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The Journal of Immunology, 2005, 174: 3432-3439.
Copyright © 2005 by The American Association of Immunologists

Cross-Priming of T Cell Responses by Synthetic Microspheres Carrying a CD8+ T Cell Epitope Requires an Adjuvant Signal1

Florence Boisgérault*, Paloma Rueda{dagger}, Cheng Ming Sun*, Sandra Hervas-Stubbs*, Marie Rojas* and Claude Leclerc2,*

* Unité de Biologie des Régulations Immunitaires, Institut Pasteur, and Institut National de la Santé et de la Recherche Médicale E352, Paris, France; and {dagger} Immunología y Génetica Aplicada, Madrid, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Controlling the cross-presentation of exogenous Ags to CD8+ T cells represents a major step for designing new vaccination strategies. Whereas several recombinant pseudo-viral particles have been used as delivery systems for triggering potent CTL responses to heterologous exogenous Ags, the adjuvant properties of virus-like particles (VLPs) themselves were little questioned. Here, we analyzed the contribution of the porcine parvovirus (PPV)-VLPs to the induction of protective cellular responses to exogenous Ags carried by an independent delivery system. Microspheres, which are known to transfer exogenous Ags into the MHC class I pathway, were chosen for delivering the immunodominant OVA257–264 CD8+ T cell epitope (B-OVAp). This delivery system fulfills the requirements in terms of cross-presentation, but fails to induce cross-priming of specific CD8+ T cells. Coinjection of PPV-VLPs with B-OVAp results in the priming of potent CTL responses and type 1-biased immunity in a CD4- and CD40-independent manner, as efficiently as the recombinant PPV-VLPs carrying the same epitope (PPV-OVAp). Furthermore, vaccination with PPV-VLPs and B-OVAp was fully efficient to protect mice against the development of OVA-bearing melanoma. These findings indicate that PPV-VLPs act not only as a delivery system but also as a strong adjuvant when independently provided with exogenous Ag. Thus, dissociation between delivery system and adjuvant would provide a more flexible and reliable system to induce potent and protective CTL.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD8+ T cells are critical effector cells for the clearance of tumors, viruses, and intracellular bacteria (1). These cells recognize MHC class I molecules associated with peptides, which usually result from the processing of Ags localized inside the APCs. However, in many instances, viruses or tumors do not invade APCs, rendering this endogenous pathway of presentation totally inefficient to prime CD8+ T cells. Another alternative pathway, termed cross-presentation, enables APCs to process exogenous Ags and present them in the context of MHC class I molecules (2, 3). Such cross-presentation has been shown to be involved in the induction of tolerogeneic as well as immunogeneic T cell responses in vivo (4). Although most APCs have the capacity to cross-present exogenous Ags in vitro, dendritic cells (DCs)3 are much more efficient in performing this task (5, 6, 7). In addition, evidence has been provided showing that DCs are both able to cross-prime and cross-tolerize CTL, and they are sufficient to cross-present self-Ag to CD8+ T cells in vivo (8, 9). Although it appears that cross-presentation by DCs is central both for inducing tolerance and immunity, the activation status of the APCs is important in determining the outcome of the response (10).

To find the best strategy to deliver exogenous Ags into the MHC class I pathway and induce potent MHC class I-restricted CTL responses to exogenous Ags, a broad panel of soluble and particulate vectors has been engineered (reviewed in Ref.11). Virus-like particles (VLPs) represent promising vaccine candidates since they display an exceptional capacity to trigger protective cellular and humoral responses (reviewed in Ref.12). Based on these properties, chimeric VLPs were designed and used as Ag delivery systems for heterologous epitopes (reviewed in Ref.12). We previously developed a new delivery system based on the 25-nm porcine parvovirus pseudo-VLPs (PPV-VLPs) resulting from the assembly of the major structural VP2 protein. Insertion of T cell epitopes at the N terminus of the protein provides an efficient strategy to stimulate potent Th and CTL responses to foreign epitopes without additional adjuvant in mice. Particularly, we have shown that PPV-VLPs carrying the NP118–132 epitopes from the lymphocytic choriomeningitis virus nucleoprotein induce high frequency of specific CTL associated with viral protection after a single i.p. injection (13, 14). In addition, insertion of a CD8+ T cell epitope, like OVA257–264, in PPV-VLPs (PPV-OVAp) is sufficient to induce specific CTL responses in mice after a single i.v. or i.p. injection (15). However, some limitations prevent the genetic insertion of any antigenic sequences into VLPs. Indeed, size and properties of the heterologous inserted Ag can alter the assembly of structural proteins into immunogeneic particles, and the ability of chimera to self-assemble is highly unpredictable (16). Therefore, a more flexible and reliable system to induce potent and protective CTL responses to exogenous CD8+ T cell epitopes is required.

To optimize the potential of PPV-VLPs as vaccine, it was crucial to understand whether this particle could display an adjuvant activity per se. We addressed this issue by analyzing the ability of native PPV-VLPs to trigger efficient and protective cellular responses to exogenous Ags carried by an independent delivery system. Microspheres, which are known to transfer exogenous Ags into the MHC class I pathway (17), were chosen to deliver the immunodominant OVA257–264 CD8+ T cell epitope (B-OVAp).

In this report, we establish that microspheres can be used to deliver Ag to DCs either in vitro or in vivo without inducing their phenotypic maturation. As a consequence, B-OVAp failed to induce CTL responses unless a second signal was delivered. Importantly, we demonstrate that PPV-VLPs when injected with B-OVAp can restore a specific T cell response, even in the absence of CD4+ T cells or CD40 expression, and can protect mice from the development of OVA-expressing tumors. Our results indicate that PPV-VLPs are not only potent delivery systems but also a promising adjuvant for the development of antiviral and anticancer vaccines.


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

Female C57BL/6 (H-2b) mice (6- to 8-wk old) were purchased from Janvier. CD40 knockout mice were obtained from The Jackson Laboratory and bred in our pathogen-free animal facility. MHC class II knockout mice were purchased from Taconic Farms. These knockout mice were bred onto C57BL/6 background.

The C57BL/6 thymoma, EL-4, was obtained from the American Type Culture Collection. The C57BL/6-derived melanoma B16 and the OVA-transfected B16, named MO5 were kindly provided, respectively, by L. Rosthein and L. Sigal (University of Massachusetts, Worcester, MA). B3Z, a Kb-restricted CD8+ T cell hybridoma specific for the OVA257–264 epitope (18) was kindly given by N. Shastri (University of California, Berkeley, CA). Cells were grown in RPMI 1640 Glutamax-I (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5 x 10–5 M 2-ME. B3Z and MO5 cells were maintained under selective conditions, in medium containing 1 mg/ml G418 and 400 µg/ml hygromycin B for B3Z, and in medium containing 2 mg/ml G418 and 60 µg/ml hygromycin B for MO5 cells.

Peptides and microspheres

The OVA257–264 synthetic peptide (SIINFEKL) corresponding to the immunodominant H-2b-restricted CD8 epitope from the chicken egg OVA was purchased from Neosystem. To generate particulate delivery systems, the OVA257–264 peptide (OVAp) was covalently linked to the surface of 1-µm diameter synthetic latex particles (Polysciences) using glutaraldehyde (Sigma-Aldrich) as previously described (19). Similar microspheres labeled with the yellow-green fluorochrome (B-YG) (Polysciences) were used as tracers for in vivo uptake studies. The carbodiimide kit (Polysciences) was used for covalently coupling the OVA257–264 peptide to fluorescent microparticles according to the manufacturer’s instructions. The loading efficiency of peptides or proteins to microspheres was evaluated by measuring the absorbance between the solution before and after the linkage or by using radiolabeled material. This estimation was made using different peptides containing a Y residue in their sequence. Typically, the amount of peptide bound to beads varied from 10 to 30 ng/106 beads.

Recombinant and native PPV-VLPs

The construction, characterization, and purification of chimeric and nonrecombinant PPV-VLPs were previously described in detail (13). PPV-VLPs are composed of 60 copies of the VP2 protein. PPV-OVAp particles result from the self-assembly of the VP2 protein carrying the OVA257–264 epitope. Wild-type (wt) PPV-VLPs and chimeric PPV-OVAp particles were produced in Sf9 cells using a baculovirus expression system. VLPs were purified by salt precipitation with 20% ammonium sulfate followed by dialysis. The concentration of VLPs was determined by densitometry. The endotoxin content was determined in samples using the Limulus amebocyte lysate test (BioWhittaker). Endotoxin values were below 0.5 ng/mg protein in PPV-VLPs samples.

Purification of APCs

Naive spleens were treated with 400 U/ml collagenase type IV and 50 µg/ml DNase I (Boehringer Mannheim) in RPMI 1640 Glutamax-I (Invitrogen Life Technologies) for 45 min at 37°C. Cells were dissociated before being incubated with MACS anti-CD11c colloidal paramagnetic beads (N418 clone; Miltenyi Biotec) according to the manufacturer’s instructions. CD11c+ cells were positively selected using high-speed MACS (AutoMACS; Miltenyi Biotec). Beyond 96% pure CD11c+ cells were obtained. The negative fraction was then collected and CD11b+CD11c– cells were stained with MACS anti-CD11b beads (Mac-1{alpha} clone; Miltenyi Biotec) and purified as well. Average of 85% pure CD11b+ cells was obtained. The negative fraction was kept as the CD11b–CD11c– population, or submitted to a third round of purification using MACS-anti-B220 beads (RA3-6B2 clone; Miltenyi Biotec) to select B cells.

Ag presentation assay

From 1.25 x 104 to 105 CD11c+CD11b+ and CD11b–CD11c– cells were plated onto 96-well culture microplates to which PBS, B-OVAp (108 microspheres/ml) and OVA257–264 peptide (1 µg/ml) were added. B3Z hybridoma cells (105) were added per well in a 0.2-ml final volume. Cells were incubated overnight at 37°C, and quantification of IL-2 released in supernatants by B3Z was determined using the CTLL-2 cell line. Supernatant (100 µl) was transferred to flat-bottom 96-well microplates and cultured with 104 CTLL-2 cells per well. [3H]Thymidine was added 2 days later, and the experiment was stopped and harvested 6 h later. The incorporated [3H]thymidine was detected with a LKB Wallac cell scintillation counter and expressed in cpm. Results are representative of the mean of duplicates.

Uptake and maturation analysis

Mice were injected i.v. with either PBS, 109 B-YG, or 25 µg of LPS from Escherichia coli serotype 5 purchased from Sigma-Aldrich. APCs were purified as described above 15 h later. To analyze the uptake, the different cell populations were preincubated with a rat anti-CD16/32 mAb (2.4G2 clone) and stained with allophycocyanin-conjugated anti-CD11c (HL-3 clone), -CD11b (Mac-1{alpha}, M1/70 clone), -CD45R (B220, RA3-6B2 clone), -Gr1 (Ly-6G, RB6-8C5 clone) mAbs, and isotype controls for 30 min. For maturation analysis, 30-min incubation was performed with biotinylated anti-CD80 (B7.1, 16-10A1 clone), -CD86 (B7.2, GL1 clone), -CD40 (3/23 clone) mAbs, and isotypes controls. Abs were purchased from BD Pharmingen. After washing, cells were stained with allophycocyanin-streptavidin conjugate for 15 min. Propidium iodide was added to exclude dead cells. Each sample was acquired on the FACSCalibur cytometer and data were analyzed using the CellQuest Software.

Mice immunization

Mice were immunized once i.v. with 109 B-OVAp injected alone or together with 10 µg of PPV-VLPs. PBS, PPV-VLPs, and unloaded microspheres were used as controls. In the experiments performed with anti-CD40 mAbs, mice were first immunized with PBS and beads, and, immediately afterward, were injected i.v. with 100 µg of anti-CD40 mAb (3/23 clone, BD Pharmingen) or 100 µg of rat IgG isotype control.

Cytotoxic T cell assay

Spleens were removed surgically 7 days after priming. Spleen cells were stimulated in vitro with 0.1 µM OVA257–264 peptide for 5 days in the presence of gamma-irradiated (3000 rad) syngeneic naive splenocytes. Effector cells were then assayed for cytotoxic activity on 51Cr-labeled EL4 target cells pulsed or not with 50 µM OVA257–264 peptide. The radioactivity released was measured in the supernatant. The percentage of lysis was calculated as 100 x (experimental release – spontaneous release)/(maximum release – spontaneous release). Maximum release was evaluated by adding 10% Triton X-100 to EL4 cells, whereas spontaneous release was determined by incubating EL4 in medium. The percentage of specific lysis was calculated after deduction of the nonspecific lysis (obtained for unloaded EL4) from the total lysis of OVA257–264-loaded EL4 cells at multiple E:T ratio. Lysis of EL4 was always <10%.

ELISPOT assays

Multiscreen filtration plates (96-well; Millipore) were coated with either anti-IL-4 (2 µg/ml) or anti-IFN-{gamma} (4 µg/ml) capture mAbs (BD Pharmingen) in sterile PBS overnight. Plates were then washed twice in sterile PBS, blocked with RPMI 1640 supplemented with 10% heat-inactivated FCS for 1.5 h, and then washed three times with sterile PBS. Spleen cells from immunized mice were added to wells previously filled with gamma-irradiated (3000 rad) syngeneic naive splenocytes (ratio 2:1) either with or without the OVA257–264 peptide (1 µg/ml). Cells were then incubated for 42 h at 37°C, 7% CO2, and plates were washed extensively with PBS and PBS containing 0.025% Tween 20. Biotinylated rat anti-mouse IL-4 or IFN-{gamma} detection mAbs (BD Pharmingen) were added at 2 µg/ml and incubated for 4–5 h at room temperature, then plates were treated for 2 h with streptavidin-alkaline phosphatase (BD Pharmingen). Spots were revealed by the addition of the 5-bromo-4-chloro-3-indolylphosphate-NBT (Sigma-Aldrich). Frequency of IL-4- and IFN-{gamma}-producing cells was determined by counting the number of spots per well with a computer-assisted ELISPOT image analyzer (Bioreader-3000 Pro). The results were expressed as the number of spot-forming cells (SFC) per million splenocytes.

Tumor protection experiments

Mice were first immunized i.v. with PBS, PPV-VLPs (10 µg), B-OVAp (109), B-OVAp/PPV-VLPs (109 beads + 10 µg of PPV-VLPs), and PPV-OVAp (10 µg). Mice were injected s.c. 11 days later on the right flank with 2 x 104 syngeneic melanoma cells from either the parental B16 or the OVA-transfected B16, designed as MO5. Mice were followed for tumor growth and survival. Tumor diameters were measured to appreciate tumor growth. When tumors reached 30-mm diameter, mice were killed and recorded as "not surviving" mice. To compare the survival between each treated group, statistical analyses were performed based upon the Kaplan and Meier method (20).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Although microparticles can deliver exogenous Ags into the MHC class I pathway, we have previously demonstrated that they require CD4+ T cells to trigger CTL responses (14). In contrast, recombinant PPV-VLPs carrying CD8+ T cell epitopes induce potent CTL responses in the absence of adjuvant and without requirement of CD4+ T cells (14). This suggests that bead-targeted APCs do not directly deliver activation signals to CD8+ T cells, whereas PPV-targeted APCs do. Therefore, we investigated which of the events leading to cross-priming of CD8+ T cells is missing when CD8+ T cell epitopes are delivered by microspheres, i.e., APCs targeting, cross-presentation, or maturation signals.

DCs present efficiently OVA257–264 delivered by microspheres in vitro

Priming of naive CD8+ T cells either in vivo or in vitro requires Ag presentation by specialized APCs, namely DCs. Therefore, the first requirement for a delivery system is to target the Ag to DCs and be processed efficiently. We checked whether OVA257–264 peptide bound to microspheres (B-OVAp) is presented in vitro by spleen DCs, purified on the basis of CD11c expression. We also compared the efficacy of DCs and other APCs, like macrophages and B cells, to present the OVA257–264 peptide delivered by microspheres. Presentation of the OVA257–264 epitope to MHC I-restricted T cells was evaluated by monitoring stimulation of the B3Z hybridoma. As illustrated in Fig. 1, DCs incubated with B-OVAp for 15 h efficiently presented the OVA257–264 epitope even when a low number of cells was used. Conversely, the remaining phagocytic cells (granulocytes and macrophages), selected on the basis of CD11b expression, were much less efficient in presenting the OVA257–264 peptide bound to microspheres. At least 10-fold more CD11b+ than CD11c+ cells was required to stimulate B3Z in the presence of equal quantities of B-OVAp. Finally, the CD11b–CD11c– population, composed of 60% B cells, was globally inefficient in presenting OVA257–264 peptide delivered by microspheres. These observations cannot be attributed to differences in the availability of MHC I molecules since both CD11c+CD11b+ and CD11b–CD11c– cells were equally efficient in presenting saturating doses of OVA257–264 peptide. These results clearly show that DCs are much more efficient than any other APCs in presenting CD8+ T cell epitopes linked to microparticles.



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  		FIGURE 1. The OVA257–264 peptide delivered by microspheres is efficiently presented by DCs in vitro. C57BL/6 splenic CD11c+ cells were positively selected and the CD11clow fraction was then stained with anti-CD11b to purify CD11b+ cells. The negative fraction was kept as CD11b–c– cells. Different concentrations of purified cells were then incubated with the OVA257–264 peptide ({circ}) (1 µg/ml), B-OVAp (•) (108/ml), or control lymphocytic choriomeningitis virus118–126 peptide ({diamondsuit}), and cocultured overnight with the anti-OVA B3Z hybridoma (105/well). The release of IL-2 was monitored by the proliferation of CTLL-2 cell line. One representative experiment of three is depicted.

 
In vivo capture of microspheres by DCs does not induce maturation

Whereas we have shown that microspheres can efficiently deliver CD8+ T cell epitopes to DCs in vitro, it was important to investigate whether microspheres are also able to target DCs in vivo and whether this capture results in DC maturation. To address the first issue, we analyzed the in vivo uptake of fluorescent-labeled beads by different APCs. Naive C57BL/6 mice were injected with either PBS or 109 YG-labeled microspheres (B-YG). CD11c+ (DCs), CD11b+ (granulocytes and macrophages), and B220+ (B cells) populations of spleen APCs were sorted out 15 h later. As expected, B cells were almost unable to capture microparticles since <1% of the B220+ cells were YG+ (Fig. 2). In contrast, the phagocytic populations including CD11c+ and CD11b+ showed a weak but significant capacity to take up B-YG, since ~3–4% of each population was YG+. However, this uptake efficacy is very low compared with what was observed for PPV-VLPs, which is captured by 50–60% spleen DCs (15).



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  		FIGURE 2. Analysis of microspheres capture following i.v. injection into mice. C57BL/6 mice were injected i.v. with 109 fluorescent microspheres coupled to OVAp (B-YG) or PBS. Fifteen hours later, CD11c+ and CD11b+ spleen cells were purified as described above. B220+ cells were obtained after a third round of purification using MACS anti-B220 beads on the CD11c– fraction. The uptake of beads was analyzed on a FACSCalibur cytometer. Purified CD11c+ (left), CD11b+ (middle), and B220+ (right) cells from PBS- (top) or B-YG- (bottom) injected mice were, respectively, stained for CD11c, CD11b, and B220 molecules with allophycocyanin-labeled mAbs (FL4 channel), and B-YG+ cells were caught on the FL1 channel. The percentage of YG+ cells was represented on the dot plot.

 
Although these synthetic beads are composed of inert materials, we could not exclude that phagocytic stimuli trigger DC maturation. To investigate whether beads induce maturation of splenic DCs, we analyzed the phenotypic markers that are up-regulated upon DC maturation, i.e., MHC II molecules and costimulatory molecules such as CD40, CD80, and CD86. Naive C57BL/6 mice were injected with PBS, 109 B-YG or beads carrying the OVA257–264 epitope (B-OVAp), or 25 µg of LPS; 15 h later, DCs were purified. Although DCs costimulatory and MHC class II molecules were up-regulated following LPS administration, they were identical in B-YG- and PBS-injected mice (Fig. 3A). To rule out a specific effect of beads carrying the OVA257–264 peptide (B-OVAp) on DCs, maturation was also assessed after injection of B-OVAp. As shown in Fig. 3, B and C, we did not detect any significant differences in the level of costimulatory and MHC II molecules in either the B-OVAp+ or B-OVAp– DC populations of B-OVAp-injected mice. Thus, the uptake of beads by DCs did not induce maturation of these cells.



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  		FIGURE 3. Microspheres are taken up by DCs, following i.v. injection in mice, but do not induce DC maturation. C57BL/6 mice were injected i.v. with 109 fluorescent microspheres, PBS, or 25 µg of LPS. Fifteen hours later, CD11c+ spleen cells were purified as described above and stained with CD11c PE mAb in combination with CD40, CD80, CD86, and MHC II biotin mAb and then with streptavidin APC. DC staining was analyzed on a FACSCalibur cytometer. CD40, CD80, CD86, and MHC II molecules expressed on DCs purified from PBS- (filled histogram), B-YG- (bold line), and LPS- (dotted line) injected mice were compared (A). Dot plots (B) illustrate the uptake of beads coupled to OVA257–264 peptide (B-OVAp) (y-axis) and DC maturation (x-axis). The maturation profile of B-OVAp– DCs (gray histogram), B-OVAp+ DCs (bold line) was further depicted on histograms (C).

 
Microspheres delivering OVA257–264 do not activate nor tolerize specific T cells

We then examined whether targeting these few immature spleen DCs with B-OVAp was sufficient to prime specific CTL responses, or whether a "licensing" signal such as an agonist anti-CD40 mAb was required. C57BL/6 mice were injected i.v. with 109 beads carrying the OVA257–264 epitope (B-OVAp). Unloaded beads (B) and PBS were used as negative controls, whereas PPV-OVAp was used as a positive control (data not shown). Spleen cells were harvested 7 days later and stimulated in vitro with the OVA257–264 peptide for 5 days. Their CTL activity against OVA257–264 peptide-coated EL4 target cells was then tested in a 51Cr release assay. We observed that, in the absence of adjuvant, i.v. injection of beads carrying the CD8+ OVA257–264 epitope was unable to trigger anti-OVA-specific CTL responses in C57BL/6 mice (Fig. 4A). The same observation was done following i.p. injection of B-OVAp (data not shown). Conversely, when mice were immunized with 109 B-OVAp and were given anti-CD40 mAb (clone 3/23) immediately afterward, a potent CTL response was observed (Fig. 4B). Therefore, in steady-state conditions, B-OVAp do not trigger CTL responses, but they are able to do so if a further signal is delivered to DCs.



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  		FIGURE 4. Microspheres delivering OVA257–264 do not trigger CTL responses unless CD40 signaling is provided. C57BL/6 mice received an i.v. injection of PBS or 109 B-OVAp (A), or were administered i.v. with PBS, 109 B-OVAp and 100 µg of anti-CD40 mAb (clone 3/23), 109 B-OVAp and 100 µg of control rat IgG, or 109 unloaded beads (B) and 100 µg of anti-CD40 mAb. Seven days later, spleen cells were harvested and restimulated for 5 days with the OVA257–264 peptide and irradiated syngeneic cells. Effector cells were then assayed for cytotoxic activity using 51Cr-labeled EL4 incubated with medium alone or with the OVA257–264 peptide. The percentage of 51Cr specifically released by OVA257–264-loaded target cells at varying E:T ratios was calculated after deduction of the nonspecific release by unloaded EL4. The nonspecific release was below 10%. Data are representative of one of three independent experiments performed with individual mice.

 
Since B-OVAp do no cross-prime specific T cells, induction of cross-tolerance should be ruled out especially because immature DCs were shown to induce tolerance (8, 9). To address this question, we first inoculated C57BL/6 mice with either PBS or 109 B-OVAp. Fifteen days later, mice from each group received a s.c. injection of the recombinant PPV-OVAp carrying the OVA epitope. Twelve days after this last immunization, the CTL activity was determined after restimulation of effector cells in vitro with the OVA257–264 peptide. As shown in Fig. 5, mice immunized with PPV-OVAp developed a specific CTL response to the OVA257–264 epitope. Moreover, we did not observe any decrease in the intensity of the CTL response when mice had received a former immunization with the B-OVAp compared with the PBS-injected ones. The same observation was done in mice that received a second immunization with the OVA peptide mixed in IFA (data not shown). Thus, these results demonstrate that B-OVAp do not tolerize specific T cells following i.v. injection but are rather ignored by the immune system. Altogether, these data show that microspheres can deliver peptide to DCs but are devoid of stimulatory or anergizing capacity.



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  		FIGURE 5. Microspheres delivering the OVA257–264 peptide do not tolerize specific T cells. C57BL/6 mice were injected i.v. with PBS or 109 B-OVAp. Fifteen days later, each group of mice received s.c. either 10 µg of PPV-OVAp or PPV-VLPs. Ten days after the last immunization, spleen cells were stimulated in vitro with the OVA257–264 peptide, and the cytotoxic activity of effector cells was assayed on 51Cr-labeled EL4 incubated with medium alone or with OVA257–264 peptide. The percentage of 51Cr specifically released by OVA257–264-loaded target cells at varying E:T ratios was calculated after deduction of the nonspecific release, as described above. Results are expressed as the mean ± SEM of the percentage of specific lysis obtained with individual mice in one of two representative experiments.

 
Microspheres delivering OVA257–264 induce specific cytotoxic T cell responses in the presence of PPV-VLP

In a previous report, we have established that PPV-VLPs carrying the OVA257–264 epitope (PPV-OVAp) induce potent CTL responses in the absence of adjuvant when injected i.v. one time in mice (15) as confirmed in the present study. We have also shown that PPV-VLPs are very efficient to deliver heterologous CD8+ T cell epitopes into the MHC class I pathway of DCs and are potent maturating agents for DCs (15). To investigate whether the signals supplied by PPV-VLPs could rescue T cell immunity to CD8+ T cell epitopes provided by a nonimmunogeneic exogenous delivery system, mice were immunized by various routes (i.v., s.c., or i.m.) with both B-OVAp (109 beads) and PPV-VLPs (10 µg). Control mice received a single dose of PPV-OVAp (10 µg), 109 B-OVAp, or 10 µg of PPV-VLPs. Spleen cells were harvested 2 wk later and stimulated in vitro with the OVA257–264 peptide to be tested for their cytotoxic activity in a 51Cr release assay (Fig. 6). Whereas neither B-OVAp nor PPV-VLPs injected alone primed anti-OVA257–264-specific CTL, coinjection of PPV-VLPs and B-OVAp (B-OVAp/PPV), i.v., s.c., or i.m., induced a strong specific CTL response as efficiently as following immunization with PPV-OVAp. In contrast, no specific CTL response was detected following coinjection of equivalent amounts of soluble OVA257–264 peptide and PPV-VLPs (data not shown). These data demonstrate that signals supplied by PPV-VLPs are sufficient to complement the signals provided by the CD8+ T cell epitopes delivered by nonimmunogeneic synthetic particles. Interestingly, this effect was also observed when B-OVAp were replaced by OVA (unpublished data). Therefore, PPV-VLPs have the ability to trigger CTL responses to exogenous Ags either in a particulate or soluble form.



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  		FIGURE 6. Injection with B-OVAp and PPV-VLP induce a specific anti-OVA257–264 CTL response in mice. C57BL/6 mice were injected i.v. (A), s.c. (B), or i.m. (C) with 109 B-OVAp or 10 µg of PPV-VLPs, or coinjected with the same doses of B-OVAp and PPV-VLPs. PPV-OVAp (10 µg) was used as a positive control. Two weeks later, spleen cells were harvested and incubated with irradiated syngeneic splenocytes and OVA257–264 peptide for 5 days. Effector cells were then assayed for cytotoxic activity on 51Cr-labeled EL4 incubated with medium alone or with OVA257–264 peptide. The percentage of 51Cr specifically released by OVA257–264-loaded target cells at varying E:T ratios was calculated after deduction of the nonspecific release, as described above. Data obtained with individual mice in one of three representative experiments are illustrated.

 
Induction of a Tc1 response to OVA257–264 by B-OVAp and PPV-VLPs

Since both Tc1 and Tc2 have been generated in vitro against the OVA257–264 epitope, we analyzed whether PPV-VLPs could polarize the response to OVA257–264 delivered by beads. We next determined the frequency of anti-OVAp IFN-{gamma}- and IL-4-producing cells found in the spleen of mice injected either with both 109 B-OVAp and 10 µg of PPV-VLPs (B-OVAp/PPV) or with 10 µg of PPV-OVAp. The results obtained by ELISPOT are illustrated in Fig. 7. In mice immunized with either B-OVAp or PPV-VLPs, the frequencies of specific IFN-{gamma}-producing cells were equivalent to those found in naive animals. On the contrary, the number of specific IFN-{gamma}-producing cells was strongly increased in mice injected with B-OVAp/PPV compared with naive mice. Interestingly, the high frequency of OVA-specific IFN-{gamma}-producing cells was similar in PPV-OVAp and B-OVAp/PPV-VLP-injected mice. In turn, we did not detect any specific IL-4-producing cells in the spleen of immunized mice. Thus, these data show that the simultaneous administration of B-OVAp and PPV-VLPs elicits a vigorous Tc1 response as potent as the one induced by injection of PPV-OVAp.



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  		FIGURE 7. High frequency of IFN-{gamma}-producing cells is induced following B-OVAp/PPV-VLP injection in mice. C57BL/6 mice were immunized i.v. with 10 µg of PPV-VLPs, 109 B-OVAp, or with both B-OVAp and PPV-VLPs as indicated on the x-axis. PPV-OVAp was used as a positive control. Seven days later, the frequency of IFN-{gamma} and IL-4-producing splenocytes was measured by ELISPOT assay. Results are expressed as the number of SFC per million spleen cells. Data obtained with individual mice are representative of three independent experiments.

 
Induction of Tc1 response by B-OVAp and PPV-VLPs in MHC class II–/– and CD40–/– mice

To determine the contribution of Th cells to the induction of the anti-OVA response generated after immunization with B-OVAp/PPV, we analyzed whether disrupting MHC class II presentation or CD40/CD40L signaling affects the frequency of specific IFN-{gamma}-producing cells. We immunized MHC class II–/–, CD40–/–, and wt C57BL/6 mice according to the protocol described earlier. As expected, the number of SFC obtained in MHC class II–/– and CD40–/– mice injected with PBS, PPV-VLPs, and B-OVAp was similar to what is seen in naive mice (Fig. 8 and data not shown). Following immunization with B-OVAp/PPV, we found a similar high frequency of specific IFN-{gamma}-producing cells in MHC class II–/–, CD40–/–, and wt mice (Fig. 8). These results demonstrate that the high frequency of Tc1 effector cells, which is found in the spleen of mice immunized with B-OVAp/PPV, is neither dependent on CD4+ Th cells nor CD40 signaling.



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  		FIGURE 8. The anti-OVA257–264 T cell response induced following B-OVAp/PPV-VLP injection is CD40- and CD4-independent. CD40 KO, CD4 KO, and C57BL/6 wt mice were injected i.v. with 10 µg of PPV-VLPs, 109 B-OVAp, or coinjected with the same dose of B-OVAp and PPV-VLPs. Seven days later, spleen cells were harvested, and the frequency of IFN-{gamma}-producing splenocytes was measured by ELISPOT assay. The specific response to OVA257–264 peptide was calculated after deduction of the background obtained in the absence of peptide and is expressed as the {Delta} number of SFC ({Delta}SFC) per million cells. Results are expressed as the mean ± SEM of the number of SFC obtained with individual mice tested in two to three independent experiments.

 
Immunization with B-OVAp and PPV-VLPs induce protective immunity to OVA-expressing tumors

To evaluate the capacity of B-OVAp and PPV-VLPs to induce protective immunity, C57BL/6 mice were immunized with both B-OVAp (109 beads) and PPV-VLPs (B-OVAp/PPV), and mice were grafted 11 days later s.c. with 2 x 104 OVA-expressing melanoma cells, MO5 (Fig. 9A). Control mice, injected with a single dose of PBS, 109 B-OVAp, 10 µg of PPV-VLPs, or 10 µg of PPV-OVAp, were also inoculated with MO5 tumor cells in the same conditions (Fig. 9B). We observed that mice injected with PBS, B-OVAp, or PPV-VLPs displayed a progressive tumor growth and died between days 30 and 70 (Fig. 9A). In contrast, onset of tumors was delayed in mice immunized with either PPV-OVAp or B-OVAp/PPV, especially in B-OVAp/PPV-injected mice, in which no tumor formation was detected 40 days after tumor graft. A single immunization with either PPV-OVAp or B-OVAp/PPV conferred a survival advantage to mice challenged with MO5, since 60 and 80% had survived, respectively. Although survivals of the PPV-OVAp- and the B-OVAp/PPV-immunized mice were slightly different when compared with the PPV-treated group (p = 0.02 vs p = 0.002), the level of protection achieved with PPV-OVAp and B-OVAp/PPV was similar (p = 0.3).



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  		FIGURE 9. Immunization with B-OVAp/PPV-VLPs protect mice from the growth of the MO5 OVA expressing tumor. C57BL/6 mice were first immunized i.v. with PBS, 10 µg of PPV-VLPs, 109 B-OVAp, or with the same doses of B-OVAp and PPV-VLPs or with 10 µg of PPV-OVAp as positive control. Eleven days later, mice were grafted with 2 x 104 OVA-transfected B16 (MO5) cells (A) or with B16 (B) melanoma cells. Mice were then followed for tumor growth (right) and survival (left). The results represent cumulative data of two experiments each performed with five mice per group (n = 10). *1 and *2, p < 0.05 compared with the PPV-VLPs-treated group.

 
To investigate whether the protection conferred by B-OVAp/PPV and PPV-OVAp was Ag-specific, mice immunized with B-OVAp/PPV-VLPs or PPV-OVAp were challenged with the parental B16 melanoma cells, which do not express OVA. As shown in Fig. 9B, all mice developed tumors and died within 40 days. Thus, the protective immunity conferred by a vaccination with PPV-OVAp and B-OVAp/PPV is highly specific and directed to OVA-expressing tumor cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
VLPs are widely used as exogenous delivery systems, and have proven to be potent CTL inducers when compared with other vectors (21). Whereas some VLPs do require additional adjuvant to be functional, most of them do not, suggesting that not only VLPs transfer Ags into the MHC class I pathway but also hold intrinsic adjuvant properties. Since Ag delivery and adjuvant activity involve different aspects of the immune response, it was important to dissociate them. We describe herein a nonimmunogeneic exogenous delivery system that allows analysis of the adjuvant properties of coinjected compounds. Using this system, we provide evidence that PPV-VLPs per se is a potent adjuvant for priming CD8+ T cells to exogenous Ags.

DCs play a crucial role in the initiation of primary response, making them an absolutely required target for exogenous Ags. They have the ability to cross-present Ags derived from cell-associated Ags and soluble or particulate Ags that they have engulfed (3, 5, 9). In this study, we demonstrated that DCs were much more efficient in presenting OVAp delivered by microspheres than other APCs. These results are in accordance with in vitro findings showing that DCs are more potent in cross-presenting Ag acquired from apoptotic cells than macrophages (5). Whereas the accessibility to APCs is different in vitro and in vivo, we also found that the relative number of spleen DCs holding beads 15 h after i.v. administration is higher compared with other spleen APCs. Although DCs could have captured particles elsewhere, either in the blood or the liver, they have the ability to recirculate into distinct compartments of lymphoid tissues (22). The overall capacity of DCs to capture beads is very low at the concentration used in this study. Interestingly, we detected even less beads in the CD11b+ Gr1– population (data not shown), suggesting that macrophages do not play a major role in the clearance of beads in the spleen. We cannot exclude that Kupffer cells are mainly targeted in the liver as it was previously established upon i.v. immunization with Ags entrapped in liposomes (23). However, it is unlikely that microspheres had targeted macrophages first and then end up in DCs, since we and others did not notice any difference in the percentage of bead-containing DCs 2 and 15 h postinjection (unpublished results) (24). In addition, it was shown that resident spleen macrophages could transfer only small fragments loaded onto particles to DCs, but not the particle itself (25). Randolph et al. (26) have also reported the ability of monocytes to phagocytose particles and differentiate into DCs following migration to lymphoid organs. However, none of the bead-containing DCs displayed the high level of costimulatory molecules that were observed upon differentiation or stimulation with LPS (26). Thus, it is likely that either resident or blood-borne immature DCs phagocytose directly exogenous particles and present CD8+ T cell epitopes without being converted into fully competent mature DCs.

In the absence of adequate costimulation, repeated exposure of T cells to Ag presented by DCs tends to result in T cell tolerization (8, 27, 28). Although microspheres do not trigger DC maturation, CD8+ T cell epitopes delivered by beads do not induce T cell tolerance but rather result in immunological ignorance. Our data fit with recent observations showing that cross-presentation on immature DCs results in T cell ignorance but not in tolerance (29). Accumulating evidence indeed indicates that maturation stimuli are required to induce T cell anergy (30).

The type and the level of costimulation received during first encounter with Ag are key determinants in the outcome of immune response. The active costimulatory state of DCs is promoted by activated CD4+ T cells, in particular by interaction between CD40 on DCs and CD40L on T cells (31, 32). It probably explains why CD8+ T cell epitope-laden microspheres are not immunogeneic unless a second signal, like CD40-triggering, is provided. However, the requirement for Th functions in the generation of CTL can be bypassed by licensing DCs (33). Very interestingly, we found that PPV-VLPs can replace this licensing signal in a CD40-independent manner and drive the differentiation of specific T cells toward a type 1 phenotype. It should be emphasized that beads delivering both CD8+ and CD4+ T cell epitopes induce specific CTL responses as well, but these responses are not polarized (13). Therefore, PPV-VLPs do not only have the ability to rescue CD8+ T cell immunity but also the potency to polarize the response, suggesting that PPV-VLPs provide T cells with commitment signals. Previous observations have indicated that PPV-VLPs are efficiently captured by DCs in vivo, and induce a broad range of phenotypic changes associated with DC maturation (15).

On the other hand, we demonstrated that the cellular and the protective responses induced to CD8+ T cell epitope delivered by microspheres in the presence of PPV-VLPs are similar to those induced when the epitope is directly inserted into PPV-VLPs. We have previously determined that ~50–60% spleen DCs are targeted by PPV-VLPs (15) whereas ~4% can engulf beads, meaning overall that ~2% DCs received both the Ags delivered by beads and the conditioning signals. Considering that really few activated DCs are required to induce a potent CTL response, both the PPV-OVAp and the B-OVAp/PPV systems fulfill these requirements. However, we expect that decreasing the dose of PPV-VLPs and B-OVAp would have more dramatic effects on the CD8+ T cell response compared with PPV-OVAp. Consistent with this hypothesis, Cho et al. (34) have shown that DNA rich in nonmethylated CG motifs (CpG) is much more efficient when linked to the Ag that needs to be delivered. Our data also suggest that an efficient targeting of DCs by exogenous Ags is not a prerequisite to induce cross-priming as soon as potent licensing signals could be codelivered. Therefore, the strong immune responses induced by recombinant PPV-VLPs might not be due to their efficient Ag delivery to DCs, but to their ability to provide DCs with maturation signals that are actually sufficient to convert a nonimmunogeneic MHC class I delivery system into a potent vaccine.

PPV-VLPs hold potent adjuvant properties that are not shared by all other VLPs. For instance, CTL responses are not generated by either recombinant hepatitis B core Ag (HBcAg) carrying heterologous CD8 epitopes or hepatitis B surface Ag (HBsAg) in CD4-deficient host, in the absence of further signals such as DNA rich in nonmethylated CG motifs (CpG) (35). The lack of adequate APC activation was thought critical to induce CTL to VLPs (36). Interestingly, purified papillomavirus (PV)-VLPs were also shown to induce acute activation of bone marrow-derived DCs, characterized by up-regulation of costimulatory molecules, secretion of proinflammatory cytokines, and production of IL-12 following T cell encounter (37). In addition, PV-VLPs can bind to human DCs, trigger their maturation, and subsequently activate specific human T cells in vitro (38). However, to our knowledge, the contribution of CD40-CD40L interactions to the CD8+ T cell responses induced by PV-VLPs has not been studied so far. Therefore, we cannot exclude that PV-VLPs and PPV-VLPs share common adjuvant properties that would explain their strong immunogenicity. Many microorganism-derived molecular structures have the ability to stimulate DCs, especially through TLRs expressed on DCs (39). Thus, to understand whether TLR4 and TLR9 play a role in the adjuvant effect generated by PPV-VLPs, we have replaced PPV-VLPs by their ligands, LPS and CpG, respectively. We observed that, in our conditions, none of them were able to induce CTL responses to OVA257–264 carried by microspheres (unpublished results). In addition, our preliminary results also indicate that the adjuvant effect of PPV-VLPs does not require TLR2, TLR4, or TLR9. However, it remains to be determined whether some viral components of the PPV-VLPs do activate DCs through other TLRs, such as TLR3, TLR7, or TLR8.

In conclusion, the results presented here highlight the properties of PPV-VLPs as a strong adjuvant of the T cell response to exogenous Ags. Obviously, such adjuvants could be combined to well defined and reliable delivery systems to trigger protective immunity. Understanding in detail the adjuvant properties of PPV-VLPs is now the next step required to design a new generation of adjuvants aimed at eliciting CTL functions.


   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 a grant from the European Community (FP6 503582, Theravac). Back

2 Address correspondence and reprint requests to Dr. Claude Leclerc, Unité de Biologie des Régulations Immunitaires, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France. E-mail address:cleclerc{at}pasteur.fr Back

3 Abbreviations used in this paper: DC, dendritic cell; VLP, virus-like particle; PPV, porcine parvovirus; SFC, spot-forming cells; wt, wild type; PV, papillomavirus. Back

Received for publication May 5, 2004. Accepted for publication January 7, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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