Antigen-loaded exosomes alone induce Th1-type memory through a B-cell-dependent mechanism

doi:10.1182/blood-2008-04-153536Prepublished online January 27, 2009;2009 113: 2673-2683   

 GabrielssonKhaleda Rahman Qazi, Ulf Gehrmann, Emilie Domange Jordö, Mikael C. I. Karlsson and Susanne 

dependent mechanism−cellAntigen-loaded exosomes alone induce Th1-type memory through a B

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IMMUNOBIOLOGY

Antigen-loaded exosomes alone induce Th1-type memory through aB cell–dependent mechanismKhaleda Rahman Qazi,1 Ulf Gehrmann,1 Emilie Domange Jordo,1 Mikael C. I. Karlsson,1 and Susanne Gabrielsson1

1Department of Medicine, Clinical Allergy Research Unit, Karolinska University Hospital Solna, Stockholm, Sweden

Exosomes are nanovesicles harboringproteins important for antigen presenta-tion. We compared the potency of differ-ently loaded exosomes, directly loadedwith OVA323-339 peptide (Pep-Exo) or exo-somes from OVA-pulsed DCs (OVA-Exo),for their ability to induce specific T-cellproliferation in vitro and in vivo. BothPep-Exo and OVA-Exo elicited specifictransgenic T-cell proliferation in vitro, withthe Pep-Exo being more efficient. In con-trast, only OVA-Exo induced specific T-cell responses in vivo highlighting the

importance of indirect loading strategiesin clinical applications. Coadministrationof whole OVA overcame the unresponsive-ness with Pep-Exo but still elicited alower response compared with OVA-Exo.In parallel, we found that OVA-Exo notonly augmented the specific T-cell re-sponse but also gave a Th1-type shift andan antibody response even in the ab-sence of whole OVA. We detected IgG2aand interferon-� production from spleno-cytes showing the capability of exo-somes to provide antigen for B-cell activa-

tion. Furthermore, we found that B cellsare needed for exosomal T-cell stimula-tion because Bruton tyrosine kinase–deficient mice showed abrogated B- andT-cell responses after OVA-Exo immuniza-tion. These findings reveal that exosomesare potent immune regulators and arerelevant for the design of vaccine adju-vants and therapeutic intervention strate-gies to modulate immune responses.(Blood. 2009;113:2673-2683)

Introduction

Dendritic cells (DCs) are professional antigen-presenting cells thatregulate the induction and outcome of the immune response. DCsprocess exogenous antigens in the endosomal compartment and multive-sicular bodies are formed, which contain vesicles with peptide/majorhistocompatibility complex class II (MHC II) complexes on theirsurface.1,2 These small vesicles are known as exosomes when they aresecreted from the cells. Exosomes are actively secreted by a diverserange of cells, and especially DC-derived exosomes have acquiredmuch attention because they harbor all the necessary molecules requiredfor the activation of potent immune responses, for example, MHC I andMHC II, CD54, CD80, and CD86, on their surface.2 The initialinvestigations have shown the biologic significance of exosomes indifferent areas of research, such as tumor and transplantation immunol-ogy,3-7 vaccine therapy against infection,8-10 as well as a biomarker fordiagnostic purposes.11 Exosome-based tumor vaccines have recentlybeen tested in phase 1 clinical trials in melanoma, nonsmall cell lungcancer, and colorectal cancer patients.12-14 To fully understand thepotential of exosome-based immunotherapy, there is a need to furtherexplore the fundamental mechanisms of exosome-mediated immunestimulation and regulation. Several studies have demonstrated thepotency of antigen-pulsed DC-derived exosomes (indirectly loadedexosomes) to elicit in vitro and in vivo antigen-specific activation ofT cells.15-17 Hsu et al20 developed a direct peptide-loading method onexosomes and have shown a superiority of the direct peptide loadingover indirect loading regarding exosome immunogenicity in vitro;however, they were not compared in vivo.

Based on the studies that antigen-loaded exosomes can serve toamplify DC function and stimulate T cells, we aimed to evaluate andcompare the potency of both directly and indirectly antigen-loadedexosomes for their ability to stimulate antigen-specific T cells in vitro

and in vivo. As model antigens, we used native ovalbumin (OVA) andthe OVA323-339 peptide to load indirectly or directly on exosomes,respectively, and T cells from the OVAT-cell receptor (TCR) transgenic(Tg) mouse DO11.10 were used. We demonstrate that both directly andindirectly loaded exosomes can induce the proliferation of transgenicT cells in vitro. However, on the contrary, only indirectly loaded, but notdirectly loaded, exosomes elicited T-cell proliferative responses in vivo.Coadministration of the whole OVA overcame the unresponsivenesswith the directly loaded exosomes in wild-type mice, indicating that B-and T-cell collaboration is crucial for exosome-mediated specificimmune activation in vivo. Interestingly, indirectly loaded exosomesexerted their effect in the absence of whole OVA and modulated thespecific response toward Th1 type, which may imply the presence ofrecycled antigen on indirectly loaded exosomes. The need for B-cellhelp for T-cell stimulation was verified in Bruton tyrosine kinase (Btk)knockout (KO) mice, which show decreased antibody production andsplenocyte proliferation in response to OVA-Exo. Our results suggest anadditional role for indirectly loaded exosomes in the presentation of notonly MHC II/pep but also native antigen to B cells, thus assistingactivation of B cells, which in turn stimulates the efficient priming ofantigen-specific T cells.

Methods

Mice

BALB/c and DO11.10 OVA TCR transgenic mice (The Jackson Laboratory,Bar Harbor, ME)18 were kept and bred at the animal facility at theDepartment of Microbiology, Tumor and Cell Biology (MTC; KarolinskaInstitutet, Stockholm, Sweden). Age- and sex-matched mice of 6 to 8 weeks

SubmittedApril 25, 2008; accepted January 15, 2009. Prepublished online as BloodFirst Edition paper, January 27, 2009; DOI 10.1182/blood-2008-04-153536.

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were used for experiments. Btk KO mice were kindly provided by EdvardSmith (Clinical Research Center, Karolinska Institutet). Btk KO mice wereon a C57BL/6 background and hence were used as wild-type controls inthese experiments. All animal experiments were approved by the EthicsCommittee at Karolinska University Hospital Solna.

Generation of BMDCs

Bone marrow–derived dendritic cells (BMDCs) were generated as describedpreviously with some modifications.19 Bone marrow cells were cultured incomplete RPMI 1640 medium (Invitrogen, Carlsbad, CA; 10% exosome-depleted fetal calf serum, 1 mM sodium pyruvate, 100 IU/mL penicillinstreptomycin, 200 mM L-glutamine, 50 �M �-mercaptoethanol) in the presenceof 10 ng/mL interleukin-4 (IL-4; Invitrogen) and 10% granulocyte macrophagecolony-stimulating factor conditioned medium (Ag8653/X63 clone, a kind giftfrom Mattias Svensson, Center for Infectious Medicine, Karolinska Institutet).Atday 6, 50% of the culture supernatant was replaced with fresh medium. Formaturation, lipopolysaccharide (LPS; Sigma-Aldrich, St Louis, MO) or inter-feron-� (IFN-�; Invitrogen) at 30 ng/mL was added on day 6 of culture followedby 48 hours of incubation, and the supernatant was collected and kept at �80°C.

Preparation of exosomes from DC culture supernatants

Exosomes were isolated by differential ultracentrifugation (BeckmanCoulter, Fullerton, CA) as described previously1 with modifications. Thesupernatants were subjected to centrifugation at 3000g, followed by10 000g for 30 minutes. Exosomes were pelleted at 100 000g for 2 hoursand washed at 100 000g. Pelleted exosomes were dissolved in phosphate-buffered saline (PBS). The protein contents were measured by a DC proteinassay (Bio-Rad, Hercules, CA).

Sucrose gradient

Exosomes were layered on a linear sucrose gradient (0.25-2 mM sucroseand 20 mM N-2-hydroxyethylpiperazine-N�-2-ethanesulfonic acid/NaOH,pH 7.4; Sigma-Aldrich).1 The gradients were centrifuged for 21 hours at79 000g at 4°C. Eighteen fractions were collected, and the density wasdetermined by refraction index measurements.

Phenotypic analysis of exosomes by FACS

A total of 30 �g exosomes was incubated with 10 �L aldehyde/sulfate latexbeads (Invitrogen) and rotated overnight at room temperature. The reactionwas stopped by 1 mL of 100 mM glycine (Sigma-Aldrich). Beads withexosomes were labeled with a panel of fluorescein isothiocyanate (FITC)–or phycoerythrin (PE)–conjugated antibodies specific for H-2Kd, CD9,CD54, CD80, CD81, and CD86 (BD Biosciences, San Jose, CA) and thecorresponding isotype-matched antibodies.

Western blot analysis

Thirty micrograms of OVA-Exo, Exo, or 1 �g OVA was loaded on 8% to16% Tris-HCl gel (Bio-Rad) and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins were transferred to a polyvi-nylidene difluoride membrane (Millipore, Billerica, MA) using semidryblotting. OVA was detected using immune sera from OVA-alum sensitizedmice (1:500 diluted). Bands were detected using alkaline phosphatase(ALP)–conjugated anti–mouse IgG (Southern Biotechnology, Birmingham,AL; 1:2000 diluted) and ALP substrate kit (Bio-Rad).

Direct and indirect loading of OVA on exosomes

Direct loading of exosomes with OVA peptide (ISQAVHAAHAEINEAGR;OVA323-339; Innovagen, Lund, Sweden) was done using the acid elution methodas described.20 Exosomes were mixed with 0.2 M sodium acetate at pH 5.2 andwith OVA323-339 peptide at the concentration 10 �g/mL. The mixture wasneutralized to pH 7.0 with 2 M Tris-HCl (2.6% of total volume; Bio-Rad) of pH11 and incubated at room temperature to allow reassembly of MHC II/peptide onexosomes. Unbound peptides were removed by filtering through 100-kDaUltrafree Biomax filters (Millipore). Directly loaded exosomes were termedPep-Exo. The same amount of OVA323-339 peptide as loaded on exosomes was

filtrated in parallel and the fraction above the filter was used as control for theremoval of free peptides and was termed pep-cont.

For indirect loading, OVA (Serotec, Oxford, United Kingdom) orbovine serum albumin (BSA; Sigma-Aldrich; 300 �g/mL) proteins wereadded to DC cultures at day 6, for overnight followed by washing once andthen LPS was added to the culture. After 48 hours, exosomes were purified.Indirectly loaded exosomes were termed OVA-Exo or BSA-Exo.

DO11.10 CD4� T-cell isolation and in vitro T-cell proliferationassay

DO11.10 CD4� T cells from spleen were isolated by positive selection onanti-CD4 beads by magnetic-activated cell sorter (Miltenyi Biotec, AuburnCA) according to the manufacturer’s instructions. Purity was checked byfluorescence-activated cell sorter (FACS). To assess T-cell proliferation invitro, magnetic-activated cell sorter–purified CD4� DO11.10 T cells wereincubated with 5 �M carboxy fluoroscein succinimidyl ester (CFSE;Invitrogen) for 10 minutes at room temperature. Labeling was stopped byadding cold PBS/10% fetal calf serum. Cells were then washed 3 times inPBS and cocultured at a concentration of 106 cells/mL with differentconcentrations of Pep-Exo, OVA-Exo, and the respective controls followedby incubation at 37°C in a humid incubator with 5% CO2 for 5 days.

In vivo T-cell proliferation assay

Purified DO11.10 CD4� T cells were adoptively transferred to BALB/cmice intravenously with 5.5 � 106 cells/ mouse at day 0. On day 1, micewere immunized intravenously with Pep-Exo, OVA-Exo, or with respectivecontrols. On day 4, mice were killed and splenocytes were stained withanti–CD3-allophycocyanin together with anti–KJ1-26�-FITC antibodiesspecific for OVA TCR and the number of KJ1-26� cells assessed by FACS.In some experiments, DO11.10 splenocytes were labeled with CFSE andadoptively transferred to BALB/c mice before exosome injection. Lympho-cyte early activation was checked using biotinylated anti-CD69 anddetected using streptavidin-PE by FACS.

Immunohistochemistry

Spleens from the adoptively transferred mice were frozen in OCT medium(Sakura Finetek, Zoeterwoude, The Netherlands) and 8-�m-thin sections werecut in a cryostat microtome. After overnight drying, the slides were fixed inacetone, blocked with 5% goat serum (Dako North America, Carpinteria, CA)together with avidin and followed by biotin (Vector Laboratories, Burlingame,CA). The following antibodies were used: biotinylated KJ1-26, FITC-conjugatedanti-B220 and CD11c, allophycocyanin-conjugated anti-B220 (BD PharMingen,San Diego, CA), streptavidin-Qdot605 (Invitrogen/Molecular Probes, Eugene,OR), and biotinylated anti–FDC-M2 (ImmunoKontact, Abingdon, UnitedKingdom). Images were collected using a Leica DM IRBE confocal laserscanning microscope (Leica Microsystems, Heidelberg, Germany) equippedwith 1 argon and 2 HeNe lasers, using an HC PL APO lens at 20�/0.70 IMMCORR and 100�/1.40-0.7 oil and 90% glycerol (MP Biomedicals, Solon, OH).Images were processed with Adobe Photoshop CS3 Extended 10.0.1 (AdobeSystems, San Jose, CA).

Immunization of mice

Female BALB/c mice, or where indicated, C57BL/6 or Btk mice, wereprimed intravenously with 50 �g OVA/mouse formulated with 50 �g/mouse of different exosome preparations in 100 �L PBS. Priming was doneonce with loaded exosomes, followed by boosting with 50 �g OVA/mouse4 weeks later. Animals were bled from the tail vein 7 days after primaryimmunization and 7 days after boosting.

Determination of serum antibody levels by ELISA

To determine specific antibody responses, microtiter plates (Costar, Corning,Corning, NY) were coated with 10 �g/mL OVA protein. Plates were thenincubated overnight at room temperature followed by incubation overnight withserial dilutions of sera. Isotypes of the reactive antibodies were determined byincubating for 2 hours at room temperature with alkaline phosphatase–

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conjugated goat immunoglobulin specific for mouse � and � isotypes andsubclasses (Southern Biotechnology). Development was done at room tempera-ture with p-nitrophenyl phosphate disodium (Sigma-Aldrich), and the absorbancewas measured at 405 nm at different time points by an enzyme-linkedimmunosorbent assay (ELISA) reader.

Proliferation and Th1/Th2 cytokine assay

Supernatants, obtained from the 48-hour cocultures of immunized spleno-cytes with in vitro stimuli, were analyzed for proliferation by thymidineincorporation and for T-helper cytokine production using the Th1/Th2 CBAassay kit measuring IL-2, IL-4, IL-5, IFN-�, and TNF-� in a single sample.The results were analyzed using the BD Biosciences CBA analysissoftware.

Statistical analyses

Results were expressed either as individual data and mean or as mean plusor minus SEM from individual mice from each group. NonparametricMann-Whitney U test was performed to identify significant differencesbetween experimental groups.

Results

Characterization of exosomes derived from immature andmatured DCs

We compared differently matured (LPS- or IFN-�–matured) orimmatured BMDC exosomes by FACS. We obtained approxi-

mately 2 �g exosomes/million cells from immature DCs and0.5 to 1 �g/million cells for LPS- or IFN-�–matured DCs.Exosomes from both immature and LPS- or IFN-�–maturedDCs displayed the tetraspanins CD9 and CD81, the costimula-tory molecule CD80, as well as MHC II and CD54 (Figure 1A).However, LPS and IFN-�–matured DC exosomes exhibitedhigher expression of CD80 and CD81 on their surface, whereasMHC II and CD86 were more highly expressed on LPS-maturedDC exosomes compared with the other types (Figure 1A).

To further verify the exosomal nature of BMDC exosomes,exosome preparations were layered on a continuous sucrosegradient, and the distribution of the MHC II and CD81 over thegradient fractions was analyzed by direct coating of eachfraction on the latex beads and visualized by flow cytometry.21

As shown in Figure 1B, MHC II and CD81 molecules from eachexosome preparation were distributed in fractions with densitiesbetween 1.12 and 1.15 g/mL, indicating their exosomal nature.

OVA-loaded exosomes stimulate DO11.10 T-cell proliferationin vitro

MHC on exosomes can be directly loaded with peptides in a mildlyacidic condition, which generates higher numbers of MHC/peptidecomplexes.20 We compared the efficiency of directly and indirectlyOVA-loaded exosomes for their ability to elicit OVA-specific T-cellresponses in vitro, in a TCR Tg mouse model (DO11.10). Weevaluated the proliferative responses of CFSE-labeled DO11.10

IFN-γγmatured

LPS matured

Immature

MHCII CD9 CD54 CD80 CD81 CD86A

CD81

MHC II

B

Figure 1. Exosomes from LPS and IFN-�–matured BMDCs display higher expression of MHC II and CD80 molecules on their surface than exosomes from immatureBMDCs. (A) Exosomes derived from immature, LPS, or IFN-�–matured BMDCs were coated on aldehyde/sulfate latex beads, stained with a panel of PE- andFITC-conjugated specific (open histogram) and isotype-matched (solid histogram) antibodies, and then analyzed by flow cytometry. One representative experiment of 3 isdisplayed. (B) BMDC-derived exosomes have similar densities as previously reported for exosomes. Pelleted (100 000g) exosomes from immature, LPS, or IFN-�–maturedBMDCs were loaded on continuous sucrose density gradients and ultracentrifuged. Fractions were collected and directly analyzed by flow cytometry after coating on latexbeads and staining with fluorochrome-conjugated antibodies against MHC II (grid histogram) and CD81 (open histogram).

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T cells induced by exosomes, indirectly loaded (OVA-Exo) ordirectly loaded with the OVA peptide (Pep-Exo) by detecting theDO11.10 TCR with the mAb KJ1-26. KJ1-26 is a monoclonalantibody that specifically recognizes DO11.10 Tg TCR. Weassessed the potency of directly loaded, differently matured (LPSor IFN-�) exosomes to stimulate T-cell proliferation by CFSEstaining. Pep-Exo could stimulate the expansion of DO11.10 CD4�

T cells in a dose-dependent manner (Figure 2A). Similarly, as seenby us and others,22-24 LPS-matured Pep-Exo was 8 times morepotent in stimulating OVA-specific T cells compared with imma-ture Pep-Exo. In addition, IFN-�–stimulated exosomes were morepotent than exosomes from immature DCs but only approximately2 times more potent than immature DC exosomes (Figure 2A).Peptide control (pep-cont) failed to trigger a measurable T-cellresponse (Figure 2A). OVA-Exo also induced proliferation ofDO11.10 T cells in a dose-dependent manner, which was signifi-cantly higher than the response achieved with unloaded LPS-matured exosomes (Exo) or exosomes indirectly loaded with acontrol protein BSA (BSA-Exo; Figure 2B). However, it isnoteworthy that 75% of DO11.10 T cells proliferated in response tothe highest concentration (10 �g/mL) of LPS-matured Pep-Exoadded to the culture (Figure 2A), whereas only 11% of OVA323-339-specific cell populations were expanded in response to the sameconcentration of OVA-Exo (Figure 2B). Polymyxin treatment didnot affect the proliferative response (not shown). LPS content ofeach exosome preparation was determined, the correspondingamount of LPS was added to the culture, and this gave undetectableresponse (not shown). Because LPS-matured BMDC exosomeselicited stronger T-cell activation, we used LPS-matured exosomesin the following experiments.

OVA-loaded exosomes enhance the proliferation of DO11.10T cells in vivo

To compare the different loading methods in vivo, BALB/c mice wereadoptively transferred with DO11.10 Tg CD4� T cells and immunizedintravenously with differently loaded or unloaded exosomes (Figure3A). The percentage of OVA-specific T cells in the spleens wasdetermined as CD3�/KJ1-26� cells by FACS 3 days later. As shown inFigure 3B and D, OVA-Exo potently stimulated the proliferation ofOVA-specific DO11.10 T cells as evidenced by an 8- to 10-fold increase

in the percentage of KJ1-26� cells (1.64% 0.14%) in thespleen compared with the PBS (0.20% 0.018%) or unloadedexosome (0.16% 0.028%) treated mice, respectively. Surpris-ingly, only a trace population (0.22% 0.027%) of KJ1-26�

cells was observed for the Pep-Exo–immunized group, whichcorresponded to the values obtained with PBS or unloadedexosome control (Figure 3B,D). This suggests that an additionalfactor is needed for the T-cell proliferative response in vivo.

Although the early activation marker CD69 expression wasobserved on DO11.10 spleen cells from both Pep-Exo– andOVA-Exo–treated groups, the expression of CD69 was signifi-cantly higher in the spleens of the OVA-Exo group (Figure 3C). Nosignificant differences in the CD69 expression could be detected inthe splenocytes between mice injected with unloaded exosomes orPBS (Figure 3C).

To visualize T-cell expansion, spleens from the adoptively trans-ferred, exosome-treated mice were sectioned and stained for OVA-specific T cells (KJ1-26 antibody) and for B cells (anti-B220). Thenumber of OVA-specific T cells in the T-cell zone remained low in micegiven PBS or unloaded exosomes (Figure 3D), whereas the number wasmarkedly increased in the mouse group treated with OVA-Exo. Nochange in the number of the specific T cells was observed for thePep-Exo–treated group (Figure 3E). To further verify that the KJ1-26�

cells had proliferated in response to OVA-Exo, we also adoptivelytransferred CFSE-labeled splenocytes from DO11.10 mice to wild-typemice followed by OVA-Exo injection. Cells stained for KJ1-26 hadproliferated 4 to 8 times at day 4 after OVA-Exo injection (Figure 3F).We also stained the spleen sections of OVA-Exo–treated mice withantibody against the DC marker CD11c together with the B- (B220) andT- (KJ1-26�) cell markers to be able to observe the interaction betweenthese cell types on exosome treatment. We observed that both B andT cells were in close proximity with the DCs (Figure 3G). Thisobservation indicates the involvement of B cells and DCs in the earlyevents in exosome-mediated stimulation of T cells.

OVA-loaded exosomes have the potency to prime the immunesystem to induce OVA-specific antibody responses innaive mice

To test whether B-cell help is needed to overcome the nonrespon-siveness of Pep-Exo, we delivered the loaded exosomes, thought to

1 µg/mL 5

10

A B

5 10 50

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CFSE100 101 102 103 104

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Pep-Exo OVA-Exo

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Figure 2. Directly loaded LPS-matured exosomes aremore potent in stimulating T-cell proliferation in vitrothan indirectly loaded exosomes. (A) Exosomes de-rived from LPS, IFN-�–matured, or immature BMDCswere directly loaded with OVA323-339 peptide by acidelution or (B) indirectly loaded by pulsing BMDCs withOVA or BSA protein and cocultured in different concentra-tions with CFSE-labeled CD4� T cells, sorted from thesplenocytes of DO11.10 mice. Proliferation was detected5 days later by flow cytometry, and results are expressedas the mean percentage plus or minus SEM of proliferat-ing cells cultured in triplicate according to the dimmingintensity of the CFSE-positive cells. One representativeexperiment of 3 is displayed. (C) Representative FACSplots from experiments shown in panels A and B (shownfor 10 �g/mL).

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contain T-cell epitopes, together with the whole OVA protein,containing B-cell epitopes, to wild-type mice. BALB/c mice wereprimed intravenously with 50 �g OVA administered either alone ortogether with OVA-Exo or Pep-Exo. As an exosome control, agroup of mice was immunized only with OVA-Exo. We comparedthe antibody responses induced by loaded exosomes with theresponse induced by OVA with aluminium hydroxide (Alum) orLPS. The anti-OVA antibody titers were determined 7 days afterpriming. Figure 4A shows that both Pep-Exo and OVA-Exo giventogether with OVA promoted the generation of primary IgM

antibodies against OVA, which was comparable with the responseinduced in the presence of Alum and LPS. Nearly undetectableamounts of IgM antibodies were produced in mice immunized withOVA alone (Figure 4A). Interestingly, the mouse group immunizedonly with OVA-Exo also elicited OVA-specific IgM responses(Figure 4A). IgG antibodies were also detected in the serum ofmice primed with OVA plus OVA-Exo or only with OVA-Exo(Figure 4B). Negligible amounts of IgG were induced in the mousegroup primed with OVA plus Pep-Exo or only with OVA. Theseresults show the efficiency of OVA-Exo to induce a humoral

Figure 3. Only indirectly loaded exosomes are able tostimulate OVA-specific T-cell proliferation in vivo.(A) BALB/c mice were adoptively transferred with a totalof 5.5 � 106 purified CD4� T cells from DO11.10 miceand immunized either with PBS or 50 �g unloaded (Exo),directly (Pep-Exo) or indirectly (OVA-Exo) loaded exo-somes. After 3 days of immunization, the percentage of(B,D) KJ1-26�/CD3� or (C) KJ1-26�/CD69� DO11.10cells per spleen was determined by flow cytometry or(E) the spleens were sectioned and stained with KJ1-26(DO11.10 T cells; red) and anti-B220 (B cells; green). Thepictures shown are representative of 2 independentexperiments. Bar represents 150 �m. For panel C,***P .001. (F) Splenocytes from DO11.10 mice werestained with CFSE and adoptively transferred to theBALB/c mice followed by OVA-Exo injection the day after.Spleens were taken 3 days later, and CFSE division wasassessed in the flow cytometry. A representative experi-ment with 1 mouse of 4 is shown. (G) Spleen sectionsfrom mice adoptively transferred with DO11.10 CD4�

T cells and immunized with OVA-Exo were also stainedwith KJ1-26 (DO11.10 T cells; red), anti-B220 (B cells;pseudo-colored blue) and anti-CD11c (DCs; green). Origi-nal magnification: left, �20; bar represents 150 �m;right, �100; bar represents 15 �m.

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response in the absence of whole OVA and suggest that indirectlyloaded exosomes may carry intact OVA for stimulation of B cells.It also suggests that the B-cell stimulatory function of exosomesis needed for them to be able to evoke a specific T-cell responsein vivo.

To confirm the presence of B-cell epitopes on OVA-Exo,Western blot analysis was performed with OVA-Exo, Exo, orwith purified OVA using anti-OVA immune sera. As shown inFigure 4C, the band appeared on the OVA-Exo lane correspond-ing to that of the band with OVA protein-loaded lane, which isapproximately 45 kDa. In addition, we could detect the presenceof OVA on the surface of exosomes by FACS analysis (Figure4D). These results indicate the presence of conformationallyintact OVA on the OVA-Exo.

OVA-loaded exosomes can prime for the induction ofsecondary responses to OVA

Given the ability of indirectly loaded OVA-Exo to prime a primaryhumoral response, we next addressed whether the primed immunesystem could induce a memory response after a second injectionwith OVA. We therefore boosted the aforementioned primed mousegroups with OVA alone 4 weeks after the priming. As shown inFigure 5A, when mice were primed either with OVA plus OVA-Exoor only with OVA-Exo, followed by boosting with OVA alone,anti-OVA IgG antibodies were produced. The antibody titer forthose groups attained similar levels as obtained from the group of

mice primed with OVA plus Alum or OVA plus LPS and boostedwith OVA. The IgG level in the serum of the OVA plus Pep-Exogroup was comparatively lower than in the other groups. Thecontrol group, primed and boosted only with OVA, producednegligible amounts of IgG anti-OVA antibodies (Figure 5A). Todetermine whether the priming with loaded exosomes and boostingwith OVA conditioned the nature of OVA-specific humoral re-sponse, the IgG1 and IgG2a profiles were analyzed. As illustratedin Figure 5B and C, whereas IgG1 was induced in all immunizedgroups except for the group that received OVA alone, onlyOVA-Exo–immunized mice elicited strong IgG2a response, deliv-ered either alone or in conjunction with OVA. This indicates thepotency of OVA-Exo as an immune-modulating vaccine adjuvant.

OVA-loaded exosomes can trigger the polarization of T cells tothe Th1 type

The efficacy of adjuvants in the induction of optimal immune responseshas been judged by the isotype and levels of antibodies elicited as well asthe cytokine milieu. We sought to determine the immunomodulatoryeffect of OVA-Exo on splenocyte proliferation and cytokine productionin vitro to observe the polarization of T cells. Comparisons were madewith the OVA-primed group or OVA plus OVA-Exo– or OVA plusAlum-primed groups. Splenocytes isolated 2 weeks after the OVAboostwere restimulated in vitro either with whole OVA or with OVA-Exo orleft unstimulated. Proliferation was observed for all groups of mice withdifferent intensities when restimulated with OVA-Exo or whole OVA

Figure 4. Indirectly loaded exosomes alone can induce potent OVA-specific primary antibody responses. BMDCs were stimulated with LPS on day 6, and exosomeswere harvested on day 8. BALB/c mice were primed intravenously with 50 �g OVA per mouse alone or together with 10 �g LPS, Alum (1:1 ratio with OVA), 50 �g OVA-Exo, orPep-Exo or only with 50 �g OVA-Exo. Sera were collected 7 days after priming, and the presence of OVA-specific (A) IgM and (B) IgG antibodies was detected by ELISA usingserial dilution of the sera. Results are expressed as optical density at 1:100 serum dilutions. Individual data and mean are presented (n � 7 per group, for Alum group n � 5).(C) A total of 30 �g OVA-Exo and Exo or 1 �g native OVA protein was separated on 8% to 16% SDS-acrylamide gel, transferred to polyvinylidene difluoride membrane, andincubated with OVA-specific immune sera (1:500 dilutions), followed by incubation with horseradish peroxidase–conjugated secondary antibodies, and detected bychemiluminescence kit. (D) A total of 30 �g OVA-Exo was coated on anti-CD9–coated latex beads, treated with OVA immune sera (open histogram) or preimmune sera (solidhistogram), followed by addition of PE-conjugated anti–mouse Ig, and then analyzed by flow cytometry.

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protein, which was significantly higher than the proliferation achievedwith unstimulated cells for each group of mice (Figure 6A). In vitrorestimulation with OVA-Exo gave a 1.8 to 3 times higher proliferativeresponse compared with OVA alone (Figure 6A).

Cytokine responses, analyzed by CBA assay, were used todetermine the T-cell regulatory effect of exosomes. Neither OVAplus OVA Exo- nor OVA-immunized groups mounted a visibleIL-4 and IL-5 response (data not shown). As illustrated in Figure6B through D, splenocytes from the mouse group primed with OVAalone generated low levels of IL-2, TNF-�, and IFN-� in responseto OVA or OVA-Exo. However, all these cytokines were signifi-cantly up-regulated for OVA plus OVA-Exo–primed splenocytes in

response to OVA-Exo. In particular, higher levels of IFN-� werereleased by stimulation with OVA-Exo, whereas OVA stimulationgave low amounts. Taken together, these data suggest that OVA-Exo can influence the polarization of T-cell responses toward Th1.

Exosome-induced T-cell responses are B-cell dependent

Because T-cell proliferation was only detected where a B-cellresponse was seen, we speculated that activation of B cells isneeded for exosomal T-cell activation. By injecting B-cell signaling-deficient Btk mice with OVA-Exo, we verified this hypothesis.Immunization of Btk KO mice with OVA-Exo resulted, as ex-pected, in a significant reduction in primary antibody production toOVA compared with the wild-type controls (IgM and IgG, P � .001;data not shown). After the second immunization with OVA protein,wild-type C57BL/6 mice generated significantly higher titers ofIgG (P � .001) as well IgG1 (P � .017) and IgG2a (P � .001)antibodies, whereas the Btk KO mice responded with very low ornegligible amounts of antibodies (Figure 7A-C). Splenocytes fromBtk KO mice showed diminished ability to proliferate whenrestimulated in vitro with OVA (P � .001) or OVA-Exo (P � .001)compared with the wild-type controls (Figure 7D). This shows therequirements of B-cell help for effective activation of T cells byOVA-loaded exosomes.

FDCs accumulate C4 molecules on OVA-Exo injection

According to Denzer et al,25 follicular DCs (FDCs) can bindexosomes on their surface, which might support antigen-specificT and B cells for activation. Because FDCs play an important roleduring the germinal center reaction and antibody class switch, wewanted to investigate whether OVA-Exo targets the FDCs in thespleen to modulate the immune response. BALB/c mice wereinjected either with OVA-Exo or with PBS, and spleens werecollected after 24 hours. Immunostaining showed that FDCsaccumulated C4 components of complement on OVA-Exo immuni-zation, which was comparatively lower in the PBS control group(Figure 7E). This observation suggests that exosomes, besidesbinding to FDCs, also focus complement components to these cellsthat might be involved in the OVA-Exo–mediated immune activa-tion and adjuvant effect.

Discussion

The interaction between T cells and DCs is necessary to generateeffective T-cell help for the production of high-affinity B cells andlong-lived plasma cells. In the present study, we show thatexosome-mediated immune activation needs the assistance ofB cells in vivo for generating antigen-specific T-cell responses. Weevaluated the influence of both directly (Pep-Exo) and indirectly(OVA-Exo) OVA-loaded exosomes on the immune response. Theincreased efficiency in vitro of Pep-Exo, as seen by others,20 mightlead to the conclusion that Pep-Exo should be more optimal also inclinical settings. However, to our surprise, in contrast to the in vitroresults, the OVA-Exo induced a potent antigen-specific T-cellresponse in adoptively transferred mice, whereas no such prolifera-tion was seen after injection of Pep-Exo. This nonresponsivenesswith Pep-Exo suggests that additional factors are involved for invivo activation of T cells with Pep-Exo. Based on the understand-ing that activation of the immune response is achieved through thecoordination of 3 classes of cells, B and T cells and DCs, we

Figure 5. Indirectly loaded exosomes induce a potent memory response to OVAthat is distinct from the response obtained with other adjuvants. All primedmouse groups were boosted with 50 �g OVA alone per mouse 4 weeks later and bled7 days after boosting. The presence of OVA-specific (A) IgG, (B) IgG1, and (C) IgG2aantibodies was detected by ELISA using serial dilution of the sera. The results fromthe 1:2700 dilutions are presented, showing OD (A 405 nm) values for each subjectand the mean. Significant differences between groups after nonparametric Mann-Whitney U test are depicted with asterisks: *P .01, **P .002.

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therefore designed a new experiment to explore whether B-cellhelp is crucial to overcome the nonresponsiveness with Pep-Exo invivo. We combined Pep-Exo with the native OVA as a source ofB-cell epitopes, injected to BALB/c mice, and measured thehumoral response. For making comparison, we also treated micewith OVA-Exo codelivered with the whole OVA. Together with thenative OVA, Pep-Exo could prime the immune system to produceantibodies, although the magnitude of the response was lower thanthe one achieved with OVA-Exo or with Alum or LPS.

Surprisingly, we observed that OVA-Exo alone also generated apotent primary antibody response in the absence of whole antigen,and this response was boosted after OVA challenge. This findingcorroborates what we have noticed in the experiment with in vivoTg T-cell proliferation with OVA-Exo. Because B cells recognizethe conformationally intact antigen, it is thus evident that OVA-Exocarries B-cell epitopes either inside or on the surface of them. It hasbeen shown previously that DCs have an antigen retention compart-ment enabling DCs to internalize, store, and recycle antigen for thedirect presentation of native antigen to B cells.26 Indeed, ourWestern blot analysis confirmed the presence of conformationalOVA on OVA-Exo, and the FACS analyses confirmed its presenceon the surface of the exosomes. Colino and Snapper have failed toidentify the whole antigen in exosome preparations by ELISA.15

However, Skokos et al could detect the presence of OVA on themast cell–derived exosomes by Western blot.27 Hence, it isprobable that a minimal amount of intact antigen might stimulatethe B cells, suggesting an efficient mechanism for exosome-mediated enhancement of the immune response. In addition, onepossibility could be that sequential B-cell epitopes might beexposed on the MHC28 of the OVA-Exo. Regardless of thisquestion, we show that activation of antigen-specific B cells isneeded for the induction of efficient T-cell proliferation in thissystem. According to previous studies, mice given repeated injec-

tions of anti-� antisera (�sm mice) showed total lack of B cells andthose mice displayed functional T-cell defects.29 Later, Ron andSprent showed that the impaired T-cell proliferation in �sm micecan be restored by injecting purified B cells before antigenadministration.30,31 In support of these findings, we show that BtkKO mice, lacking mature conventional B cells,32 failed to generateantibody and T-cell proliferative responses in the spleen. The B-celldependence of T-cell activation reveals that not only DCs areneeded to help T-cell activation in vivo; however, we think thatDCs probably also contribute to full T-cell activation. Denzer et al25

have demonstrated that B cell–derived exosomes specifically bindto FDCs. In line with this, we show an increased C4 staining onFDCs after OVA-Exo injection. FDCs are accessory cells of theimmune system essential for affinity maturation and immunoglobu-lin isotype switching of B-cell clones during the germinal centerreaction,33-35 in which they present antigen to B and T lympho-cytes.36 FDCs are also pivotal for selection for high-affinityB-lymphocytes. In this line, our data propose a model in whichOVA-Exo that display peptide/MHC II molecules as well asconformationally intact antigen dock on FDCs. Thus, in coordina-tion with FDCs, exosomes might facilitate interaction betweenspecific B and T cells, ultimately leading to isotype switching anddifferentiation into plasma and memory B cells (Figure 7F).Thereby, OVA-Exo served to efficiently prime the immune systemto amplify and modulate the humoral response to IgG2a and theT-cell response to the Th1 type.

Our observation that OVA-Exo but not OVA plus Pep-Exoelicited a detectable IgG antibody response on primary immuniza-tion is striking. One can speculate that the B-cell epitopes onexosomes are more efficiently presented compared with the nativeprotein, which makes them more accessible to the B cells torecognize, thus lowering the threshold for their activation. Inaddition to effective presentation of antigen to B cells, it is possible

Figure 6. In vitro stimulation with OVA-Exo induces a potent Th1 response in mice sensitized with OVA-Exo and challenged with OVA in vivo. OVA � OVA-Exo,OVA � Alum, or OVA alone primed mice groups were boosted with OVA, and 2 weeks later splenocytes were restimulated for 48 hours. (A) Proliferation was detected bythymidine incorporation assay, and the production of (B) IL-2, (C) TNF-�, and (D) IFN-� was measured by cytometric bead array from the culture supernatant. Results areexpressed as picograms per milliliter. Recombinant cytokines were used for standard curves for the quantification of sample cytokines by software provided by BD Biosciences.Data are presented as mean of 7 mice per group plus or minus SEM.

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that the OVA-Exo carry B-cell costimulatory molecules, eg, C337 orpossibly C4, as shown on the FDCs by us, engaging the CD21complement receptor and CD54,38 which bind LFA-1 on B cells,which leads to stronger primary IgG response to OVA.

Our findings that immunization with OVA plus Pep-Exoresulted in a comparatively low magnitude secondary antibodyresponse of the IgG1 type whereas OVA-Exo induced strongsecondary IgG2a antibodies could be the result of a higher TCRbinding affinity of the MHC/OVA-Exo. It has been demonstratedpreviously that OVA323-339 can bind to the MHC in a differentconfiguration, leading to altered TCR-exposed residues.39 In ourcase, processing of OVA by DCs may favor a specific peptideorientation on the MHC on exosomes with relatively higher affinityfor the TCR than the synthetic OVA323-339 peptide directly loadedon exosomes.40 Thus, the higher binding affinity of the OVA-Exo toTCR might be associated with a prolonged interaction with theTCR, which leads to the induction of a Th1 phenotype and IgG2aproduction.41,42 An alternative explanation can be that OVA loading

might promote distinct sorting of cargo proteins involved inTh1-biased responses. Our results demonstrate that OVA-Exo notonly can function as adjuvant to enhance the humoral response butalso contribute to the deviation of the response to Th1 type in naivemice, which is different from that obtained with the commonlyused adjuvants Alum and LPS. These data support the findings byColino and Snapper where they have shown that DT-pulsedDC-derived exosomes induce a Th1 type of response; however,they induced a systemic proinflammatory response by injectingcomplete Freund adjuvant before exosome treatment.15 We showthat exosomes are so potent that an additional adjuvant is notneeded. In another approach, Toxoplasma gondii antigen-pulsedDC2.4 cell-derived exosomes were reported to trigger Th1 typehumoral immunity and IFN-� production, which they found to beassociated with the protection against T gondii infection.8 Recently,Beauvillain et al have reported that Toxoplasma antigen-pulsedDC-derived exosomes induce protective immunity against parasiteinfection both in syngeneic and allogeneic mice.10 In these systems,

A D

B

C

APC

T-cell B-cell

Exo

E

F

PBS

Figure 7. Btk-deficient mice show abrogated production of antibodies and splenocytes proliferation compared with the wild-type control. C57BL/6 and Btk-deficientmice were primed intravenously with 50 �g per mouse with OVA-Exo and boosted with the OVA protein 4 weeks later. Sera were collected 7 days after boosting, and thepresence of OVA-specific (A) IgG and (B) IgG1, and (C) IgG2a antibodies was detected by ELISA using serial dilution of the sera. Results are expressed as optical density at1:900 serum dilutions. Individual data and mean are presented (n � 7 per group). (D) Splenocytes from the aforementioned boosted mice were restimulated in vitro with OVA orOVA-Exo for 48 hours, and proliferation was detected by thymidine incorporation assay. (E) Spleen sections from mice immunized with OVA-Exo stained with anti–FDC-M2(follicular dendritic cells; red) and anti-B220 (B cells; pseudo-colored blue). Bar represents 150 �m. (F) A proposed model describing the mechanism of how indirectly loadedexosomes facilitate interaction between B and T cells for efficient activation of the immune response. APC indicates antigen-presenting cell; Exo, exosome.

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whole antigen is added and exosomes probably contain both B- andT-cell epitopes.

Our results are in contrast to the findings by Thery et al,16 whoshowed that Pep-Exo could induce T-cell responses in vivo. Thediscrepancy between their and our results might be the result of thenature of the antigen they used, which is a male-specific naturalself-antigen. It is probable that that there already exists self-reactive B cells to this antigen, inherited in the B-cell repertoire.

The production of different antibody isotypes shows that theadjuvant effect of exosomes is not the result of LPS contaminationof the exosome preparations. Previously, we have shown that LPSstimulated the production of IgG1 antibody in C57BL/6 mice whenused as adjuvant.43 We used LPS-matured DC-derived exosomes inthis study because exosomes from LPS stimulated DC displayhigher expression of MHC II and costimulatory molecules thanexosomes from immature DCs, and are more potent stimulatorsthan immature exosomes in vitro 23 (our results). Exosomes fromLPS-matured DC were also more potent than the IFN-�–maturedexosomes, however, whether the cytokine and immunoglobulinprofile after administration of IFN-� exosomes might be the focusof another study, especially given the discrepancy between in vitroand in vivo results.

The rationale of using DC-derived exosomes in tumor vaccinedesign is now relatively well established. Because antigen-loadedexosomes are now being considered to have strong implications forthe use in clinical settings, especially in the cancer therapy, theloading method should be considered and optimized accordingly. Ithas been observed that the combination of indirect loading throughDC incubation for class II peptides and direct loading for MHC Ipeptides could be performed for stimulation of both CD8� andCD4� T cells.44 Our data show that the indirect loading is essentialfor inducing CD4� T-cell proliferation to exosomes in vivo. Inaddition, in other therapeutic approaches, such as vaccine develop-ment for infectious diseases, indirectly loaded exosomes should be

considered when Th1-like responses are desired. Vaccine adjuvantsare an attractive option to overcome poor immunogenicity ofcandidate antigens and offer the opportunity to improve vaccineefficacy; thus, antigen-loaded exosomes may mimic the naturaladjuvant to enhance and modulate the immune response. Further-more, using exosomes as adjuvant instead of Alum might reducethe risk of Th2-biasing of the immune response. Our findings mightlead to the design of more efficient exosome-based vaccines in thenear future.

Acknowledgments

This work was supported by the Swedish Research Council (grant57X-15 242-05-02), the Karolinska Institutet, the Hesselman, ÅkeWiberg, and Magnus Bergvall foundations, the Swedish Heart-Lung Foundation, and the Center for Allergy Research at theKarolinska Institutet.

Authorship

Contribution: K.R.Q., M.C.I.K., and S.G. contributed ideas andhypotheses; K.R.Q. performed most of the experiments and wrotethe major part of the manuscript; U.G. provided help in someexperiments and gave suggestions to the manuscript; E.D.J.performed the immunohistochemistry and wrote part of the manu-script; M.C.I.K. guided in setting up in vivo experiments andhelped edit the manuscript; and S.G. provided guidance for theexperimental design and cowrote the manuscript.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Khaleda Rahman Qazi, Department of Medicine,Clinical Allergy Research Unit L2:04, Karolinska University HospitalSolna, 171 76 Stockholm, Sweden; e-mail: [email protected].

References

1. Raposo G, Nijman HW, Stoorvogel W, et al. Blymphocytes secrete antigen-presenting vesicles.J Exp Med. 1996;183:1161-1172.

2. Thery C, Zitvogel L, Amigorena S. Exosomes:composition, biogenesis and function. Nat RevImmunol. 2002;2:569-579.

3. Zitvogel L, Regnault A, Lozier A, et al. Eradicationof established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes.Nat Med. 1998;4:594-600.

4. Hao S, Moyana T, Xiang J. Review: cancer immu-notherapy by exosome-based vaccines. CancerBiother Radiopharm. 2007;22:692-703.

5. Peche H, Heslan M, Usal C, Amigorena S, CuturiMC. Presentation of donor major histocompatibil-ity complex antigens by bone marrow dendriticcell-derived exosomes modulates allograft rejec-tion. Transplantation. 2003;76:1503-1510.

6. Peche H, Renaudin K, Beriou G, Merieau E,Amigorena S, Cuturi MC. Induction of toleranceby exosomes and short-term immunosuppressionin a fully MHC-mismatched rat cardiac allograftmodel. Am J Transplant. 2006;6:1541-1550.

7. Luketic L, Delanghe J, Sobol PT, et al. Antigenpresentation by exosomes released from peptide-pulsed dendritic cells is not suppressed by thepresence of active CTL. J Immunol. 2007;179:5024-5032.

8. Aline F, Bout D, Amigorena S, Roingeard P,Dimier-Poisson I. Toxoplasma gondii antigen-pulsed-dendritic cell-derived exosomes induce aprotective immune response against T. gondii in-fection. Infect Immun. 2004;72:4127-4137.

9. Kuate S, Cinatl J, Doerr HW, Uberla K. Exosomalvaccines containing the S protein of the SARScoronavirus induce high levels of neutralizing an-tibodies. Virology. 2007;362:26-37.

10. Beauvillain C, Ruiz S, Guiton R, Bout D, Dimier-Poisson I. A vaccine based on exosomes se-creted by a dendritic cell line confers protectionagainst T. gondii infection in syngeneic and allo-geneic mice. Microbes Infect. 2007;9:1614-1622.

11. Zhou H, Yuen PS, Pisitkun T, et al. Collection,storage, preservation, and normalization of hu-man urinary exosomes for biomarker discovery.Kidney Int. 2006;69:1471-1476.

12. Escudier B, Dorval T, Chaput N, et al. Vaccinationof metastatic melanoma patients with autologousdendritic cell (DC) derived-exosomes: results ofthe first phase I clinical trial. J Transl Med. 2005;3-10.

13. Morse MA, Garst J, Osada T, et al. A phase Istudy of dexosome immunotherapy in patientswith advanced non-small cell lung cancer. JTransl Med. 2005;3-9.

14. Dai S, Wei D, Wu Z, et al. Phase I clinical trial ofautologous ascites-derived exosomes combinedwith GM-CSF for colorectal cancer. Mol Ther.2008;16:782-790.

15. Colino J, Snapper CM. Exosomes from bonemarrow dendritic cells pulsed with diphtheria tox-oid preferentially induce type 1 antigen-specificIgG responses in naive recipients in the absenceof free antigen. J Immunol. 2006;177:3757-3762.

16. Thery C, Duban L, Segura E, Veron P, Lantz O,Amigorena S. Indirect activation of naive CD4� T

cells by dendritic cell-derived exosomes. Nat Im-munol. 2002;3:1156-1162.

17. Hao S, Yuan J, Xiang J. Nonspecific CD4(�) Tcells with uptake of antigen-specific dendritic cell-released exosomes stimulate antigen-specificCD8(�) CTL responses and long-term T cellmemory. J Leukoc Biol. 2007;82:829-838.

18. Getahun A, Hjelm F, Heyman B. IgE enhancesantibody and T cell responses in vivo via CD23�B cells. J Immunol. 2005;175:1473-1482.

19. Lutz MB, Kukutsch N, Ogilvie AL, et al. An ad-vanced culture method for generating large quan-tities of highly pure dendritic cells from mousebone marrow. J Immunol Methods. 1999;223:77-92.

20. Hsu DH, Paz P, Villaflor G, et al. Exosomes as atumor vaccine: enhancing potency through directloading of antigenic peptides. J Immunother.2003;26:440-450.

21. Admyre C, Johansson SM, Qazi KR, et al. Exo-somes with immune modulatory features arepresent in human breast milk. J Immunol. 2007;179:1969-1978.

22. Utsugi-Kobukai S, Fujimaki H, Hotta C,Nakazawa M, Minami M. MHC class I-mediatedexogenous antigen presentation by exosomessecreted from immature and mature bone marrowderived dendritic cells. Immunol Lett. 2003;89:125-131.

23. Segura E, Amigorena S, Thery C. Mature den-dritic cells secrete exosomes with strong ability toinduce antigen-specific effector immune re-sponses. Blood Cells Mol Dis. 2005;35:89-93.

2682 QAZI et al BLOOD, 19 MARCH 2009 � VOLUME 113, NUMBER 12 only.For personal use at KAROLINSKA INSTITUTET on January 28, 2013. bloodjournal.hematologylibrary.orgFrom

24. Admyre C, Johansson SM, Paulie S, GabrielssonS. Direct exosome stimulation of peripheral hu-man T cells detected by ELISPOT. Eur J Immu-nol. 2006;36:1772-1781.

25. Denzer K, van Eijk M, Kleijmeer MJ, Jakobson E,de Groot C, Geuze HJ. Follicular dendritic cellscarry MHC class II-expressing microvesicles attheir surface. J Immunol. 2000;165:1259-1265.

26. Bergtold A, Desai DD, Gavhane A, Clynes R. Cellsurface recycling of internalized antigen permitsdendritic cell priming of B cells. Immunity. 2005;23:503-514.

27. Skokos D, Botros HG, Demeure C, et al. Mastcell-derived exosomes induce phenotypic andfunctional maturation of dendritic cells and elicitspecific immune responses in vivo. J Immunol.2003;170:3037-3045.

28. Jarvinen KM, Beyer K, Vila L, Bardina L, MishoeM, Sampson HA. Specificity of IgE antibodies tosequential epitopes of hen’s egg ovomucoid as amarker for persistence of egg allergy. Allergy.2007;62:758-765.

29. Cooper MD, Kearney JF, Gathings WE, LawtonAR. Effects of anti-Ig antibodies on the develop-ment and differentiation of B cells. Immunol Rev.1980;52:29-53.

30. Ron Y, Sprent J. T cell priming in vivo: a majorrole for B cells in presenting antigen to T cells inlymph nodes. J Immunol. 1987;138:2848-2856.

31. Ron Y, De Baetselier P, Gordon J, Feldman M,

Segal S. Defective induction of antigen-reactiveproliferating T cells in B cell-deprived mice. EurJ Immunol. 1981;11:964-968.

32. Khan WN, Alt FW, Gerstein RM, et al. Defective Bcell development and function in Btk-deficientmice. Immunity. 1995;3:283-299.

33. Liu YJ, Grouard G, de Bouteiller O, BanchereauJ. Follicular dendritic cells and germinal centers.Int Rev Cytol. 1996;166:139-179.

34. Tew JG, Burton GF, Kupp LI, Szakal A. Folliculardendritic cells in germinal center reactions. AdvExp Med Biol. 1993;329:461-465.

35. Lindhout E, Koopman G, Pals ST, de Groot C.Triple check for antigen specificity of B cells dur-ing germinal centre reactions. Immunol Today.1997;18:573-577.

36. Gray D, Kosco M, Stockinger B. Novel pathwaysof antigen presentation for the maintenance ofmemory. Int Immunol. 1991;3:141-148.

37. Papp K, Vegh P, Prechl J, et al. B lymphocytesand macrophages release cell membrane depos-ited C3-fragments on exosomes with T cell re-sponse-enhancing capacity. Mol Immunol. 2008;45:2343-2351.

38. Hao S, Bai O, Li F, Yuan J, Laferte S, Xiang J.Mature dendritic cells pulsed with exosomesstimulate efficient cytotoxic T-lymphocyte re-sponses and antitumour immunity. Immunology.2007;120:90-102.

39. Scott CA, Peterson PA, Teyton L, Wilson IA. Crys-tal structures of two I-Ad-peptide complexes re-veal that high affinity can be achieved withoutlarge anchor residues. Immunity.1998;8:319-329.

40. Adorini L, Guery JC, Fuchs S, Ortiz-Navarrete V,Hammerling GJ, Momburg F. Processing of endo-genously synthesized hen egg-white lysozymeretained in the endoplasmic reticulum or in secre-tory form gives rise to a similar but not identicalset of epitopes recognized by class II-restricted Tcells. J Immunol. 1993;151:3576-3586.

41. Boutin Y, Leitenberg D, Tao X, Bottomly K. Dis-tinct biochemical signals characterize agonist-and altered peptide ligand-induced differentiationof naive CD4� T cells into Th1 and Th2 subsets.J Immunol. 1997;159:5802-5809.

42. Kumar V, Bhardwaj V, Soares L, Alexander J,Sette A, Sercarz E. Major histocompatibility com-plex binding affinity of an antigenic determinant iscrucial for the differential secretion of interleukin4/5 or interferon gamma by T cells. Proc NatlAcad Sci U S A. 1995;92:9510-9514.

43. Qazi KR, Oehlmann W, Singh M, Lopez MC,Fernandez C. Microbial heat shock protein 70stimulatory properties have different TLR require-ments. Vaccine. 2007;25:1096-1103.

44. Le Pecq JB. Dexosomes as a therapeutic cancervaccine: from bench to bedside. Blood Cells MolDis. 2005;35:129-135.

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