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In Vivo, Dendritic Cells Can Cross-Present Virus-Like Particles Using an Endosome-to-Cytosol Pathway ¹

Víctor Gabriel Morón^*, Paloma Rueda, Christine Sedlik and Claude Leclerc^2,*

^* Unité de Biologie des Régulations Immunitaires, Institut National de la Santé et de la Recherche Médicale, E352, Institut Pasteur, Paris, France; Inmunología y Genética Aplicada S.A., Madrid, Spain; and Institut National de la Santé et de la Recherche Médicale Unité 520, Institut Curie, Paris, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recombinant parvovirus-like particles (PPV-VLPs) are particulate exogenous Ags that induce strong CTL response in the absence of adjuvant. In the present report to decipher the mechanisms responsible for CTL activation by such exogenous Ag, we analyzed ex vivo and in vitro the mechanisms of capture and processing of PPV-VLPs by dendritic cells (DCs). In vivo, PPV-VLPs are very efficiently captured by CD8^- and CD8⁺ DCs and then localize in late endosomes of DCs. Macropinocytosis and lipid rafts participate in PPV-VLPs capture. Processing of PPV-VLPs does not depend upon recycling of MHC class I molecules, but requires vacuolar acidification as well as proteasome activity, TAP translocation, and neosynthesis of MHC class I molecules. This study therefore shows that in vivo DCs can cross-present PPV-VLPs using an endosome-to-cytosol processing pathway.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The priming of T cell responses relies on the presentation of Ag-derived peptides by MHC molecules. MHC class I-restricted T cell responses were, until recently, thought to target only Ags synthesized within the APC, for instance when they are infected by a pathogen. Thus, Ags that do not reach the cytosol of APCs should not elicit a CTL response. However, not all pathogens are capable of infecting APCs, and moreover, this mechanism cannot explain how CTL responses can be induced against tumor cells. It is now well established that at least some exogenous, noncytosolic Ags can gain access to the MHC class I pathway of APCs by cross-presentation (1) through several alternative pathways of processing (2). Both macrophages (M)³ (3) and dendritic cells (DCs) (4) cross-present Ags, but only DCs are capable of stimulating naive CD8⁺ T cells (5). Two main routes of cross-presentation have been proposed. One route involves the passage of Ags from endosomes to cytosol (also named cytosolic diversion) (6), while in the other route the Ags do not escape from endosomes, but are processed inside these vesicles (7, 8). The first route seems to be used mostly by DCs, and the second one by M (9). A third route, which uses the properties of certain viruses and bacterial toxins to gain direct access to cytosol, is based on the translocation of Ag from cell surface to cytosol (10, 11).

We have developed an Ag delivery system based on nonreplicative, recombinant parvovirus-virus-like particles (PPV-VLPs) formed by the self-assembly of the VP2 capsid protein of porcine parvovirus (PPV) (12, 13). The VP2 protein (14), carrying foreign CD8⁺ T cell epitopes, self-assembles into 25-nm VLPs after expression in insect cells (13). Mice immunized with PPV-VLPs carrying a CD8⁺ T cell epitope, in the absence of an adjuvant, develop a strong and specific MHC class I-restricted CTL response (13, 15). This CTL response protects against a viral challenge and is based on the induction of a high frequency of high avidity CTLs (16). PPV-VLPs target DCs with very high efficiency, whereas M and B cells have a poor capacity to capture these particles. Following an in vivo injection of PPV-VLPs, both CD8^- and CD8⁺ DCs capture and process these particles, although they have different kinetics and T cell requirements (15). DC stimulation by PPV-VLPs induces phenotypic changes on CD8^- DCs, leading to the acquisition of CD8 and CD205 and the loss of CD4 molecules as well as expression of costimulatory molecules on both DC subsets (15).

Most studies that aimed at studying the different steps of processing of exogenous Ags into the MHC class I pathway have been conducted using unpurified peritoneal exudate cells (17) (which contain various cell populations, including M and DCs) or bone-marrow derived DCs (18, 19, 20), which do not represent the whole spectrum of peripheral DCs. In the present report to decipher the mechanisms responsible for CTL activation by exogenous Ags, we analyze ex vivo and in vitro the mechanisms of capture and processing of PPV-VLPs by DCs, using DCs freshly purified from spleen. This study shows that DCs can use the endosome-to-cytosol pathway in vivo to cross-present exogenous Ags such as PPV-VLPs.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Six- to 8-wk-old female C57BL/6 (H-2^b) mice were obtained from Janvier (Le Genet St. Isle, France). Female TAP1 knockout mice, bred onto a C57BL/6 background, were a gift from Dr. A. Bandeira (Institut Pasteur, Paris, France). All animals were maintained under specific pathogen-free conditions.

Peptides and cell lines

The peptide SIINFEKL, corresponding to aa 257–264 from chicken OVA (OVA_257–264 peptide), an immunodominant H-2^b-restricted CTL epitope, was purchased from Neosystem (Strasbourg, France). B3Z, a CD8⁺ T cell hybridoma specific for the OVA_257–264 epitope in the context of K^b (21), was a gift from Dr. N. Shastri (University of California, Berkeley, CA).

PPV particles

The construction, characterization, and purification of recombinant and control PPV-VLPs were previously described in detail (13, 15). Briefly, the VP2 gene was expressed with the OVA_257–264 peptide plus natural flanking sequences (LEQLESIINFEKLTE) (the underlined sequence corresponds to the CTL epitope, and the nonunderlined sequences correspond to flanking sequences to the CTL epitope) from chicken egg OVA in its 5' end (PPV-VLPs-OVA) or without this sequence (PPV-VLPs) using a baculovirus vector system. After infection of Sf9 insect cells, the recombinant VLPs were purified by salt precipitation with 20% ammonium sulfate, followed by dialysis. Characterization of PPV-VLPs-OVA and PPV-VLPs by CsCl sedimentation analysis and electron microscopy revealed properties identical to those of native PPV virions. In some experiments PPV-VLPs-OVA were labeled with the fluorochrome Alexa488, using the Alexa Fluor 488 Protein Labeling Kit (Molecular Probes Europe, Leiden, The Netherlands) following the manufacturer’s instructions.

The concentration of PPV-VLPs-OVA was determined by densitometry and by double-Ab sandwich ELISA. The densitometric assay was conducted with 1D Image Analysis software 2.0.1. (Eastman Kodak, Rochester, NY) using BSA as reference. The double-Ab sandwich ELISA was performed as previously described (22), using as capture Ab the anti-PPV mAb 15C5 and as detection Ab the anti-PPV biotinylated mAb 13C6 (23). Highly purified PPV-VLPs from size exclusion chromatography were used as the standard reference.

Endotoxin values were determined in each sample of VLPs using the Limulus amebocyte lysate test (BioWhittaker, Walkersville, MD). For PPV-VLPs, endotoxin values were <0.5 ng/mg of protein (5 endotoxin units/mg), and for PPV-VLPs-OVA, they were <10 ng/mg (100 endotoxin units/mg). PPV-VLPs preparations contained minimal traces of DNA, as determined after DNAzol (Invitrogen, Paisley, U.K.) or phenol extractions and electrophoresis in agarose gel, although not enough to be quantified by spectrophotometry at A_260/280.

CyaA-E5-OVA

CyaA-E5-OVA is a genetically detoxified CyaA toxin carrying the OVA_257–264 peptide plus natural flanking sequences (PASIINFEKLGT) between Arg²²⁴ and Ala²²⁵ of the amino acid sequence (11).

Preparation of splenic DCs

Spleens were removed from mice and treated for 45 min at 37°C with 400 U/ml of collagenase type IV and 50 µg/ml of DNase I (Roche, Mannheim, Germany) in RPMI 1640. After inhibition of collagenase activity with 6 mM EDTA in PBS, spleens were dissociated in Ca²⁺- and Mg²⁺-free PBS in the presence of 2.5 mM EDTA and 0.5% FCS (Life Technologies, Paisley, Scotland). In all assays involving DCs, the same batch of endotoxin-free FCS (as determined by Limulus amebocyte lysate test) was used. Furthermore, all reagents were also tested for endotoxins. Single spleen cell suspensions were prepared and incubated with anti-CD16/32 (2.4G2 clone; BD PharMingen, San Diego, CA) and with colloidal superparamagnetic microbeads conjugated to anti-CD11c mAb (MACS-anti-CD11c, N418 clone; Miltenyi Biotec, Bergisch-Gladbach, Germany) following the manufacturer’s instructions. CD11c⁺ cells were positively selected with high speed magnetic cell sorting (program posseld, AutoMACS; Miltenyi Biotec). The purified DC preparations contained 3–10% autofluorescent cells (defined as double-positive cells in a FL2 vs FL3 dot plot, without Ab labeling). The purity of DC preparations (excluding autofluorescent cells) was always 95–99% (Fig. 1A). CD11c⁺ cells were H-2 K^b+, I-A^{b low}, CD40^low, CD80^low, and CD86^-. Twenty-five to 30% were CD8⁺, and 60–70% were CD8^- CD11b⁺.

View larger version (28K): [in this window] [in a new window]   FIGURE 1. In vivo, PPV-VLPs-OVA are captured and presented by DCs, delivering the OVA257–264 epitope into the MHC class I pathway. A, One C57BL/6 mouse was i.v. injected with 50 µg of Alexa488-PPV-VLPs. One PBS-injected mouse was used as a control. Ninety minutes later, CD11c+ cells were purified from spleen by magnetic sorting, stained with PE-anti-CD11c, and analyzed on a FACStar cytometer. A representative dot-plot analysis of cells purified from PPV-VLP-injected or control mice is shown. B, C57BL/6 mice were i.v. injected with 100 µg of PPV-VLPs-OVA (•) or PPV-VLPs (), and 90 min later, various numbers of DCs purified from their spleens were cocultured overnight with 1 x 105 B3Z cells/well. The presentation of OVA257–264 peptide to B3Z cells was monitored by IL-2 production, measured by a CTLL-2 proliferation assay, and expressed as the mean ± SEM counts per minute of duplicate wells. C, Purified splenic DCs (105 cells/well) were incubated in vitro with PPV-VLPs-OVA (•), control PPV-VLPs (), or OVA257–264 peptide () for 4 h, washed, and cultured overnight with 1 x 105 cells/well of B3Z cells. The presentation of OVA257–264 peptide to B3Z cells was monitored by IL-2 production, measured by a CTLL-2 proliferation assay, and expressed as the mean ± SEM counts per minute of duplicate wells.

CD11c⁺ spleen cells (10⁵ cells/well) were first pulsed with Ag (PPV-VLPs-OVA, PPV-VLPs, or OVA_257–264 peptide) for 4 h in 96-well culture microplates in a final volume of 0.2 ml of RPMI 1640 Glutamax-I plus 5 x 10^-5 M 2-ME, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10% FCS (RPMI 10%; all from Life Technologies). The Ag concentration used in each experiment is indicated in the figure legends. Then DCs were washed three times with RPMI 10%, and 10⁵ cells/well of B3Z hybridoma were added and incubated overnight at 37°C in 95% CO₂. The stimulation of B3Z cells was monitored by IL-2 release in the supernatants, which was measured using the CTLL-2 bioassay. Cells (10⁴ /well) of the CTLL-2 cell line were cultured with 100 µl of supernatant in a final volume of 200 µl. Two days later, [³H]thymidine (NEN Life Science, Boston, MA) was added, and the cells were harvested 18 h later with an automated cell harvester (Skatron, Lier, Norway). Incorporated thymidine was detected by cell scintillation counting. In all experiments each point was determined in duplicate. Results are expressed as counts per minute. In some experiments APCs were fixed before or after Ag pulse with 0.05% glutaraldehyde/PBS, then treated with 0.2 M lysine/RPMI and washed three times with RPMI 10%.

Inhibition studies

For most inhibition studies, APCs (10⁵ cells/well) were first incubated in 0.1 ml with each drug for 1 h. Then Ag diluted in 0.1 ml was added to the wells (0.2 ml final volume) at the final concentration indicated in the continuous presence of inhibitors for 4 h. APC were then washed three times and fixed with 0.05% glutaraldehyde, and the experiments were continued as described above. The inhibitors used were brefeldin A (BFA), N-acetyl-L-leucinal-L-norleucinal (LLnL), N-acetyl-L-leucinal-L-methioninal (LLmL), dimethylamiloride (DMA), chloroquin, cytochalasin B (CCB), primaquin, cycloheximide (CHX), chlorpromazine, filipin III (all from Sigma-Aldrich, St. Louis, MO), lactacystin (BIOMOL, Plymouth Meeting, PA), leupeptin, and pepstatin (Roche, Indianapolis, IN).

For inhibition of clathrin-mediated endocytosis by K⁺ depletion following hypotonic shock, DCs (10⁵ cells/well) were preincubated for 30 min in the presence of serum-free synthetic OptiMEM medium (Life Technologies), supplemented with 5 x 10^-5 M 2-ME, 100 IU/ml penicillin, and 100 µg/ml streptomycin. DCs were then incubated for 5 min in hypotonic medium (OptiMEM mixed with Ultrapure H₂O, 50/50) and finally for 30 min in isotonic K⁺-free (140 mM NaCl, 20 mM HEPES-NaOH, 1 mM CaCl₂, 1 mM MgCl₂, 1 mg/ml glucose, and 0.5% BSA) or K⁺-containing (10 mM KCl, 130 mM NaCl, 20 mM HEPES-NaOH, 1 mM CaCl₂, 1 mM MgCl₂, and 0.5% BSA) buffer. Then 1.23 nM PPV-VLPs-OVA or 1.1 nM OVA_257–264 were added. DCs were incubated for 1 h in the continued presence of the drugs and Ags. Then DCs were washed and cocultured overnight with 1 x 10⁵ cells/well of OVA-specific B3Z cells in RPMI 10%. As control, cells were maintained in OptiMEM medium and incubated with Ag before washing and culture with B3Z cells in RPMI 10% as indicated above.

Confocal microscopy and in vitro internalization assay

CD11c⁺ spleen cells from PPV-VLP-injected mice were allowed to adhere to glass slides coated with poly-L-lysine (Sigma-Aldrich). Cells were then fixed with 4% paraformaldehyde and in some cases labeled with wheat-germ agglutinin (WGA; Molecular Probes Europe,) directly conjugated to Alexa Fluor 488 for 30 min without permeabilization of the cells to detect plasma membrane. Intracellular immunofluorescence was performed on cells permeabilized in PBS containing 1% saponin and 5% BSA with Abs against CD8 (53-6.7 clone; BD PharMingen), Lamp2 (BD PharMingen), H2-M (DM5 clone) (24), Rab7 (supplied by P. Chavrier, Institut Curie, Paris, France) (24, 25), Rab11 (26), or biotinylated anti-VP2 Ab (mixture of 11D1, 13C4, 13C5, 13C6, and 15G4 mAbs) (23). After incubation with the appropriate secondary Abs, cells were mounted and analyzed by confocal microscopy using a Leica TCS SP2 microscope (Leica, Deerfield, IL) equipped with a x100 1.4 NA HCX PL APO oil immersion objective.

For in vitro internalization assay, CD11c⁺ spleen cells from noninjected mice were incubated with Alexa488-PPV-VLPs for 1 h at 37°C, followed by rhodamine-WGA (Molecular Probes Europe) staining as described above.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In vivo and in vitro, DCs capture and process PPV-VLPs-OVA

To characterize the in vivo and in vitro processing of PPV-VLPs, we first studied the in vivo capture of PPV-VLPs by DCs. Naive C57BL/6 mice were injected with PPV-VLPs-OVA coupled to Alexa488, a strong green fluorescent dye, which fluorescence does not extinguish at low pH. Alexa488-PPV-VLPs-OVA preserved their biological activity after labeling, as demonstrated by their capacity to be processed and presented by DCs to B3Z cells (data not shown). Ninety minutes after injection, the CD11c⁺ spleen cells were sorted out and labeled with an anti-CD11c mAb. As shown in a representative experiment depicted in Fig. 1A, 53% of CD11c⁺ cells were Alexa488⁺ showing that in vivo, DCs captured PPV-VLPs-OVA very efficiently. Fifteen hours after injection, DCs remained strongly positive for Alexa488 (52% of Alexa488⁺ DCs; data not shown).

We then examined whether DCs can process PPV-VLPs in vivo, using an ex vivo Ag presentation assay. C57BL/6 mice were i.v. injected with 50 µg (13 pmol/mouse) of PPV-VLPs-OVA or PPV-VLPs. Ninety minutes later, splenic CD11c⁺ cells were purified and cocultured with B3Z CD8⁺ T cell hybridoma. DCs purified from PPV-VLPs-OVA-injected mice were capable of presenting the OVA_257–264 epitope to B3Z cells, whereas DCs purified from control PPV-VLP-injected mice failed to stimulate this hybridoma (Fig. 1B). Thus, in vivo, DCs efficiently capture and process PPV-VLPs-OVA.

To evaluate the capacity of DCs in vitro to process PPV-VLPs-OVA, splenic CD11c⁺ spleen cells purified from naive mice were incubated with PPV-VLPs-OVA or PPV-VLPs or the OVA_257–264 peptide for 4 h and then cocultured overnight with the B3Z hybridoma. DCs incubated with PPV-VLPs-OVA or the OVA_257–264 peptide efficiently presented the OVA_257–264 epitope, whereas DCs incubated with PPV-VLPs did not stimulate B3Z cells (Fig. 1C). Moreover, presentation of PPV-VLPs-OVA requires cellular processing, because DCs fixed with glutaraldehyde before incubation with PPV-VLPs-OVA did not stimulate B3Z. In contrast, DCs fixed after PPV-VLPs-OVA incubation were fully capable of stimulating this hybridoma (data not shown).

After capture, PPV-VLPs are localized in late endosomes of DCs

To analyze in detail the capture of PPV-VLPs by DCs, we studied by confocal microscopy the intracellular localization of PPV-VLPs in DCs. Mice were injected with Alexa488-PPV-VLPs-OVA, and their splenic DCs were purified, permeabilized or not with saponin, and stained with a mixture of anti-VP2 mAbs. This mix of mAbs colocalized with Alexa488 labeling in saponin-permeabilized DCs from mice injected with Alexa488-PPV-VLPs, whereas no colocalization was observed in nonpermeabilized DCs from the same mice (Fig. 2A), clearly showing that PPV-VLPs were effectively found inside DCs and demonstrating that in vivo, DCs endocytose PPV-VLPs. Furthermore, PPV-VLPs were found inside both CD8^- and CD8⁺ DCs (Fig. 2B). Inside DCs, PPV-VLPs were observed in vesicle-shaped structures of very different sizes (Fig. 2, A–C). Therefore, to characterize these vesicles, DCs from mice injected with PPV-VLPs were stained with anti-VP2 mAbs and with mAbs against several molecules recognized as markers of endosomal compartments. The confocal microscopy revealed that PPV-VLPs colocalized with Lamp2⁺, H2-M⁺, and rab7⁺ labeling (Fig. 2C), which are associated with late endosomes (27, 28, 29). Furthermore, no colocalization of PPV-VLPs was observed with rab11, a GTPase of the Rab family that is a crucial element in the control of traffic through the recycling endosome (30, 31) (Fig. 2C). Therefore, after endocytosis, PPV-VLPs reach late endosomes. To determine whether this capture can be also observed in vitro, purified splenic DCs from naive mice were incubated with Alexa488-PPV-VLPs. The confocal analysis of DCs stained with WGA (which recognizes (GlcNAc)₂ residues in cell surface glycoproteins) clearly showed that PPV-VLPs were effectively found inside DCs (Fig. 2D), demonstrating that in vitro, DCs can also endocytose PPV-VLPs. Moreover, in vitro endocytosed PPV-VLPs were also found inside Lamp2⁺, H2-M⁺, and rab7⁺ vesicles, confirming the in vivo results (data not shown).

View larger version (30K): [in this window] [in a new window]   FIGURE 2. In vivo, captured PPV-VLPs-OVA are found in late endosomes/lysosomes in DCs. A, Intracellular location of PPV-VLPs. Mice were injected with 50 µg of Alexa488-PPV-VLPs, and 90 min later, splenic DCs were purified. Cells were then stained in the presence or the absence of saponin to permeabilize the cells, with biotinylated anti-PPV-VLPs mAbs detected with Cy3-streptavidin. Far left figures show Alexa488 fluorescence, left images show Cy3 fluorescence, while right figures show the merged images. Far right figures show the transmission image. B, Capture of PPV-VLPs by CD8+ and CD8- DCs. Mice were injected with 50 µg of Alexa488-PPV-VLPs, and 90 min later, splenic DCs were purified. Cells were then permeabilized and labeled with an anti-CD8 mAb (in red), which was detected with a Cy3-anti-rat secondary Ab. The transmission light image shows the body of the cell. C, Mice were injected with unlabeled PPV-VLPs, and 90 min later, splenic DCs were fixed, and different intracellular markers were identified by specific antibodies detected with Alexa488-conjugated secondary Abs. Intracellular location of PPV-VLPs was detected on permeabilized cells with anti-PPV mAb, followed by Cy3-streptavidin. D, Purified splenic DCs from naive mice were incubated with 5 µg/ml of Alexa488-PPV-VLPs for 1 h at 37°C, stained with rhodamine-WGA, and analyzed by confocal microscopy to visualize the plasma membrane.

Endocytic pathways may include clathrin-coated pits, macropinocytosis, phagocytosis, or lipid rafts containing or not containing caveolin. To define the pathways involved in PPV-VLP capture, we used several drugs that are known to inhibit with a relative specificity a particular step of these various pathways. Clathrin-mediated endocytosis was assessed by K⁺ depletion following hypotonic shock and by inhibition with chlorpromazine. K⁺ depletion following hypotonic shock was performed by cell exposure to hypotonic medium, followed by incubation in the absence of extracellular potassium (32, 33, 34). This treatment results in dissociation of clathrin coats from the plasma membrane and nonproductive assembly of clathrin cages in the cytoplasm adjacent to coated pits. As consequence, internalization is impaired for all membrane proteins carrying cytoplasmic amino acid sequences that interact with the clathrin adapter complex AP2. Upon this treatment, DCs incubated with PPV-VLPs-OVA or OVA_257–264 peptide did not show any inhibition of OVA_257–264 presentation (Fig. 3A).

View larger version (41K): [in this window] [in a new window]   FIGURE 3. PPV-VLP uptake involves macropinocytosis, lipid rafts, and actin cytoskeleton polymerization, but not clathrin-coated pits. A, Purified splenic DCs (105 cells/well) were preincubated for 30 min in the presence of serum-free synthetic OptiMEM, then for 5 min in hypotonic medium and finally for 30 min in isotonic K+-free or K+-containing buffer. Then, 1.2 nM PPV-VLPs-OVA, 27 nM CyaA-E5-MalE-OVA, or 1.1 nM OVA257–264 was added. DCs were then incubated for 1 h in the continued presence of the drugs and Ags. Then DCs were washed and cocultured overnight with 1 x 105 cells/well of OVA-specific B3Z cells in RPMI 10%. As control, cells were incubated with OptiMEM and the above-indicated Ags before washing and culture of B3Z cells. B, Purified splenic DCs (105 cells/well) were preincubated for 1 h in the presence of the indicated concentrations of chlorpromazine. Then 1.2 nM PPV-VLPs-OVA, 27 nM CyaA-E5-MalE-OVA, or 1.1 nM OVA257–264 was added. DCs were incubated for 1 h in the continued presence of the drugs and Ags. C–E, Purified splenic DCs (105 cells/well) were preincubated for 1 h in the presence of the indicated concentrations of DMA (C), filipin III (D), or 10 µg/ml cytochalasin B (E). Then 1.2 nM PPV-VLPs-OVA or 1.1 nM OVA257–264 was added. DCs were incubated for 4 h in the continued presence of the drugs and Ags. The DCs were cocultured overnight with 105 cells/well of B3Z-specific hybridoma. The presentation of OVA257–264 epitope to B3Z cells was monitored by IL-2 production and expressed as the percentage of [3H]thymidine incorporation compared with DCs incubated with PPV-VLPs-OVA or OVA257–264 peptide in the absence of inhibitor.

To confirm these results, we used chlorpromazine as an inhibitor of clathrin-coated pit formation. Chlorpromazine is a cationic amphiphilic drug that has been extensively used to analyze the role of clathrin-mediated endocytosis in virus entry (40, 41). OVA_257–264 epitope presentation by DCs pretreated with chlorpromazine and then incubated with PPV-VLPs-OVA was not altered, whereas CyaA-E5-OVA presentation was reduced by 50% compared with untreated control DCs (Fig. 3B). This result confirmed that clathrin-coated pits do not play a significant role in the capture of PPV-VLPs.

To study whether PPV-VLP uptake is mediated by macropinocytosis, DCs were preincubated with DMA, which inhibits Na⁺/H⁺ exchange (42) and, therefore, macropinocytosis (43). As shown in Fig. 3C, DMA strongly inhibited PPV-VLPs-OVA presentation, whereas it slightly affected the OVA_257–264 peptide presentation at a very high concentration of DMA.

Lipid rafts also participate in PPV-VLPs uptake, as revealed by the use of filipin III inhibitor. Filipin III is a sterol-binding agent that binds to cholesterol, disrupting lipid raft formation, including caveolin-containing rafts, without affecting clathrin-mediated endocytosis (44). OVA_257–264 epitope presentation by DCs pretreated with filipin III and then incubated with PPV-VLPs-OVA was severely reduced, whereas this treatment did not affect the presentation of the OVA_257–264 peptide (Fig. 3D).

Finally, we studied PPV-VLPs processing by DCs preincubated with CCB, a known inhibitor of actin filament polymerization (45), which, in turn, can alter macropinocytosis, phagocytosis, as well as caveolae-mediated endocytosis. DCs incubated with PPV-VLPs-OVA in the presence of CCB were not able to present the OVA_257–264 epitope (Fig. 3E), whereas DCs incubated with the OVA_257–264 peptide in the presence of CCB were fully able to activate B3Z cells.

The OVA_257–264 epitope processed from PPV-VLPs-OVA does not bind to recycling MHC class I molecules

Most endocytosed molecules, including recycling receptors, are delivered to early endosomes, where efficient sorting occurs (31). Receptors and some ligands segregate to recycling compartments, whereas other molecules are delivered into late endosomes-lysosomes. Recycling of endocytosed MHC class I molecules back to the cell surface has been observed (46). Some of the recycling MHC class I molecules can be loaded into endosomes with peptide derived from endocytosed molecules (20, 47). Therefore, to establish whether PPV-VLP processing involves MHC class I recycling, we studied PPV-VLPs-OVA presentation by DCs incubated in the presence of primaquin, which inhibits the recycling of MHC class I and II molecules (46). DCs incubated in the presence of this drug presented efficiently both PPV-VLPs-OVA and the OVA_257–264 peptide (Fig. 4A). This result is in accord with the absence of colocalization of PPV-VLPs with rab11 by confocal microscopy (Fig. 2C). Therefore, PPV-VLPs would not enter into recycling compartments. In contrast, OVA_257–264 presentation was fully inhibited when DCs were incubated with PPV-VLPs-OVA in the presence of cycloheximide (Fig. 4B), a potent inhibitor of protein synthesis, suggesting that newly synthesized proteins, particularly MHC class I molecules, are required for efficient presentation of PPV-VLPs-OVA to B3Z cells.

View larger version (27K): [in this window] [in a new window]   FIGURE 4. Presentation of PPV-VLPs-OVA requires the neosynthesis of MHC class I molecules. Purified splenic DCs (105 cells/well) were preincubated for 1 h with medium alone or in the presence of 0.2 nM primaquin (A) or 4 µg/ml cycloheximide (B). Then 1.2 nM PPV-VLPs-OVA or 110 nM OVA257–264 was added, and DCs were incubated for 4 h in the continued presence of the drugs. After glutaraldehyde fixation, DCs were cocultured overnight with 105 cells/well of B3Z hybridoma. The presentation of OVA257–264 epitope to B3Z cells was monitored by IL-2 production and expressed as the percentage of [3H]thymidine incorporation compared with DCs incubated with PPV-VLPs-OVA or OVA257–264 peptide in the absence of inhibitor.

Purified DCs were incubated with PPV-VLPs-OVA in the presence of chloroquin, a known inhibitor of acidification of late endosomes and lysosomes. Chloroquin strongly inhibited PPV-VLPs-OVA presentation, without altering OVA_257–264 peptide presentation, showing that acidification of late endosomes is necessary for PPV-VLPs processing (Fig. 5A).

View larger version (30K): [in this window] [in a new window]   FIGURE 5. PPV-VLP processing depends on vacuolar acidification and protease activity. A, Purified splenic DCs (105 cells/well) were preincubated for 1 h with medium alone or in the presence of various amounts of chloroquin. Then 1.2 nM PPV-VLPs-OVA (•) or 110 nM OVA257–264 peptide () was added; DCs were incubated for 4 h in the continued presence of the drugs. B, DCs (105 cells/well) were preincubated for 1 h with medium alone, in the presence of 50 µg/ml of leupeptin or pepstatin, or in the absence of drugs. Then 1.2 nM PPV-VLPs-OVA (left) or 110 nM OVA257–264 (right) was added; DCs were incubated for 4 h in the continued presence of the drugs. After glutaraldehyde fixation, DCs were cocultured overnight with 1 x 105 cells/well of B3Z hybridoma. The presentation of OVA257–264 epitope to B3Z cells was monitored by IL-2 production and expressed as the percentage of [3H]thymidine incorporation compared with DCs incubated with PPV-VLPs-OVA or OVA257–264 peptide in the absence of inhibitor.

PPV-VLP processing requires proteasome activity

To evaluate whether the proteosomal complex also participates in PPV-VLPs processing, CD11c⁺ spleen cells were treated with two 20S proteasome inhibitors, lactacystin (50, 51) and LLnL (52). OVA_257–264 presentation by DCs incubated with PPV-VLPs-OVA in the presence of these inhibitors was severely diminished (Fig. 6, A and B), demonstrating that PPV-VLPs or derived peptides translocate to the cytosol of DCs and are degraded by the proteasome. The lack of inhibition of PPV-VLPs-OVA presentation in the presence of LLmL (a calpain I inhibitor) confirmed the specificity of the inhibition observed with LLnL (Fig. 6C). DCs incubated with the OVA_257–264 peptide in the presence of these inhibitors fully stimulated B3Z cells, showing that these inhibitors did not block steps of the MHC class I processing pathway (such as peptide transport, synthesis of MHC molecules, or exocytosis of peptide-MHC complexes) other than proteasome activity.

View larger version (40K): [in this window] [in a new window]   FIGURE 6. PPV-VLP processing requires proteasome activity. Purified splenic DCs (105 cells/well) were preincubated for 1 h with medium alone or in the presence of lactacystin (A), LLnL (B), or LLmL (C). Then 1.2 nM PPV-VLPs-OVA or 110 nM OVA257–264 was added and incubated for 4 h in the continued presence of the drugs. After glutaraldehyde fixation, the DCs were cocultured overnight with 105 cells/well of B3Z hybridoma. The presentation of OVA257–264 epitope to B3Z cells was monitored by IL-2 production and is expressed as the percentage of [3H]thymidine incorporation compared with DCs incubated with PPV-VLPs-OVA or OVA257–264 peptide in the absence of inhibitor.

We then analyzed whether translocation to endoplasmic reticulum (ER)-Golgi complex is necessary for PPV-VLPs processing. We thus analyzed the in vitro PPV-VLP-OVA presentation by DCs from TAP1^-/- mice, which do not have a functional TAP system (53). DCs from TAP1^-/- mice incubated with PPV-VLPs-OVA were unable to present the OVA_257–264 epitope, in contrast to TAP1^+/+ DCs (Fig. 7A). Although TAP1^-/- cells have a diminished expression of class I MHC molecules (53), their capacity to present OVA_257–264-K^b complexes was not affected, as shown for DCs incubated with the OVA_257–264 peptide (Fig. 7B). This result clearly shows that the processing of PPV-VLPs depends upon the translocation of derived peptides from cytosol to endoplasmic reticulum through TAP molecules, confirming previous in vivo results (15).

View larger version (18K): [in this window] [in a new window]   FIGURE 7. PPV-VLPs-OVA are presented in vitro by DCs in a TAP-dependent way. Purified splenic DCs (105 cells/well), purified from TAP1+/+ (•) or TAP1-/- () mice, were incubated with various amounts of PPV-VLPs-OVA (A) or OVA257–264 (B) for 4 h. Then pulsed DCs were washed and cocultured overnight with 1 x 105 cells/well of B3Z cells. The presentation of OVA257–264 epitope to B3Z cells was monitored by IL-2 production and is expressed as the mean ± SEM counts per minute of duplicate wells.

BFA disorganizes the Golgi complex and then inhibits the exocytosis of proteins, blocking the secretion of newly synthesized proteins (54, 55). Using this drug, we therefore determined whether PPV-VLPs-OVA processing involves the transport of processed peptides through Golgi complex to the DC membrane. BFA did not block presentation by DCs of the OVA_257–264 synthetic peptide (Fig. 8) but, as shown in Fig. 8, BFA inhibited B3Z stimulation by DCs incubated with PPV-VLPs-OVA. This strongly suggests that after translocation to ER, PPV-VLP-derived peptides are transported to DC surface through the Golgi complex.

View larger version (33K): [in this window] [in a new window]   FIGURE 8. PPV-VLP processing is sensitive to BFA. DCs (105 cells/well) were preincubated for 1 h with medium alone or in the presence of BFA. Then 0.25 nM PPV-VLPs-OVA or 110 nM OVA257–264 was added and incubated for 4 h in the continued presence of the drugs. After glutaraldehyde fixation, the DCs were cocultured overnight with 105 cells/well of B3Z-specific hybridoma. The presentation of OVA257–264 epitope to B3Z cells was monitored by IL-2 production and is expressed as the percentage of [3H]thymidine incorporation compared with DCs incubated with PPV-VLPs-OVA or OVA257–264 peptide in the absence of inhibitor.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The results of this study clearly show that PPV-VLPs can be processed by DCs through an endosome-to-cytosol pathway. Indeed, after i.v. injection, PPV-VLPs are found in late endosomal vesicles of DCs, as revealed by confocal microscopy. The use of protease inhibitors and cloroquin suggests that vacuolar acidification and lysosomal proteases are involved in PPV-VLPs processing. However, proteasome, TAP-mediated translocation, and transport through Golgi complex are also required for processing of these particles, steps that belong to the cytosolic processing pathway. Protein synthesis is strictly necessary for presentation of the OVA_257–264 epitope contained in PPV-VLPs-OVA.

The capture of PPV-VLPs seems to be mediated by macropinocytosis rather than by clathrin-coated pits, based on the inhibition of OVA_257–264 presentation in the presence of DMA and on the lack of inhibition when cells were deprived of K⁺ or treated with chlorpromazine. Sequestration of cholesterol in plasma membrane by filipin III, which disrupts lipid rafts, also inhibited PPV-VLPs-OVA presentation. It remains to be determined whether these lipids rafts contain caveolin. However, most vesicles containing PPV-VLPs were large, in contrast to the typical small size of caveolin-containing vesicles (61).

Interestingly, the processing pathway of hepatitis B virus small envelope protein (HBsAg) VLPs, another model of VLPs, has been shown to be completely different. Indeed, HBsAg particles are presented to CD8⁺ T lymphocytes by several cell types, including mastocytoma cells, fibroblasts, DC lines, M lines, and B and T lymphocytes, indicating that these particles do not follow a pathway exclusive to DCs (56, 62, 63). In contrast, in vivo, PPV-VLPs are only processed by DCs, whereas M and B cells can neither present PPV-VLPs nor induce CTL responses (15). The internalization of both HBsAg and PPV particles is inhibited by amiloride-derived drugs, and both types of VLPs are initially processed in acidic compartments (20, 63). However, after this step these particles follow different routes. Indeed, HBsAg follow a BFA-resistant and TAP-independent pathway in both M and DCs (20, 63). The presentation of HBsAg VLPs involves binding of antigenic peptides derived from endocytosed HBsAg particles to empty L^d molecules in endosomal compartments and recycling to the cell surface of APCs (20, 47). In contrast, PPV-VLPs translocate to the cytosol of DCs, as demonstrated by their lack of presentation by DCs treated with proteasome inhibitors or from TAP1^-/- mice (15).

An intermediate model is the processing of HBcAg particles, which are 30-nm particles formed by the HBV core Ag. Indeed, in vitro as well as in vivo, the presentation of HBcAg particles by DCs is partially TAP dependent, whereas their presentation by Ms is fully TAP independent, showing that these APCs use different pathways for HBcAg processing (64).

Therefore, DCs can cross-present particulate Ags such as VLPs by different pathways. The translocation from endosome to cytosol remains the key event that determines the pathway used by APCs for presentation to CD8⁺ T cells. If the exogenous Ag has the ability to cross the endosome barrier, it will follow an intracytosolic processing. If not, it will end its processing within vesicles, and the resulting peptides could bind to recycling MHC class I molecules. The lack of inhibition of PPV-VLPs presentation by primaquin as well as the lack of colocalization of PPV-VLPs and rab11 strongly suggest that PPV-VLPs (or derived peptides) are not delivered into recycling vesicles, but remain in the endosomes to deliver the OVA_257–264 epitope into the cytosol. This conclusion is consistent with the inhibition of OVA_257–264 presentation in the presence of BFA, which inhibits protein transport (including nascent MHC class I molecules) from the ER, without affecting recycling MHC class I and II molecules (65), showing that OVA_257–264 presentation is mediated by neosynthesized MHC class I molecules.

After uptake, PPV-VLPs are localized in late endosomal compartments. Acidification of late endosomes is necessary for receptor recycling, movement/maturation of organelles, activation of endolysosomal hydrolases, and activation of membrane transporters (66). The inhibitory effect of chloroquin as well as the partial inhibition of PPV-VLPs processing by pepstatin (an inhibitor of aspartate proteases, included cathepsins D and E) (48, 49) indicates that proteolytic degradation could be necessary in the initial steps of PPV-VLP degradation. However, acidification could also play a role in PPV-VLP translocation to cytosol. Indeed, translocation from endosomes to cytosol is a crucial event for the entry of some viruses. Porcine parvovirus is a nonenveloped virus, and the mechanisms by which it enters the host cell are unknown, as they are for most nonenveloped viruses. However, it seems that interactions between hydrophobic portions of capside proteins participate in the entry. Furthermore, the entry is pH dependent, because endosome acidification blocks virus infection and replication (67).

Translocation to the cytosol is a key event observed only in DCs during endosome-to-cytosol transport (68). The mechanisms involved in this translocation are still unknown, but such a transport has been also proposed for soluble OVA (69), bacteria (19), DC-targeted liposomes (70), heat shock proteins (71), immune complexes (68, 72), exosomes (73), and apoptotic (4) and necrotic (74) cells. This mechanism seems to be specific for internalized Ags and selective for the size of the transported molecules (68). Therefore, PPV-VLPs may take also advantage of this pathway, which has been described as more efficient than TAP-independent pathways (75). This could explain why PPV-VLPs, which need cytosolic processing to allow OVA_257–264 presentation, can only be presented by DCs in vivo.

In the cytosol of DCs, PPV-VLPs or, more likely, products of its degradation are processed by proteasome, as shown by experiments on inhibition with lactacystin and LLnL. Peptides generated from proteolysis in the cytosol are then translocated into the lumen of ER by TAP molecules, where they can bind MHC class I molecules, which can then transit to the cell surface for recognition by CD8⁺ T cells.

Very recently, the existence of cross-presentation and cross-priming as general, physiological processes to induce CD8⁺ T cell response has been doubted (76) on the basis of the lack of convincing in vivo solid experimental evidence. Previous studies have clearly showed that exogenous PPV-VLPs can induce a protective antiviral response, based on the induction of a high frequency of CTLs of high affinity (12, 13, 16, 77). The evidence presented in this report clearly establishes that PPV-VLPs processing by DCs involves cross-presentation. Thus, our results show that cross-presentation can occur in vivo under physiological conditions and lead to the activation of protective CTL responses.

	Acknowledgments

 
We thank Daniel Ladant for the gift of CyaA-E5-OVA, and Bruno Goud and Fransesca Senic-Matuglia for the gift of the anti-Rab11 Ab. We thank Sebastián Amigorena for his helpful discussion. We are grateful to Catherine Fayolle for endotoxin dosage, and to Anne Louise, Anne-Marie Balazuc, and Javier Sarraseca for their excellent technical assistance.

	Footnotes

 
¹ This project is a collaborative work between Pasteur Institute and INGENASA, supported by European Commission Grant QLK2-CT-1999-00318. G.M. was supported by a postdoctoral fellowship from the Consejo Nacional de Investigaciones Científicas y Tecnológicas (Argentine), European Economic Community Grant QLK2-CT-1999-00429, and the Fondation de la Recherche Medicale (France). C.S. was supported by grants from the European Union (Contract BMH4-CT 98-3703).

² Address correspondence and reprint requests to Dr. Claude Leclerc, Departement d’Immunologie, Institut Pasteur, 25 rue du Docteur Roux 75724, Paris Cedex 15, France. E-mail address: cleclerc{at}pasteur.fr

³ Abbreviations used in this paper: M, macrophages; BFA, brefeldin A; CCB, cytochalasin B; CHX, cycloheximide; DC, dendritic cell; DMA, dimethylamiloride; ER, endoplasmic reticulum; LLmL, N-acetyl-L-leucinal-L-methioninal; LLnL, N-acetyl-L-leucinal-L-norleucinal; OVA_257–264, a K^b-restricted epitope corresponding to aa 257–264 of chicken egg albumin; PPV-VLP, porcine parvovirus-virus-like particle; PPV-VLPs-OVA, porcine parvovirus-virus-like particles carrying the OVA_257–264 epitope; WGA, wheat-germ agglutinin; VLP, virus-like particle; HBsAg, hepatitis B virus small envelope protein.

Received for publication December 31, 2002. Accepted for publication June 24, 2003.

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