time points p.i. were always directly compared in the same experiment with the same set of CD8 T cell lines, the observed differences in the peptide presentation patterns cannot be attributed to different sensitivities of the CD8 T cell lines used. In contrast to acidic peptide extraction the Ag presentation assay detects selectively cell surface peptide presentation. This is an important advantage if the relevance of Ag presentation for the induction of T cells shall be studied because some microbial immune escape mechanisms selectively prevent cell surface presentation of antigenic peptides (20). However, after infection of mice with L. monocytogenes, the peptide presentation patterns of splenocytes and infected cell lines were principally confirmed by peptide extraction of whole organs and cells, respectively. The Ag presentation pattern of in vitro infected P388 or J774 cells confirmed data reported by Pamer’s group (14). Compared with acidic peptide extraction the ELISPOT-based Ag presentation assay requires less material and is more sensitive. The ELISPOT-based assay also enables measurement of the Ag presentation pattern of defined, ex vivo isolated cell populations. This gives the assay a broad range of possible applications for the study of animal and probably also human disease states.

The quantitative aspects of Ag processing are generally analyzed in long term cell cultures. A number of previous studies compared the abundance of naturally processed peptides in infected cell lines with the frequency of peptide-specific CD8 T cells in vivo. In the murine L. monocytogenes infection model an inverse correlation between the abundance of antigenic peptides in infected cell lines and the frequency of peptide-specific CD8 T cells has been noted (13, 14). Similarly, immunodominance among EBV-derived MHC class I-restricted epitopes does not correlate with the abundance of antigenic peptides in EBV-transformed cell lines (21). Also, a detailed study of the CD8 T cell response against influenza virus has shown that inefficient Ag processing accounts only for the poor immunogenicity of one subdominant determinant, while in other instances the limitation is located on the side of the T cell (22). Taken together, from these experiments no obvious general correlation between the frequency of peptide-specific CD8 T cells in vivo and the abundance of antigenic peptides in infected cells is evident. However, it should be noted that those studies generally analyzed Ag presentation in vitro. When long-term in vitro propagated cell lines are studied, the most obvious differences to the in vivo situation include the cell type and the absence of the physiological cytokine environment. Additionally, it has to be considered that compared with an in vitro cell culture model, microorganisms in vivo generally exhibit different growth kinetics and thus also the rate and kinetics of protein expression might not be identical. Analysis of the Ag processing of pp89168–176, an antigenic peptide derived from the murine CMV, has shown that in vivo Ag processing is strongly influenced by IFN-{gamma} (5) and also by the infected cell type (23). The important influence of the cell type on Ag presentation has been shown in a study of virus-infected dendritic and fibroblast cell lines (24). A possible explanation for these observations is that cell type-specific or IFN-{gamma}-mediated differences in the composition of the proteasome (reviewed in Ref. 25) alter Ag presentation in vivo.

In the current study also the peptide presentation pattern of in vivo-infected macrophages was analyzed. On a per cell basis a significantly stronger Ag presentation by ex vivo isolated macrophages was observed, while the peptide presentation pattern was identical with unseparated splenocytes. As it is known that in the spleen the majority of Listeriae resides in macrophages this result could be expected (26, 27). Thus, either the peptide presentation of other infected cell types in the spleen, e.g., dendritic cells or sinusoidal lining cells is similar to macrophages or the quantitative contribution of these cell types to the overall Ag presentation pattern of unseparated splenocytes is to low to be detected.

LLO91–99 and p60217–225 form relatively stable peptide/Kd complexes with a half-life of ~6 h, while p60449–457/Kd complexes have a half-life of <1 h (28, 29). The relative strength of the CD8 T cell response against these peptides correlates with the stability of the corresponding MHC class I/peptide complexes (29). Similar results were obtained with EBV-specific CD8 T cells (30) and in a CD8 T cell immunization study with a large number of synthetic peptides (31). In the context of these data it is remarkable that in L. monocytogenes-infected spleens 6 h p.i. all peptides were presented with similar strength, while at later time points p.i. the presentation of peptides that form stable MHC/peptide complexes was significantly stronger than the presentation of peptides that form unstable complexes. The remarkable correlation between the stability of MHC class I/peptide complexes, prolonged peptide presentation, and the frequency of peptide-specific CD8 T cells in vivo suggests a model for the observed changes of the in vivo Ag presentation. In infected cells p60 protein secretion is limiting for the generation of p60-derived epitopes (32). Thus, over an extended period of time it has to be expected that peptides that form stable MHC/peptide complexes outnumber peptides that form less stable complexes. Finally, this could result in the preferential stimulation and expansion of T cells directed against the more stable peptide/MHC complexes. The importance of peptide stability for the Ag presentation pattern is obvious when protein secretion is inhibited. Sijts et al. have shown that after inhibition of p60 biosynthesis by tetracycline treatment p60449–457 that forms unstable MHC class I/peptide complexes diminishes quickly, while p60217–225 that forms stable complexes persists over an extended time period (28). Accumulation of stable peptides over time could also at least in part explain the observed differences of the Ag presentation patterns of in vitro- and in vivo-infected APC. For peptide extraction macrophage-like cells were harvested 6 h p,i,, while spleens were removed 48 h p.i.. Thus, the accumulation of stable peptides in the spleen 48 h p.i. is clearly plausible. However, the peptide presentation patterns of splenocytes and macrophage-like cells differed also at an early time point 6 h after infection. At this early time point after infection, for example, LLO91–99 was presented clearly stronger on in vivo-infected APC than on in vitro-infected APC.

The relevance of prolonged Ag presentation for the induction of protective anti-listerial immunity has been demonstrated by the antibiotic abridgement of bacterial replication in vivo. Abridgement by ampicillin treatment during the first 5 days p.i. results in a diminished protective T cell response (33). Recently, Mercado et al. have shown that the magnitude and the kinetics of the p60217–225 and LLO91–99-specific T cell response against L. monocytogenes are determined during the first 24 h of bacterial infection independently from the infectious dose (34). Additionally they showed that the transfer of naive, p60449–457-specific CD8 T cells results in a strongly enhanced CD8 T cell response after subsequent L. monocytogenes infection, suggesting that Ag is also not limiting for the expansion of p60449–457-specific CD8 T cells in vivo. Therefore, and also because dendritic cells that possibly contribute only very few to the overall Ag presentation pattern of unseparated splenocytes play an important role in the primary stimulation of naive CD8 T cells, caution has to be exercised in the interpretation of the observed correlation between the Ag presentation pattern and the frequency of L. monocytogenes-specific CD8 T cells in vivo. However, the timing requirements described by Mercado et al. are not strikingly different from the time frame of the changing Ag presentation pattern in L. monocytogenes-infected mice, as by 24 h p.i. the presentation of the subdominant epitopes p60449–457 and p60476–484 started to decrease in relation to that of the dominant CD8 T cell epitopes.

In vivo, CD8 T cells mediate protection against L. monocytogenes (35, 36). To exert their protective function CD8 T cells must recognize infected target cells. A number of observations suggest that the Ag presentation requirements for the primary stimulation of naive T cells differ from the antigenic stimulus that is necessary to stimulate the effector function of experienced T cells. Shen et al. have analyzed the CD8 T cell response against a model T cell Ag expressed by recombinant L. monocytogenes either as secreted or nonsecreted fusion protein (37). Remarkably, they have found that the compartmentalization of bacterial Ags has differential effects on priming of CD8 T cells and protective immunity. Dichotomous requirements for CD8 T cell effector cell function and expansion are also implicated by the observation that subdominant CD8 T cell populations can have an important contribution to protective immunity against diverse intracellular microorganisms (38, 39, 40, 41, 42). The good protection obtained after adoptive transfer of p60449–457-specific CD8 T cells extents these results to the infection with L. monocytogenes. The observed kinetic changes in the in vivo peptide presentation pattern during L. monocytogenes infection further suggest an explanation for this dichotomy between the quantitative and protective hierarchy of CD8 T cells. The equal adoptive protection mediated by different peptide-specific CD8 T cell lines correlated with the equal peptide presentation of these peptides 6 h p.i.. In contrast to the protective function, the frequency of L. monocytogenes-specific CD8 T cells correlated with the Ag presentation pattern 48 h p.i.. Taken together these correlations suggest distinct Ag presentation requirements for CD8 T cell expansion and CD8 T cell effector function.

The current study represents the first study of the dynamics of Ag presentation in vivo. The obtained results were unexpected and highlight the limitations of in vitro experiments to predict Ag presentation in vivo. Therefore, it will be important to better define the quantitative, cell type-specific, and regulatory aspects of Ag presentation in vivo. The answer to these questions should help to facilitate the design of T cell vaccines and the therapy of T cell-mediated autoimmune diseases.


   Acknowledgments
 
We thank S. Schenk for excellent technical assistance, and W. Meister for his valuable help while preparing the figures.


   Footnotes
 
1 This work was supported by Deutsche Forschungsgemeinschaft Grant GE 1081/1-1. M.S. was supported by the Slovenian Ministry of Science and the Medical Faculty of the University of Ljubljana. Back

2 Address correspondence and reprint requests to Dr. Gernot 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: geginat{at}rumms.uni-mannheim.de Back

3 Abbreviations used in this paper: LLO, listeriolysin O; AcN, acetonitrile; TFA, trifluoroacetic acid; p.i., postinfection. Back

Received for publication February 15, 2001. Accepted for publication June 5, 2001.


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 Introduction
 Materials and Methods
 Results
 Discussion
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