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The Journal of Immunology, 2005, 175: 796-805.
Copyright © 2005 by The American Association of Immunologists

Cross-Presentation of the Long-Lived Lymphocytic Choriomeningitis Virus Nucleoprotein Does Not Require Neosynthesis and Is Enhanced via Heat Shock Proteins1

Sameh Basta2,3,*, Ricarda Stoessel2,*, Michael Basler*, Maries van den Broek{dagger} and Marcus Groettrup*

* Department of Biology, Division of Immunology, University of Constance, Konstanz, Germany; and{dagger} Institute of Experimental Immunology, Department of Pathology, University Hospital Zurich, Zurich, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Many viral proteins that contain MHC class I-restricted peptides are long-lived, and it is elusive how they can give rise to class I epitopes. Recently, we showed that direct presentation of an epitope of the long-lived lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP) required neosynthesis in accordance with the defective ribosomal products hypothesis. In this study, we report that LCMV-NP can be cross-primed in mice using either LCMV-NP-transfected human HEK293 or BALB/c-derived B8 cells as Ag donor cells. In addition, we establish that contrary to direct presentation, cross-presentation required accumulation of the mature LCMV-NP and could not be sustained by the newly synthesized LCMV-NP protein, intermediate proteasomal degradation products, or the minimal NP396 epitope. Nevertheless, NP cross-presentation was enhanced by heat shock and was blunted by inhibitors of heat shock protein 90 and gp96. We propose that cross-presentation has evolved to sustain the presentation of stable viral proteins when their neosynthesis has ceased in infected donor cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Peptides presented on MHC class I molecules are normally generated through fragmentation of proteins that have been synthesized within the cell. It is therefore remarkable that several structural proteins of viruses are quite stable in cells and nevertheless give rise to sufficient quantities of peptide to induce a CTL response (1, 2, 3). A hypothesis which reconciles this apparent contradiction is the defective ribosomal products (DRiPs)4 hypothesis that states that CTL epitopes are not derived from properly folded proteins but from newly synthesized polypeptides that are degraded within a few minutes after their translation (4). The lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP) is an example of such proteins since it is stable over 3–4 days but nevertheless contains immunodominant epitopes like NP396. Epitopes generated from LCMV-NP shortly after neosynthesis were the sole factor for its direct presentation on MHC class I (5). However, the mechanisms involved in the presentation of DRiPs via the MHC class I pathway are only just surfacing (6, 7, 8).

Professional APCs (pAPCs) can generate class I peptides by two different mechanisms. Either from internally synthesized proteins (9) via "direct-presentation" or by acquiring exogenous Ags through "cross-presentation." Cross-presentation represents a vital pathway for the initiation of immune responses against tumors and viruses that do not have access to, or interfere with, the classical pathway for MHC class I presentation (10, 11, 12, 13, 14, 15). Dendritic cells and macrophages are pAPCs that have the unique capability of generating and presenting peptides on MHC class I molecules from exogenous Ags (16, 17, 18). Activation of naive TCD8+ cells by such pAPCs that have cross-processed Ags into the MHC class I pathway is known as cross-priming (13).

Although the DRiP hypothesis explicates how direct presentation of stable proteins in general can occur, it remains unknown whether stable viral proteins such as LCMV-NP can be cross-presented by pAPCs. This is particularly important since in vivo evidence questioned the ability of LCMV-NP to access the cross-priming pathway (19), yet, it was recently shown that proteins are the mediators of in vivo cross-priming (20, 21, 22). Moreover, immediate proteasomal products were reported to be the main source of Ags transferred to the pAPC in another virus system (23), while others substantiate that Ags could be transferred as peptides bound to heat shock proteins (HSPs) (24). It is possible that virus-specific differences, depending on the nature of virus proteins and their cellular localization, could exist during Ag processing. We therefore started our study by exploring the following avenues; are stable proteins, such as LCMV-NP, predisposed to access the cross-presentation pathway and, if so, what are the antigenic elements responsible for that (20, 21, 22, 23, 24, 25)?

In this study, we report on the cross-presentation of LCMV-NP in vitro and in vivo. We show that accumulated mature proteins probably aided by molecular chaperones are the source of Ags fueling the cross-presentation pathway. Interestingly, neither proteasomal products nor nascent proteins nor DRiPs per se were capable of providing Ags to the cross-presentation-pathway. Our data highlight the significant role of the cross-presentation machinery in degrading stable proteins that are otherwise resistant to processing via the proteasome in virus-infected cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, viruses, cells, and media

Female C57BL/6 (H-2b) mice were originally obtained from Charles River Breeding Laboratories. The animals were kept at the University of Constance in a pathogen-free facility in accordance with the rules of the veterinarian authority of Regierungspräsidium Freiburg and used at 6–10 wk of age.

LCMV-WE was originally obtained from F. Lehmann-Grube (Hamburg, Germany) and propagated on the fibroblast line L929. Recombinant vaccinia virus encoding the LCMV nucleoprotein (rVV-NP) or the NP396 minigene was propagated on BSC40 cells (26). All media were purchased from Invitrogen Life Technologies and contained GlutaMAX, 10% FCS, and 100 U/ml penicillin/streptomycin.

HEK293 is a human embryonic kidney cell line and B8 is a BALB/c-derived fibroblast line (H-2d) obtained by SV40 infection in vitro (5). BMC-2 and DC2.4 are macrophage and dendritic cell lines, respectively (a kind gift from Dr. K. Rock, University of Massachusetts Medical School, Worcester, MA).

Plasmids

The plasmid pAT153.H-2Db was a kind gift from F. Momburg (German Cancer Research Center (DKFZ), Heidelberg, Germany). pCMV-NP and pCMV-U-NP were a kind gift from Dr. L. Whitton (The Scripps Research Institute, La Jolla, CA).

Transfection and establishment of stable cell lines

HEK293 cells were transfected with the following plasmids pCMV-NP or pCMV-Ub-NP (3 µg) and the pLXSP plasmid for selection marker puromycin (1 µg). The transfection was conducted with FuGENE 6 according to the manufacturer’s instructions (Roche Diagnostics). Cells were selected for growth with 2.5 µg/ml puromycin (Sigma-Aldrich), and several clones were tested positive for NP expression. Immunostaining revealed that the protein localized to the cytosol in the transfected cells. In the case of pCMV-U-NP, the protein was only detected after inhibition of the proteasome with the inhibitor N-acetyl-L-leucinyl-L-leucinyl-norleucinal (LLnL; Sigma-Aldrich), 50 µM, overnight. For MHC expression, HEK-Ub-NP (2 x 106 cells) were transfected with the plasmid pAT153.H-2Db (2 µg) using FuGENE 6 reagent. The transfection efficiency was measured with the FITC-labeled anti-H-2Db Ab (clone KH95; BD Biosciences).

Western blotting

HEK293 cells (2 x 106, wild type or transfected) were lysed in 500 µl of lysis buffer (50 mM HEPES (pH 7.5), 2 mM MgCl2, 1 mM EDTA, 1% Triton X-100, and 10% glycerol) on ice for 10 min. After sonification and centrifugation, 10-µl aliquots of the crude lysates were boiled for 5 min at 95°C in 10 µl of 2x reducing Laemmli sample buffer and separated by SDS-PAGE (10% gel). Proteins were blotted onto nitrocellulose (Schleicher & Schüll BioSciences). After blocking overnight at 4°C, the membrane was incubated with the mouse Ab (anti-LCMV/NP, clone 2539 (27), kindly contributed by Dr. M. Buchmeier, The Scripps Research Institute, La Jolla, CA) in PBS in 0.2% Tween 20 and 5% milk for at least 2 h at room temperature, washed three times in PBS/0.2% Tween 20, and then exposed to the HRP-conjugated goat anti-mouse Ab (diluted 1/1000 in PBS/5% milk; DakoCytomation).

Intracellular detection of LCMV-NP

For the detection of LCMV-NP, cells (1 x 105) were harvested and resuspended in 200 µl of PBS, followed by fixation with 4% Formalin for 20 min at room temperature. After washing with 1x PBS, the cells were permeabilized with 1% Triton X-100 for 20 min at room temperature, followed by incubation overnight at 4°C with rat anti-LCMV-NP Ab (clone VL4) (28) in PBS/2% FCS. After two further washing steps, FITC-conjugated polyclonal mouse anti-rat Ab (diluted 1/150 in PBS; BD Biosciences) was left with the cells for at least 1 h at room temperature. Data were acquired with a FACScan flow cytometer (BD Biosciences) using CellQuest (BD Biosciences) and analyzed with FlowJo software (TreeStar).

Intracellular cytokine staining (ICS)

In vitro To detect TCD8+ cell activation with ICS, effector cells were coincubated in round-bottom 96-well plates with (0.1 µM) peptide-loaded APCs (3 x 105/well). Either DMSO or irrelevant peptide (gp33) were used as controls for background activation in all assays. The coincubation ratio of 1:1 APCs (BMC-2) to responders was chosen when T cells were expanded for 5–6 days in vitro. When TCD8+ cell lines were used, using the DC2.4 cell line as stimulator cells, brefeldin A (BFA) was added directly and the cells were left together for 3 h before staining.

In vivo Ex vivo responders were incubated with the specific peptides for 2 h at 37°C and then left in the presence of BFA (Sigma-Aldrich) at 10 µg/ml for 3 h. The coincubation ratio of 1:10 APCs (BMC-2) to responders was chosen in the case of direct analysis ex vivo. In general, cells were then stained with PE-Cy5 anti-CD8 rat IgG Ab clone 53-6.7 (BD Biosciences) on ice for 20 min, washed, and fixed with 1% paraformaldehyde in PBS at room temperature for 20 min. After washing, we labeled the cells with FITC-conjugated rat anti-IFN-{gamma} Ab clone XGM1 (BD Pharmingen) in PBS/0.1% saponin at 4°C overnight. Stained cells were analyzed with a live gate on the CD8+ cells after 80,000 gated cells were acquired.

Tetrameric MHC class I peptide analysis

Tetrameric complexes containing biotinylated H-2Db {beta}2-microglobulin, the NP396 peptide, and extravidin-PE were generated as described previously (26, 29). Splenocytes (5 x 105 cells) were stained with 0.5–1 µg of tetramer in 50 µl of FACS buffer (PBS, 2% FCS, 0.01% NaN3, and 20 mM EDTA) at room temperature for 30 min, followed by addition of PE-Cy5 anti-CD8 rat IgG Ab clone 53-6.7 (BD Biosciences) on ice for 30 min. Cells were washed twice and data were acquired using CellQuest (BD Biosciences) as before by gating on CD8+ lymphocytes.

TCD8+ cell priming and induction of T cell lines

For in vivo priming, 8- to 10-wk-old female C57BL/6J mice were injected i.p. with 1 x 107 cells in PBS. In the case of primary responses, ICS measurements were performed directly 8 days after injection. In some experiments, spleens were harvested and restimulated with peptide-loaded APCs. On day 5 or 6 of the in vitro expansion, T cells were tested for IFN-{gamma} production by ICS as before.

For generating NP396-specific T cell lines, C57BL/6 mice were injected i.v. with 200 µl of LCMV-WE (1000 PFU/ml). The spleens were harvested 4 wk after injection and the splenocytes were purified by density centrifugation (Ficoll-Paque). The splenocytes were cultured in RPMI 1640 containing mouse IL-2 (40 U/ml) along with gamma-irradiated peptide-loaded APCs. An additional density centrifugation step was conducted 5–6 days later when they were used for in vitro Ag presentation experiments. In all assays, we used the T cell lines at this time point and their specificity corresponded to at least 85% when tested with peptide (50 nM). For the detection of the epitope NP396, we resuspended the APCs in RPMI 1640 (1 x 106/ml), added them to NP396-specific TCD8+ cells, and stained for CD8 and IFN-{gamma} as described above.

Preparation of Ag donor or presenting cells (ADCs and APCs)

ADCs were detached with trypsin-EDTA, washed with PBS, and resuspended in RPMI 1640 (1 x 106 cells/ml) and treated with different inhibitors where indicated. For interfering with HSPs, the HSP90-specific inhibitors geldanamycin (5 µM) and herbimycin A (5 µM) were used (Sigma-Aldrich). Cycloheximide (250 µg/ml) was obtained from Calbiochem. For testing different conditions, the cells were either further heated (14 min at ~45°C), or UV irradiated (8 min), or snap frozen in liquid nitrogen (–180°C) and UV irradiated for 8 min (lysed and UV treated (LyUV)). UV-triggered apoptosis was induced using the Stratalinker 2400 UV cross-linker that provides a radiation intensity of 4 mW/cm2 per s. Interestingly, one round of lysis resulted in intact cells incorporating trypan blue, whereas with LyUV cells only 40% incorporated trypan blue. If the cells were subjected to repeated cycles of freezing and thawing, the outcome was mainly cell fragments.

As APCs, we used the macrophage cells line BMC-2 that was recently shown to posses competent phagosomes as organelles for Ag cross-presentation (30). Where indicated, the following inhibitors were used: BFA (10 µg/ml), chloroquine (100 µM), leupeptin (100 µM), pepstatin A (50 µg/ml), fucoidin (250 µg/ml; Sigma-Aldrich), lactacystin (LC, 50 µM; Biomol). During the cross-presentation assays, coincubation of ADCs with BMCs was conducted in round-bottom 96-well plates (greiner bio-one; Cellstar) at 37°C at a ratio of 1:1 for 16 h.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Expression of LCMV-NP in transfected cells

To study LCMV-NP cross-presentation, we used human HEK293 cells as ADCs since they cannot directly present the H-2Db-restricted NP396 epitope to specific T cell lines that we used to monitor cross-presentation. We generated stable transfectants expressing either the full-length LCMV-NP (designated HEK-NP) or a ubiquitin-LCMV-NP fusion protein (HEK-Ub-NP). The N-terminal ubiquitin moiety is a G76A mutant that cannot be cleaved off by ubiquitin-specific proteases and targets the fusion protein for rapid degradation by the proteasome (31). Western blot analyses of LCMV-NP in either LCMV-infected HEK293 cells (HEKi) or HEK-NP cells demonstrated that LCMV-NP was abundantly expressed (Fig. 1A). LCMV-NP expression was very low in untreated HEK-Ub-NP cells but proteasome inhibitors markedly enhanced the expression level, thus confirming rapid proteasomal degradation of the fusion protein (Fig. 1A, HEK-Ub-NP + LLnL) as previously demonstrated in transiently transfected cells (31). The relative NP expression levels as assessed by Western blot analysis were further confirmed by pulse-chase analysis as well as intracellular NP staining and flow cytometry (data not shown and Fig. 1B).



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  		FIGURE 1. Characterization of stable transfectants expressing the NP (LCMV-NP) and a Ub-LCMV-NP fusion protein. A, Western blot analysis of the LCMV-NP steady-state levels. HEK293 cells untransfected (wild type (WT)), LCMV-infected (HEKi), and stably transfected with either NP (HEK-NP) or Ub-NP (HEK-Ub-NP) were lysed, and their NP content was determined by immunoblotting with anti-NP Ab. HEK-Ub-NP cells were also left overnight in the presence of the proteasome inhibitor LLnL (50 µM, HEK-Ub-NP + LLnL). As a positive control, LCMV-infected L929 cells were used (L929i). B, The intensity of NP expression as determined by flow cytometry. HEK-Ub-NP cells are represented by the black-filled histogram that overlapped the negative control. L929i are depicted by the filled histogram with the black line, HEK-NP by the unfilled histogram with the black line, and HEK-Ub-NP + LLnL depicted by unfilled histogram with the gray line.

 
The long-lived form of LCMV-NP is needed for cross-presentation in vivo and in vitro

Initially, we compared the ability of HEK-NP and HEK-Ub-NP cells to induce T cells in vivo. T cell activation was higher in the spleen and peritoneum (Fig. 2A) when mice were injected with HEK-NP than with the HEK-Ub-NP cells. We detected the priming of T cells by coincubating the responder cells with peptide-loaded APCs for a total of 5 h in a standard ICS assay. For controls, we used either the same concentration of DMSO or an irrelevant peptide, gp-33 in all assays. In some circumstances, ex vivo-activated T cells are underestimated with a functional assay such as ICS. We therefore conducted tetramer analysis ex vivo using the specific H-2 Db-NP396-PE tetramer to measure the induced T cell frequencies. Interestingly we could detect significantly higher numbers in the HEK-NP condition compared with the HEK-Ub-NP cells (Fig. 2B).



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  		FIGURE 2. Cross-presentation of LCMV-NP in vivo is mediated primarily via the long-lived form of NP. The stable transfectants HEK-NP and HEK-Ub-NP were injected i.p. (1 x 107/mouse), and spleen and peritoneal exudates cells (PECs) were recovered after 8 days to examine direct ex vivo TCD8+ activation after addition of the NP396 peptide (396) with an IFN-{gamma}/ICS assay; as a negative control, either DMSO or the gp33 peptide was added to the cells (A). T cell frequencies were estimated in B after staining isolated splenocytes from the injected animals with anti-CD8 and the specific H-2Db-NP396-PE tetramer. Data represent one of three experiments. C, Analysis of conditions influencing LCMV-NP cross-presentation in vivo. Untransfected HEK293 cells (HEK) as well as HEK-NP and HEK-Ub-NP transfectants were injected in three different states (whole cells, lysates, and LyUV). Seven days later, splenocytes were restimulated for 5 days in vitro with the dendritic cell line DC2.4 pulsed with NP396 peptide before determining the percentage of activated T cells by IFN-{gamma}/ICS after stimulation with the NP396 peptide-pulsed macrophage line BMC-2. Data represent the means of three experiments ± SEM.

 
However, because of the low signal intensity obtained ex vivo, we assessed the induction of TCD8+ after restimulation and expansion for 5 days in vitro using ICS (Fig. 2C). Before animal inoculation, the ADCs were either left untreated (whole), or lysed to become necrotic (lysed), or LyUV; but for all three protocols, we found after in vitro expansion that HEK-NP cells expressing stable NP induced higher levels of activated TCD8+ cells up to five times more than HEK-Ub-NP cells expressing the short-lived NP fusion protein. As stated in the legend to Fig. 2, we have resorted to two different pulsed APC lines (irradiated DC2.4 for expanding the T cells in vitro and BMCs as APCs for the ICS assay) to avoid any possible cross-reactive T cells and therefore reduce background noise.

To further explore the in vivo phenomena, we performed experiments in vitro where ADCs were left with APCs for 16 h before the addition of NP396-specific T cell lines. The data confirmed the observations made before: HEK-NP cells were a better source of Ag than HEK-Ub-NP cells (Fig. 3A). In addition, when protein degradation was blocked in HEK-Ub-NP cells with the proteasome inhibitor LC, we recovered the ability of the ADCs to cross-present (HEK-Ub-NP + LC), demonstrating that proteasomal products were less efficient than the stabilized protein as a source of Ag for cross-presentation. As expected, the addition of LC to HEK-NP cells did not significantly alter the cross-presentation signals, probably due to the fact that LCMV-NP is an inherently long-lived protein. To make sure that the inferior response in HEK-Ub-NP cells was not due to a defect in epitope generation, we transiently transfected these cells with an H-2Db expression plasmid and confirmed NP396 direct presentation which correlated with the H-2Db expression level (data not shown).



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  		FIGURE 3. Analysis of LCMV-NP cross-presentation in vitro. A, Requirement of a stable form of NP. HEK293 wild-type cells as well as the transfectants HEK-NP and HEK-Ub-NP were used as ADCs for incubation with BMC-2 cells as APCs for 16 h at a ratio of 1:1. NP396 presentation was then determined with a NP396-specific T cell line by IFN-{gamma}/ICS. Where indicated (+LC), ADCs were treated with LC (50 µM) for 8 h. B, Minimal cytosolic peptide is inefficient. HEK293 or J774 cells were infected with vaccinia virus (Vac) or rVV encoding either full-length NP (rVV-NP) or the Db-restricted minimal epitope NP396 (rVV-NP396). After UV treatment, the ADCs were left with APCs (BMC-2) for 16 h at a ratio of 1:1 before IFN-{gamma}/ICS measurement as described above. Data represent the means of three experiments ± SEM. C, Induction of heat shock or lysis and UV irradiation increases the efficiency of LCMV-NP cross-presentation. ADCs (HEK293, HEK-NP, and HEK-Ub-NP were either heat shocked for 14 min at 45°C (heated), lysed and UV irradiated (LyUV), or left untreated (W). The ADCs were added to APCs (BMC-2) for 16 h at a ratio of 1:1 before the addition of NP396-specific TCD8+ cells for IFN-{gamma}/ICS. D, Proteasome inhibition in ADCs allows cross-presentation of the unstable NP. Protein degradation in HEK-Ub-NP was inhibited by adding LC (50 µM) for 8 h (+LC), after which the ADCs were treated as in C. Data represent the means of three experiments ± SEM.

 
To confirm these findings independently and to assess the role of peptide epitopes for cross-presentation, we infected either HEK293 cells or J774 cells (H-2d) with rVV encoding either the full-length LCMV-NP protein or the NP396 minigene. In this situation, donor cells were subjected to UV irradiation to prevent secondary reinfection of the APCs. As shown in Fig. 3B, only ADCs that were expressing the full-length protein served as an efficient source of Ag for the activation of NP396-specific TCD8+ cells. In marked contrast, the rVV encoding the NP396 minigene failed to induce activation via cross-presentation, suggesting that the minimal peptide Ags cannot be efficiently cross-presented.

Heat shock or lysis and UV irradiation increases cross-presentation of LCMV-NP but not of LCMV-Ub-NP

To test different factors that may affect cross-presentation, we subjected HEK-NP and HEK-Ub-NP cells to either heat shock or to lysis by freezing/thawing followed by UV irradiation (LyUV). The heat shock treatment (14 min at 45°C) did not significantly affect the viability of HEK cells as determined by propidium iodide exclusion staining (11 ± 4% dead cells in both control or heated cells) and trypan blue (data not shown). As shown in Fig. 3C, both protocols enhanced cross-presentation of the ADCs with LyUV showing a superior outcome. Remarkably, none of the two ADC treatment protocols were able to enhance cross-presentation of HEK-Ub-NP. However, cross-presentation of HEK-Ub-NP could be recovered when the ADCs were treated with LC (Fig. 3D). Incubation with LC for 8 h did not significantly affect cell viability (propidium iodide) exclusion staining (14 ± 6% dead cells for LC-treated control or heated cells). This suggests that HSPs can enhance cross-presentation that is biased toward proteins but not peptides as an Ag source in this pathway.

The HSP90 family enhances cross-presentation of LCMV-NP

Because initial experiments indicated that treatment of the ADCs leading to increased expression of HSPs enhanced cross-presentation, we examined the outcome of interfering with a prominent class of the HSP families. HEK-NP cells were treated with the very well-characterized HSP90 inhibitors geldanamycin and herbimycina before heat shock or LyUV treatment. As shown before, when ADCs were either heat shocked or LyUV treated, there was an increase in the signals (Fig. 3C). When we used the inhibitors that are specific for both HSP90 and gp96, the signals were almost at background levels (Fig. 4A, heated or LyUV). The effect of these inhibitors on intact cells was not apparent, as no significant differences were detected (Fig. 4A, intact). The possibility that the effect of HSP90 inhibitors was due to a direct inhibition of the responder T cell lines was ruled out as peptide-loaded APCs incubated with either untreated or inhibitor-treated ADCs induced similar activation of the T cell lines (activation was almost 85%, data not shown). In addition, we ruled out possible negative effects of these inhibitors on the NP levels in ADCs by intracellular NP staining and flow cytometry (Fig. 4B).



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  		FIGURE 4. Interference with the activity of HSP90 family HSPs diminishes LCMV-NP cross-presentation. A, HEK-NP cells were treated with two specific inhibitors of HSP90 and gp96 (geldanamycina 5 µM or herbimycina 5 µM) for 6 h at 37°C followed by washing in PBS. The cells were then resuspended in RPMI 1640 (1 x 106ml) and either left untreated, heat shocked for 14 min at 45°C, or lysed and UV irradiated before they were used as ADCs. BMC-2 cells were then added as APCs for 16 h at a ratio of 1:1 before the addition of NP396-specific TCD8+, cells and measurement of cell activation with IFN-{gamma}/ICS. The results were averaged from three experiments; bars represent SEM. B, The intensity of NP expression as determined by flow cytometry. HEK-NP cells are represented by the gray-filled histogram whereas geldanamycin-treated cells are depicted by the unfilled histogram with the black line, and herbimycin-treated cells are depicted by the unfilled histogram with the gray line. The black-filled histogram is the background control.

 
Analysis of LCMV-NP uptake, processing, and degradation in APCs

The capacity of the APC to present Ags is dependent on its ability to internalize the Ags where receptors such as the scavenger receptor A (SR-A) may be playing a role. We therefore examined the contribution of SR-A in the uptake of Ags by incubating APCs with the SR-A inhibitor fucoidin. The ADCs encoding the LCMV-NP were treated as before (intact, heated, or LyUV), followed by incubation with fucoidin-treated or untreated APCs. Interestingly, inhibition of SR-A binding by fucoidin resulted in profound inhibition of cross-presentation (Fig. 5A, heated or LyUV, {square}). Addition of fucoidin did not affect responder T cell lines in a negative manner since peptide-loaded APCs incubated with fucoidin induced similar activation of the T cell lines as control APCs (data not shown). In addition, if we infected the APCs at the same time point when we added the ADCs, there were no significant differences in the activation of the T cell lines (responses were 65 ± 5% in both conditions). Moreover, we tested for the NP expression in ADCs at the same time point (intact cells conditions) and found no significant differences between ADCs incubated with untreated or treated BMCs (data not shown).



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  		FIGURE 5. Blocking SR-A, proteasome activity, ER to Golgi transport, and endosome acidification interferes with NP cross-presentation. A, The majority of uptake is mediated via SR-A receptors. APCs (BMC-2) were either left untreated or were treated with the SR-A inhibitor fucoidin (250 µg/ml) for 30 min followed by washing steps in PBS. HEK-NP cells (intact, heated, or lysed and UV irradiated) were added to these APCs for 16 h at a ratio of 1:1 before the addition of NP396-specific TCD8+ cells for IFN-{gamma}/ICS. B, The processing pathway follows mainly the cytosolic route. The APCs were incubated with either 10 µg/ml BFA or 50 µM LC during the cross-presentation period before the addition of NP396-specific TCD8+ cells as before. C, Role of vacuolar acidification and protease activity. During the coincubation period, chloroquine (100 µM), leupeptin (100 µM), or pepstatin (50 µg/ml) was present as indicated. The wells were washed extensively before the addition of NP396-specific TCD8+ cells as before. Data represent the means of three experiments ± SEM.

 
After examining LCMV-NP uptake, we studied the contribution of the processing pathways involved in its cross-presentation. By using BFA, which inhibits the export of proteins from the endoplasmic reticulum (ER), we tested whether the cross-presentation of LCMV-NP epitopes requires transport through the ER-Golgi complex. BFA blocked the presentation in all three conditions to variable degrees (Fig. 5B,

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). Although cross-presentation of intact or heat shocked but viable cells were inhibited to nearly background levels, presentation of LyUV-treated cells showed possible minor contributions of other pathways, e.g., the vacuolar or the recently discovered new ER-phagosome fusion pathway (30, 32, 33). Nevertheless, the strong inhibition by BFA suggests that Ag processing is occurring mainly through the classical cytosolic route in this system. There were no significant differences in NP expression in ADCs incubated with untreated or treated BMCs (data not shown) when cells were examined 24 h after treatment.

To assess the relative contribution of the proteasome-independent vacuolar processing pathway, we made use of the proteasome inhibitor LC. When we used LC-treated APCs, the presentation of LCMV-NP was significantly diminished in all three conditions (Fig. 5B,

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), suggesting that the NP presentation pathway involves translocation to the cytosol and degradation by the proteasome. Flow cytometric analysis of NP expression revealed no adversary effect of these inhibitors (data not shown). Peptide-loaded APCs incubated with the inhibitors (BFA or LC) induced typical T cell stimulation, showing that these inhibitors did not interfere with presentation by surface expressed MHC class I molecules (data not shown).

Thus far, we have examined the processing routes involved in the presentation of LCMV-NP; however, one essential issue was to delineate the crucial factors responsible for the initial degradation of the long-lived protein during cross-presentation. To address this, we incubated the APCs with chloroquine, an inhibitor of endo/lysosomal acidification. Presentation was significantly inhibited in either the heated or LyUV conditions (Fig. 5C, hatched bars#), showing that acidification is required. To investigate whether endosomal enzymes were also required, we treated APCs with two different protease inhibitors, leupeptin and pepstatin. Incubation of APCs in the presence of leupeptin reduced presentation, whereas pepstatin slightly increased the presentation signals (Fig. 5C, filled or dotted bars). However, the increase was found not to be significant (p > 0.05 as calculated using the paired Student’s t test). Accordingly, endosomal acidification and serine and cysteine proteases (cathepsin B, L, or S) play an important role in LCMV-NP processing, whereas aspartate proteases (cathepsin D or E) do not significantly affect this epitope generation during cross-presentation. In all of these inhibition studies, presentation by peptide-loaded APCs treated in the same manner yielded T cell responses similar to the untreated APCs, suggesting that the inhibitors specifically affected the processing within the APCs (data not shown).

Defective ribosomal products are not the mediators of cross-presentation

A fundamental issue regarding the presentation of LCMV-NP through the cross-presentation pathway centers on whether DRiPs per se are a source of Ag for cross-presentation. To address this point, we tried two different approaches: the first one was based on inhibition of de novo protein synthesis before injecting the ADCs in vivo. As shown in Fig. 6A, we could not detect any significant differences if ADCs were treated with cycloheximide, suggesting that the mature folded protein is responsible for the cross-presentation activity. In vitro, when de novo protein synthesis in ADCs was inhibited by cycloheximide, an inhibitory effect on cross-presentation in vitro was observed (data not shown). Unfortunately, using this approach is inadequate because translation inhibitors can affect a variety of factors in the cell.



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  		FIGURE 6. Cross-presentation of LCMV-NP is not the result of protein neosynthesis. A, HEK-NP cells were incubated with cycloheximide (CHX, 50 µg/ml) for 2 h where indicated. The cells were then washed, resuspended in PBS, and injected i.p. (1 x 107/mouse). Seven days later, splenocytes were restimulated in vitro with DC2.4 cells pulsed with NP396 peptide (pep) for 5 days before determining the percentage of activated T cells by IFN-{gamma}/ICS. As a specificity control, stimulator cells (BMC-2) pulsed with the LCMV-gp33 epitope were used as indicated in parentheses. Data represent means and SDs of triplicate mice from two experiments. B, Influence of LCMV-NP de novo synthesis on the efficiency of cross-presentation. The tet-regulated LCMV-NP transfectants B8tNP64 (NP64) were grown in the absence (at least 24 h and represented by (–) below the graph) or presence of tet to control NP expression, and then lysed and UV irradiated. Switching on NP neosynthesis only for 5 h (–5 h) is not sufficient for NP cross-presentation, whereas eliminating NP neosynthesis for 5 h (±5 h) barely reduced NP cross-presentation. Treatment of B8tNP64 cells for 6 h with 5 µM of the HSP90 inhibitors geldanamycin (geld) and herbimycina (herb) after protein induction for 24 h as indicated prevents cross-presentation. For controls, untreated or inhibitor-treated B8tNP64 cells were incubated with the NP396 peptide-pulsed macrophage line BMC-2 as indicated below. Data are representative of one of three experiments. Bars represent SEM from triplicate samples. C, The intensity of NP expression in cells used in B was determined by flow cytometry and compared with HEK-NP cells. The 24-h time point is depicted by the gray-filled histogram, whereas geldanamycin-treated cells are depicted by the unfilled histogram (thin black line), and herbimycina-treated cells by the unfilled histogram (thick black line).

 
To resolve these issues, we used the tight tetracycline (tet)-repressor system where systemic effects of inhibiting protein neosynthesis are ruled out. This system relies on the tet-repressible expression of LCMV-NP in the fibroblast transfectant B8tNP64. In these cells, NP neosynthesis can be switched on and off without using inhibitors, which has been instrumental in identifying LCMV-NP as a DRiP substrate (5). First, we checked whether LCMV-NP induced for 24–72 h in B8tNP64 cells was cross-presented in vivo. This occurred to a similar degree as we have observed for HEK-NP cells (Fig. 2 and data not shown). We have thus chosen the 24-h time point for further in vitro analyses. To assess the control of LCMV-NP expression and the specificity of cross-presentation in vitro, we used either B8 recipient cells (B8-wt) or B8tNP64 cells (NP64) that were tet suppressed and confirmed that cross-presentation was undetectable when LCMV-NP was not expressed (Fig. 6B). To examine the effect of neosynthesis, we removed tet for 5 h (Fig. 6B, –5 h) so that we could analyze the role of DRiPs as an Ag source, but we failed to detect NP396 presentation after exogenous processing, unlike direct presentation (data not shown). We could recover the activity if tet had been removed for a period of 24 h, thus allowing LCMV-NP accumulation (Fig. 6B, fourth column, tet–), strongly indicating that the accumulated mature protein rather than DRiPs or nascent proteins is responsible for cross-presentation. The LCMV-NP expression levels detected by FACS and immunoblot analyses in either B8tNP64 or HEK-NP cells support this proposition (Fig. 6C and data not shown). The measurements obtained by flow cytometry reflect the nature of the fully folded protein since this Ab does not detect native forms of NP in Western blots. Importantly, as indicated before with the HEK-NP cells, when we awaited NP expression to build up for 24 h and then applied the chaperone inhibitors, no significant negative effects on NP expressions were found (Fig. 6C, histogram overlays).

The ability to cross-present LCMV-NP was significantly reduced when chaperones of the HSP90 family were inhibited in B8tNP64 cells with geldanamycin and herbimycina that were added to cells after tet removal for 24 h (Fig. 6B, columns 5 and 6). To exclude that HSP90 inhibitors negatively affected NP396 presentation by APCs, we used peptide-pulsed APCs as a control (Fig. 6B, columns 8–10). To further scrutinize the requirement for neosynthesis in cross-presentation, we allowed protein synthesis to proceed for 24 h and then added tet for 5 h to block any further neosynthesis (Fig. 6B, ±5 h). If DRiPs were playing a role, then the signal would be drastically reduced. This experiment revealed that, when only the mature LCMV-NP protein was present but no DRiPs, cross-presentation was as efficient as if LCMV-NP synthesis was permitted over 24 h. Taken together, it is evident from our results that NP neosynthesis is not required as an Ag source in LCMV-NP cross-presentation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Better understanding of factors contributing to the cross-presentation pathway is an important issue at present in the field of Ag presentation and immune surveillance. Recently, a novel phagosomal pathway involved in cross-presentation, which merits further assessment, has been discovered (30, 32, 33). Furthermore, the involvement of DRiPs as a source of Ags is a new paradigm in direct presentation (4). These new concepts have prompted us to examine whether cross-presentation of the long-lived LCMV-NP (5) is attainable in APCs after its uptake, since it is not readily degraded in ADCs. Consequently, we set our goals to scrutinize the parameters involved in its degradation in APCs and investigated the relative contribution of proteins vs DRiPs or peptides as Ag sources and the role of HSPs therein.

We tested the ability of this long-lived protein and its degradable products to cross-present in vitro and in vivo to investigate whether it conforms with the recent findings regarding the role of proteins in cross-priming (20, 21, 22) or its stability does not allow it to access the cross-presentation pathway (19). Although the in vitro assays proved to be a more sensitive readout system compared with in vivo, the overall conclusion reached was similar in both systems. Accordingly, and contrary to previously reported data (19), we found that NP transfectants (xeno- or alloresponses) can prime TCD8+ cells in vivo via the exogenous pathway. Perhaps, our system was more sensitive to detect such activity because our transfectants expressed sufficient amounts of NP. Cross-priming and presentation of this long-lived protein was considerably reduced when it was targeted for rapid degradation by the proteasome. This conclusion was confirmed by different means; T cell frequencies were estimated using tetramer analysis and their activation was examined directly ex vivo or after expansion in vitro. Considering all of the data combined, it is clear that less efficient T cell activation, at least four times less, occurred when the NP was targeted for degradation. We hypothesized at this stage that the long-lived form of NP is a better source for cross-priming than the unstable ubiquitin (Ub)-NP fusion protein. This finding concurs with and further extends the concept that proteasomal substrates in general rather than proteasomal products are the main source of cross-priming in vivo (20, 21, 22).

Although the Ub-NP transfectants generate more peptides or polypeptides than full-length protein in the donor cells, they nevertheless were poor inducers of TCD8+ cells via cross-priming. We confirmed these findings with rVV encoding either the full-length protein or the minigene, where the full-length protein was superior in its ability to cross-present compared with the cytosolic peptide. These conclusions are supported by earlier reports showing that cytosolic peptides, unless bound to chromatin, are unstable with a very short half-life due to cytosolic proteases (34). Furthermore, cross-priming of T cell responses in vivo does not require antigenic peptides in the ER (35). According to our results, degraded proteins are unlikely to play a major role in cross-priming of T cell responses in agreement with recently published data (20, 21, 22). Yet, HSPs bound to antigenic peptides were suggested to be a potent source of Ag for cross-presentation (24, 36). In contrast, the circumstances under which chaperone-peptide interactions play a specific role in immunity has been put in question recently and need to be thoroughly investigated (36, 37). Our findings reconcile the proposed role of HSPs in cross-presentation on the one hand and the inability of peptides to cross-prime on the other hand. Based on our results, the ability of HSPs to enhance cross-presentation is evidently biased toward proteins interactions and not proteasomal products.

To examine conditions where possible danger signals may affect the activation of pAPCs (36, 37, 38), we compared intact HEK-NP cells with other stress settings where induction or release of chaperones is envisaged. Either LyUV or heat shock protocols led to a marked enhancement of cross-presentation. This was not the case if we just UV treated intact ADCs (data not shown), indicating that the differential cell treatment can influence immunogenicity (39, 40). In our system, the use of HSP90 and gp96 inhibitors impeded cross-presentation markedly, thus indicating the involvement of one or both of these chaperones after the induction of stress. A very large body of strong biochemical and pharmacological evidence has established geldanamycin and herbimycin A as specific inhibitors of HSP90 family chaperones (41, 42). The chaperone function of HSP90 relies on ATP hydrolysis (43), which is blocked by geldanamycin and herbimycin A because they bind to the ATP binding site of HSP90 according to x-ray crystallographic analysis (44). In addition, a very recent finding reported on reduced cross-priming in mice that were defective in heat shock factor 1 (Hsf1). These mice have a decreased expression of several HSPs including HSP90 and HSP70 (45).

There are several reasons, currently under investigation by us, why these chaperones could have led to increased presentation. It is possible that the effect is indirect due to stimulation of APCs via cell surface signaling receptors like TLRs or increased uptake of ADCs via HSP receptors (36, 37, 38). The chaperones may be required to maintain the proper folding or function of other unknown interacting factors that are generally needed for the cross-presentation pathway. Alternatively, if HSPs are tightly bound to NP, especially under stress conditions, then one could envisage a direct role of chaperones as Ag carrier molecules. Therefore, a possible model could be based on protein-bound HSPs rather than peptide-bound HSPs in enhancing cross-presentation. We also examined certain aspects of the uptake mechanism and the downstream events that follow. We looked into the role of SR-A since it has an array of ligands (46), including certain molecular chaperones (47). A strong reduction of the cross-presentation signal was observed when fucoidin was incubated with APCs. Since fucoidin, a polysaccharide that competitively binds to the ligand-binding domain of SR-A (48), interfered with the presentation signal, it is likely that SR-A is playing an important role in Ag uptake. However, in this instance we could not single out whether the fucoidin was competing out stress chaperones (47) or other undefined SR-A ligands. Recent studies have revealed that stressed/apoptotic or heated cells display HSPs on their surface (36, 40) and thus their recognition could be a factor in the internalization of ADCs by pAPCs.

Hitherto, three pathways have been described for MHC class I presentation of exogenous Ags by pAPCs. In addition to the phagosome-cytosol-ER pathway, and the vacuolar TAP-independent pathway, a new ER-phagosome fusion pathway has recently been discovered (7). It was therefore interesting for us to examine which pathway may be involved in LCMV-NP cross-presentation. NP cross-presentation was markedly reduced in LC- or BFA-treated APCs, indicating that the classical phagosome-cytosol-ER pathway is the major route for the presentation of this Ag. Furthermore, we made use of several inhibitors to examine the proteolytic pathways involved in NP396 cross-presentation (Fig. 7). This was an important point in our study, since this long-lived protein is not degraded in ADCs.



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  		FIGURE 7. A model describing the different presentation pathways for long-lived viral proteins. In an infected APC that primes TCD8+ cells, virus proteins are translated by ribosomes soon after infection (1 h). A significant portion of newly synthesized proteins that are DRiPs (1) get degraded by the proteasome and contribute to the direct presentation of LCMV-NP within 3 h (2). The mature full-length protein (i) can still interact with HSPs and accumulates for at least 24 h (ii) before it can contribute Ags to noninfected cross-presenting APCs via SR-A (iii). In the acceptor APC, these long-lived proteins are preprocessed with the aid of endosomal acidification (iv) and cysteine proteases (v) before their release from endosomes and degradation via the proteasome in the cytosol and transport via the classical cytosol-ER-MHC I presentation route (vi). We propose, based on our data, that components of the DRiP presentation pathway (1 and 2 depicted here by the interrupted dotted arrows) fail to supply Ags to the cross-presenting cells and suggest that direct and cross-presentation pathways are mutually exclusive concerning DRiPs processing and presentation.

 
Accordingly, we examined what enzymes or organelles during cross-presentation are facilitating the processing before proteasomal degradation. Cross-presentation was affected by lowering the endo/lysosomal pH and cysteine and serine proteases, but not aspartate proteases. It appears that acid-dependent endolysosomal Ag preprocessing is required, and it may very well make LCMV-NP amenable to processing by the proteasome in the cytosol. These inhibition studies were controlled as described in Results, where peptide-pulsed cells (with limiting amounts of the peptide) gave similar responses to the control. In addition, viability tests clearly showed no significant differences to untreated cells. Ultimately, we addressed the role of de novo protein synthesis in ADCs in cross-presentation. Using the tight tet-repressor system where protein synthesis is controlled without causing other general inhibitory effects, we unequivocally proved that neosynthesis is not responsible for the generation of NP396 epitopes during cross-presentation of LCMV-NP.

In this study, we have addressed key questions regarding the processing pathways of virus proteins in APCs as shown in Fig. 7. We have scrutinized the potential for proteins in various cytosolic steps (newly synthesized, mature, proteasome-degraded products, and minimal epitopes) as a source of Ag in cross-presentation. Recently published data have uncovered some very interesting findings about the cross-presentation pathway, but at the same time exposed discrepancies regarding the role of proteasomal intermediates and DRiPs as a source of Ag. Besides, a role for HSP in cross-presentation was either not addressed or indirectly looked at (20, 21, 22, 23, 49), while others have reported that LCMV-NP does not access the cross-priming pathway (19). Of a particular interest is the data reported by Janda et al. (49) examining secreted bacterial proteins with a half-life of 90 min in ADCs (49) and reporting, in contrast to our findings, that unstable bacterial translation products were more efficient than mature proteins as an antigenic source for cross-presentation. In this system, the proteins examined for cross-presentation were synthesized in the bacterium before being secreted in the cytosol of infected cells. Consequently, it is difficult to directly compare their source of the cross-presenting Ag to our system wherein newly synthesized Ags are made by the cell’s own translation machinery. It is also possible that their findings reflect an alternative mechanism when fully folded newly synthesized proteins can directly access the cytosol in bacterially infected cells.

In this study, we highlight the extent of DRiPs’ contributions to the presentation of a stable virus protein via the exogenous Ag-processing pathway. Our study clearly shows that although proteasomal substrates are important in cross-presentation as previously reported (20, 21, 22), neither DRiPs per se nor de novo synthesized proteins can contribute to this pathway. It was the accumulated LCMV-NP protein possibly aided by a specific class of molecular chaperones, which was capable of providing Ags.

Since newly synthesized DRiPs constitute a large fraction of the proteasome’s substrates (50), it makes sense that DRiPs are crucial for the immediate direct NP presentation while cross-presentation awaits the build up of stable proteins that are likely to reflect heavily infected cells. Thus, cross-presentation can make long-lived viral proteins accessible for T cell priming, even if protein neosynthesis is curtailed in infected cells after the onset of the IFN response. Our data provide further insights into significant aspects regarding generation of viral immunity (10, 11, 12, 13, 14, 15) and highlight how the direct and cross-presentation pathways complement each other during viral infection (Fig. 7).


   Acknowledgments
 
We thank U. Beck for excellent technical assistance, K. Tschannen for tetramer production, and Drs. L. Whitton, M. Buchmeier, and K. Rock for providing plasmids, Abs, and cell lines.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 Marie Curie Fellowship Grant MCFI-2002-01548from the European Commission (to S.B.). Back

2 S.B. and R.S. contributed equally. Back

3 Address correspondence and reprint requests to his current address: Department of Microbiology and Immunology, Queen’s University, Kingston K7L 3N6, Canada. E-mail address: bastas{at}post.queensu.ca Back

4 Abbreviations used in this paper: DRiP, defective ribosomal product; LCMV, lymphocytic choriomeningitis virus; NP, nucleoprotein; pAPC, professional APC; HSP, heat shock protein; ADC, Ag donor cell; rVV, recombinant vaccinia virus; LLnL, N-acetyl-L-leucinyl-L-leuciyl-norleucinal; ICS, intracellular staining; LC, lactacystin; BFA, brefeldin A; ER, endoplasmic reticulum; SR-A, scavenger receptor A; tet, tetracycline; LyUV, lysed and UV treated; Ub, ubiquitin. Back

Received for publication December 2, 2004. Accepted for publication May 16, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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