The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Janda, J.
Right arrow Articles by Geginat, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Janda, J.
Right arrow Articles by Geginat, G.
The Journal of Immunology, 2004, 173: 5644-5651.
Copyright © 2004 by The American Association of Immunologists

Cross-Presentation of Listeria-Derived CD8 T Cell Epitopes Requires Unstable Bacterial Translation Products1

Jozef Janda*, Petra Schöneberger*, Mojca Skoberne2,*, Martin Messerle{dagger}, Holger Rüssmann{ddagger} and Gernot Geginat3,*

* Institut für Medizinische Mikrobiologie und Hygiene, Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Mannheim, Germany; {dagger} Medizinische Fakultät, Martin Luther Universität Halle-Wittenberg, Halle, Germany; and {ddagger} Max von Pettenkofer Institut für Hygiene und Medizinische Mikrobiologie, Ludwig Maximilians Universität, Munich, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Presentation of bacteria-derived CD8 T cell epitopes by dendritic cells (DC) requires either their direct infection or that DC acquire and cross-present Ags from other infected cells. We found that cross-presentation of Listeria monocytogenes-derived CD8 T cell epitopes was much stronger than direct Ag presentation by infected murine DC. Cross-presentation of Listeria-derived CD8 T cell epitopes showed unique physiological requirements. It was dependent upon the delivery of unstable bacterial translation products by infected, but still viable, Ag donor cells. Cross-presentation was enhanced both when unstable translation products in infected Ag donor cells were protected from proteasomal degradation and when the production of misfolded bacterial proteins was increased. The requirement of unstable translation products for cross-presentation may represent a novel pathway that functions to focus the CD8 T cell response toward epitopes derived from newly synthesized proteins.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The control of viral and intracellular bacterial pathogens requires the rapid recognition of infected cells by CD8 T cells that recognize peptides of eight to 11 aa presented on the cell surface by self-MHC class I molecules. Infected cells generate antigenic peptides after proteasome-mediated intracellular degradation of pathogen-derived proteins. In bone marrow-derived professional APC, an alternative MHC class I Ag presentation pathway is also present; dendritic cells (DC)4 and macrophages have the exclusive ability to cross-present Ags taken up from other infected cells (1, 2). For the APC, the advantage of cross-presentation is that it is not infected, and therefore, it is protected from any cytopathic effect exerted by the intracellular pathogen. The mechanisms involved in cross-presentation are not fully understood, and a number of alternative models exist. Antigenic material is taken up either in the form of peptides bound to heat shock proteins (3) or in the form of complete proteins that subsequently are processed and presented in the classical cytoplasmic MHC class I presentation pathway (4) or by an alternative endosomal pathway (5).

In BALB/c mice, Listeria monocytogenes infection stimulates a strong protective CD8 T cell response against p60217–225 and aa 91–99 of listeriolysin O (LLO) (6). The induction of the L. monocytogenes-specific CD8 T cell response requires the stimulation of naive CD8 T cells by DC (7). Both immunodominant epitopes, p60217–225 and LLO91–99, originate from secreted bacterial proteins (6). In infected cells, L. monocytogenes multiplies with an intracellular doubling time between 30 min and 1 h, and during bacterial replication secreted bacterial proteins accumulate intracellularly (8, 9). This protein pool represents a source of Ags for the cytoplasmic MHC class I Ag processing and presentation pathway. It was calculated that generation of a single H-2Kd-bound p60217–225 or LLO91–99 peptide requires approximately the degradation of 35 p60 molecules (8) or four to 11 LLO molecules (9), respectively. The intracellular protein pool that accumulates in infected cells also acts as an Ag depot for the cross-presentation of MHC class II-restricted Ags by DC. We have previously shown that CD4 T cell epitopes derived from the p60 and LLO of L. monocytogenes are cross-presented by DC, a pathway that circumvents the adverse effect of LLO on MHC class II-restricted Ag presentation (10). The cross-presentation of Listeria-derived CD4 T cell epitopes by DC suggests that cross-presentation could also be a pathway for the presentation by DC of Listeria-derived Ags to CD8 T cells.

In this study we investigate the MHC class I-restricted cross-presentation of L. monocytogenes in an in vitro model. Remarkably, in contrast to cross-presentation of CD4 T cell epitopes, MHC class I-restricted cross-presentation required viable Ag donor cells and continuous bacterial protein biosynthesis during cross-presentation. Our results indicate the existence of a cross-presentation pathway that specifically requires unstable bacterial translation products as the main substrate.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice, cell lines, and, bacteria

Female BALB/cOlaHsd (H-2d) mice were purchased (Janvier, Le Geneste St. Isle, France), kept under conventional conditions, and used at 8–10 wk of age. Macrophage-like IC21 cells (H-2b) were used as Ag donor cells and were kept in DMEM supplemented with 10% FCS without antibiotics. P388D1 (H-2d) cells were used as APC for the analysis of direct Ag presentation by infected cells.

L. monocytogenes serovar 1/2a EGD and L. monocytogenes {Delta}actA (11) were grown in brain heart infusion broth (BD Biosciences, Heidelberg, Germany) and were used in the logarithmic growth phase. The bacterial concentration was estimated from the OD at 600 nm.

Construction of recombinant vaccinia virus

The open reading frame encoding L. monocytogenes p60 was amplified by PCR using primers p60 forward (5'-cgg ctc tag acc acc atg agc act gta gta gtc gaa gct ggt-3') and p60 reverse (5-ccg agg atc cgt ata cgc gac cga agc caa ct-3') and plasmid pSK5 (12) (provided by Dr. I. Gentschev, Biozentrum, University Würzburg, Wurzburg, Germany) as a template and was subcloned into pUC19. An XbaI site in front of the ATG start codon was converted to a BamHI site by insertion of a self-complementary oligonucleotide (5'-cta gcg gca gga tcc tgc gc-3'), then the p60 open reading frame was transferred as a 1.5-kbp BamHI fragment into the vaccinia recombination vector pCS43 (13). Construction of the recombinant vaccinia virus was performed using the vaccinia virus strain Copenhagen and its temperature-sensitive mutant ts7 according to standard procedures as described previously (13).

T cell lines

CD8 T cell lines specific for p60217–225 and LLO91–99 (both Kd-restricted) and a CD4 T cell line directed against the H-2Ad-restricted epitope p60301–312 were established and propagated as described previously (10, 14).

Bone marrow-derived DC

DC were obtained from GM-CSF-supplemented (Tebu-Bio, Offenbach, Germany) bone marrow cultures as described previously (10). DC were used after 5 days of culture. In experiments in which optimal purity of DC was required, DC were further purified with anti-CD11c-coated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). After CD11c separation, cell populations were >90% CD11c+,CD8a–,CD80low,CD86 low, a phenotype typical for immature DC. For cytofluorometric analysis, cells were stained with FITC-labeled hamster anti-rat CD11c, PE-labeled hamster anti-mouse CD80-PE (IgG2, clone 16-10A1; BD Biosciences), PE-labeled rat anti-mouse CD86-PE (IgG2a mAb, clone GL1; BD Biosciences), PE-labeled rat anti-mouse CD8a (IgG2a mAb, clone 53-6.7; BD Biosciences), or isotope-matched control mAb (BD Biosciences).

Direct Ag presentation assay

Direct Ag presentation assays were performed as described previously (10) with some modifications. Either bone marrow-derived DC or P388D1 cells were infected for 1 h at a multiplicity of infection of 10. Viable extracellular bacteria were removed from P388 monolayers by repeated washing with medium containing 15 µg/ml gentamicin and from DC cultures by separation with anti-CD11c-coated magnetic beads. CD8 T cells were added 4 h after infection, and after overnight incubation T cell activation was assessed by measuring the IFN-{gamma} concentration in culture supernatants with an IFN-{gamma}-specific ELISA that binds and detects IFN-{gamma} with a pair of specific mAb. Results were corrected for dilution of the sample to yield the sample concentration in nanograms per milliliter.

Cross-presentation assay

Cross-presentation assays were performed with IC21 (H-2b) cells infected with L. monocytogenes wild type (wt) or L. monocytogenes {Delta}actA for 1 h at a multiplicity of infection of 10 as Ag donor cells. Cells were washed twice with gentamicin (50 µg/ml) and were kept in medium supplemented with 15 µg/ml gentamicin to inhibit extracellular replication of bacteria. Per well of a six-well plate, 1–2 x 106 Ag donor cells were mixed with 2–4 x 106 DC and incubated overnight at 37°C in most experiments. At the end of the incubation period, DC were recovered by immunomagnetic separation with anti-CD11c-coated magnetic beads (Miltenyi Biotec). In some experiments the proteasome inhibitor epoxomicin (1 µM; Sigma-Aldrich, Deisenhofen, Germany) was used to inhibit cytoplasmic Ag processing by Ag donor cells or by DC, respectively. Cells were pretreated with epoxomycin for 1 h, washed, and infected as described. Puromycin (1 µg/ml; Sigma-Aldrich) and L-canavanine (15 mM; Sigma-Aldrich) were used to enhance the formation of misfolded unstable proteins in bacterially infected cells (15, 16). Both inhibitors were added during the first 4 h postinfection (p.i.). If Ag donor cells were pretreated with epoxomycin, L-canavanine, or puromycin, the coculture of DC and infected Ag donor cells was limited to 5 h. In all experiments gentamicin (15 µg/ml) was added to prevent growth of extracellular bacteria. Clinafloxacin (10 µg/ml) was used to inhibit protein expression by intracellular bacteria in Ag donor cells (17). Bacterial protein synthesis in DC, e.g., possibly by viable bacteria taken up from Ag donor cells, was prevented by culturing DC in medium containing 10 µg/ml azithromycin hydrochloride (gift from Pfizer, Karlsruhe, Germany). Azithromycin is a hydrophobic macrolid antibiotic that is accumulated 200- to 300-fold in cells (18) and is active against Listeriae in both the cytoplasm as well as phagocytic vacuoles. Ag presentation by DC was assessed in an Ag presentation assay with peptide-specific T cell lines. Graded numbers of DC were mixed with 3 x 104 T cells, and activation of CD8 T cells after overnight incubation was measured as described above.

Immunofluorescence analysis

Immunofluorescence analysis of cross-presenting DC was performed by labeling DC with the green fluorescent membrane dye PKH67 (19) (Sigma-Aldrich). Cocultures with L. monocytogenes {Delta}actA-infected Ag donor cells were set up as described above. After 7 h, cytospin preparations of cells were prepared, and intracellular bacteria were stained. After fixation of cells with 2% paraformaldehyde and Triton X-100 treatment (0.05% for 30 s), intracellular bacteria were stained with a polyclonal rabbit anti-Listeria type 1 and 4 antiserum (BD Biosciences) and a donkey anti-rabbit Cy5-conjugated IgG F(ab')2 (Jackson ImmunoResearch Laboratories, West Grove, PA). Cellular actin filaments were visualized with Alexa Fluor 488 phalloidin (Molecular Probes, Leiden, The Netherlands). All samples were mounted with PROLONG mounting medium (Molecular Probes). Images were acquired using a conventional DMRE fluorescence microscope (filter blocks N2.1 and Y5 for green and far red fluorescence, respectively; Leica Microsystems, Wetzlar, Germany).

TUNEL assay

The degradation of nuclear DNA in apoptotic cells was detected using a commercial APO-BrdU TUNEL assay kit (Molecular Probes). Briefly, IC21 cells were grown in six-well plates and were harvested 9 h after infection. As a positive control, apoptosis of IC21 cells was induced by UV irradiation using two OSRAM HNS 15-W OFR bulbs (254 nm) for 10 min with a distance of 4 cm between bulb and cells (0.04 µW/cm2). Approximately 1–2 x 106 cells were fixed with 1% paraformaldehyde in PBS and permeabilized for 30 min in 70% ethanol. After washing, break sites in the DNA were labeled with 5-bromo-dUTP in the presence of TdT. Incorporated BrdU was detected with an Alexa Fluor 488-conjugated mouse-anti-BrdU mAb by flow cytometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cross-presentation of L. monocytogenes-derived CD8 T cell epitopes requires bacterial replication in the Ag donor cell

In contrast to directly infected cells, cross-presenting DC take up Ags from infected cells without being infected themselves. Because direct Ag presentation requires bacterial protein synthesis (8, 9), we examined whether cross-presentation of L. monocytogenes-derived Ags has different requirements. DC poorly presented p60- or LLO-derived CD8 T cell epitopes after infection with L. monocytogenes, but much stronger presentation occurred after incubation of DC (H-2d) with L. monocytogenes-infected MHC-disparate IC21 cells (H-2b; Fig. 1). After infection at a multiplicity of infection of 10 for 1 h, replicating bacteria were detected in ~20–30% of cells (data not shown). Bacterial replication was obvious by the presence of groups of multiple intracellular bacteria (Fig. 2A). To prevent cross-infection by spreading of bacteria from infected cells to DC, we used Ag donor cells infected with the nonspreading L. monocytogenes {Delta}actA mutant (20, 21). No cell-to-cell spread of bacteria occurred, as demonstrated by mixing of infected unlabelled Ag donor cells with covalently labeled DC (Fig. 2B). Staining of L. monocytogenes with a polyvalent rabbit antiserum detected bacteria in the unlabeled IC21 cells, but not in fluorescently labeled DC that were added to the infected IC21 cells after killing and removal of extracellular L. monocytogenes. To detect whole bacteria or bacterial fragments in DC with maximum sensitivity, the red Listeria-specific fluorescence was deliberately overexposed, which resulted in the visual merging of individual bacteria in strongly infected IC 21 cells (Fig. 2B, lower right quadrant). The strength of cross-presentation of p60217–225 by DC was similar with Ag donor cells infected with L. monocytogenes wt or L. monocytogenes {Delta}actA, respectively (Fig. 3A, upper panel). Remarkably, no cross-presentation of p60217–225 occurred if DC and Ag donor cells were mixed in the presence of clinafloxacin and gentamicin that kill intra- and extracellular L. monocytogenes, whereas cross-presentation of the CD4 epitope p60301–312 (Fig. 3A, lower panel) was not inhibited in the presence of clinafloxacin and gentamicin. MHC class I-restricted cross-presentation was also abrogated if Ag donor cells were lysed by repeated freeze-thaw cycles (Fig. 3A). Similar to p60217–225, cross-presentation of LLO91–99 was inhibited if bacterial protein synthesis was suppressed by antibiotics (Fig. 3B). The failure to achieve cross-presentation if bacterial protein synthesis was inhibited or if Ags were delivered by cells killed by freeze-thawing could not be overcome by prolonged infection of Ag donor cells or by infection with an enhanced number of bacteria per cell (data not shown). A kinetic analysis revealed the strongest cross-presentation by DC after 7 h of coculture with Ag donor cells (Fig. 3C). Cross-presentation was significantly lower if the period was much shorter (3 h) or longer (16 h).



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 1. Direct vs indirect MHC class I-restricted presentation by DC. Bone marrow-derived DC (H-2d) were infected with L. monocytogenes wt directly ({square}) or were cocultured with L. monocytogenes-infected MHC-mismatched IC21 (H-2b) Ag donor cells ({blacksquare}). As a control, P388 cells (H-2d) were directly infected ({circ}). The presentation of L. monocytogenes-derived T cell epitopes was tested with p60217–225- and LLO91–99-specific CD8 T cell lines as indicated. T cell activation was assessed by quantification of the amount of IFN-{gamma} secreted into the culture supernatant. Shown is the IFN-{gamma} concentration in nanograms per milliliter and the SD of triplicate determinations. The dotted line indicates the detection limit of the IFN-{gamma} ELISA, which was 0.05 ng/ml. Similar results were obtained in two independent experiments.

 


View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 2. Infection of DC. A, Intracellular replication of L. monocytogenes {Delta}actaA after direct infection of DC. After immunomagnetic separation of DC, the actin cytoskeleton was visualized by Alexa Fluor 488 phalloidin (green, left panel), and intracellular bacteria were detected with an anti-Listeria antiserum and a Cy5-conjugated secondary Ab (red, right panel). B, The possible cross-infection of DC during cross-presentation was monitored after labeling DC with the green fluorescent membrane dye PKH67. After coincubation with L. monocytogenes {Delta}actA-infected IC21 cells, intracellular bacteria were stained as described above. Microphotographs were obtained with filter combinations selective for green IC21-specific PKH67 fluorescence (upper left) and red Listeria-specific Cy5 fluorescence (upper right) or with conventional phase contrast lightning (lower left) and were overlaid (lower right). The red fluorescent signal was deliberately overexposed to detect possible transfer of bacteria or bacterial fragments from infected cells to DC with maximum sensitivity.

 


View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 3. Cross-presentation by DC depends on continuous bacterial protein synthesis in Ag donor cells. The cross-presentation assay was performed with BALB/c DC and MHC-mismatched IC21 Ag donor cells infected with L. monocytogenes wt (LM wt) or L. monocytogenes {Delta}actA (LM {Delta}actA) as indicated. Ag donor cells and DC were cultured with gentamicin alone or with gentamicin and clinafloxacin, or Ag donor cells were repeatedly frozen and thawed before addition to cross-presentation cultures. A, Cross-presentation by DC of the MHC class I-restricted epitope p60217–225 and the MHC class II-restricted epitope p60301–312 was tested with T cell lines of corresponding specificity. B, Cross-presentation by DC was tested with LLO91–99-specific CD8 T cells. C, DC were isolated after 3, 7, and 18 h of coculture with infected Ag donor cells. Ag presentation by DC (1 x 104 DC/well) was tested with p60217–225- and LLO91–99-specific CD8 T cell lines. Shown is the IFN-{gamma} concentration in nanograms per milliliter and the SD of triplicate determinations The dotted line indicates the detection limit of the IFN-{gamma} ELISA, which was 0.05 ng/ml. Similar results were obtained in two independent experiments.

 
To exclude a possible bias toward Ag donor cells harboring viable Listeria due to the accumulation of additional proteins during the coculture of DC, we tested cross-presentation under conditions where the total time allowed for infection, and thus the total amount of bacterial Ags accumulated in infected Ag donor cells, were kept constant (Fig. 4). Infected Ag donor cells were cocultured with DC 30 min before, 1 h before, immediately after, or 30 min after blocking of protein synthesis by addition of clinafloxacin. Under these conditions, a part of the coculture of Ag donor cells and DC occurred in the presence of gentamicin only (Fig. 4; coculture starting 4 or 4.5 h p.i.) or clinafloxacin was present during the whole coculture of Ag donor cells and DC (Fig. 4; coculture starting at 5 or 5.5 h p.i.). In all groups the total time allowed for bacterial replication was kept constant (5 h) as was the duration of the coculture of Ag donor cells and DC (5 h). Under these experimental conditions, cross-presentation correlated clearly with the presence of viable bacteria in the Ag donor cells during the coculture with DC (Fig. 4B). If bacterial protein synthesis was blocked by clinafloxacin before the addition of DC, cross-presentation of p60217–225 and LLO91–99 was significantly reduced. These data indicate that efficient cross-presentation requires a form of Ag that is provided only in cells infected with viable, replicating bacteria.



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 4. The requirement of bacterial protein synthesis in Ag donor cells for cross-presentation does not depend on the total Ag load. Cross-presentation assays were performed with BALB/c DC and IC21 Ag donor cells infected with L. monocytogenes {Delta}actA. A, The settings for the cross-presentation assays are indicated. Ag donor cells were infected, cultured in the presence of gentamicin (open areas; GENTA), and 5 h p.i. the culture medium was supplemented with clinafloxacin (shaded areas, CLINA). Ag donor cells and DC were cocultured for the indicated periods (DC+ADC), totaling in all groups 5 h, and subsequently, DC were purified by immunomagnetic separation. B, Ag presentation of purified DC (6 x 104 DC/well) was tested with p60217–225- and LLO91–99-specific CD8 T cell lines. Shown is the IFN-{gamma} concentration in nanograms per milliliter and the SD of triplicate determinations. Asterisks denote statistically significant differences (by t test, p < 0.01) compared with the experimental group that was cocultured with DC for 5 h. Similar results were obtained in two independent experiments.

 
It has been reported that in certain experimental systems, apoptosis of the Ag donor cells (22) or immune complexes (23) are required for cross-presentation. A TUNEL assay with L. monocytogenes {Delta}actA-infected IC21 cells revealed no DNA fragmentation in infected cells, indicating that bacteria did not induce apoptosis in infected cells (Fig. 5A). The viability of infected cells 9 h p.i. was still ~80%. If bacterial protein synthesis was inhibited by clinafloxacin, neither UV radiation of infected Ag donor cells (Fig. 5B) nor serum from L. monocytogenes-immune mice (data not shown) restored cross-presentation of p60217–225 or LLO91–99. DC engulf Ag donor cells and actively take up antigenic material. As shown in Fig. 2B, neither whole bacteria nor large bacterial fragments were transferred from infected Ag donor cells to DC during cross-presentation. However, because bacteria rapidly multiplied in DC (Fig. 2A), even a low percentage of infected DC might be sufficient to stimulate T cells. To prevent the principal possibility of bacterial infection and intracellular protein expression, DC were pretreated with azithromycin, a highly hydrophobic macrolid antibiotic that accumulates intracellularly and prevents growth of bacteria in L. monocytogenes-infected cells (18). Intracellular L. monocytogenes wt did not show actin polymerization after infection of azithromycin-treated P388 cells (data not shown), and azithromycin-treated P388 cells infected with L. monocytogenes did not present L. monocytogenes-derived CD8 T cell epitopes even if cells were infected 4 h after azithromycin was washed out (Fig. 6A). A possible interference of azithromycin or clinafloxacin with the cytosolic Ag presentation pathway was excluded by a control experiment with APC infected with a recombinant vaccinia virus expressing p60 (Fig. 6B). Cross-presentation of L. monocytogenes-derived Ags by azithromycin-loaded DC showed similar results as cross-presentation by untreated DC (Fig. 6C), excluding bacterial infection and intracellular protein synthesis as the source of Ags in cross-presenting DC.



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 5. Effect of apoptosis on cross-presentation. A, Apoptotic death of L. monocytogenes-infected IC21 cells was assessed with a TUNEL assay. Cells were uninfected (no infection), uninfected and UV-irradiated (no infection + UV), infected for 9 h in the presence of gentamicin only (LM {Delta}actaA + GENTA), or infected for 9 h in the presence of clinafloxacin for the last 5 h (LM {Delta}actaA + CLINA). B, Cross-presentation of L. monocytogenes {Delta}actA-infected IC21 cells was tested after induction of apoptosis after UV irradiation (+UV). The coculture with DC was performed in the presence of gentamicin (+GENTA) or clinafloxacin (+CLINA). After isolation of DC, Ag presentation (3 x 104 DC/well) was tested with p60217–225- and LLO91–99-specific CD8 T cell lines. Shown is the IFN-{gamma} concentration in nanograms per milliliter and the SD of triplicate determinations. Similar results were obtained in two independent experiments.

 


View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 6. Intracellularly accumulated azithromycin prevents bacterial replication and MHC class I-restricted Ag presentation in infected cells. A, P388 cells were cultured overnight in the presence (+AZ) or the absence (ØAZ) of azithromycin. Cells were extensively washed; 4 h later, cells were infected with L. monocytogenes {Delta}actA and fixed. Five hours p.i. Ag presentation was tested with p60217–225- and LLO91–99-specific CD8 T cells. B, P388 cells were infected with a p60-expressing recombinant vaccinia virus in the presence of either azithromycin (10 µg/ml) or clinafloxacin (10 µg/ml) or in the absence of antibiotics. Ag presentation was tested with p60217–225-specific CD8 T cells. C, Cross-presentation assay with L. monocytogenes {Delta}actA-infected Ag donor cells and DC that were cultured with (+AZ) or without (ØAZ) azithromycin. Cross-presentation was tested with p60217–225- and LLO91–99-specific CD8 T cell lines. Shown is the IFN-{gamma} concentration in nanograms per milliliter and the SD of triplicate determinations.

 
Regulation of costimulatory molecules on DC during cross-presentation

Cross-presentation requires uptake of Ags and activation of DC. Uptake of Ags by DC is followed by the maturation of DC, down-regulation of phagocytotic activity, and concomitant up-regulation of costimulatory molecules (1). Differences between Ag donor cells that harbor dead or viable bacteria, respectively, in their ability to activate DC and trigger the expression of costimulatory molecules could explain why cross-presentation is blocked by antibiotic treatment of Ag donor cells. However, cytofluorometric analysis of DC from cross-presentation cultures revealed similar strong up-regulation of CD80 and CD86 on DC independent of bacterial replication in Ag donor cells (Fig. 7), indicating that bacterial replication in Ag donor cells was not required for the maturation of DC.



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 7. DC maturation during cross-presentation. The expression of CD80 and CD86 was monitored on DC in the presence of different stimuli. DC were cultured overnight with LPS or were cocultured with IC 21 cells infected with L. monocytogenes {Delta}actA. The cultures with infected cells were performed in the presence of gentamicin alone or in the presence of both gentamicin and clinafloxacin to prevent either selectively extracellular or both extra- and intracellular bacterial replication and protein synthesis, respectively. DC were isolated by immunomagnetic separation, stained, and subjected to cytofluorometric analysis. Similar results were obtained in two independent experiments.

 
Processing of cross-presented Ags

During cross-presentation, Ags could be transferred from the Ag donor cell to DC either unprocessed, partially processed, or finally processed. To learn more about the state of the transferred Ags, we tested the effect of the proteasome inhibitor epoxomycin on cross-presentation (Fig. 8A). Epoxomycin treatment of DC suppressed cross-presentation, indicating that the Ags taken up by DC are not finally processed. Quantitatively, the inhibitory effect of epoxomycin pretreatment of DC was on the same order of magnitude as the inhibition of cross-presentation by clinafloxacin (Fig. 8A). The strict requirement for proteasome-mediated Ag processing by DC also ruled out peptide regurgitation (5), a scenario that is also highly unlikely, because in our system the Ag donor cell and the APC did not share MHC class I molecules. Transfer of soluble material was also not observed in experiments in which DC and Ag donor cells were separated by a semipermeable membrane (2-µm pore diameter) that totally inhibited cross-presentation (data not shown). In contrast, epoxomycin treatment of Ag donor cells did not inhibit cross-presentation, indicating that the Ags transferred from the infected cells to DC did not require proteasomal preprocessing in the Ag donor cell (Fig. 8A).



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 8. Proteasome-mediated processing of Ags transferred during cross-presentation of L. monocytogenes-derived CD8 T cell epitopes. A and B, Standard cross-presentation assays were performed with BALB/c DC and IC21 Ag donor cells infected with L. monocytogenes {Delta}actA. Ag donor cells (EPOX ADC) or DC (EPOX DC) were treated with epoxomycin (+) or remained untreated (–). Cross-presentation was tested in the presence (+) or the absence (–) of clinafloxacin (CLINA). After 5-h incubation with Ag donor cells, the Ag presentation of DC (6 x 104 DC/well) was assessed with p60217–225- and LLO91–99-specific CD8 T cell lines. Shown is the IFN-{gamma} concentration in nanograms per milliliter and the SD of triplicate determinations. Similar results were obtained in four independent experiments.

 
Cross-presentation of L. monocytogenes-derived CD8 T cell epitopes was strictly inhibited by the blocking of bacterial protein synthesis during coculture with DC. This requirement is consistent with the Ag presentation model developed by Yewdell and colleagues that suggests that short-lived defective ribosomal products (DRiPs) are an important substrate for the cytosolic Ag presentation pathway (24). If unstable proteins are transferred from infected Ag donor cells to DC during cross-presentation, stabilization of these proteins should enable cross-presentation in the absence of bacterial protein biosynthesis. Therefore, a cross-presentation experiment with epoxomycin-treated, L. monocytogenes-infected Ag donor cells was performed. Although under conditions of continuous bacterial protein biosynthesis, epoxomycin did not influence the level of cross-presentation (Fig. 8A) in the presence of clinafloxacin, which inhibits bacterial protein synthesis, the protection of presumptive unstable p60 and LLO translation products by epoxomycin resulted in enhanced cross-presentation of p60217–225 and LLO91–99 (Fig. 8B).

The percentage of defective unstable proteins formed during protein biosynthesis can be increased by agents such as puromycin (25) or L-canavanine (26), which are incorporated into growing polypeptide chains and result in the generation of misfolded unstable proteins. Remarkably, the treatment of Ag donor cells with either 1 µg/ml puromycin or 15 mM L-canavanine resulted in a strong increase in cross-presentation of p60217–225 and LLO91–99 (Fig. 9). At these concentrations, neither puromycin nor L-canavanine interfered with intracellular or extracellular replication of L. monocytogenes {Delta}actA (data not shown). These results show that both the protection and the increased production of unstable bacterial proteins enhance cross-presentation, indicating the requirement of an unstable form of Ag for cross-presentation of L. monocytogenes-derived CD8 T cell epitopes by DC.



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 9. Enhanced synthesis of unstable bacterial translation products by Ag donor cells. After infection with Listeria monocytogenes {Delta}actA, IC21 cells were treated with puromycin (PURO ADC) or canavanine (CANA ADC) for 3 h or remained untreated. Both agents are known to increase the percentage of misfolded proteins generated during protein biosynthesis. Subsequently, Ag donor cells were incubated for 5 h with DC in the absence of clinafloxacin. Ag presentation by DC was tested with p60217–225- and LLO91–99-specific CD8 T cell lines. Shown is the IFN-{gamma} concentration in nanograms per milliliter and the SD of triplicate determinations. Similar results were obtained in two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In a previous report we demonstrated that the MHC class II-restricted presentation of L. monocytogenes-derived proteins by DC requires cross-presentation of Ags taken up from infected Ag donor cells (10). In this report we extend these findings and demonstrate that MHC class I-restricted Ag presentation by DC similarly is much more effective if DC remain uninfected and cross-present antigenic material taken up from other infected cells. We also show that MHC class I- and MHC class II-restricted cross-presentation have different physiological requirements. MHC class I-restricted cross-presentation of L. monocytogenes-derived proteins specifically requires that an unstable form of bacterial protein be taken up form viable Ag donor cells, whereas MHC class II-restricted cross-presentation is dependent upon neither viable Ag donor cells nor newly synthesized bacterial proteins.

The induction of an L. monocytogenes-specific CD8 T cell response in mice requires the presence of DC (7). DC presenting L. monocytogenes-derived Ags could either be infected directly or present exogenous material by cross-presentation. Our in vitro data are clearly in favor of the cross-presentation model, because directly infected DC poorly presented LLO- or p60-derived CD8 T cell epitopes, whereas efficient presentation occurred if DC were cocultured with infected Ag donor cells. This is probably due to the apoptogenic effect of LLO on DC and lymphocytes (27, 28). By taking up Ags from other infected cells, DC and T cells avoid the detrimental effect of LLO. Because not all DC were infected, Ag from infected DC could be taken up and cross-presented by uninfected DC. However, we found that bacteria residing in dead or apoptotic cells were cross-presented weakly (see Figs. 3A and 5B), probably due to the rapid inhibition of bacterial protein synthesis after influx of extracellular gentamicin into dying cells. Second, the cell density in the direct infection experiments was also different. In cross-presentation cultures, a high cell density (1 x 106 Ag donor cells together with 2 x 106 DC in 1 ml of medium) ensured the direct cell-to-cell contact of Ag donor cells and DC.

Professional APC have the ability to present exogenous nonviable Ags in the context of MHC class I molecules (4, 5, 29, 30). In DC, cross-presentation occurs in an endoplasmic reticulum-phagosome fusion compartment in which the functions of protein processing and peptide loading are present in direct neighborhood (31, 32). It is not known whether this processing pathway is identical with the pathway involved in the cross-presentation of cell-bound Ags. Ags from a nonreplicating source do not reflect the physiological situation of cross-presentation in vivo, in which intracellular replication of the pathogen and uptake of Ags from infected cells by DC are most likely synchronous events. The cross-presentation pathway that we studied strictly required continuous protein synthesis in Ag donor cells. In this respect, the pathway engaged in cross-presentation of L. monocytogenes-derived CD8 T cell epitopes is clearly different from the pathway described in a number of cellular (29), viral (22, 33), and bacterial (34) cross-presentation models in which Ags were delivered by apoptotic or necrotic cells, and continuous protein biosynthesis was not required. This difference is also reflected by the observation that a relatively long time (7 h) was necessary to obtain optimal cross-presentation, whereas efficient cross-presentation of Ags from a nonreplicating source has been reported to occur after 1 h of coculture of DC and Ag donor cells (33).

Why does MHC class I-restricted cross-presentation of L. monocytogenes-derived antigenic peptides require continuous bacterial protein synthesis? In general, accumulated Listeria-derived proteins in Ag donor cells could be present in an insufficient amount or in a state that does not support cross-presentation. If bacteria double intracellularly every 60 min, it could be expected that after an extended growth period of, e.g., 5 h, ~30-fold more bacteria and bacterial proteins would be present in the infected cell. Three lines of evidence indicate that the cross-presentation pathway described in this report does not simply depend on the pure amount of protein that accumulated in Ag donor cells, but requires the presence of an unstable form of Ag. First, the cross-presentation of Ags from Ag donor cells harboring replicating bacteria was stronger compared with nonreplicating bacteria independent of the total Ag load of the Ag donor cells (see Fig. 4), and the failure to achieve cross-presentation could not be overcome by prolonged infection or by infection of Ag donor cells with an enhanced number of bacteria per cell. Second, after inhibition of bacterial protein synthesis in Ag donor cells, the inhibition of the proteasome by epoxomycin restored cross-presentation. This effect of epoxomycin can only be understood if it is assumed that besides the normal stable bacterial proteins (that in the case of p60 are degraded with a t1/2 of ~90 min) (8) unstable translation products with a shorter half-life are also generated. If bacteria and thus also the protein production rate grow exponentially, and new proteins decay with a t1/2 of 90 min, ~25% of the synthesized proteins would be degraded during the first 4 h of infection. Thus, if epoxomycin protects only this 25% of proteins, this would not significantly change the total load of bacterial proteins in infected cells. The third line of evidence is that the induction of DRiP formation in Ag donor cells enhanced cross-presentation. Puromycin (25, 35) and canavanine (26) are erroneously integrated into the growing polypeptide chain by the ribosome in place of normal amino acids and result in truncated and aberrantly folded nascent polypeptides that are rapidly degraded by the proteasome. These truncated nascent proteins are considered equivalent to naturally occurring DRiPs (15, 16) that in eukaryotic cells constitute ~30% of newly synthesized proteins (36). In contrast to the role of DRiPs during direct Ag presentation (24), DRiPs have not yet been reported to play a role as an Ag source during cross-presentation. Because most cross-presentation studies used noninfectious Ags to avoid cross-infection, there have been few chances to observe this specific requirement. Two studies show that virally infected viable cells can be effective Ag donors (37, 38). However, in these two experimental systems, Ags from apoptotic cells were also cross-presented, indicating that continuous viral protein synthesis was not an absolute requirement for cross-presentation. A possible pathway for the uptake of DRiPs from Ag donor cells would be DC nibbling, a process that involves scavenger receptor A and enables DC to take up Ags from viable cells (39, 40). The failure to detect bacteria or bacterial fragments in DC suggests that cross-presentation did not depend on cross-infection or the uptake of whole bacteria or large bacterial fragments by DC.

The current study focused on an in vitro Ag presentation system. Only the in vitro manipulation of Ag donor cells and DC allowed us to pinpoint the physiological requirements necessary for cross-presentation of L. monocytogenes-derived Ags. Preliminary in vivo tests revealed that immunization of mice with Ag donor cells harboring no viable bacteria failed to stimulate a strong LLO- or p60 Listeria-specific CD8 T cell response (J. Janda and G. Geginat, unpublished observations). However, the alternative situation, i.e., immunization with Ag donor cells that harbor viable bacteria that are strictly confined to their host cells, cannot accurately be tested in vivo. This is a principle problem if studying cross-presentation of infectious microorganism that could directly infect professional APC in vivo. It also must be noted that the requirement of bacterial protein synthesis for the induction of an L. monocytogenes-specific CD8 T cell is not absolute in vivo, as demonstrated by the antigenic potential of very high doses of heat-killed Listeriae (41, 42). In a recent report, Norbury et al. (43) showed that in vivo cross-presentation favors stable Ags. Similar to the results reported by Norbury at al. (43) and in contrast to the study by Serna et al. (37), we found that Ags transferred during cross-presentation do not require proteasome-mediated Ag processing in the Ag donor cells. Norbury et al. (43) immunized mice with vaccinia virus-infected cells that were UV-inactivated. Between UV irradiation and contact with DC in secondary lymphatic organs, unstable proteins might already be degraded, which might explain why stable Ags were required for cross-presentation in vivo.

Taken together, our data suggest the existence of a specific cross-presentation pathway that requires that during cross-presentation, unstable bacterial translation products be transferred from viable Ag donor cells to DC in which proteasomal Ag processing occurs. By this pathway, Ag presentation by DC that cross-present Ags is focused on antigenic peptides derived from freshly synthesized proteins. In light of the short time required for intracellular multiplication of bacteria or viruses, this is an important advantage for the infected host, because only the prompt recognition and elimination of infected cells can prevent the spread of an intracellular infection.


   Acknowledgments
 
We thank H. Hof and R. Holtappels for reading the manuscript, and S. Schenk for expert technical assistance.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Deutsche Forschungsgemeinschaft Grants GE 1081/1-1 and GE 1081/2-1. M.S. was supported by the Slovenian Ministry of Science and the Medical Faculty of the University of Ljubljana. Back

2 Current address: New York University School of Medicine, New York, NY 10016. Back

3 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

4 Abbreviations used in this paper: DC, dendritic cell; DRiPs, defective ribosomal product; LLO, listeriolysin O; p.i., postinfection; wt, wild type. Back

Received for publication May 18, 2004. Accepted for publication August 19, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Guermonprez, P., J. Valladeau, L. Zitvogel, C. Thery, S. Amigorena. 2002. Antigen presentation and T cell stimulation by dendritic cells. Annu. Rev. Immunol. 20:621.[Medline]
  2. Heath, W. R., F. R. Carbone. 2001. Cross-presentation, dendritic cells, tolerance and immunity. Annu. Rev. Immunol. 19:47.[Medline]
  3. Srivastava, P.. 2002. Interaction of heat shock proteins with peptides and antigen presenting cells: chaperoning of the innate and adaptive immune responses. Annu. Rev. Immunol. 20:395.[Medline]
  4. Kovacsovics-Bankowski, M., K. L. Rock. 1995. A phagosome-to-cytosol pathway for exogenous antigens presented on MHC class I molecules. Science 267:243.[Abstract/Free Full Text]
  5. Pfeifer, J. D., M. J. Wick, R. L. Roberts, K. Findlay, S. J. Normark, C. V. Harding. 1993. Phagocytic processing of bacterial antigens for class I MHC presentation to T cells. Nature 361:359.[Medline]
  6. Busch, D. H., K. Kerksiek, E. G. Pamer. 1999. Processing of Listeria monocytogenes antigens and the in vivo T-cell response to bacterial infection. Immunol. Rev. 172:163.[Medline]
  7. Jung, S., D. Unutmaz, P. Wong, G. Sano, K. De los Santos, T. Sparwasser, S. Wu, S. Vuthoori, K. Ko, F. Zavala, et al 2002. In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens. Immunity 17:211.[Medline]
  8. Villanueva, M. S., P. Fischer, K. Feen, E. G. Pamer. 1994. Efficiency of MHC class I antigen processing: a quantitative analysis. Immunity 1:479.[Medline]
  9. Villanueva, M. S., A. J. Sijts, E. G. Pamer. 1995. Listeriolysin is processed efficiently into an MHC class I-associated epitope in Listeria monocytogenes-infected cells. J. Immunol. 155:5227.[Abstract]
  10. Skoberne, M., S. Schenk, H. Hof, G. Geginat. 2002. Cross-presentation of Listeria monocytogenes-derived CD4 T cell epitopes. J. Immunol. 169:1410.[Abstract/Free Full Text]
  11. Hauf, N., W. Goebel, F. Fiedler, Z. Sokolovic, M. Kuhn. 1997. Listeria monocytogenes infection of P388D1 macrophages results in a biphasic NF-{kappa}B (RelA/p50) activation induced by lipoteichoic acid and bacterial phospholipases and mediated by I{kappa}B{alpha} and I{kappa}B{beta} degradation. Proc. Natl. Acad. Sci. USA 94:9394.[Abstract/Free Full Text]
  12. Köhler, S., M. Leimeister-Wachter, T. Chakraborty, F. Lottspeich, W. Goebel. 1990. The gene coding for protein p60 of Listeria monocytogenes and its use as a specific probe for Listeria monocytogenes. Infect. Immun. 58:1943.[Abstract/Free Full Text]
  13. Arnold, B., M. Messerle, L. Jatsch, G. Kublbeck, U. Koszinowski. 1990. Transgenic mice expressing a soluble foreign H-2 class I antigen are tolerant to allogeneic fragments presented by self class I but not to the whole membrane-bound alloantigen. Proc. Natl. Acad. Sci. USA 87:1762.[Abstract/Free Full Text]
  14. Skoberne, M., R. Holtappels, H. Hof, G. Geginat. 2001. Dynamic antigen-presentation patterns of Listeria monocytogenes-derived CD8 T cell epitopes in vivo. J. Immunol. 167:2209.[Abstract/Free Full Text]
  15. Lelouard, H., E. Gatti, F. Cappello, O. Gresser, V. Camosseto, P. Pierre. 2002. Transient aggregation of ubiquitinated proteins during dendritic cell maturation. Nature 417:177.[Medline]
  16. Lelouard, H., V. Ferrand, D. Marguet, J. Bania, V. Camosseto, A. David, E. Gatti, P. Pierre. 2004. Dendritic cell aggresome-like induced structures are dedicated areas for ubiquitination and storage of newly synthesized defective proteins. J. Cell Biol. 164:667.[Abstract/Free Full Text]
  17. Nichterlein, T., F. Bornitz, M. Kretschmar, H. Hof. 1997. Clinafloxacin (CL 960) is superior to standard therapeutics in the treatment of murine listeriosis and salmonellosis. Zentralbl. Bakteriol. Mikrobiol. Hyg. Ser. A 286:401.
  18. Carryn, S., F. Van Bambeke, M. P. Mingeot-Leclercq, P. M. Tulkens. 2002. Comparative intracellular (THP-1 macrophage) and extracellular activities of {beta}-lactams, azithromycin, gentamicin, and fluoroquinolones against Listeria monocytogenes at clinically relevant concentrations. Antimicrob. Agents Chemother. 46:2095.[Abstract/Free Full Text]
  19. Horan, P. K., S. E. Slezak. 1989. Stable cell membrane labelling. Nature 340:167.[Medline]
  20. Kocks, C., E. Gouin, M. Tabouret, P. Berche, H. Ohayon, P. Cossart. 1992. L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein. Cell 68:521.[Medline]
  21. Domann, E., J. Wehland, M. Rohde, S. Pistor, M. Hartl, W. Goebel, M. Leimeister-Wachter, M. Wuenscher, T. Chakraborty. 1992. A novel bacterial virulence gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin. EMBO J. 11:1981.[Medline]
  22. Albert, M. L., B. Sauter, N. Bhardwaj. 1998. Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 392:86.[Medline]
  23. Regnault, A., D. Lankar, V. Lacabanne, A. Rodriguez, C. Thery, M. Rescigno, T. Saito, S. Verbeek, C. Bonnerot, P. Ricciardi-Castagnoli, et al 1999. Fc{gamma} receptor-mediated induction of dendritic cell maturation and major histocompatibility complex class I-restricted antigen presentation after immune complex internalization. J. Exp. Med. 189:371.[Abstract/Free Full Text]
  24. Princiotta, M. F., D. Finzi, S. B. Qian, J. Gibbs, S. Schuchmann, F. Buttgereit, J. R. Bennink, J. W. Yewdell. 2003. Quantitating protein synthesis, degradation, and endogenous antigen processing. Immunity 18:343.[Medline]
  25. Goldberg, A. L.. 1972. Degradation of abnormal proteins in Escherichia coli (protein breakdown-protein structure-mistranslation-amino acid analogs-puromycin). Proc. Natl. Acad. Sci. USA 69:422.[Abstract/Free Full Text]
  26. Anton, L. C., U. Schubert, I. Bacik, M. F. Princiotta, P. A. Wearsch, J. Gibbs, P. M. Day, C. Realini, M. C. Rechsteiner, J. R. Bennink, et al 1999. Intracellular localization of proteasomal degradation of a viral antigen. J. Cell Biol. 146:113.[Abstract/Free Full Text]
  27. Guzman, C. A., E. Domann, M. Rohde, D. Bruder, A. Darji, S. Weiss, J. Wehland, T. Chakraborty, K. N. Timmis. 1996. Apoptosis of mouse dendritic cells is triggered by listeriolysin, the major virulence determinant of Listeria monocytogenes. Mol. Microbiol. 20:119.[Medline]
  28. Carrero, J. A., B. Calderon, E. R. Unanue. 2004. Listeriolysin O from Listeria monocytogenes is a lymphocyte apoptogenic molecule. J. Immunol. 172:4866.[Abstract/Free Full Text]
  29. Carbone, F. R., M. J. Bevan. 1990. Class I-restricted processing and presentation of exogenous cell-associated antigen in vivo. J. Exp. Med. 171:377.[Abstract/Free Full Text]
  30. Speidel, K., W. Osen, S. Faath, I. Hilgert, R. Obst, J. Braspenning, F. Momburg, G. J. Hammerling, H. G. Rammensee. 1997. Priming of cytotoxic T lymphocytes by five heat-aggregated antigens in vivo: conditions, efficiency, and relation to antibody responses. Eur. J. Immunol. 27:2391.[Medline]
  31. Houde, M., S. Bertholet, E. Gagnon, S. Brunet, G. Goyette, A. Laplante, M. F. Princiotta, P. Thibault, D. Sacks, M. Desjardins. 2003. Phagosomes are competent organelles for antigen cross-presentation. Nature 425:402.[Medline]
  32. Guermonprez, P., L. Saveanu, M. Kleijmeer, J. Davoust, P. Van Endert, S. Amigorena. 2003. ER-phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells. Nature 425:397.[Medline]
  33. Ramirez, M. C., L. J. Sigal. 2002. Macrophages and dendritic cells use the cytosolic pathway to rapidly cross-present antigen from live, vaccinia-infected cells. J. Immunol. 169:6733.[Abstract/Free Full Text]
  34. Yrlid, U., M. J. Wick. 2000. Salmonella-induced apoptosis of infected macrophages results in presentation of a bacteria-encoded antigen after uptake by bystander dendritic cells. J. Exp. Med. 191:613.[Abstract/Free Full Text]
  35. Etlinger, J. D., A. L. Goldberg. 1980. Control of protein degradation in reticulocytes and reticulocyte extracts by hemin. J. Biol. Chem. 255:4563.[Free Full Text]
  36. Schubert, U., L. C. Anton, J. Gibbs, C. C. Norbury, J. W. Yewdell, J. R. Bennink. 2000. Rapid degradation of a large fraction of newly synthesized proteins by proteasomes. Nature 404:770.[Medline]
  37. Serna, A., M. C. Ramirez, A. Soukhanova, L. J. Sigal. 2003. Efficient MHC class I cross-presentation during early vaccinia infection requires the transfer of proteasomal intermediates between antigen donor and presenting cells. J. Immunol. 171:5668.[Abstract/Free Full Text]
  38. Maranon, C., J. F. Desoutter, G. Hoeffel, W. Cohen, D. Hanau, A. Hosmalin. 2004. Dendritic cells cross-present HIV antigens from live as well as apoptotic infected CD4+ T lymphocytes. Proc. Natl. Acad. Sci. USA 12:12.
  39. Harshyne, L. A., S. C. Watkins, A. Gambotto, S. M. Barratt-Boyes. 2001. Dendritic cells acquire antigens from live cells for cross-presentation to CTL. J. Immunol. 166:3717.[Abstract/Free Full Text]
  40. Harshyne, L. A., M. I. Zimmer, S. C. Watkins, S. M. Barratt-Boyes. 2003. A role for class A scavenger receptor in dendritic cell nibbling from live cells. J. Immunol. 170:2302.[Abstract/Free Full Text]
  41. Szalay, G., C. H. Ladel, S. H. Kaufmann. 1995. Stimulation of protective CD8+ T lymphocytes by vaccination with nonliving bacteria. Proc. Natl. Acad. Sci. USA 92:12389.[Abstract/Free Full Text]
  42. Lauvau, G., S. Vijh, P. Kong, T. Horng, K. Kerksiek, N. Serbina, R. A. Tuma, E. G. Pamer. 2001. Priming of memory but not effector CD8 T cells by a killed bacterial vaccine. Science 294:1735.[Abstract/Free Full Text]
  43. Norbury, C. C., S. Basta, K. B. Donohue, D. C. Tscharke, M. F. Princiotta, P. Berglund, J. Gibbs, J. R. Bennink, J. W. Yewdell. 2004. CD8+ T cell cross-priming via transfer of proteasome substrates. Science 304:1318.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
S. Basta, R. Stoessel, M. Basler, M. van den Broek, and M. Groettrup
Cross-Presentation of the Long-Lived Lymphocytic Choriomeningitis Virus Nucleoprotein Does Not Require Neosynthesis and Is Enhanced via Heat Shock Proteins
J. Immunol., July 15, 2005; 175(2): 796 - 805.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Janda, J.
Right arrow Articles by Geginat, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Janda, J.
Right arrow Articles by Geginat, G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS