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The Journal of Immunology, 2007, 179, 5024-5032
Copyright © 2007 by The American Association of Immunologists, Inc.
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Antigen Presentation by Exosomes Released from Peptide-Pulsed Dendritic Cells Is not Suppressed by the Presence of Active CTL1

Lea Luketic*, Jordan Delanghe*, Paul T. Sobol{dagger}, Pingchang Yang*, Erin Frotten*, Karen L. Mossman*, Jack Gauldie*, Jonathan Bramson* and Yonghong Wan2,*

* Department of Pathology and Molecular Medicine and {dagger} Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Despite the potency of dendritic cells (DCs) as a vaccine carrier, they are short-lived and sensitive to CTL-mediated elimination. Thus, it is believed that the longevity of Ag presentation by peptide-pulsed DC is limited in vivo. Surprisingly, however, we found that although the majority of injected DCs disappeared from the draining lymph nodes within 7 days, Ag presentation persisted for at least 14 days following DC immunization. This prolonged Ag presentation was not mediated by the remaining injected DCs or through Ag transfer to endogenous APCs. We provide evidence that exosomes released by DCs might be responsible for the persistence of Ag presentation. Functional exosomes could be recovered from the draining lymph nodes of C57BL/6 mice following DC vaccination and, in contrast to DCs, T cell stimulation by exosomes in vivo was not affected by the presence of CTL. Our findings demonstrate that Ag presentation following delivery of DC vaccines persists for longer than expected and indicate that the exosome may play a previously unrecognized role in Ag presentation following DC vaccination. Furthermore, our study reinforces the application of exosomes as a vaccination platform and suggests that exosome-based vaccines may be advantageous for booster immunizations due to their resistance to CTL.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The CD8+ CTLs are a major effector population in host defense against virus infection and tumor challenge. Thus, there is currently a great effort directed toward the development of vaccines that elicit effective and sustained CTL responses. To gain full effector function, CTL must be primed by "professional" APCs, such as dendritic cells (DCs),3 which can transport and present Ag to naive T cells within secondary lymphoid organs. Primed CTL then identify antigenic peptides presented by MHC class I on virus-infected cells and tumor cells, resulting in cytolysis of the target cell. Thus, using DCs as a vaccine carrier represents a promising immunotherapeutic approach. Indeed, it has been well demonstrated in a variety of animal models that DC pulsed with a defined MHC class I peptide can effectively activate CTL response and elicit protective immunity (1, 2, 3, 4, 5).

However, several reports suggest that the lifespan of DCs in the lymph node (LN) is short (6). They undergo apoptosis shortly after interaction with Ag-specific T cells and disappear from the LN in 2–3 days (7, 8, 9). Although the mechanisms controlling this process are not fully understood, it is hypothesized that a negative feedback process exists in which the DCs become targets of the CTL that they prime (8, 9, 10). Recent studies demonstrated that enhancement of DC survival augmented T cell priming supporting the idea that prolonging Ag presentation by the DC vaccine may be advantageous (11, 12, 13, 14, 15). Furthermore, a report by Medema et al. (16) demonstrated that activation of DCs by CD40L or LPS rendered DCs resistant to the in vitro cytotoxic activity of CTLs. This manipulation provides a potential mechanism to enhance both primary and secondary immune responses by overcoming T cell-mediated DC elimination. However, these results are restricted to an in vitro system and no data from in vivo experiments have confirmed that DCs can be protected from CTL killing.

In the current study, we have examined the longevity of Ag presentation in mice immunized with peptide-pulsed DC. To our surprise, Ag presentation persisted beyond the peak CTL response (day 7). We provide evidence in this study that exosomes, which are small membrane-bound vesicles (50–90 nm) secreted by different cells including DCs (17), may mediate the prolonged Ag presentation. Exosomes derived from DCs contain MHC-peptide complexes and costimulatory molecules, capable of stimulating Ag-specific T cell responses and are currently being considered as a vaccination platform (18). Our observations provide important insight into the biology of DC vaccination and address a novel aspect of exosome vaccination that has important clinical implications, particularly in the context of booster immunizations.


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

Six- to 8-wk-old C57BL/6 (H-2b) female mice were purchased from Charles River Breeding Laboratories. OT-I transgenic mice (expressing a V{alpha}2Vbeta5 TCR specific for the peptide OVA257–264) and P14 transgenic mice (expressing a V{alpha}2Vbeta8 TCR specific for lymphocytic choriomeningitis virus (LCMV)-gp33–41) were bred in the Central Animal Facility, McMaster University (Hamilton, Ontario, Canada) (19, 20). B6.C-H-2bm-1 (bm1) mice were purchased from The Jackson Laboratory. The (bm1 x B6) F1 mice were generated by crossing bm1 mice with the C57BL/6 strain. F10-gp33 is derived from a murine melanoma B16F10 cell line that stably expresses LCMV-gp33 peptide (21). The cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 800 µg/ml G418. All of our investigations have been approved by the McMaster Animal Research Ethics Board.

Peptides and Abs

The OVA257–264 peptide (SIINFEKL) and the LCMV-gp33–41 (KAVYNFATM) were purchased from Dalton Chemical Laboratories. All flow cytometry Abs were purchased from BD Pharmingen. The mAb 25-D1.16 (specific for Kb/SIINFEKL complex) was provided by Yewdell and colleagues (22).

Preparation of DC-based vaccines

Bone marrow cells prepared from C57BL/6 mice or bm1 mice were cultured in the presence of 20 ng/ml recombinant murine GM-CSF as previously described (23). On day 7 of culture, DCs were pulsed for 4 h in culture medium with 500 ng/ml SIINFEKL or KAVYNFATM or mixtures of these. We did not further manipulate cells before injection or exosome preparation.

Exosome preparation

On day 6 of culture, DCs were incubated with the peptides for 4 h as described and then washed three times with PBS to remove excess peptides. DCs were resuspended in fresh culture medium with 10% exosome-free FBS prepared by overnight ultracentrifugation at 100,000 x g, and incubated for an additional day to allow for exosome secretion. DC supernatants were harvested and centrifuged at 300 x g (10 min), 1200 x g (20 min), and 10,000 x g (30 min) to remove cell debris (24). Exosomes were pelleted at 100,000 x g for 1 h and resuspended in PBS for subsequent studies. For some experiments, the exosome pellet was additionally purified by floatation on a sucrose density cushion (1.00–1.30 g/ml), according to methods described by Raposo (24). The exosomes were quantified by protein content using a Bradford assay (Bio-Rad). To prepare DC-derived exosomes (DCex) from the draining LN, C57BL/6 mice were injected with wild-type DCs (DCB6) pulsed with SIINFEKL into the footpads (1 x 106 cells/footpad). Nonpulsed DCB6 or bm1 DCs (DCbm1) pulsed with SIINFEKL were similarly injected in control mice. Three days later, axillary LN and popliteal LN were harvested and digested with collagenase (250 U/ml) for 1 h at 37°C. Exosomes were processed by differential centrifugation as described.

Electron microscopy

DCex were incubated with primary Ab for 30 min on ice and then washed with PBS (three times) to remove unbound Ab. Stained DCex samples were fixed (4% paraformaldehyde and 0.75% glutaraldehyde in 0.1 M cacodylate buffer) for 2 h and resuspended in 0.1 M cacodylate buffer. One drop of the suspension was placed on a Formvar/carbon-coated grid and incubated with a 12 nm gold particle-conjugated anti-IgG (1/30 dilution; Jackson ImmunoResearch Laboratories) for 1 h, followed by 6 10-min washes. Samples were counterstained with 2% aqueous uranyl acetate, followed by 0.2% lead citrate. Particle visualization was performed using a JEOL JEM-1200 EX transmission electron microscopy.

Ag presentation assays

LN were harvested from OT-I mice and CD8+ T cells were positively selected using MACS magnetic beads (Miltenyi Biotec). Purified OT-I CD8+ T cells were cocultured with peptide-pulsed DC or DCex in 96-well plates for 72 h. Cells were pulsed with 0.1 µCi of [3H]thymidine per well for the last 18 h of culture. The amount of incorporated [3H]thymidine was measured by a beta counter. For blocking experiments, 0.2 µg/ml 25-D1.16 mAb or irrelevant IgG1 was added into the cultures.

To measure the presence of Ag in vivo, we monitored the proliferation of OT-I CD8+ T cells, or P14 CD8+ T cells in some experiments, transferred into mice at various times postimmunization with DCs or DCex. Purified TCR transgenic T lymphocytes were labeled with 5 µM CFSE and injected i.v. into immunized syngeneic mice at 1–2 x 106 cells per mouse. Recipients were sacrificed 72 h later and proliferation of transgenic T cells in the draining popliteal LN was determined by dilution of CFSE fluorescence using flow cytometry.

DC migration

Peptide-pulsed DCs were labeled with 5 µM CFSE and injected into the footpads of syngeneic mice at 0.5 x 106 cells per footpad. Alternatively, for studies comparing the recovery rate of two different populations of DCs from the same animal, PKH-26 labeling was used for the second population. PKH-26 staining was performed according to the manufacturer’s instructions (Sigma-Aldrich). Briefly, the DCs were resuspended in 1 ml the PKH diluent and then mixed with 4 µM PKH-26 (for 5 min at room temperature). After incubation, 2 ml of 5% FBS/PBS was added to neutralize staining reaction. For these studies, 0.5 x 106 cells from each population were mixed immediately before injection into each footpad.

Popliteal LN were collected at various times and digested with collagenase (250 U/ml) for 1 h at 37°C. Cells were stained with CD11c Ab and analyzed by flow cytometry to determine the number of migrating DCs.

Western blot

Cell lysates and exosome fractions, purified from the same cell culture supernatants, were resuspended in RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 100 mM sodium orthovanadate, 1 mM PMSF, 2 mM DTT) and 1x protease inhibitor cocktail (Sigma-Aldrich), and incubated for 20 min at 4°C. Protein quantification of clarified extracts was performed with the Bradford assay (Bio-Rad). Twenty microgram equivalents of DC lysate or DCex fractions were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). Although MHC class II Western blots were blocked in 5% BSA, 0.1% Tween 20 Tris-buffered saline (TBST), all other blots were blocked in 5% skim milk TBST. Primary and secondary Ab hybridizations were completed for 1 h at room temperature in blocking solution: rat anti-LAMP-1 (1/500, clone 1D4B; BD Pharmingen), mouse anti-AIP1 (1/500; BD Biosciences), mouse anti-human calnexin (1/1000; BD Biosciences), mouse anti-TSG101 (1/500; BD Biosciences), and rat anti-mouse MHC class II purified from M5 ascites fluid (1/5000). All secondary Abs were conjugated to HRP. Blots were visualized using a chemiluminescence kit (Amersham Biosciences) and developed on Hyperfilm (Kodak).

In vivo CTL

C57BL/6 mice were s.c. immunized with 1 x 106 SIINFEKL-pulsed DC and cytolytic activity against SIINFEKL-pulsed target cells were measured in vivo at various times according to our published method (25).

Induction of Ag-specific immunity by DCex

C57BL/6 mice were immunized s.c. with 1 x 106 SIINFEKL-pulsed DC to establish a SIINFEKL-specific CTL response. Seven days later, mice were immunized via footpad injection with 1 x 106 DC pulsed with both SIINFEKL and KAVYNFATM or ~1 µg exosomes derived from copulsed DCs. Three days after the secondary immunization, KAVYNFATM-specific immune response was determined by proliferation of adoptively transferred P14 CD8+ T cells as described in "Ag presentation assays". Alternatively, mice were challenged with 106 F10-gp33 tumor cells injected s.c. into the flank. Mice were monitored for tumor growth twice a week for 8 wk.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Ag presentation persists beyond the peak of the CTL response following immunization with DC pulsed with SIINFEKL peptide

To understand the relationship between DC persistence and Ag presentation during the activation of CTL, we set out to establish the kinetics of CTL activation and DC clearance following immunization with DC pulsed with SIINFEKL peptide (DC/SIINFEKL). As shown in Fig. 1A, using an in vivo CTL assay we demonstrated that Ag-specific cytotoxicity was observed within the spleen and local LN (data not shown) as early as 3 days following immunization. Cytotoxic activity continued to increase, reaching its peak at day 7 followed by a gradual loss of activity over the subsequent 2 wk. To examine how the survival of Ag-loaded DC was affected by the appearance of activated CTL, we injected equal quantities of CFSE-labeled, nonpulsed and PKH-26-labeled, SIINFEKL-pulsed DC into the footpads (0.5 x 106 cells each/footpad) of C57BL/6 mice to monitor the persistence of injected DCs in the draining popliteal LN over a period of 14 days. DC accumulation in the LN reached a maximum around 24–36 h postinjection, at which time ~2000 injected DCs could be recovered from the popliteal LN (Fig. 1B). By day 3, a reduction in the number of SIINFEKL-pulsed DC was detected and 90% disappeared by day 7, consistent with the peak of the CD8+ T cell response observed in Fig. 1A and other reports (9, 10). Together with the fact that the disappearance of coinjected nonpulsed DC was much slower (Fig. 1B), our results confirmed that CTL played a role in eliminating Ag-presenting DC. However, a small number of Ag-pulsed DC (40 ± 8) were consistently detected until day 11, indicating that some DCs escaped CTL-mediated elimination.


Figure 1
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  		FIGURE 1. Ag presentation persists beyond the peak of CTL response following immunization with DC/SIINFEKL. A, C57BL/6 mice were s.c. immunized with 1 x 106 DC pulsed with SIINFEKL. At different time points, CFSE-labeled target cells were adoptively transferred into mice for an in vivo CTL assay. Spleens were harvested 6 h after target cell transfer and the percentage of Ag-specific killing was determined by flow cytometry. Each time point represents the mean ± SEM for n = 3–5 mice. B, An equal number of CFSE-labeled, nonpulsed, and PKH-26-labeled SIINFEKL-pulsed DC was injected into the footpads (0.5 x 106 cells each/footpad) of C57BL/6 mice. The total number of CFSE+ and PKH-26+ DC in the draining popliteal LN was analyzed at the indicated time points. Values are from pooled LN of n = 3 mice per time point. C–G, Mice were immunized with 5 x 105 DC/SIINFEKL per footpad. CFSE-labeled CD8+ OT-I cells were transferred into the mice 7, 9, 11, 14, or 21 days postimmunization as indicated. As a control, a group of mice were injected with PBS (H). Three days after OT-I transfer, popliteal LN were harvested and OT-I proliferation was assessed by flow cytometry. Data are representative of three experiments.

 
This latter observation prompted us to reassess the durability of Ag presentation during CTL activation. To this end, we transferred CFSE-labeled OT-I CD8+ T cells to mice at various times postimmunization with DCs, and the presence of Ag presentation was determined by proliferation of transferred transgenic T cells (Fig. 1, C–E). Surprisingly, although the majority of Ag-pulsed DC were cleared from the draining LN within 7 days, proliferation of OT-I CD8+ T cells was detected up to 14 days after immunization; 1 wk beyond the peak CTL response (Fig. 1F). No proliferation was observed in mice immunized with unpulsed DC (data not shown) or PBS (Fig. 1H). This result suggested that Ag presentation following DC immunization persisted through a mechanism resistant to CTL-mediated clearance.

Prolonged Ag presentation is not mediated by residual DCs present beyond day 7

Because a small number of Ag-pulsed DC could be detected from the draining LN up to 11 days postinjection, we first sought to determine whether residual DCs from the injection were responsible for prolonged Ag presentation. To address this possibility, we immunized mice with graded doses of DC/SIINFEKL, ranging from 1 x 104 to 1 x 106 cells per mouse, to determine the minimal number of injected DCs required to stimulate naive transgenic T cell proliferation. Consistent with the results in Fig. 1B, an injection of 1 x 106 DCs per mouse resulted in ~1500–2000 DCs in the draining LN after 24 h, the peak time of DC accumulation, and recovery of migrating DCs was observed in a dose-dependent manner at the same time (Fig. 2A). When naive OT-I CD8+ T cells were adoptively transferred into immunized mice, we found that doses of 1–5 x 104 DCs per mouse failed to stimulate T cell proliferation (Fig. 2, B and C), even though >100 DCs could be detected in the LN after injection with 5 x 104 DCs, which was comparable to the number of residual DCs observed between day 7 and 11 following injection of 5 x 105 DCs in Fig. 1B. Therefore, the Ag presentation observed in Fig. 1C does not appear to be a result of residual injected DCs.


Figure 2
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  		FIGURE 2. Dose relationship between number of injected DC and OT-I CD8+ T cell proliferation. C57BL/6 mice (n = 3) were injected in the footpad with varying amounts of CFSE-labeled DC pulsed with SIINFEKL as indicated (on the x-axis (A) and above each histogram). A, One day after injection, CFSE-labeled DCs were enumerated in the draining LN. B–F, CFSE-labeled CD8+ OT-I cells were transferred to mice 1 day after immunization with 104–106 DC, as indicated in each experiment. As a control, a group of mice were injected with PBS (G). Three days after OT-I transfer, popliteal draining LN were harvested and OT-I proliferation was assessed by flow cytometry. The experiment was repeated once with similar results.

 
Peptide-pulsed DC vaccine does not transfer Ag to endogenous APC

Another possibility is that injected peptide-loaded DC may transfer Ag to host APCs following lysis by CTL. To test this hypothesis, we generated bone marrow-derived DC from mice carrying the bm1 mutation of Kb. Previous studies have demonstrated that the SIINFEKL peptide can bind to Kbm1 with relatively high affinity but these complexes are unable to properly engage the TCR of CD8+ T lymphocytes (22). Thus, DCbm1 pulsed with SIINFEKL would not be able to directly activate Ag-specific T cell responses. To avoid the allogenic response against mutated Kb, we injected SIINFEKL-pulsed DC into (bm1 x B6) F1 mice and subsequently measured the proliferation of adoptively transferred OT-I cells and in vivo CTL activity. Whereas DCs from wild-type mice (DCB6) induced OT-I proliferation (Fig. 3A), DCbm1 failed to elicit measurable OT-I responses (Fig. 3B), suggesting that Ag transfer to endogenous APC was not likely the case. This result is consistent with previous reports that only protein- but not peptide-loaded donor cells can transfer Ag to host APCs for cross-priming (22, 26). The lack of Ag transfer by peptide-pulsed DC was further confirmed through an in vivo CTL assay in which no Ag-specific killing was seen in mice immunized with DCbm1 (Fig. 3E). In contrast, SIINFEKL-coated DCB6 stimulated a strong CTL response (Fig. 3D).


Figure 3
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  		FIGURE 3. OT-I proliferation and activation of CTL is not due to Ag transfer to endogenous DC. (C57BL/6 x bm1) F1 mice were immunized in each hind footpad with 5 x 105 SIINFEKL-pulsed DCB6 (A and D) or DCbm1 (B and E) or were given a sham injection of PBS (No DC) (C and F). A–C, OT-I cells were transferred into immunized mice 7 days after DC injection. Three days after OT-I transfer, popliteal draining LN were harvested and OT-I proliferation was assessed by flow cytometry. D–F, CFSE-labeled C57BL/6 splenocytes pulsed with SIINFEKL (specific targets) or KAVYNFATM (nonspecific targets) were adoptively transferred into mice 7 days after DC immunization. The percentage of Ag-specific killing was assessed 18 h later by flow cytometry. Data are representative of two independent experiments.

 
Ag presentation by DCex

Because neither the injected nor the endogenous DCs appeared to be responsible for the prolonged Ag presentation, we investigated other possible mechanisms that could trigger proliferation of naive OT-I cells in our model. Recent studies have demonstrated that Ag-pulsed DC can release small membrane-bound vesicles called exosomes that contain MHC-peptide complexes and costimulatory molecules, and thus possess the necessary ligands to elicit Ag-specific T cell responses (27, 28). Using electron microscopy, we were able to visualize these small particles (50–100 nm) in the supernatant of DCs culture (Fig. 4A). They were stained positive for CD11c, MHC class II, and CD86, reflecting their DC origin (Fig. 4, B–D). More specifically, the Kb/SIINFEKL complexes could be stained with a specific mAb, 25-D1.16 (Fig. 4E), indicating that exosomes indeed carried exogenously loaded Ag. We also confirmed in this study by Western blot analysis that exosome-associated proteins including LAMP-1, Alix/AIP1, TSG101, and MHC class II were abundant in both DC lysates and exosome samples (Fig. 4F) (29, 30, 31). In contrast, calnexin, a protein found in the endoplasmic reticulum (32), was only detectable in DC lysates but essentially absent in the exosome samples indicating that the exosome preparations were not contaminated with other vesicles (Fig. 4F).


Figure 4
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  		FIGURE 4. Characterization of DCex. A–E, Immunoelectron microscopic analysis of DC-specific markers on the exosomal surface. Exosomes were prepared from SIINFEKL-pulsed DC and stained using specific Abs directed against CD11c, I-Ab, CD86, and Kb/SIINFEKL complex (B–E). The specific staining was visualized by 12 nm gold particles (arrows) using a JEOL JEM-1200 EX transmission electron microscopy. No staining was found on exosomes incubated with an isotype-matched negative control Ab (A). One representative experiment of three was shown. F, Western blot analysis of DCs and DCex. DC lysates or their exosomal products were prepared and quantified as described in Materials & Methods. Twenty micrograms of protein were separated on SDS-PAGE and analyzed for the expression of LAMP-1, Alix/AIP1, calnexin, TSG101, and MHC class II. Results are representative of three independent experiments.

 
Exosomes derived from SIINFEKL-pulsed wild-type DCs provoked robust OT-I proliferation confirming that DCex possess a capacity for direct Ag presentation (Fig. 5A). We ruled out the possibility that proliferation might be stimulated by free-peptides carried over from DC supernatants or contaminating endogenous DCs from positive T cell selection (i.e., CD8+ DC) because similarly prepared exosomes from SIINFEKL-pulsed DCbm1 failed to trigger a measurable T cell response (data not shown). Furthermore, T cell proliferation was completely blocked by addition of 25-D1.16 but not an irrelevant control Ab, indicating that engagement of OT-I CD8+ T cells by exosomes was Ag-specific (Fig. 5A). Finally, the ability of DCex to present Ag and stimulate immune responses in vivo was evaluated by immunizing mice with exosomes derived from SIINFEKL-pulsed DC that resulted in strong proliferation of adoptively transferred naive OT-I CD8+ T cells (Fig. 5C).


Figure 5
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  		FIGURE 5. DCex stimulate T cell response in vitro and in vivo. A, Purified OT-I CD8+ T cells (2 x 105 cells per well) were stimulated with DCex/SIINFEKL (0.25 µg per well) in 96-well plate for 3 days and proliferation was determined by [3H]thymidine incorporation. Proliferation was blocked by 25-D1.16 but not control Ab. B–D, C57BL/6 mice received 1 x 106 CFSE-labeled OT-I CD8+ T cells and were subsequently immunized with 106 DC pulsed with SIINFEKL (B) or 1 µg DCex from DC cultures pulsed with SIINFEKL (C). As a control, a group of mice were injected with PBS (D). Three days later, draining LN were harvested and examined for T cell proliferation. One representative mouse of three is shown from each group and the experiment was repeated twice.

 
Ag-carrying DCex can be recovered from the draining LN after DC/SIINFEKL immunization

Although our data and results reported by others have suggested a role of DCex in the generation of CD8+ T cell responses, the evidence that peptide-loaded DC could release exosomes in vivo has not been established. We next evaluated the presence of Ag-specific exosomes in the draining LN following DC immunization. Mice were injected with 1 x 106 SIINFEKL-pulsed or nonpulsed DC per footpad and 3 days later the draining popliteal LN were harvested and processed. The exosomes were purified and characterized using the same methods described for in vitro studies. Examination of the exosome preparations by electron microscopy revealed that exosomes carrying the Kb-SIINFEKL complexes could be found in mice immunized with Ag-pulsed DCB6 (Fig. 6A) but not DCbm1 (data not shown) or unpulsed DCB6 (Fig. 6B). Furthermore, Western blot analysis indicated that in vivo recovered exosomes (Fig. 6C) had a very similar protein composition to exosome fractions prepared in vitro (Fig. 4F), confirming their proposed origin as exosomes and not apoptotic blebs.


Figure 6
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  		FIGURE 6. Ag carrying DCex can be recovered from the draining LN following DC/SIINFEKL immunization. C57BL/6 mice were injected with C57BL/6 DC (DCB6) pulsed with SIINFEKL in the footpads (1 x 106 DC/footpad). Nonpulsed DCB6 or bm1 DC (DCbm1) pulsed with SIINFEKL were similarly injected in control mice. Three days later, popliteal LN were harvested and pooled for the preparation of exosomes and DCs. A–B, Purified exosomes from mice immunized with SIINFEKL-pulsed (A) or unpulsed (B) DCs were stained with the mAb 25-D1.16 for Kb/SIINFEKL expression and visualized by 12 nm gold particles (arrows) using electron microscopy. One representative of two experiments was shown. C, Western blot analysis of in vivo recovered DCs and exosomes. The DC and exosome fractions were analyzed for the expression of LAMP-1, Alix/AIP1, calnexin, TSG101, and MHC class II as described in Fig. 4. D, Proliferation of OT-I CD8+ T cells stimulated by the exosome and DC fractions was evaluated by [3H]thymidine incorporation. The mAb 25-D1.16 was used to determine the specificity of Ag presentation by exosomes and DCs. Results are shown as mean ± SEM of three mice per group.

 
To assess the ability of in vivo recovered exosomes to stimulate T cells, OT-I CD8+ T lymphocytes were incubated with either LN-derived exosomes (10 µg/well) or DCs (1/100 ratio) purified from the same LN. Fig. 6D demonstrates that both CD11c+ DCs and exosomes from mice that received peptide-pulsed wild-type DCB6 were able to stimulate OT-I proliferation, whereas neither component prepared from mice injected with peptide-pulsed DCbm1 or nonpulsed DCB6 elicited any responses. Furthermore, proliferation of OT-I T cells elicited by exosomes prepared from the LN was completely abrogated after addition of the Kb-SIINFEKL specific blocking Ab, confirming that stimulation was Ag-specific (Fig. 6D).

Pre-existing CTL affects DC- but not DCex-mediated Ag presentation

Because exosomes lack a nucleus and do not appear to be metabolically active, they would not be expected to be sensitive to either perforin- or fas-mediated lysis. In that regard, the release of Ag-loaded exosomes following injection of DCs may explain the observation of prolonged Ag presentation shown in Fig. 1C. To demonstrate that Ag presentation by exosomes is resistant to CTL-mediated clearance, we pulsed DCs with two different peptides: SIINFEKL (presented on Kb) and KAVYNFATM (LCMV-gp33 peptide presented on Db). Using this model, we can monitor Ag presentation to P14 CD8+ T cells (responsive to KAVYNFATM) in the presence of SIINFEKL-specific CTL. To verify that DCs and exosomes pulsed with two peptides were able to present both antigenic epitopes, CFSE-labeled OT-I or CFSE-labeled P14 transgenic T cells were adoptively transferred into mice concomitant with DCs or exosome injection. DCs and DCex were both able to stimulate OT-I and P14 CD8+ T cell proliferation confirming that both epitopes are presented using this approach (Fig. 7 A–D). When DC pulsed with KAVYNFATM and SIINFEKL were injected into mice that had been immunized with DC/SIINFEKL 7 days earlier, no P14 CD8+ T cell proliferation was observed (Fig. 7E), consistent with previous observations by Hermans et al. (9). To directly examine the impact of pre-existing CTL on DC survival, we injected equal numbers of DC/SIINFEKL, labeled with PKH-26 and nonpulsed DC, labeled with CFSE into naive mice or mice preimmunized with DC/SIINFEKL for 7 days. Two days later, the recovery of injected DCs from the draining LN was analyzed. As shown in Fig. 7H, both Ag-pulsed and nonpulsed DC reached the local LN with the same efficiency in naive animals, whereas the recovered Ag-loaded DCs were only 10% of the number of nonpulsed DC in preimmunized mice, indicating that DC are specifically eliminated by pre-existing CTL. In contrast, P14 CD8+ T cell stimulation by exosomes from DC pulsed with both peptides was not affected by pre-existing immunity (Fig. 7F). This result supports the notion that Ag presentation by exosomes is not influenced by the presence of activated CTL.


Figure 7
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  		FIGURE 7. Ag presentation by DCex is not affected by pre-existing CTL. A–D, C57BL/6 mice (three mice per group) were injected with CFSE-labeled OT-I (A and C) or P14 (B and D) CD8+ T cells and subsequently immunized with 106 DC pulsed with SIINFEKL and KAVYNFATM (A and B) or 1 µg of exosomes derived from similarly pulsed DC (C and D). Three days later, popliteal draining LN were harvested and examined for proliferation by flow cytometry. E–G, C57BL/6 mice were immunized with 106 DC pulsed with SIINFEKL. Seven days later, mice received a secondary immunization with DCs (E) or exosomes (F) carrying both SIINFEKL and KAVYNFATM peptides. Control mice received no secondary immunization (G). Draining LN were harvested 3 days after secondary immunization and P14 T cell proliferation was assessed by flow cytometry. H, C57BL/6 mice were immunized with 106 DC pulsed with SIINFEKL or left untreated. Seven days later, mice received equal numbers of CFSE-labeled, nonpulsed, and PKH-26-labeled, SIINFEKL-pulsed DC through footpad injection (0.5 x 106 cells each/footpad). One day later, cell suspensions from the draining popliteal LN were prepared and the recovery rate of coinjected DCs was determined by FACS. The average proportion of recovered SIINFEKL-pulsed DC (PKH-26+) was presented as a percentage of nonpulsed DC (CFSE+) + SD for three mice from each group.

 
To determine whether T cells activated by exosomes become functional effectors, we examined the antitumor activity of CD8+ T cells elicited by exosomes. For these studies, we examined the response of endogenous polyclonal T cells rather than T cells from TCR transgenic mice. Mice were first immunized with DC loaded with SIINFEKL, to induce SIINFEKL-specific CTL responses, and 7 days later mice were immunized with DCs or exosomes pulsed with KAVYNFATM and SIINFEKL. One week after the second immunization, mice were challenged with a murine melanoma cell line (B16F10) expressing gp33 (F10-gp33) and tumor growth was monitored. Consistent with proliferation data, DC pulsed with both peptides were unable to elicit protective immunity in the presence of SIINFEKL-specific CTL, whereas exosomes were able to elicit protective immunity in 80% of the mice under the same conditions (Fig. 8A). The fact that DC pulsed with KAVYNFATM alone (Fig. 8A) could induce a protective response ruled out the possibility that pre-existing CTL may affect subsequent DC immunization in a nonspecific manner. Furthermore, DC pulsed with two peptides were able to generate antitumor protective immunity in animals without preimmunization confirming the quality of DC vaccine preparation (Fig. 8B). No tumor protection was observed using DCs or DCex alone (without loaded peptide) either in preimmunized or nonimmunized mice (Fig. 8B). These data demonstrate clearly that DCs are sensitive to CTL-mediated elimination but exosomes can effectively present Ag to naive T cells even in the presence of CTL.


Figure 8
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  		FIGURE 8. Exosomes but not DCs can elicit antitumor immunity in the presence of activated CTL. C57BL/6 mice were immunized with 106 DC pulsed with SIINFEKL (A) or left untreated (B). Seven days later, mice received a secondary immunization with DCs or exosomes carrying both SIINFEKL and KAVYNFATM peptides. One week after secondary immunization, tumor challenge was performed using F10-gp33 (1 x 106 cells) via s.c. injection on the right flank. Results are representative of two experiments with five mice for each group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
DCs have been intensively studied as a vaccine platform in preclinical models and clinical trials (5). Although the mechanisms that regulate the ability of DCs to stimulate naive T cells are still being elucidated, a number of studies support the concept of a negative feedback model in which recently activated CTL clear the Ag-loaded DC (33, 34). In this study, we have observed that although the majority of the inoculated DCs are cleared from the draining LN within a few days, a small number of DCs persist until at least day 9. Most interestingly, Ag presentation following DC immunization persists for at least 1 wk following the peak of the CTL response. Further examination revealed that this prolonged Ag presentation was not due to the residual DCs or transfer of Ag to endogenous APC. Thus, we considered alternate explanations for the prolonged Ag presentation despite the presence of activated CTL.

Exosomes are small vesicles secreted by multiple cell types including DCs, B cells, and tumor cells (17, 24, 35). Exosomes derived from DCs carry many of the surface markers found on DCs, such as MHC and costimulatory molecules, and have been shown to possess the ability to activate naive T cells (36, 37, 38). Because exosomes are merely vesicles, they should not be affected by the presence of activated CTL. Based on this understanding, we reasoned that exosomes may explain the prolonged Ag presentation observed in our model. Indeed, DCex were able to activate Ag-specific CD8+ T cells in vitro and in vivo and we have demonstrated that functional exosomes can be recovered in the draining LN following DC immunization. Furthermore, our data demonstrate that exosomes can stimulate naive T cells in the presence of established CTL against another Ag that is simultaneously presented on the same exosome particles, in contrast to Ag-bearing DCs, which are susceptible to CTL-mediated elimination. These data support the hypothesis that exosomes secreted by DCs may mediate Ag presentation after peptide-bearing DCs are cleared from the system.

The mechanisms by which exosomes activate naive T cells remain to be fully elucidated. Some studies show that exosomes or exosome-like membrane vesicles from APC can stimulate T cell clones or naive T cells on their own (24, 38, 39, 40), whereas other reports suggest that bystander DCs are required to increase the immunogenicity of exosomes, especially in eliciting CD4+ T cell response (28, 38, 41, 42). To minimize the possible contamination of DCs (i.e., CD8+ DC) in the in vitro proliferation assays, we performed negative selection of OT-I CD8+ T cells that were stimulated by SIINFEKL-carrying exosomes. No difference was observed between positive and negative selections (data not shown) supporting the idea that exosomes are able to directly stimulate T cells at least under culture conditions. It is likely that both host DC-dependent and -independent pathways coexist in vivo. Several studies have shown that exogenously transferred exosomes can be processed by host DC and presented again in the context of endogenous MHC molecules (35, 43, 44). However, it has also been observed that exosomes at the surface of recipient DC can be recognized by T cells without a need for Ag reprocessing, though the function of DCs in this scenario is not known (42, 45). In that regard, we cannot rule out the possibility that exosomes may also be presented by T cells themselves as demonstrated in other studies in which T cells are able to present Ags by obtaining membrane fragments from APCs (46, 47, 48). It is possible that DCs or T cells provide bystander help or simply serve as a carrier to enhance the contact of T cells with exosomes (41, 47). However, because endogenous cells that take up the exosomes in our model would be susceptible to CTL lysis, we believe that our results are consistent with the concept that exosomes can directly present Ag to naive T cells. Alternatively, the exosomes may be recycled between host cells and thereby amplify or prolong Ag presentation.

Our data indicate that actual stimulation of T cells following immunization with DC-based vaccines is longer than previously interpreted and this process may involve Ag presentation by both the inoculated DC and DCex. Particularly, these data offer a mechanism by which Ag presentation can persist in the presence of robust CTL. At present, however, the impact of exosome-mediated Ag presentation on the outcome of T cell responses remains elusive because the requisite duration of antigenic stimulation for optimal CD8+ T cell function is unclear and controversial results have been reported in model systems. Using an in vitro culture system with transgenic T cells, van Stipdonk et al. (49) demonstrated that only 20 h of stimulation is required to induce CD8+ T cells to acquire full effector function, whereas Storni et al. (11) showed that Ag had to be presented for several days by activated APCs to trigger protective T cell responses in nontransgenic mice. These observations suggest that the induction of effective T cell immunity under physiological conditions may require Ag presentation for an extended period of time and together with our data, exosomes released by Ag-carrying DCs may play a role in this process. Current studies in our laboratory are focused on the development of a model in which we can control the survival of injected DCs to dissect DC- and DCex-mediated Ag presentation in vivo.

In addition to the primary immune response, resistance of exosomes to CTL lysis is likely to have a much greater impact on secondary immune responses. Vaccines designed to elicit cellular immunity typically require repeated vaccination to generate a protective number of memory T cells. Previous studies have demonstrated that the efficacy of readministered DCs is severely reduced by the presence of established CTL (34, 50). Thus, using exosomes as vaccine carrier may provide a novel platform for booster immunizations.


   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 grants from the Canadian Institutes of Health Research and the Ontario Cancer Research Network. Y.W. is a recipient of a Canadian Institutes of Health Research Scholarship. J.L.B. is supported by an Rx&D Health Research Foundation/Canadian Institutes of Health Research Career Award in Health Research. L.L. was supported by a Canada Institutes of Health Research Master’s Award. Back

2 Address correspondence and reprint requests to Dr. Yonghong Wan, Department of Pathology and Molecular Medicine, 5024 Michael DeGroote Centre for Learning & Discovery, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada Back

3 Abbreviations used in this paper: DC, dendritic cell; DCex, DC-derived exosome; LN, lymph node; LCMV, lymphocytic choriomeningitis virus. Back

Received for publication July 12, 2007. Accepted for publication August 8, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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