Synergistic Induction of Adaptive Antitumor Immunity by ... · CD8þ T cells, and gd T cells can induce cell death, whereas antibodies are important for antibody-dependent cellular

Microenvironment and Immunology

Synergistic Induction of Adaptive Antitumor Immunity byCodelivery of Antigen with a-Galactosylceramide onExosomes

Ulf Gehrmann, Stefanie Hiltbrunner, Anna-Maria Georgoudaki, Mikael C. Karlsson, Tanja I. N€aslund, andSusanne Gabrielsson

AbstractExosomes and the invariant NKT (iNKT) immune cell ligand a-galactosylceramide (aGC) may offer novel

tools for cancer immunotherapy. In this study, we investigated whether exosomes loaded with aGC can activateiNKT cells and potentiate a cancer-specific adaptive immune response. aGC loaded exosomes readily activatediNKT cells both in vitro and in vivo. Exosomes loaded withaGC plus themodel antigen ovalbumin (OVA) inducedpotent NK and gd T-cell innate immune responses, and they also synergistically amplified T- and B-cell responsesthat were OVA specific. In contrast to soluble aGC, which anergizes iNKT cells, we found that aGC/OVA-loadedexosomes did not induce iNKT cell anergy but were more potent than soluble aGCþ OVA in inducing adaptiveimmune responses. In an OVA-expressing mouse model of melanoma, treatment of tumor-bearing mice withaGC/OVA-loaded exosomes decreased tumor growth, increased antigen-specific CD8þ T-cell tumor infiltration,and increased median survival, relative to control mice immunized with soluble aGC þ OVA alone. Notably,an additional injection of aGC/OVA-loaded exosomes further augmented the treatment effects. Our findingsshow that exosomes loaded with protein antigen and aGC will activate adaptive immunity in the absence oftriggering iNKT-cell anergy, supporting their application in the design of a broad variety of cancer immuno-therapy trials. Cancer Res; 73(13); 3865–76. �2013 AACR.

IntroductionEffective antitumor immunity needs activation of both the

innate and adaptive immune system to overcome the immuneevasion strategies used by tumors. Furthermore, a long-lastingadaptive immune response needs a boost by the innateimmune system (1). Natural killer (NK) cells, cytotoxicCD8þ T cells, and gd T cells can induce cell death, whereasantibodies are important for antibody-dependent cellularcytotoxicity (ADCC; ref. 2). Thus, effective antitumor therapyneeds to activate multiple players of the immune system tomount an effective, multifaceted, and long-lasting immuneresponse (3).Exosomes are small membrane vesicles around 100 nm,

derived from the endosomal compartment and are secreted bymany different cell types, among other cancer cells and den-

dritic cells (4). They can express immunostimulatory mole-cules such as MHC molecules, CD80 and CD86 (5) and elicitantigen-specific CD4þ (6), CD8þ T cell (7), B-cell responses (8),and NK-cell (9) responses in vivo. Exosomes from melanomapeptide-pulsed dendritic cells were able to reverse tumorgrowth in mice (7), which led to the evaluation of exosomesas therapeutic agents and vaccine vehicles (10). However, twophase I clinical trials using peptide-loaded dendritic cell exo-somes in patients withmelanoma (11) and non–small cell lungcancer (12) showed that exosomes were well tolerated but hadlimited immunostimulatory effects. Thus, a better understand-ing of the exosomal immune response is needed to increaseexosomal immunogenicity and thereby improving the chancesof therapeutic success. We have recently shown that exosomesinduce CD4þ and CD8þ T-cell responses in a B cell–dependentmanner in vivo (13, 14) where protein loading, but not peptideloading of exosomes enhanced their immunogenicity. We nowasked whether dendritic cell–derived exosomes, carrying thelipid antigen–presentingmolecule CD1d (15, 16), can be loadedwith glycolipid antigen to activate invariant NKT (iNKT) cells,a T-cell subset that has been shown to be important for anti-cancer responses (17) and whether this would boost T- andB-cell responses to a protein antigen on the same exosome.

iNKT cells are a cell type that share characteristics with bothinnate and adaptive immune cells (18), which recognizes selfand bacterial glycolipids in a CD1d-dependent manner (19).Upon activation, iNKT cells rapidly release cytokines such asIFN-g and interleukin (IL)-4 (19) and have impact on

Authors' Affiliation: Department of Medicine Solna, Karolinska Institutet,Translational Immunology Unit, Stockholm, Sweden

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

T.I. N€aslund and S. Gabrielsson contributed equally to this work.

Corresponding Author: Susanne Gabrielsson, Department of MedicineSolna, Translational Immunology Unit, Karolinska Institutet, KarolinskaUniversity Hospital Solna L2:04, SE-171 76 Stockholm, Sweden. Phone:46-8-51776441; Fax: 46-8-335724; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-3918

�2013 American Association for Cancer Research.

CancerResearch

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Published OnlineFirst May 8, 2013; DOI: 10.1158/0008-5472.CAN-12-3918

http://cancerres.aacrjournals.org/

subsequent NK-, T- and B-cell responses (20–22). Alpha-galac-tosylceramide (aGC) is a glycolipid that induces a rapidactivation of iNKT cells in vivo (19). However, injection ofsoluble aGC causes anergy of iNKT cells (23) and multipleinjections of aGC in humans have had limited therapeuticeffects (24). Because coupling of aGC-loaded CD1d moleculesto antigenic protein or nanoparticles (25) was suggested toovercome anergy induction (26), we speculated that dendriticcell–derived exosomes could serve as an endogenous deliveryplatform for protein and glycolipid antigens without inducinganergy.

We report that exosomes loaded with aGC and ovalbumin(OVA) activate iNKT cells, overcome anergy induction, andamplify tumor-specific adaptive immune responseswith impli-cations for the development of novel cancer immunotherapy.

Materials and MethodsMice and antibodies

C57Bl/6, Va14-Ca�/� (kindly donated by Dr. G. Berne,Karolinska Institutet), and CD1d�/� mice (kindly donated byProf. K. K€arre, Karolinska Institutet) were bred andmaintainedunder pathogen-free conditions at Karolinska Institutet's ani-mal facility. All experiments were approved by the StockholmRegional Ethics Committee. A list of antibodies used is avail-able in Supplementary Table S1.

Bone marrow–derived dendritic cell culturesBone marrow cells were prepared from female C57Bl/6 or

CD1d�/�mice as previously described (13). On day 6, cells wereincubated overnight with 300 mg/mL OVA (grade V; Sigma), 2mg/mL SIINFEKL (Innovagen), and/or 100 ng/mL aGC (KRN-7000; Larodan Fine Chemicals). On day 7, supernatants werediscarded and cells were grown in new medium containingexosome-depleted FCS (27), GM-CSF, IL-4, and 30 ng/mL LPS(Sigma). On day 9, supernatants were used for exosomepreparation.

Exosome preparationExosomes were prepared as described before (27), with

some modifications. After centrifugation for 30 minutes at3,000 � g, supernatants were filtered through a 0.22-mm cut-off filter (Nordic Biosite), pelleted and washed in PBS at100,000 � g for 2 hours and 10 minutes. The final pellet wasre-suspended in a small volume of PBS and protein contentwas measured using a DC protein assay according to themanufacturer's instructions (Bio-Rad). Exosomes were ali-quoted and frozen at �80�C.

Exosome phenotypingSulfate–aldehyde latex microspheres (4 mm; Invitrogen)

were coated with anti-CD9 (BDBiosciences) antibodies toenrich for exosomes on the beads as previously described(28). Exosomes from wt or CD1d�/� mice were coated ontoanti-CD9 latex beads at a ratio of 50 mg protein per mL beadsand phenotyped as described before (13) using specific anti-bodies and corresponding isotype controls (SupplementaryTable S1). Data were acquired using a FACSCalibur (BDBios-

ciences) and analysis was done using FlowJo software (TreeStar Inc.).

In vitro proliferationA total of 7.5� 105 splenocytes were labeled with 5 mmol/L

carboxyfluorescein succinimidyl ester (CFSE; Invitrogen) andstimulated with exosomes at different concentrations (0.05,0.5, 5, and 50 mg/mL) for 72 hours. DimerX (BD Biosciences)was loaded overnight with sonicatedaGC at 37�C and labeledwith antimouse IgG1 Alexa 647 (Invitrogen). To stain for iNKTcells, cells were Fc-blocked (BDBiosciences), incubated withLive/Dead viability marker (Invitrogen), DimerX, and anti-bodies against B220, CD4, NK1.1, T-cell receptor b chain(TCR-b; all Biolegend). Data were acquired using a FACSAria(BDBiosciences).

In vivo proliferationC57Bl/6 wild-type mice (wt) or CD1d�/� female mice were

injected on day 0 or on days 0 and 14 i.v. with soluble aGC,soluble OVA, or 40 mg of exosomes from wt or CD1d�/�

dendritic cells. Mice were fed with 0.8 mg/mL 5-bromo-20-deoxyuridine (BrdU; Sigma) in drinking water either for 7 daysor in intervals fromday 0 to 1, day 1 to 3, day 3 to 5, or day 5 to 7.Mice were sacrificed on days 1, 3, 5, 7, or 21 and blood, liver,and spleen were removed. Hepatic lymphocyte preparationsand splenocyte single-cell suspensions were prepared asdescribed previously (13, 29) and serum was prepared fromcoagulated blood and frozen at �20�C. BrdU incorporationwas measured using a BrdU staining kit (BDBiosciences),according to themanufacturer's instructions. For OVA-specificCD8þ T-cell staining, cells were incubated with phycoerythrin(PE)-labeled H-2Kb/SIINFEKL pentamer (ProImmune). Datawere acquired using FACSAria (BDBiosciences).

B16/OVA melanoma tumor modelA total of 1 � 105 B16/OVA melanoma cells were injected

s.c. in the right flank of C57Bl/6 mice. Mice were treated i.v.either with PBS or with 40 mg of exosomes 11 days aftertumor injection, when tumors were palpable, or on day 4, oron day 4 and 11 as indicated in figure legends. Tumorgrowth was monitored regularly and mice were euthanizedwhen tumors reached 1,000 mm3 in size. Tumors wereremoved, and either embedded in Killik (Bio-Optica) andfrozen at�80�C for immunohistochemistry or homogenizedfor FACS analysis using antibodies against CD45, B220, TCR-b, CD8 (Supplementary Table S1) and PE-labeled H-2Kb/SIINFEKL pentamer (ProImmune).

Tumor immunohistochemistryEight-micrometer sections of Killik-embedded tumors were

fixed in acetone, dried overnight, and blocked with goat serum,avidin, and biotin (Vector Laboratories). Samples were stainedwith anti-TCR-b-APC and analyzed using a Leica DM IRBEmicroscope.

Intracellular cytokine stainingSplenocytes were restimulated ex vivo for 4 hours using

50 ng/mL Phorbol 12-myristate 13-acetate (PMA), 500 ng/mL

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Cancer Res; 73(13) July 1, 2013 Cancer Research3866




ionomycin, and 1 mg/mL Brefeldin A (all from Sigma) aspreviously described (30).

ELISPOTEnzyme-linked immunospot assay (ELISPOT) for IFN-g was

done according to the manufacturer's instructions (Mabtech)using 200,000 splenocytes per well. Splenocytes from Va14-transgenic mice were stimulated with a serial dilution ofsoluble aGC or 0.05, 0.5, 5, and 50 mg/mL of exosomes for72 hours. Splenocytes were stimulated in vitro with 2 mg/mLaGC, 2 mg/mL SIINFEKL, 2 mg/mLOVA323-339 peptide (Innova-gen), 2 mg/mLConcanavalin A (Sigma), or left unstimulated for22 hours.

ELISAIFN-g and IL-17A concentrations were determined in serum

or in supernatants from ELISPOT experiments using ELISAaccording to the manufacturer's instructions (Mabtech).Mouse sera from in vivo experiments were analyzed for con-centrations of OVA-specific IgG, total IgG, IgG2c, and IgG1 asdescribed before (13).

Statistical analysisStudent t test or one-way ANOVA with Bonferroni's correc-

tion was used for normally distributed data. Mann–Whitney orKruskal–Wallis with Dunn's correction was used for nonpara-metric data. For kinetic experiments, 2-way ANOVA withBonferroni's correction was used. Statistical analysis was con-ducted using GraphPad software (GraphPad Inc.).

ResultsDendritic cell–derived exosomes express CD1d andinduce iNKT-cell activation in vitroTo confirm that exosomes from bone marrow–derived

dendritic cells express CD1d (16), exosomes from C57Bl/6 wtor CD1d�/� dendritic cells were generated and phenotypedusing anti-CD9–coated latex beads and flow cytometry. Bothwt and CD1d�/� exosomes expressed similar amounts ofMHC class II (I-A/I-E), CD9, CD11c, CD40, CD54, CD80, CD81,and CD86, but only exosomes from wt dendritic cells express-ed CD1d (P ¼ 0.004; Fig. 1A).Next, we investigated whether exosomes loaded with aGC

could activate iNKT cells in vitro. OVA-loaded (Exo-OVA) oraGC-loaded exosomes (Exo-aGC) from wt mice or Exo-aGCfrom CD1d�/� mice (CD1d�/� Exo-aGC) were added tosplenocytes from Va14 mice, transgenic for the iNKT cellreceptor. Exo-aGC induced proliferation in the majority ofiNKT cells whereas CD1d�/� Exo-aGC induced substantiallyless iNKT cell proliferation and Exo-OVA induced none (Fig. 1Band C) shown by CFSE dilution assay. Exo-aGC also inducedincreased numbers of IL-4-producing splenocytes as deter-mined by ELISPOT (Fig. 1D) and higher levels of IFN-g and IL-17A using ELISA than did CD1d�/� Exo-aGC and Exo-OVA(Fig. 1E and F). However, CD1d�/� Exo-aGC induced highercytokine responses than Exo-OVA (Fig. 1D–F), indicating thataGC is not exclusively loaded onto CD1d molecules in exo-somes. Together, these results suggest that exosomes from

aGC-pulsed dendritic cells can induce potent iNKT cell pro-liferation and cytokine production mainly, but not exclusively,via exosomal CD1d in vitro.

Exosomes loaded with aGC activate iNKT, NK, and gd Tcells in vivo

Next, we investigated the effect of exosomes loaded withaGCand themodel antigenOVA [Exo(aGC-OVA)] or theCD8þ

T-cell OVA-specific peptide SIINFEKL [Exo(aGC-SIINFEKL)]in vivo. We injected 40 mg of Exo-OVA, Exo-SIINFEKL, Exo(aGC-SIINFEKL), Exo(aGC-OVA), or PBS i.v. into wt recipientmice, which were fed with BrdU for 7 days. Splenic iNKT cellsproliferated in response to Exo(aGC-OVA) and Exo(aGC-SIINFEKL) but not to Exo-OVA, Exo-SIINFEKL, and PBS asdetermined by flow cytometry (Fig. 2A and B). iNKT cellsupregulated the activation marker CD69 on day 1 (Fig. 2C)and proliferated up to day 5 (Fig. 2D) in response to Exo(aGC-OVA), but not to Exo-OVA or PBS. Hepatic iNKT cells showedsimilar patterns of activation and proliferation as their spleniccounterparts (data not shown). Intracellular cytokine stainingshowed that splenic iNKT cells produce IFN-g during the first5 days (Fig. 2E), whereas IL-4 was produced during the first3 days (Fig. 2F) in response to Exo(aGC-OVA) but not to PBSor Exo-OVA.

As reported by others (20, 31), activation of iNKT cells led toan early activation and proliferation of dendritic cells, NK, andgd T cells, the latter two being innate-like lymphocytes withimportant functions in anticancer immunity (SupplementaryFig. S1, data not shown). Interestingly, we also detected sig-nificantly lower levels of the complement receptor CD21 onmarginal zone B (MZB) cells on day 1 after Exo(aGC-OVA)injection and increased proliferation between days 1 and 3 incomparison to Exo-OVA immunization (Supplementary Fig.S2A and S2B). These data indicate that aGC-loaded exosomesinduce an early iNKT-cell response, dendritic cell, MZB cellactivation as well as NK- and gd T-cell activation and prolif-eration in vivo.

Exosomes loaded with aGC potentiate OVA-specificCD8þ T-cell responses

CD8þ T cells are adaptive immune cells that are crucial forexosome-induced antitumor immunity (7). We detected thatExo(aGC-OVA) stimulated OVA-specific CD8þ T-cell prolif-eration and led to a larger pool of OVA-specific CD8þ T cells,7 days after exosome injection in comparison to PBS andExo-OVA–immunized groups (Fig. 3A–C). This effect wasnot due to an aGC-induced polyclonal CD8þ T-cell expan-sion because Exo(aGC-SIINFEKL) immunized mice did nothave increased numbers of OVA-specific CD8þ T cells (Fig.3B). This is in agreement with previous data where SIINFEKLpeptide-loaded exosomes are not sufficient to stimulateproliferation of OVA-specific CD8þ T cells in vivo, becauseB-cell activation is needed for exosome-induced T-cell acti-vation in vivo (14). The increase in OVA-specific CD8þ T cellswas iNKT cell dependent (Fig. 3C). Proliferation of OVA-specific CD8þ T cells was detected on days 5 and 7 in Exo(aGC-OVA)-immunized mice (Fig. 3D), leading to a signif-icantly increased OVA-specific CD8þ T-cell pool on days 5

aGC on Exosomes Amplifies Antitumor Immunity

www.aacrjournals.org Cancer Res; 73(13) July 1, 2013 3867




and 7 when compared with PBS and Exo-OVA (Fig. 3E).Similar kinetics for OVA-specific CD8þ T cells were observedin the liver (data not shown). Exo(aGC-OVA) immunizationgenerated increased numbers of SIINFEKL-specific IFN-gproducing cells as detected by ELISPOT (Fig. 3F), an effectthat was dependent on exosomal CD1d (Fig. 3G). Theseresults show that aGC-loaded exosomes amplify antigen-specific CD8þ T-cell responses via iNKT cells in vivo and thatexosomal CD1d has an important role in that response.

aGC-loaded exosomes boost CD4þ T- and B-cellresponses

Antibodies are crucial in initiating ADCC, which is animportantmechanism in antitumor immunity. Thus, we inves-tigated the effect of aGC-loaded exosomes on T-helper cellresponses and humoral immunity. We observed that prolifer-ation of CD4þ T cells was significantly increased in the spleenin response to Exo(aGC-OVA) compared with PBS and Exo-OVA7days after exosome injection (Fig. 4A) and that this effect

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Figure 1. Exosomes from bone marrow–derived dendritic cells loaded with aGC activate iNKT cells in vitro. A, exosomes from dendritic cell of wt mice(wt) or CD1d�/� mice (CD1d�/�) loaded with aGC and/or OVA were coupled to anti-CD9–coated latex beads and analyzed for surface molecule expressionusing flow cytometry. Data are from 20 (wt) or 15 (CD1d�/�) exosome batches. Data are expressed as the MFI ratio between specific antibody andcorresponding isotype control. Statistical analysis was done using nonparametrical Mann–Whitney test. B, representative CFSE dilution histogram plots andquantification ofCFSEdilution assay using splenocytes fromVa14 transgenicmice stimulatedwith different concentrations of exosomes fromOVA-pulsedwtdendritic cell (Exo-OVA) or from aGC-pulsed wt dendritic cell (Exo-aGC) or CD1d�/� dendritic cell (CD1d�/� Exo-aGC). C, numbers indicate thepercentage of proliferated iNKT cells, defined as B220�, TCR-bþ, and DimerXþ cells. Data are from 3 independent experiments using duplicates in eachexperiment. D, Va14 splenocytes were stimulated with increasing amounts of exosomes in an IL-4 ELISPOT assay for 72 hours. E and F, supernatantsfrom ELISPOT experiments were analyzed for IFN-g and IL-17A using ELISA. Data are pooled from 3 independent (D) or 5 independent experiments (D–F).Two-way ANOVA was used to test for statistical significance. Bars indicate mean þ SEM. �, P < 0.05; ��, P < 0.01; ��, P < 0.001.

Gehrmann et al.





was iNKT cell–dependent because responses were lower inCD1d�/� mice (Fig. 4B). Injection of Exo(aGC-OVA) alsoinduced proliferation of follicular helper T cells (Tfh-cells;Supplementary Fig. S3), which are important for antibodyclass switching. In line with this, we detected increased num-bers of total and proliferated germinal center B cells andplasma cells (Supplementary Fig. S4B–S4G), but not of follic-ular B cells (Supplementary Fig. S4A), and OVA-specific IgGlevels were significantly higher already 7 days after immuni-zation with Exo(aGC-OVA) when compared with all othergroups (Fig. 4C). Intriguingly, only the Th1 antibody subclassIgG2c, but not IgG1 or total IgG antibodies, were significantlyincreased in serum of Exo(aGC-OVA)-treated mice comparedwith both PBS and Exo-OVA (Fig. 4D). Thus, Exo(aGC-OVA)induce CD4þ T-cell activation and increased B-cell responses,

possibly via the induction of T-follicular helper cells andgerminal center formation.

Antigen-loaded exosomes are more potent than solubleantigens in inducing adaptive immunity

It has been suggested that codelivery of aGC together witha protein antigen to the same APC is important to potentiateadaptive immune responses (32). To compare the efficiencyof exosome-bound aGC and soluble aGC as adjuvant, weestimated the OVA and aGC content on Exo(aGC-OVA),using IFN-g ELISPOT and ELISA (Fig. 5A and B). We injected40 mg of Exo(aGC-OVA) or the equivalent amount of solubleOVA (range: 32–320 ng) and aGC (range: 22–200 ng) andmeasured proliferation of innate and adaptive immune cells.Importantly, Exo(aGC-OVA) were less potent in inducing

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Figure 2. Exosomes loadedwithaGCactivate iNKT cells in vivo. C57Bl/6micewere injected i.v. with PBSor 40mgExo-OVA, Exo(aGC-OVA) orwith exosomesfromSIINFEKL (Exo-SIINFEKL) oraGCandSIINFEKL [Exo(aGC-SIINFEKL)] pulsed dendritic cells and fedBrdU in drinkingwater for 7 days andeuthanizedonday 7 (A, B) or for 1 to 2 days on day 0, 1, 3, or 5 and euthanized on day 1, 3, 5, or 7 (C–E). A and B, representative BrdU–histogram plots (A) and proliferationof splenic iNKT cells (defined as BrdUþ of B220�, TCR-bþ, DimerXþ live lymphocytes; B). Data are pooled from 4 independent experiments. One-wayANOVA with Bonferroni's multiple comparison test was used to determine statistical significance. Dots represent single mice and lines indicate themean � SEM. C and D, expression of CD69 (C) or proliferation of splenic iNKT (D) cells as assessed by flow cytometry. Data are pooled from 2 independentexperiments. Dots represent mean � SEM. Two-way ANOVA with Bonferroni's multiple comparison test was used to test for statistical significance. E,intracellular flow cytometry for IFN-g and IL-4–expressing splenic iNKT cells after ex vivo restimulation with PMA/ionomycin/Brefeldin A for 4 hours. Dataare pooled from 2 independent experiments. Dots represent mean � SEM. Two-way ANOVA with Bonferroni's multiple comparison test was used totest for statistical significance. ��,P < 0.01; ��,P

iNKT- and NK-cell proliferation compared with soluble aGCand OVA, whereas the opposite was true for gd T cells, CD4þ

T cells, and OVA-specific CD8þ T cells (Fig. 5C). After asecond injection, we also observed stronger B-cell responseswith significantly higher numbers of GC B cells and OVA-specific IgG levels in exosome-treated mice (Fig. 5D). Thesefindings show that aGC is a more effective adjuvant foradaptive immune responses when loaded to exosomes com-pared to soluble ligand.

Exo(aGC-OVA) does not induce iNKT-cell anergySoluble aGC induces iNKT-cell anergy already after 1 injec-

tion, a possible reason for the limited clinical success of aGC-based immunotherapies (23, 25). To test whether aGC-loadedexosomes were less prone to induce iNKT-cell anergy, weinjected PBS, soluble aGC þ OVA, or Exo(aGC-OVA) 2 timeswith 2-week interval (Fig. 6A) and measured serum IFN-g ,iNKT-cell proliferation, and IFN-g production in ELISPOT todetermine anergy induction. As expected, soluble aGC and

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Figure 3. aGC on exosomes increases OVA-specific CD8þ T-cell responses via iNKT cells. C57Bl/6 mice were injected i.v. with PBS or 40 mg Exo-OVA,Exo(aGC-OVA), Exo-SIINFEKL, or Exo(aGC-SIINFEKL) and fed BrdU in drinking water for 7 days and euthanized on day 7 (A, B, C, F, G) or for 1 to2 days on day 0, 1, 3, or 5 and euthanized on day 1, 3, 5, or 7 (D, E). A and B, representative dot plots (A) and quantification of splenic OVA-PentamerþCD8þ

T cells (defined as OVA-Pentamerþ of B220�, CD3þ, CD8þ live lymphocytes; B). Data are pooled from 4 experiments and one-way ANOVA withBonferroni's multiple comparison test was used to determine statistical significance. Dots represent single mice and lines mean � SEM. C, percentage ofOVA-specific CD8þ T cells in wt and CD1d�/� mice in response to Exo-OVA and Exo(aGC-OVA). Data are from 3 independent experiments and Studentt test was used to test for significance. Dots represent single mice and lines mean � SEM. D and E, proliferation (defined as BrdUþ cells; D) andpercentage (E) of splenic OVA-Pentamerþ CD8þ T cells. Data are pooled from 2 independent experiments. Two-way ANOVA with Bonferroni's multiplecomparison test was used to test for statistical significance. Dots represent mean � SEM. F, IFN-g ELISPOT after ex vivo stimulation of splenocytes withSIINFEKL-peptide for 22 hours. Data are pooled from 4 experiments and one-way ANOVAwith Bonferroni's multiple comparison test was used to determinestatistical significance. Dots represent single mice and lines mean � SEM. G, IFN-g ELISPOT of splenocytes from wt mice in response to Exo(aGC-OVA)from wt and CD1d�/� mice after ex vivo restimulation with SIINFEKL-peptide for 22 hours. Data are from 2 independent experiments and Student t testwas used to test for significance. Dots represent single mice and lines mean� SEM. �, P < 0.05; ��, P < 0.01; ��, P < 0.001 when compared with PBS group;#, P < 0.05; ##, P < 0.01; ###, P < 0.001 when compared with the Exo-OVA group. For all experiments, at least 3 mice were used per group and time point.

Gehrmann et al.





OVA induced significantly higher levels of serum IFN-g afterthe first injection when compared with both PBS and Exo(aGC-OVA) groups. However, only Exo(aGC-OVA) inducedincreased levels of serum IFN-g after both injections (Fig. 6B).After boosting, iNKT cells from soluble aGC þ OVA, but notfrom Exo(aGC-OVA)-injected mice had decreased CD69expression (Fig. 6C) and were refractory to ex vivo restimula-tion with aGC (Fig. 6D). Accordingly, we detected significantlymore IFN-g producing OVA-specific CD8þ T cells in spleno-cytes from twice exosome-injected mice (Fig. 6D). Theseresults indicate that aGC-loaded exosomes can stimulateIFN-g secretion by iNKT cells even after a second injection,in contrast to soluble aGC, and strikingly, boost adaptiveimmune responses.

Exo(aGC-OVA) decrease tumor growth and induce T-cellinfiltration in a mouse melanoma modelHaving shown that exosomes codelivering glycolipid and

protein antigen potentiate adaptive immune responses with-out inducing iNKT-cell anergy, we investigated the potency ofExo(aGC-OVA) to improve antitumor immunity. We injectedmice with OVA-expressing B16 melanoma cells and treatedon day 4 and/or 11 with PBS, Exo-OVA, soluble aGC þ OVA,or Exo(aGC-OVA). Tumor bearing mice receiving 1 (day 4 orday 11) or 2 (day 4 and 11) injections of Exo(aGC-OVA) hada significantly increased median survival compared with PBS,Exo-OVA, and soluble aGC þ OVA groups (Fig. 7A and B;Supplementary Fig. S5A and S5B).Moreover, 2 injections of Exo(aGC-OVA) significantly prolonged survival while slowingdown tumor growth when compared with 1 injection of

exosomes (Fig. 7A and B). Using flow cytometry and immu-nofluorescence, we observed more tumor T-cell infiltrates anddetected higher levels of OVA-specific CD8þ T cells in tumortissues (Fig. 7C; Supplementary Fig. S5C). Similarly, OVA-specific IgG levels in serum of Exo(aGC-OVA)-treated micecompared with Exo-OVA or PBS-treated mice were signifi-cantly elevated (Fig. 7D; Supplementary Fig. S5D). Togetherthese results show that Exo(aGC-OVA) but not soluble aGCþ OVA induce tumor-specific B- and T-cell responses thatlead to potent antitumor immunity and that treatmenteffects can be amplified by 2 injections of Exo(aGC-OVA).

DiscussionWe show in this study that aGC-loaded exosomes can

induce iNKT-cell activation in vitro and in vivo, without induc-ing iNKT-cell anergy, leading to enhanced adaptive immuneresponses and increased antitumor immunity. Importantly,only aGC codelivered with a protein antigen on exosomesboosted adaptive immune responses and could activate iNKTcells upon a second injection. These are important findings toconsider when designing future cancer immunotherapy.

Exosomes from wt BMDC loaded with aGC induced stron-ger iNKT-cell proliferation and proinflammatory cytokineproduction than exosomes from CD1d�/� BMDC in vitro,arguing for an important role of exosomal CD1d. In vivo,CD1d�/� exosomes induced significantly lower numbers ofIFN-g producing OVA-specific CD8þ T cells, supporting thatexosomal CD1d is important for the induced adaptive immuneresponse. However, our in vitro data suggest that CD1d�/�

exosomes also carry aGC in a CD1d independent fashion,

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T-cell responses and increasedIgG2c antibody production. C57Bl/6or CD1d�/� mice were injected i.v.with Exo-OVA, Exo(aGC-OVA) orPBS and fed BrdU in drinking waterfor 7 days and euthanized on day 7.A and B, percentage of proliferatedsplenic CD4þ T cells (defined asTCR-bþ, CD4þ live lymphocytes) inwt mice (A) or in wt and CD1d�/�

mice (B) in response to Exo-OVAand Exo(aGC-OVA). Data arepooled from 6 (A) or 3 (B)independent experiments and one-way ANOVA with Bonferroni'smultiple comparison test was usedto determine statistical significance.Dots represent single mice andlines mean � SEM. Serum levels ofOVA-specific IgG (C), total IgG,IgG1, and IgG2c levels (D) inwtmice.Data are pooled from 5 (C) or 4 (D)experiments and one-wayANOVA with Bonferroni's multiplecomparison test was used todetermine statistical significance.Dots represent single mice andlines mean � SEM. �, P < 0.05;��, P < 0.01; ��, P < 0.001.






perhaps on uptake receptors such as LDL receptors (33). Weand others reported previously thatwhole protein antigens canbe retained on exosomes after dendritic cell pulsing (8, 13, 14)and lipid antigens might be retained on exosomes in a similarfashion. Alternatively, the aGC not loaded on CD1d could belocated within the exosome. However, the mechanism of howexternally added antigen is retained in membrane vesiclesremains a matter of investigation.

In this study, we confirmed our recent finding that B-cellactivation is needed for a potent CD8þ T-cell activation in vivo(14). Here, aGC loading of exosomes was not able to overcome

the inability of peptide-loaded exosomes to induce antigen-specific CD8þ T-cell responses. We speculate that the adjuvantproperties of aGC cannot compensate for the need of CD4þ

andB-cell epitopes to induce cytotoxic T-cell responses. In fact,our data suggest that aGC enhances the involvement ofdendritic cells and MZBs during the induction of the exo-some-induced immune response. Early iNKT-cell activationcoincided with upregulation of the costimulatory moleculeCD86 on dendritic cells (26, 34, 35) and downregulation of theC3-binding complement receptor CD21 on MZBs. Dendriticcells are considered important cells for exosome-induced

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Figure 5. Exosome-bound aGC/OVA are more potent in stimulating T-cell responses than soluble aGC and OVA. A, splenocytes from Va14 mice werestimulated with increasing amounts of soluble aGC to form a standard curve in an IL-4 ELISPOT assay for 72 hours and with Exo(aGC-OVA) toestimate the exosomal content of aGC. Dots indicate mean � SEM of triplicates. B, the OVA content of Exo(aGC-OVA) was estimated using a-OVA ELISA.Dots indicatemean�SEMof triplicates. C,micewere injected, i.v., with Exo(aGC-OVA) or corresponding amounts of solubleaGCandOVAand fedwithBrdUin drinking water for 7 days. Mice were euthanized after 7 days and proliferation (as defined as the percentage of BrdUþ cells) was analyzed for spleniciNKT cells, NK cells, gd T cells, OVA-specific CD8þ T cells, and CD4þ T cells. Dots represent single mice and lines indicate mean � SEM values. Data arefrom 2 independent experiments using 4 mice per group and Mann–Whitney test was used to test for statistical significance. D, mice were injectedi.v. on day 0 and 14with PBS, 40 mg Exo(aGC-OVA) or 100 ng/mouse solubleaGC and 150 ng/mouse soluble OVA.Mice were bled on days 1, 10, and 15 andeuthanizedonday 21. Spleenswere analyzed for the percentageof germinal center B cells (GC, definedasB220þ, GL-7þ, CD95þ cells) amongBcells (definedas B220þ cells) and OVA-specific IgG was determined using ELISA. Data are pooled from 2 experiments using 5 to 7 mice per experiment and group.Bars indicate meanþ SEM and dots indicate mean� SEM. Statistical comparison between groups was conducted using One-way ANOVA or Student t testwith Bonferroni's multiple test correction. �, P < 0.05; ��, P < 0.01; ��, P < 0.001 for comparisons between Exo(aGC-OVA) and soluble aGC and OVA.

Gehrmann et al.





T-cell activation (36) and our recent data suggest that also Bcells, including MZB cells, have a role in exosome-inducedimmune responses (14, 37). It is possible that, similar toimmune complexes (38), MZB-cell transport exosomal cargointo splenic B-cell follicles. Interestingly, C3 fragments havebeen found in proteomic analyses of dendritic cell–derivedexosomes preparations (39). Thus, we speculate that exosometransport by MZB cells allows for B-cell activation, whereasdendritic cell transport of exosomes into the T-cell zoneallows for T-cell activation and that both mechanisms arecrucial for potent exosome-mediated immune effects.Exosomes also induced NK- and gd-cell proliferation and

IFN-g production in an iNKT-cell–dependent manner. gd Tcells express NKG2D, which can bind to ligand-expressingtumor cells (40) and due to their cytotoxic properties havebeen attributed important functions in melanoma immunity(41). In our study, proliferation and cytokine kinetics weresimilar for NK- and gd T-cell activation by aGC-loaded exo-somes. Although NK cells were also efficiently activated bysoluble aGC, gd T cells were not. In contrast, a recent reportshowed that a high dose of soluble aGC can induce gd T-cell

activation (42). These differences might be due to exosomeslowering the threshold foraGC-induced gd T cell activation bysynergistically providing additional activation signals, such asNKG2D ligands (9). In the same study, gd T-cell activation alsoled to an increased CD8þ T-cell response (42), indicating thatthe aGC-induced innate immune response is needed foreffective adaptive immunity to develop.

Following the innate immune response, we observed pro-liferation of OVA-specific, IFN-g–producing CD8þ T cells afterinjection with Exo(aGC-OVA), an effect that was superior tothat of exosomes lacking aGC. It has recently been shown thataGC-activated iNKT cells can induce CD8aþ dendritic cells toproduce CCL-17, a CCR4 ligand that led to increased CD8þ T-cell recruitment to the activated dendritic cell (32). Thissuggests an important role for codelivery of protein andglycolipid antigen to the same dendritic cell and is in accor-dance with the iNKT-cell–dependent increase in antigen-spe-cific CD8þ T-cell numbers we detect after Exo(aGC-OVA)immunization. The cross-talk between iNKT cells and den-dritic cells then allows for a selective recruitment of CD8þ Tcells to activated, protein antigen-presenting dendritic cells.

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Figure 6. Exo(aGC-OVA), but not soluble aGC and OVA, induce IFN-g production from iNKT cells after second injection. A, mice were injected i.v. on day0 and 14 with PBS, 40 mg Exo(aGC-OVA), or 100 ng/mouse soluble aGCþ 150 ng/mouse soluble OVA. Mice were bled and sacrificed on the indicated days.B, serum IFN-g levels were measured 1 day after first or second injection using ELISA. Data are pooled from 2 experiments using 5 to 7 mice perexperiment and group. Bars indicate mean þ SEM. Student t test with Bonferroni multiple comparison test was used to determine statistical significance.�, P < 0.05; ��, P < 0.001. C, spleens were analyzed for the expression of CD69 on iNKT cells. MFI, mean fluorescence intensity. Data are pooled from2 experiments using 4 to 5mice per group. Dots represent individual mice and lines indicatemean� SEM. Student t test with Bonferroni multiple comparisontest was used to determine statistical significance. �,P < 0.05; ��,P < 0.001. D, IFN-g ELISPOT of splenocytes after second injection and ex vivo restimulatedwith aGC or SIINFEKL-peptide for 22 hours. Data are pooled from 2 experiments using 4 to 5 mice per group. Bars represent mean þ SEM.






TheCD8þT-cell responsewas accompanied by proliferationof T-helper cells and production of OVA-specific antibodies.Interestingly, we found that T follicular helper cells, importantin germinal center formation, were proliferating 5 days afterExo(aGC-OVA) injection, preceding an increase in numbers ofgerminal center B cells and plasma cells on day 7. In accor-dance with this, a second injection of Exo(aGC-OVA) led tohigher numbers of GC B cells and boosted OVA-specific IgG.We speculate that Exo(aGC-OVA) induces the formation ofTfh cells, which leads to subsequent formation of germinalcenters in which germinal center B cells undergo affinity

maturation, somatic hypermutation, and differentiate intoplasma cells that produce antigen-specific antibodies. Wefound that Exo(aGC-OVA) predominantly induced IgG2c anti-bodies, indicative of a Th1 immune response. In accordance,two studies have previously reported that exosomes induceTh1 immunity (8, 13). The production of IFN-g during the earlyphase of the immune responses by iNKT, NK, and gd T cellsmight lead to the Th1-biased ensuing adaptive immuneresponse.

An exciting finding is that aGC-loaded exosomes, incontrast to soluble aGC, do not induce iNKT-cell anergy

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Figure 7. Exo(aGC-OVA) but not soluble aGC and OVA reduces tumor growth, increases survival, and induces adaptive antitumor immunity in a mousemelanoma model. C57Bl/6 mice were injected s.c. with 1 � 105 B16/OVA melanoma cells. Tumor growth was monitored regularly and mice weretreated on day 4 (PBS, 1� Exo(aGC-OVA) and 1� aGCþOVA) or on days 4 and 11 (2� Exo(aGC-OVA) and 2� aGCþOVA). Mice were euthanized whentumors reached 1,000mm3 in volume. A and B, Kaplan–Meier survival curve ( A) and tumor size (B) for all 5 groups. Dots represent mean� SEM.Mantel–Coxtest with Bonferroni multiple test adjustment was used to test for significance in A and 2-way ANOVA was used to test for statistical significance in B.�, P < 0.05; ��, P < 0.01. C, representative dot plots of flow cytometry and quantitation of OVA-specific CD8þ T-cell infiltration (defined as CD45þ,B220�, CD3þ, OVA-Pentamerþ of live cells) using OVA-specific Pentamer. Bars represent mean þ SEM. One-way ANOVA with Bonferroni multiple testcorrection was used to test for statistical significance. �, P < 0.05; ��, P < 0.01; ��, P < 0.001. D, OVA-specific IgG levels in sera of euthanized mice asdetermined by ELISA. Dots represent mean � SEM. Two-way ANOVA was used to test for statistical significance. A–D, data are one representativeexperiment (7–8 mice per group) of 3 independent experiments using 5 to 10 mice per group. �, P < 0.05; ��, P < 0.01; ��, P < 0.001. �, significances betweenExo(aGC-OVA) and the respective aGC þ OVA groups, #, significances between 1 � Exo(aGC-OVA) and 2 � Exo(aGC-OVA) groups.

Gehrmann et al.





after a second injection. In the initial studies, low doses ofaGC (200 ng injected intraperitoneally) could induce iNKT-cell unresponsiveness up to 1 month after injection. In ourstudy, 100 ng of soluble aGC injected i.v. induced a remark-able unresponsiveness of iNKT cells 2 weeks after injectionwhereas exosomes maintained their ability to stimulatecytokine responses. This is important for the ensuing adap-tive immunity because our data show that the OVA-specificCD8þ T-cell response persists after 2 injections of Exo(aGC-OVA), but not of soluble aGC and OVA.To test whether this lack of anergy induction had a func-

tional relevance for the induction of antitumor immunity, weevaluated the therapeutic effect of Exo(aGC-OVA) treatmentin an OVA-expressing melanomamodel. We observed additivetreatment effects with two injections of Exo(aGC-OVA), sug-gesting that exosomal delivery might allow for multiple boostinjections of aGC during a treatment regimen. Based on theresults of this study, we speculate that the codelivery of lipidand a protein antigen is beneficial to induce adaptive antitu-mor immunity.In summary, we present novel data stating that aGC-loaded

exosomes boost innate and antigen-specific Th1 adaptiveimmunity and enhance antitumor immunity without inducingiNKT-cell anergy. Our approach increased the immunogenicityof dendritic cell–derived exosomes and should be taken intoaccount when designing future clinical therapies formalignantdiseases.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design:U. Gehrmann, M.C. Karlsson, T.I. N€aslund, S. GabrielssonDevelopment of methodology: U. Gehrmann, S. Hiltbrunner, A.-M. Georgou-daki, T.I. N€aslundAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): U. Gehrmann, S. Hiltbrunner, A.-M. Georgoudaki, M.C. Karlsson, S. GabrielssonAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):U. Gehrmann, S. Hiltbrunner, T.I. N€aslund, S. GabrielssonWriting, review, and/or revision of the manuscript: U. Gehrmann,S. Hiltbrunner, A.-M. Georgoudaki, M.C. Karlsson, T.I. N€aslund, S. GabrielssonAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): T.I. N€aslundStudy supervision: T.I. N€aslund, S. Gabrielsson

AcknowledgmentsThe authors thank the animal facility staff at MTC, Karolinska Institutet,

Dr. E. Lord and Dr. J. Frelinger, University of Rochester, Rochester, NY, forkindly contributing the B16/OVA melanoma cell line, F. Wermeling, S. Lind,T. H€aggl€of, E. Grasset, M. Baptista, C. Dahlberg, and L. Westerberg for sharingknowledge on iNKT and T cells and antibodies.

Grant SupportThis work was supported by Swedish Medical Research Council, Karolinska

Institutet's IMTAC consortium, Swedish Cancer Society, Swedish Heart-LungAssociation, Hesselman's and David and Astrid Hagel�en's Foundations.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 22, 2012; revised March 18, 2013; accepted April 18, 2013;published OnlineFirst May 8, 2013.

References1. Pulendran B, Ahmed R. Immunological mechanisms of vaccination.

Nat Immunol 2011;12:509–17.2. Hubert P, Heitzmann A, Viel S, Nicolas A, Sastre-Garau X, Oppezzo P,

et al. Antibody-dependent cell cytotoxicity synapses form in miceduring tumor-specific antibody immunotherapy. Cancer Res 2011;71:5134–43.

3. Zitvogel L, ApetohL,Ghiringhelli F, KroemerG. Immunological aspectsof cancer chemotherapy. Nat Rev Immunol 2008;8:59–73.

4. TheryC, Zitvogel L, Amigorena S. Exosomes: composition, biogenesisand function. Nat Rev Immunol 2002;2:569–79.

5. Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV,Melief CJ, et al. B lymphocytes secrete antigen-presenting vesicles.J Exp Med 1996;183:1161–72.

6. Thery C, Duban L, Segura E, Veron P, Lantz O, Amigorena S. Indirectactivation of naive CD4þ T cells by dendritic cell-derived exosomes.Nat Immunol 2002;3:1156–62.

7. Zitvogel L, Regnault A, Lozier A, Wolfers J, Flament C, Tenza D, et al.Eradication of established murine tumors using a novel cell-freevaccine: dendritic cell-derived exosomes. Nat Med 1998;4:594–600.

8. Colino J, Snapper CM. Exosomes from bone marrow dendritic cellspulsed with diphtheria toxoid preferentially induce type 1 antigen-specific IgG responses in naive recipients in the absence of freeantigen. J Immunol 2006;177:3757–62.

9. Viaud S, Terme M, Flament C, Taieb J, Andre F, Novault S, et al.Dendritic cell-derived exosomes promote natural killer cell activationandproliferation: a role forNKG2D ligands and IL-15Ralpha. PLoSOne2009;4:e4942.

10. Thery C, Ostrowski M, Segura E. Membrane vesicles as conveyors ofimmune responses. Nat Rev Immunol 2009;9:581–93.

11. Escudier B, Dorval T, Chaput N, Andre F, Caby MP, Novault S, et al.Vaccination of metastatic melanoma patients with autologous den-dritic cell (DC) derived-exosomes: results of the first phase I clinicaltrial. J Transl Med 2005;3:10.

12. Morse MA, Garst J, Osada T, Khan S, Hobeika A, Clay TM, et al. Aphase I study of dexosome immunotherapy in patients with advancednon-small cell lung cancer. J Transl Med 2005;3:9.

13. Qazi KR, Gehrmann U, Domange Jordo E, Karlsson MC, GabrielssonS. Antigen-loaded exosomes alone induce Th1-type memory througha B-cell-dependent mechanism. Blood 2009;113:2673–83.

14. N€aslund TI, Gehrmann U, Qazi KR, Karlsson MC, Gabrielsson S.Dendritic cell-derived exosomes need to activate both T and B cellsto induce antitumor immunity. J Immunol 2013;190:2712–9.

15. Lamparski HG, Metha-Damani A, Yao JY, Patel S, Hsu DH, RueggC, et al. Production and characterization of clinical grade exo-somes derived from dendritic cells. J Immunol Methods 2002;270:211–26.

16. Coppieters K, Barral AM, Juedes A, Wolfe T, Rodrigo E, Thery C, et al.No significant CTL cross-priming by dendritic cell-derived exosomesduringmurine lymphocytic choriomeningitis virus infection. J Immunol2009;182:2213–20.

17. Smyth MJ, Wallace ME, Nutt SL, Yagita H, Godfrey DI, Hayakawa Y.Sequential activation of NKT cells and NK cells provides effectiveinnate immunotherapy of cancer. J Exp Med 2005;201:1973–85.

18. Bendelac A, Savage PB, Teyton L. The biology of NKT cells. Annu RevImmunol 2007;25:297–336.

19. Kawano T. CD1d-restricted and TCR-mediated activation of V14 NKTcells by glycosylceramides. Science 1997;278:1626–9.

20. Carnaud C, Lee D, Donnars O, Park SH, Beavis A, Koezuka Y, et al.Cutting edge: cross-talk between cells of the innate immune system:NKT cells rapidly activate NK cells. J Immunol 1999;163:4647–50.

21. Schofield L. CD1d-restricted immunoglobulin G formation to GPI-anchored antigens mediated by NKT cells. Science 1999;283:225–9.

22. TaniguchiM, HaradaM, Kojo S, Nakayama T,WakaoH. The regulatoryrole of Valpha14 NKT cells in innate and acquired immune response.Annu Rev Immunol 2003;21:483–513.






23. Parekh VV, Wilson MT, Olivares-Villagomez D, Singh AK, Wu L, WangCR, et al. Glycolipid antigen induces long-term natural killer T cellanergy in mice. J Clin Invest 2005;115:2572–83.

24. Neparidze N, Dhodapkar MV. Harnessing CD1d-restricted T cellstoward antitumor immunity in humans. Ann N Y Acad Sci 2009;1174:61–7.

25. Thapa P, Zhang G, Xia C, Gelbard A, Overwijk WW, Liu C, et al.Nanoparticle formulated alpha-galactosylceramide activates NKTcells without inducing anergy. Vaccine 2009;27:3484–8.

26. StirnemannK,Romero JF,Baldi L, Robert B,CessonV, BesraGS, et al.Sustained activation and tumor targeting of NKT cells using a CD1d-anti-HER2-scFv fusion protein induce antitumor effects in mice. J ClinInvest 2008;118:994–1005.

27. Thery C, Amigorena S, Raposo G, Clayton A. Isolation and charac-terization of exosomes from cell culture supernatants and biologicalfluids. Curr Protoc Cell Biol 2006;Chapter 3:Unit 3;22.

28. Admyre C, Johansson SM, Qazi KR, Filen JJ, Lahesmaa R, NormanM,et al. Exosomes with immune modulatory features are present inhuman breast milk. J Immunol 2007;179:1969–78.

29. Diana J, Beaudoin L, Gautron AS, Lehuen A. NKT and tolerance.Methods Mol Biol 2011;677:193–206.

30. Wermeling F, Lind SM, Jordo ED, Cardell SL, Karlsson MCI. InvariantNKT cells limit activation of autoreactive CD1d-positive B cells. J ExpMed 2010;207:943–952.

31. Kitamura H, Iwakabe K, Yahata T, Nishimura S, Ohta A, Ohmi Y, et al.The natural killer T (NKT) cell ligand alpha-galactosylceramide demon-strates its immunopotentiating effect by inducing interleukin (IL)-12production by dendritic cells and IL-12 receptor expression on NKTcells. J Exp Med 1999;189:1121–8.

32. Semmling V, Lukacs-Kornek V, Thaiss CA, Quast T, Hochheiser K,Panzer U, et al. Alternative cross-priming through CCL17-CCR4-mediated attraction of CTLs toward NKT cell-licensed DCs. NatImmunol 2010;11:313–20.

33. van den Elzen P, Garg S, Leon L, Brigl M, Leadbetter EA, Gumperz JE,et al. Apolipoprotein-mediated pathways of lipid antigen presentation.Nature. 2005;437:906–10.

34. Fujii S, Shimizu K, Smith C, Bonifaz L, Steinman RM. Activation ofnatural killer T cells by alpha-galactosylceramide rapidly induces thefull maturation of dendritic cells in vivo and thereby acts as an adjuvantfor combined CD4 and CD8 T cell immunity to a coadministeredprotein. J Exp Med 2003;198:267–79.

35. Fujii S, LiuK, SmithC,Bonito AJ, SteinmanRM.The linkage of innate toadaptive immunity via maturing dendritic cells in vivo requires CD40ligation in addition to antigenpresentation andCD80/86 costimulation.J Exp Med 2004;199:1607–18.

36. Segura E, Guerin C, Hogg N, Amigorena S, Thery C. CD8þ dendriticcells use LFA-1 to captureMHC-peptide complexes from exosomes invivo. J Immunol 2007;179:1489–96.

37. Montecalvo A, Larregina AT, Shufesky WJ, Stolz DB, Sullivan ML,Karlsson JM, et al. Mechanism of transfer of functional microRNAsbetween mouse dendritic cells via exosomes. Blood 2012;119:756–66.

38. Carroll MC. The complement system in regulation of adaptive immu-nity. Nat Immunol 2004;5:981–6.

39. TheryC, BoussacM, VeronP, Ricciardi-Castagnoli P, RaposoG,GarinJ, et al. Proteomic analysis of dendritic cell-derived exosomes: asecreted subcellular compartment distinct from apoptotic vesicles.J Immunol 2001;166:7309–18.

40. Bauer S, Groh V,Wu J, Steinle A, Phillips JH, Lanier LL, et al. ActivationofNKcells andTcells byNKG2D, a receptor for stress-inducibleMICA.Science 1999;285:727–9.

41. Gao Y, Yang W, Pan M, Scully E, Girardi M, Augenlicht LH, et al.Gamma delta T cells provide an early source of interferon gamma intumor immunity. J Exp Med 2003;198:433–42.

42. Paget C, Chow MT, Duret H, Mattarollo SR, Smyth MJ. Role ofgammadelta T cells in alpha-galactosylceramide-mediated immunity.J Immunol 2012;188:3928–39.

Gehrmann et al.





2013;73:3865-3876. Published OnlineFirst May 8, 2013.Cancer Res Ulf Gehrmann, Stefanie Hiltbrunner, Anna-Maria Georgoudaki, et al.

-Galactosylceramide on ExosomesαCodelivery of Antigen with Synergistic Induction of Adaptive Antitumor Immunity by

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