vaccination with irradiated tumor cells pulsed with an ... · results: vaccine treatment alone was...

Cancer Therapy: Preclinical

Vaccination with Irradiated Tumor Cells Pulsed with anAdjuvant That Stimulates NKTCells Is an Effective Treatmentfor Glioma

MartinK.Hunn1,Kathryn J. Farrand1, KateW.R.Broadley1,RobertWeinkove1, Peter Ferguson1,RoseJ.Miller2,Cameron S. Field1, Troels Petersen1, Melanie J. McConnell1, and Ian F. Hermans1,3

AbstractPurpose: The prognosis for patients with glioblastoma multiforme (GBM) remains extremely poor

despite recent treatment advances. There is an urgent need to develop novel therapies for this disease.

Experimental Design: We used the implantable GL261 murine glioma model to investigate the

therapeutic potential of a vaccine consisting of intravenous injection of irradiated whole tumor cells pulsed

with the immuno-adjuvant a-galactosylceramide (a-GalCer).Results: Vaccine treatment alone was highly effective in a prophylactic setting. In a more stringent

therapeutic setting, administration of one dose of vaccine combined with depletion of regulatory T cells

(Treg) resulted in 43% long-term survival and the disappearance of mass lesions detected by MRI.

Mechanistically, the a-GalCer component was shown to act by stimulating "invariant" natural killer–like

T cells (iNKT cells) in a CD1d-restricted manner, which in turn supported the development of a CD4þ

T-cell–mediated adaptive immune response. Pulsinga-GalCer onto tumor cells avoided the profound iNKT

cell anergy induced by free a-GalCer. To investigate the potential for clinical application of this vaccine, the

number and function of iNKT cells was assessed in patients with GBM and shown to be similar to age-

matched healthy volunteers. Furthermore, irradiated GBM tumor cells pulsed with a-GalCer were able tostimulate iNKT cells and augment a T-cell response in vitro.

Conclusions: Injection of irradiated tumor cells loaded with a-GalCer is a simple procedure that

could provide effective immunotherapy for patients with high-grade glioma. Clin Cancer Res; 18(23);

6446–59. �2012 AACR.

IntroductionGlioblastoma multiforme (GBM) is a highly malignant

primary brain tumor with an extremely poor prognosis.Despite recent advances in chemotherapy, median survivalis only 15.4 months, and 5-year survival is less than 10%(1). There is therefore a pressing need to develop bettertreatments. Cellular immunotherapy is a potential thera-peutic option for GBM, with the most commonly usedapproach being injection of autologous dendritic cells (DC)loaded ex vivowith unspecified antigens derived from autol-

ogous whole tumor preparations. However, although clin-ical trials have reported some success in eliciting T-cell–mediated antitumor immune responses, survival benefit inmost studies has been modest (2).

The fundamental requirement of any active anticancerimmunotherapy is the generation of potent systemic anti-tumor immune responses with long-lasting memory. Infact, unless antigen-presenting cells (APC) such as dendriticcells are properly licensed to initiate effector T-cellresponses, vaccination can have a tolerizing effect (3). Oneway to promote optimal activity of dendritic cells is toinclude a potent immune adjuvant in the vaccine. Alpha-galactosylceramide (a-GalCer) is a glycolipid that stimu-lates invariant natural killer–like T cells (iNKT cells; ref. 4), apopulation of T cells with innate-like function found pre-dominantly in spleen, liver, and bone marrow (5). Inaddition to cell surface markers typically observed on NKcells, iNKT cells express a semi-invariant ab T-cell receptorthat recognizes glycolipid antigens (including a-GalCer)bound to the MHC class I–like molecule CD1d. CD1d isexpressed at high levels onAPCs includingdendritic cells, soinclusion of an iNKT cell ligand such as a-GalCer canencourage iNKT:DC interactions that enhance innate and

Authors' Affiliations: 1Malaghan Institute of Medical Research; 2Depart-ment of Pathology and Molecular Medicine, University of Otago; and3School of Biological Sciences, Victoria University of Wellington, Welling-ton, New Zealand

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Authors: Martin K. Hunn, Malaghan Institute of MedicalResearch, POBox 7060,Wellington 6242, NewZealand. Phone: 64-4-499-6914; Fax: 64-4-499-6915; E-mail: [email protected]; and Ian F.Hermans. E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-0704

�2012 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 18(23) December 1, 20126446

on December 29, 2020. © 2012 American Association for Cancer Research.clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst November 12, 2012; DOI: 10.1158/1078-0432.CCR-12-0704

http://clincancerres.aacrjournals.org/

adaptive immune responses (6–8). Because CD1d is non-polymorphic, the "help" provided by iNKT cells to dendriticcells is not dependent on HLA status, making this attractivefor broad clinical application. a-GalCer can also be com-binedwithother adjuvants such as Toll-like receptor ligandsto further strengthen vaccine-induced immune responses(9).Anticancer vaccines that activate iNKT cellswitha-GalCer

are highly effective in animal studies, with long-lastingimmunity induced in response to coadministration oftumor antigens and a-GalCer (10–13). This approach hasyet to be tested directly in patients with cancer. One poten-tial obstacle to clinical translation is the observation thatiNKT cell numbers seem to be reduced in many patientswith cancer (14, 15). In this study, we investigated theefficacy of a vaccine based on injection of whole tumor-derived antigens combined with a-GalCer in a murineglioma model. We also assessed whether the crucial effec-tors required for the success of a-GalCer–based vaccines arepresent and competent in a cohort of typical patients withGBM.

Materials and MethodsMiceThe inbred strainC57BL/6was obtained from theAnimal

Resource Centre (Canning Vale, WA). Also used wereCD1d�/� mice (16), MHC class II–deficient B6Aa0/Aa0

mice (17), TAP1-deficient mice (18), and langerin-DTREGFP mice that express the human Diptheria toxin(DT) receptor under control of the langerin gene (19). Inadoptive transfer experiments cells from OT-I x B6.SJL-PtprcaPepcb/BoyJ CD45.1þ mice were transferred intoC57BL/6 (CD45.2þ) hosts as previously described (29). Allmice were maintained by the Biomedical Research Unit,Malaghan Institute of Medical Research (Wellington, NewZealand). Experimental protocols were approved by Victo-ria University Animal Ethics Committee and done accord-

ing to their guidelines. Mice were 6 to 10 weeks of age andmatched for age and gender.

Cell lines and reagentsThemurine glioma cell lineGL261was obtained from the

DCTD Tumor Repository (National Cancer Institute, Fre-derick, MD, USA) and cultured in Dulbecco’s modifiedEagle’s medium (DMEM; Invitrogen) supplemented with20% FBS (Sigma-Aldrich), 2 mmol/L GlutaMax, 100 U/mLpenicillin, and 100 mg/mL streptomycin (all Invitrogen). Amethylcholanthrene-induced fibrosarcoma cell line raisedin a CD1d�/�mouse, and a CD1d-transfected C1R cell line,both used as controls in flow cytometry experiments, weregifts from Prof. Mark Smyth (Peter MacCallum CancerInstitute, University of Melbourne, Melbourne, Australia)and Prof. Vincenzo Cerundolo (Weatherall Institute ofMolecular Medicine, University of Oxford, Oxford, UnitedKingdom), respectively. The iNKT cell ligand a-a-GalCerwas manufactured by Industrial Research as previouslydescribed (20). Diphtheria toxin and doxorubicin werefrom Sigma-Aldrich. Recombinant human IFN-g was fromPeproTech.

Tumor implantation modelsGL261 cells were harvested with TrypLE (Invitrogen) and

washed. For subcutaneous implantation, 5 � 105 cells in100 mL of DMEM were injected in the left flank. Mice wereconsidered to harbor tumors when any 2 perpendiculardiameters were both at least 2 mm. For intracranialimplantation, 5 � 103 cells in 2 mL PBS were injected viaa 32 gauge needle into the right striatum at a point 2.1 mmlateral to the bregma and at a depth of 3 mm using astereotactic frame (Harvard Apparatus) under general anes-thesia by intraperitoneal injection of 100 mg/kg ketamineand 10 mg/kg xylazine (both Phoenix Pharm). Lacrilube(Allergan) was applied to the cornea to prevent dessica-tion. Buprenorphine (Renckitt Benckiser Pharmaceuticals)and Carprofen (Norbrook Laboratories) were used forperioperative analgesia. Time to symptom appearance wasdefined as time to weight loss more than 10% or overtbehavioral symptoms (reduced activity, hunching). Thepresence of brain tumors in symptomatic mice was con-firmed by necropsy, including histologic examination insome experiments.

Generation and administration ofwhole tumor vaccineTo generate "Glioma/Gal" vaccines, GL261 cells were

incubated in complete media supplemented with 200 ng/mL a-GalCer for 24 hours, g-irradiated (150 Gy) on ice,washed 3 times in PBS, and resuspended in PBS for injec-tion. Themethod for "Glioma/Gal/Dox" vaccines was iden-tical except that 0.05 mmol/L doxorubicin was also added tothe final 24 hours of culture. Unless otherwise stated, micereceived 105 cells in 200 mL PBS, injected intravenously intoa lateral tail vein. To mitigate toxicity when more than 105

cells were injected, mice were pretreated 10 minutes beforevaccination with 2 U heparin (Pfizer) delivered intrave-nously. To deplete regulatory T cells (Treg), mice received

Translational RelevanceAlpha-galactosylceramide (a-GalCer) is a glycolipid

that stimulates a population of natural killer–like T cells(NKT cells) to secrete cytokines and provide help toantigen-presenting cells (APC). It is therefore a promis-ing adjuvant that could enhance the potency of antican-cer vaccines.Herewe show for thefirst time that a vaccineconsisting of irradiated tumor cells pulsedwitha-GalCeris an effective treatment of intracranial glioma in amouse model. We also show that the NKT cell popula-tion required for this approach to succeed is present andfunctional in a group of patients with glioblastomamultiforme (GBM). In this respect, high-grade gliomaseems tobe an exception toother advanced solid cancers.This simple vaccine could be a useful treatment for agroup of patients who currently face an extremely poorprognosis.

Glioma/Gal Vaccine

www.aacrjournals.org Clin Cancer Res; 18(23) December 1, 2012 6447




125 mg of anti-CD25 antibody intraperitoneally (clonePC61, prepared in-house from hybridoma supernatant),a dose that was separately shown to deplete 40% to 50% ofFoxP3þ CD4þ T cells in na€�ve mice (data not shown).

Isolation of OT-1 cells and in vivo cross-priming assaySpleens and lymph nodes from OT-I � B6.SJL-Ptprca

Pepcb/BoyJ mice (CD45.1þ) were strained through a 70mm filter, then CD8 cells positively selected using magneticbeads (Dynabead FlowComp, Invitrogen) and analyzed byflow cytometry to check purity. OT-1 cells (104) weretransferred into C57BL/6 mice by intravenous injectionand 1 day later recipient mice were injected intravenouslywith 200 mg endotoxin-free ovalbumin (OVA; Profos AG)and 200 ng a-GalCer. Seven days later mice were bled fromthe lateral tail vein and leukocytes stained directly ex vivowith antibodies for TCR Va2, CD8, and CD45.1. Flowcytometry was used to enumerateOT-1 cells as a proportionof total CD8þ lymphocytes.

In vivo depletion studiesCD8aþ cells were depleted with antibody clone 2.43,

CD4þ cells with clone GK1.5, and NK1.1þ cells with clonePK136, all prepared in-house from hybridoma superna-tants. Appropriate intraperitoneal dosing schedules weredeveloped to achieve >95% depletion over the course of agiven experiment. Depletion of langerinþ cells in langerin-DTREGFP recipients was achieved with 350 ng of Diphthe-ria toxin given 48 and 24 hours before vaccination, and 1and 2 days after vaccination. In some experiments, antigenpresentation in spleen was prevented entirely by splenec-tomy conducted 7 days before vaccination. Splenectomieswere conducted under general anesthesia as described byReeves and colleagues (21).

Flow cytometry in animal experimentsAll antibody staining was conducted for 10 minutes at

4�C in PBS supplemented with 1% fetal calf serum (FCS),0.05% sodium azide, and 2 mmol/L EDTA. NonspecificFcR-mediated binding was blocked by incubation for 10minutes with anti-CD16/32 (clone 24G2, prepared in-house from hybridoma supernatant). Propidium iodide(Sigma-Aldrich) was used to exclude dead cells. Flow cyto-metry was conducted on a FACSCalibur (BD Biosciences)with data analysis using FlowJo software (TreeStar Inc.).Monoclonal antibodies used in mouse experiments were:CD1d (clone 1B1; PE-conjugated), CD3e (145-2C11;FITC), CD4 (GK1.5; PE or APC), CD8a (53-6.7; FITC),CD45.1 (A20; PE), CD45.2 (104; conjugated in-house toAlexa-A488 from Invitrogen), MHC class II (M5/114.15.2;PE or conjugated to Alexa-488), TCR Va2 (B20.1; conju-gated to A647), and rat IgG2b,k (149/10H5; PE) fromeBioscience; CD11c (HL3; APC), TCRb (H57-597; FITC orPE), CD45R/B220 (RA3-6B2; PerCP), CD44 (IM7; APC),CD69 (H1.2F3; FITC), and NK1.1 (PK136; conjugated toAlexa 488) from BD Biosciences. Annexin V-FITC from BDBiosciences was used according to the manufacturer’sinstructions.

Analysis of cytokine releaseBlood was collected from the lateral tail vein, allowed to

clot at room temperature and serum collected after centri-fugation. Levels of the cytokines interleukin (IL)-4, IL-12p70, and IFN-g were assessed by cytokine bead arraysanalysed on a Bio-Plex analyzer (Bio-Rad Laboratories). Foranalysis of cytokine release in spleen, liver, and lymphnodepostvaccination, tissue was removed from euthanized ani-mals and mechanically dissociated. Enrichment for liverlymphocytes was conducted using a Lymphoprep gradient(Axis-Shield). Cells were washed and incubated at 37�C inIMDM with 5% FBS, 2 mmol/L GlutaMax, 100 U/mLpenicillin, 100 mg/mL streptomycin, and 50 mmol/L 2-mercaptoethanol (all Invitrogen). Culture supernatantswere collected after 48 hours for analysis.

MRIMRI was conducted on anesthetized animals using a

clinical 1.5-T MR scanner (Philips Medical Systems),equipped with a wrist solenoid coil. T1-weighted imageswere acquired with the following parameters: TE ¼ 21.4milliseconds, TR ¼ 800 milliseconds, pixel size ¼ 300 mm� 300 mm, thickness ¼ 1 mm, 3 averages. Contrast wasenhanced by intravenous administration of 100 mL/mouseof gadolinium-DTPA (Magnevist, Bayer Schering Pharma).T2-weighted spin-echo images were acquired with the para-meters: TE ¼ 54 milliseconds, TR ¼ 2000 milliseconds.

Patient sample collectionPeripheral blood and tumor tissue was collected from 38

patients whomet histologic criteria for a diagnosis of GBM.Exclusion criteria included any other active malignancy orany previous chemotherapy for a disease other than GBM.Venous blood was drawn into CPT tubes (BD Biosciences),the peripheral blood mononuclear cells (PBMC) collectedaccording tomanufacturer’s instructions, washed, and cryo-preserved in the vapor phase of liquid N2 in 70% RPMI(Invitrogen), 20% FCS, and 10% dimethyl sulfoxide (Sig-ma-Aldrich) until use. Absolute differential blood countswere determined by separate venesection using a Sysmex XE2100 analyser (Roche Diagnostics) within 24 hours ofPBMC collection. PBMC from 35 healthy age-matchedcontrols were collected and stored according to a similarprotocol. Tumor tissue was mechanically dissociated tocreate a single-cell suspension and primary GBM cultureswere generated in RPMI supplemented with 10% FCS, 100U/mL penicillin, and 100 mg/mL streptomycin. All patientdonors gavewritten informed consent, and ethical approvalfor tissue collectionwas obtained from theCentral RegionalEthics Committee of New Zealand.

Flow cytometric analysis of human PBMC samplesPBMC were thawed, washed, and resuspended in PBS.

Antibody staining was conducted at 4�C for 20 minutes inPBS. Fc receptorswere blockedby incubation for 15minuteswith 2 mg/mL polyclonal human IgG (Intragam P, CSLLimited). Exclusion of dead cells was using LIVE/DEADFixable Blue (Molecular Probes), 40,6-diamidino-1 2-

Hunn et al.

Clin Cancer Res; 18(23) December 1, 2012 Clinical Cancer Research6448




phenylindole (DAPI; Molecular Probes), or propidiumiodide (Sigma-Aldrich). Data were acquired on an LSRIIflow cytometer or FACSCalibur apparatus (both BD Bios-ciences) and analyzed using FlowJo software (TreeStar Inc.).Compensation was conducted using the appropriate fluor-ophore-labeled antibodies bound to antimouse Ig-coatedparticles (BD Compbeads; BD Biosciences). For nonanti-body stains, compensation was conducted using stainedand unstained populations of positive control cells. Fluo-rescent monoclonal antibodies for the following moleculeswere used: CD1d (clone CD1d42; PE-conjugated), CD14(MjP9; APC) and CD19 (SJ25C1; APC from BD Bios-ciences; CD3 (SK7; APC-H7), CD11c (Bu15; APC), CD16(3G8; FITC), HLA-DR (L243; PerCP) from Biolegend. Alineage 1 cocktail with antibodies for CD3, CD14, CD16,CD19, CD20, and CD56 (BD Biosciences) was used todiscriminate dendritic cells. Human iNKT cells weredetected using CD1dmonomers (NIH Tetramer Core Facil-ity), that had been loaded with a-GalCer and tetramerizedwith streptavidin-PE (BD Biosciences).

In vitro iNKT cell proliferation assayPBMCswere thawed,washed, andplated (5�105PBMCs

perwell in a 96-well plate) in IMDMsupplementedwith 5%

human AB serum (Invitrogen), and either 100 ng/mLa-GalCer or vehicle. After 24 hours, 50 U/mL recombinanthuman IL-2 (Chiron Corporation) was added to each well.After 7 days, live cells were counted with Trypan Blue(Invitrogen) exclusion, and iNKT cells enumerated by flowcytometry.

Analysis of impact of a-GalCer–loaded tumor cells oniNKT cell proliferation and allo-response

Primary GBM cell cultures from 5 patients were eitherpulsed overnight with 200 ng/mL a-GalCer or left untreat-ed, and then g-irradiated on ice (150 Gy). After washing 3times to remove any free a-GalCer, 105 irradiated tumorcells were cocultured with 5 � 105 carboxyfluoresceinsuccinimidyl ester (CFSE)–labeled allogeneic PBMC in96-well plates. After 5 days, CFSE dilution was assessed byflow cytometry.

Assessment of MHC class II expression by GBM tumorcells

Primary GBM cell lines were cultured for more than 48hours with and without 100 U/mL recombinant humanIFN-g . Cells were assessed for HLA-DR expression by flowcytometry as described earlier.

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Figure 1. Glioma/Gal vaccine prevents glioma growth in a mouse model. A and B, mice were vaccinated intravenously with 105 Glioma/Gal cells, unpulsedirradiated tumor cells, or with 200 ng free a-GalCer, and challenged with subcutaneous GL261 7 days later. C, mice were treated intravenously on day 0 with105 Glioma/Gal cells or 200 ng free a-GalCer, or received no treatment. On day 13 104 OVA-specific CD8 T cells (CD45.2þ Va2þ OT-1 cells) weretransferred into all mice. On day 14 some groups were vaccinated intravenously with 200 mg OVA protein and 200 ng free a-GalCer. Plots (bottom) showindividual mice and group means, with representative flow cytometry plots above. D, as for A except some mice were treated with Glioma/Gal/Doxcells. E, mice were vaccinated with 5� 105 Glioma/Gal/Dox cells, Glioma/Gal cells, or unpulsed irradiated tumor cells and challenged with intracranial GL261tumor 7 days later. Four to 6mice per group for all experiments. Data are representative of 2 or 3 independent experiments with similar results except Cwhichwas carried out once. �, P < 0.05; ��, P < 0.01.

Glioma/Gal Vaccine





Statistical analysesBars and error bars depict means and standard error of

the mean except in graphs of observational patient data(Fig. 6 and Supplementary Fig. S7), which depict med-ians and quartiles. Nonparametric tests were used toassess statistical significance. For comparisons of 1 var-iable, the Mann–Whitney test was used for unpaireddata, the Wilcoxon-matched pairs test for paired data,and the Kruskal–Wallis 1-way analysis of variance(ANOVA) for experiments comparing more than 2groups, with Dunn posttest to determine statistical sig-nificance between 2 individual groups. For comparisonsof 2 variables, a 2-way ANOVA with Bonferroni posttestwas used. A log-rank test was used to determine statisticalsignificance between Kaplan–Meier survival curves. Allstatistical analyses were done with Prism 5.0 software(GraphPad Software, Inc.), with P values of <0.05 con-sidered significant (�,P < 0.05; ��, P < 0.01; ��, P <0.001). In studies comparing patient groups, all subjectswith complete data for that particular study were includ-ed. Age-matching was conducted by alternately removingthe oldest or youngest subject from each group until the

difference between the mean ages of the 2 groups was nomore than 2.5 years.

ResultsProphylactic treatmentwithana-GalCer–loadedwholetumor vaccine prevents glioma growth in a mousemodel

Responses to vaccination were studied in a murine glio-ma model in which GL261 cells were implanted by intra-cranial or subcutaneous injection into immunocompetentsyngeneic mice. To generate a vaccine, tumor cells werepulsed overnight with a-GalCer, irradiated, and thenwashed to remove excessa-GalCer ("Glioma/Gal" vaccine).Because iNKT cells are found predominantly in spleen andliver, the vaccine was delivered intravenously to provideaccess to these populations. Unlike some rodent glioma celllines (22), GL261 forms lethal tumors when implantedsubcutaneously. The a-GalCer–pulsed cells were thereforeirradiated to prevent tumor outgrowth and to potentiallyenhance their immunogenicity (23). When used in a pro-phylactic setting, the Glioma/Gal vaccine conferred signif-icant protection against a subcutaneous challenge with

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Figure 2. The a-GalCer–loaded whole tumor vaccine is dependent on the adjuvant function of activated iNKT cells. A, CD1d�/� mice or their CD1dþ/�

littermates were vaccinated with Glioma/Gal and challenged with GL261 subcutaneously 7 days later. Five mice per group, 1 of 3 independent experimentswith similar results is shown. B, serum cytokine levels after intravenous administration of 200 ng free a-GalCer, 105 Glioma/Gal cells, or 105 irradiatedtumour cells. Five mice per group, 1 experiment. C, serum cytokine levels after 5 � 105 Glioma/Gal cells administered intravenously to wild-type(WT) or CD1d�/�mice. Three to 5mice per group, 1 experiment. D, splenocytes were harvested fromWTmice 7 days after vaccination with either Glioma/Galor irradiated tumor cells, and replated in 96-well plates in complete media without restimulation. Cytokine levels in the supernatant of splenocyte cultureswere assessed48hours later. Threemiceper group, 1of 2 independent experimentswith similar results is shown. E,CD1d surface expression byGL261 tumorcells in vitro. Positive and negative controls are WT CD11cþ splenocytes and a fibrosarcoma cell line raised in a CD1d�/� mouse. ��, P < 0.01.

Hunn et al.





GL261 tumor cells (range, 60–100% of mice protected,mean 82%; Fig. 1A and D). Injection of free a-GalCer, orirradiated tumor cells alone, did not confer significantprotection, suggesting that activation of iNKT cells witha-GalCer coordinates an improved response to the tumorantigens in the Glioma/Gal vaccine (Fig. 1A and B). Avaccine consisting of tumor lysate coadministered intrave-

nouslywith freea-GalCerwas equally effective at protectingagainst subcutaneous tumor challenge (data not shown).However, free a-GalCer administered intravenously isknown to cause iNKT cell anergy lasting several weeks(24), potentially restricting its application in prime-boostregimens. To see if the Glioma/Gal vaccine also inducedanergy we vaccinated mice and 2 weeks later assessed

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Figure 3. Thea-GalCer–loadedwhole tumor vaccine elicits aCD4þT-cell–mediated immune response. A,Glioma/Gal vaccinatedmicewere challenged 7dayslater with 106 Lewis lung carcinoma cells subcutaneously. Five mice per group, 1 of 3 independent experiments with similar results is shown. B, Glioma/Galvaccinated mice that survived 60 days after an initial subcutaneous GL261 tumor challenge were rechallenged subcutaneously on the opposite flank.Three to 5mice per group, 1 experiment. C,mice vaccinatedwithGlioma/Gal on day 0were challengedonday 7withGL261 subcutaneously. Somemicewerealso treated with CD4-depleting antibody (left) on days 2 and 4 and then every 3 to 4 days, or with CD8-depleting antibody (right) on days �2 and �1and then every 3 to 4 days until the completion of the experiment. Five mice per group, 1 representative experiment of 3 independent experiments for eachantibody is shown. D, WT, MHC II–deficient, or TAP1-deficient mice were challenged subcutaneously 7 days after Glioma/Gal vaccination. Fivemice per group, 1 experiment for each strain. The ability of GL261 to form tumors in naive TAP1-deficient mice was confirmed separately (data not shown).E, Langerin-DTREGFPmicewere depleted of langerinþ cells by administeringDiphtheria toxin on days�2,�1, 1, and 3, andwere challenged subcutaneouslyon day 7. Some mice were vaccinated with Glioma/Gal on day 0. Five mice per group, 1 of 2 independent experiments with similar results is shown.F, top, surface expression of MHC class II (I-A/I-E) by GL261 in vitro; solid gray, expression after culture in normal media; solid line, after 48 hours exposure toIFN-g ; broken line, isotype stainingof IFN-g–treated cells. Bottom,MHCclass II expression in vivo. Single-cell suspensionswere prepared fromsubcutaneousGL261 tumors harvested from unvaccinated mice. Gated on live CD45.2� cells; solid gray, isotype. Representative results from 2 independent in vitroexperiments and 1 in vivo experiment with 3 mice are shown. �, P < 0.05; ��, P < 0.01.

Glioma/Gal Vaccine





whether a second vaccination consisting of soluble chickenOVA and free a-GalCer could induce expansion of OVA-specific T cells, a process that depends on the activity of iNKTcells (6). Although prior exposure to 200 ng free a-GalCercompletely abrogated the response to the second vaccine,pretreatment with the Glioma/Gal vaccine caused onlypartial attenuation (Fig. 1C). The chemotherapeutic agentdoxorubicin has been reported to increase the immunoge-nicity of tumor cells (25) even at very low noncytotoxicdoses (26). To exploit this phenomenon, we assessedtreatment of a-GalCer–pulsed tumor cells in vitro witha low dose of doxorubicin before irradiation and vacci-nation. Doxorubicin treatment resulted in vaccines with aslightly higher proportion of necrotic cells (Supplemen-tary Fig. S1). There was a trend for this Glioma/Gal/Dox

vaccine to provide improved protection in comparisonwith the Glioma/Gal vaccine (Fig. 1D). Although thisdid not reach statistical significance, this was probablybecause the Glioma/Gal vaccine was already highlypotent and we elected to use doxorubicin treatment ofthe vaccine in all intracranial tumor experiments. Accord-ingly, the Glioma/Gal/Dox vaccine was shown to conferexcellent protection against subsequent intracranialtumour challenge (Fig. 1E).

The a-GalCer–loaded whole tumor vaccine isdependent on the adjuvant function of activated iNKTcells

iNKT cells were absolutely required for the Glioma/Galvaccine to confer tumor immunity, as protection from

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Figure 4. Thea-GalCer–loadedwhole tumor vaccine primes adaptive responses in the lung-draining lymphnodes. A, splenectomisedmicewere vaccinated ornot vaccinated with Glioma/Gal and challenged 7 days later with GL261 subcutaneously. Five mice per group, 1 experiment. B, in vitro cytokine secretionby cells harvested from mice vaccinated 7 days previously, as in Fig. 2D. Five mice per group, 1 experiment. IngLN, inguinal lymph nodes; liver, liverlymphocytes. C, total cell count of mediastinal and inguinal lymph nodes from individual naïve mice and mice vaccinated 7 days previously with 5 � 105

Glioma/Gal. Data pooled from 4 independent experiments all showing the same trend. D, flow cytometry measuring CD44 expression of CD3þCD4þ

cells in mediastinal and inguinal lymph nodes from naïve mice or mice vaccinated 7 days previously with Glioma/Gal. Representative histograms (left) andsummary of 4 to 5 mice per group (right). One of 2 independent experiments with similar results is shown. E, flow cytometry of mediastinal lymphnode cells from naïve mice and from mice vaccinated 7 days previously with Glioma/Gal, gated on live cells. Shown are data concatenated from 3 mice pergroup, from 1 of 2 independent experiments with similar results. �, P < 0.05; ��, P < 0.01; ��, P < 0.001.

Hunn et al.





tumor challenge was not observed in CD1d�/� mice with-out iNKT cells (Fig. 2A). After intravenous treatment withfree a-GalCer, the glycolipid is acquired by resident APCsand then presented via CD1d to activate iNKT cells (27, 28).Activation of iNKT cells provokes release of cytokinesincluding IL-4, IL-13, and IFN-g into the serum. The inter-action with APCs is reciprocal, with the activated iNKT cellsin turn stimulating the APCs to release IL-12p70. Thisinteraction, which involves CD40/CD40L interactions,also promotes the priming of conventional T cells(6, 8). To examine whether similar interactions take placeafter intravenous administration of Glioma/Gal, serumwas collected at different time points after vaccination toanalyse cytokine secretion, and compared with animalsthat received free a-GalCer (Fig. 2B). As expected, in wild-type (WT) mice administered free a-GalCer, high levels of

IL-4, IL-12p70, and IFN-g were detected that peaked at 2,12, and approximately 24 hours, respectively. In contrast,after infusion of Glioma/Gal the systemic release of thesecytokines was much lower, and peaked later in the case ofIL-4 and IL-12p70. Notably, cytokine secretion after Gli-oma/Gal infusion was absent in serum in CD1d�/� mice,confirming that iNKT activation triggered the cytokinerelease observed in WT animals (Fig. 2C). Although thelevels of IL-12p70 were lower after Glioma/Gal comparedwith free a-GalCer (Fig. 2B), the ratio of peak IL-12p70 toIL-4 was higher (approximately 100:1 compared with3:1), possibly indicating vaccine-induced APC activationwith proportionately lower systemic iNKT cell activation.These observations suggest that a-GalCer is presented in aqualitatively different manner in vivo when incorporatedinto the vaccine. As GL261 tumor cells do not expressCD1d (Fig. 2E), and are therefore incapable of stimulat-ing iNKT cells directly, other CD1dþ cell types must haveacquired a-GalCer for presentation to iNKT cells. Cyto-kines were still being secreted in significant quantitiesfrom splenocytes isolated from mice 1 week after vacci-nation, which was not the case in mice that had receivedfree a-GalCer (Fig. 2D).

The a-GalCer–loaded whole tumor vaccine elicits aCD4þ T-cell–mediated immune response

We next defined the effector arms responsible for pro-tection from tumor challenge. Depletion of NK1.1þ cellsafter the priming phase did not abrogate protection(Supplementary Fig. S2A) suggesting that NK cells, whichare typically activated immediately downstream of iNKTcell stimulation, were not important antitumor effectors.The Glioma/Gal vaccine was not capable of protectingagainst challenge with the unrelated Lewis Lung Carci-noma cell line LLC (Fig. 3A), indicating that vaccine-induced immunity was tumor specific. The Glioma/Galvaccine also provided protection against late rechallengewith GL261 cells, indicating immunologic memory (Fig.3B). Transfer of serum to na€�ve mice from vaccinated micethat had survived a tumor challenge did not conferprotection, indicating that antibodies were not likely tobe directly involved in protection (Supplementary Fig.S2B). Experiments carried out in animals depleted ofspecific T cell subsets showed that protection was lost inthe absence of CD4þ T cells, but retained in the absence ofCD8þ T cells (Fig. 3C). This was confirmed in knockoutmice, with protection lost in MHC class II�/� hosts, whichlack CD4þ T cells, and retained in TAP1�/� hosts whichlack CD8þ T cells (Fig. 3D). It was independently shownthat iNKT cells are present and able to respond to a-Gal-Cer in these MHC class II�/� mice (data not shown). Thedominant role of CD4þ T cells was confirmed by theobservation that IFN-g secretion by splenocytes aftervaccination was abrogated by addition of an MHC classII blocking antibody and by depletion of CD4þ cells(Supplementary Fig. S3). Furthermore, supporting a lackof significant involvement of CD8þ T cells in this vacci-nation strategy, protection was retained in the absence of

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Figure 5. The a-GalCer–loaded whole tumor vaccine is effective againstestablished intracranial tumors but requires depletion of regulatory Tcells. A, mice were inoculated intracranially with 5� 103 GL261 cells andvaccinated 6 days later with 5 � 105 Glioma/Gal/Dox cells. Some micealso received anti-CD25 depleting antibody (PC61) intraperitoneally onthe day of tumor implantation. Five to 7 mice per group. The results arerepresentative of 3 independent experiments with similar results, withmean long-term survival across 3 experiments of 43%. B, as for A exceptsome mice were treated only with anti-CD25 antibody. Five mice pergroup, 1 experiment. C, in 1 of the experiments described in A miceunderwent MRI scans on day 20 after implantation. Surviving mice alsounderwent repeat scans on day 81. Shown are gadolinium-enhanced T1-weighted MRI scans of 1 vaccinated mouse at both time points.�, P < 0.05; ��, P < 0.01.

Glioma/Gal Vaccine





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langerinþ APC (Fig. 3E), a cell type recently shown to becritically involved in cross-presentation of blood-borneantigens to CD8þ T cells (29, 30).Given the requirement for CD4þ T cells in conferring

protection, it is notable that expression of MHC class IImolecules by GL261 was negligible in the steady state butreadily induced after exposure to IFN-g in vitro and spon-taneously upregulated in vivo (Fig. 3F). It is possible thatCD4þ T cells directly target tumor cells via MHC class II, ashas recently been suggested (31).

The a-GalCer–loaded whole tumor vaccine primesadaptive responses in the lung-draining lymph nodesInterestingly the Glioma/Gal vaccine conferred full pro-

tection against tumor challenge after splenectomy (Fig. 4A).Thus, despite the spleen being a significant source of thecytokines released after vaccination it was not required foreffective immunity. Different anatomic sites were thereforeexamined for cytokine production to identify possiblealternative locations of APCs mediating the protectiveimmune response. High levels of cytokine production weredetected in the mediastinal lymph nodes (MdLN) of vac-cinated mice, but not in inguinal lymph nodes or liver (Fig.4B). Therewas an increase in theoverall size ofMdLNs and asignificant increase in the proportion of CD4þ T cellsexpressing the activation marker CD44 in the MdLNs ofimmunizedmice thatwas not seen in inguinal lymphnodes(Fig. 4C and D). In addition, we observed iNKT cell acti-vation (Supplementary Fig. S4) and proliferation (Supple-mentary Fig. S5) and an increase in the proportion of MHCclass IIþCD11cþ cells in theMdLNs of vaccinatedmice (Fig.4E). We speculate that the infused tumor cells are filteredby the lung capillary bed where they are taken up by lung-resident or circulating immune cells that traffic to lung-draining lymph nodes.

The a-GalCer–loaded whole tumor vaccine is effectiveagainst established intracranial tumors but requiresdepletion of regulatory T cellsThe efficacy of the Glioma/Gal vaccine was tested in

a therapeutic setting where tumors were implanted intra-cranially, and mice vaccinated 6 days later. Although thevaccine provided effective protection against intracranial

tumors in a prophylactic model (see Fig. 1E), in a thera-peutic setting minimal or no protection was seen aftervaccination in repeated experiments (Fig. 5A). However,in line with previous studies (32–34), we found that partialdepletion of Tregs before vaccination resulted in effectivetreatment, with up to 60% long-term survival after a singlevaccination (range, 29%–60%, mean 43%). The survivalbenefit could not be explained by regulatory T-cell deple-tion alone (Fig. 5B). MRI scans at 20 days postimplantation(14 days post-vaccination) showed enhancing mass lesionsin most vaccinated mice that were radiologically similar to,but smaller than, those seen in unvaccinated mice (Sup-plementary Fig. S6A). Complete resolution of the lesionwasdocumented in 1 vaccinated mouse that exhibited long-term survival (Fig. 5C). To confirm that radiologic abnor-malities were tumors, some vaccinated mice were sacrific-ed on day 20 for histologic examination (SupplementaryFig. S6B). Overall, these data confirm that the vaccine iseffective in prolonging survival and, in some cases eradi-cating established intracranial tumors.

Patients with GBM have functional iNKT cells that canrespond to a-GalCer–loaded GBM tumor cells in vitro

To succeed in the clinic, this vaccination strategy requiresiNKT cells to be functional and present in sufficient num-bers in patients with glioma. We therefore assessed iNKTnumber and function in the peripheral blood of 38 histo-logically confirmed patients with GBM. Of these, 84%werenewly presenting and 95% were taking the corticosteroiddexamethasone at the time of blood donation (mean dailydose 13.7 mg). No patients had received chemotherapywithin 6months. Because iNKT cell counts decline with age(35), the analysis in patients with GBMwas compared withage-matched healthy controls. Flow cytometry with fluo-rescent a-GalCer–loaded CD1d tetramers showed that theproportion of circulating T cells that were iNKT cells wasslightly increased in thepatient group (Fig. 6A).However, aspreviously described (36), the patients with GBM werelymphopenic relative to healthy controls, so that the abso-lute iNKT cell counts were not significantly differentbetween the 2 groups (Fig. 6A, Supplementary Fig. S7).When we examined CD1d expression in some of the majorAPC populations in blood that could present a-GalCer to

Figure 6. The cell populations required for a-GalCer to be an effective adjuvant are preserved in patients with GBM. A, flow cytometry of peripheral blood frompatients with histologically proven GBM and from an age-matched population of healthy volunteers, showing (left) iNKT cells as a percentage of total CD3þ

CD19� lymphocytes. Mean ages of the patient and control groups were 56.1 years (range, 44–74) and 58.6 years (range, 27–78), respectively.Middle, absolute iNKT cell number, estimated bymultiplying% iNKT of total lymphocytes (on flow cytometry) and a contemporaneous automated peripheralblood lymphocyte count. Mean ages of the patient and control groupswere 59.8 (range, 44–74) and 61.1 (range, 48–70), respectively. Right, absolute numberof myeloid dendritic cells (mDC) in all subjects with sufficient remaining PBMC, estimated bymultiplying%mDC of total live PBMC (on flow cytometry) and acontemporaneous automated peripheral blood count of lymphocytes þ monocytes. mDC were defined as lineage marker low, CD11c high,HLA-DR high cells. Mean ages of the patient and control groups were 58 and 64 years, respectively. B, PBMC from patients with GBMand healthy volunteerswere cultured in vitro with a-GalCer for 7 days. Absolute iNKT cell number before and after coculture (left and center); fold change in absoluteiNKT cell number (right). C, 5 GBM cell lines were pulsed or not pulsed overnight with a-GalCer, then irradiated, washed, and cocultured for 5 days withCFSE-labeled PBMC from an allogeneic healthy volunteer. As a measure of proliferation, %CFSE cells of total cells was measured by flow cytometry.Left, representative flow cytometry plot of cell line 10/05, gated on live CD3þCD19� lymphocytes. Right, mean and SEM of triplicate wells for iNKT cells (top)andTetramer�Tcells (bottom).Bonferroni posttestP valuesof a2-wayANOVAare shownon the lower graph.D, surface expressionbyGBMcell lines ofCD1d(top) and MHC class II (HLA-DR) below as assessed by flow cytometry. Top, solid gray is isotype, solid line is CD1d antibody. C1R-CD1d, CD1d-transfectedcell line for positive control. Bottom, solid gray is expression after culture in normal media; solid line, after 48 hours exposure to IFN-g ; broken line, isotypestaining of IFN-g–treated cells. �, P < 0.05; ��, P < 0.01.

Glioma/Gal Vaccine





iNKT cells, patients had reduced numbers of circulatingmyeloid dendritic cells but monocyte numbers were pre-served and CD1d-expression by both cell types was equiv-alent to cells from healthy controls (Fig. 6A, SupplementaryFig. S7). Furthermore, iNKT cells could be expanded fromPBMC of both patients and healthy controls in response toa-GalCer in vitro, albeit to a slightly reduced level in patients(Fig. 6B). Thus, the iNKT/DC axis seems to be sufficientlyintact in patients with GBM for an a-GalCer–based vaccineto be effective.

To confirm this, a-GalCer was loaded onto primarytumor cell lines derived from 5 patients with GBM, whichwere then irradiated,washed, and cocultured for 5dayswithCFSE-labeled PBMC from a healthy donor. Proliferation ofdonor iNKT cells was observed in all cases as loss of CFSEfluorescence on cells identified with a-GalCer–loadedCD1d tetramers (Fig. 6C). Proliferation of tetramer-nega-tive cells was also observed, which occurs in this assaybecause of HLA-mismatch between tumor cells and donorPBMC. In 1 case this alloresponse was significantlyenhanced when the tumor cells had been pulsed witha-GalCer, providing "proof-of-principle" that a vaccinebased on a-GalCer–loaded tumor cells can enhance pro-liferation of non-CD1d restricted T cells as well as iNKT cells(Fig. 6C). The ability of a-GalCer–pulsed tumor cells tostimulate iNKT cells did not require the tumor to expressCD1d because, as previously observed in themurine GL261line, no surface staining of CD1d was seen on any of theprimary GBM lines tested (Fig. 6D).

Finally, we confirmed that expression of MHC class IImolecules after exposure to IFN-g also occurred in thehuman primary GBM cell lines (Fig. 6D), which may there-fore also be susceptible to direct targeting by CD4þ T cells.

DiscussionA simple vaccine composed of irradiated autologous

tumor cells pulsed with a-GalCer, an adjuvant that stimu-lates iNKT cells, is an effective treatment against tumors in amurine gliomamodel. This vaccinedesign is also effective inmurine models of melanoma and acute myeloid leukemiaand is therefore not cell line–specific (unpublished datafrom our laboratory, with permission from T. Petersen, J.Gibbins, and C. Tan). The Glioma/Gal vaccine depends onthe reciprocal activation of iNKT cells and APCs and elicitsan adaptive immune response dominated by CD4þ T cellsthat is tumor-specific and long lasting. Although evidence ofcellular activation was observed in the spleen, the MdLNwas shown to be an important anatomical site of T-cellpriming. We have further shown that the principal effectorsnecessary for the success of such a vaccine are present andfunctional in patients with high-grade glioma.

In some animal models, a-GalCer has been shown tohave significant antitumor effects when injected intrave-nously as a sole agent (37). These effects were largely due tostimulation of the innate immune system, particularly NKcells (38), but enhancement of spontaneous adaptiveresponses was also observed in some tumor-bearing ani-mals (39, 40). However, clinical trials in human patients

with cancer, using either free a-GalCer or a-GalCer loadedonto dendritic cells, have achieved limited clinical benefitdespite evidence of iNKT activation (7, 41, 42). All of theseclinical trials used a-GalCer without coadministration ofantigen. Thus, the adjuvant function of stimulating iNKTcells has yet to be tested directly in patients.

In animal models it has been reported that simple coad-ministration of a-GalCer with microbial products (43) orsoluble antigens (6, 8) can significantly enhance antigen-specific T-cell–and antibody–mediated responses. Therehave alsobeen reports that injectionofa-GalCerwithwholetumor cells can provoke effective antitumor responses,although different effectors were invoked depending on themodel studied. As reported here for glioma, a previousreport of an a-GalCer–pulsed lymphoma cell vaccineshowed that efficacy was dependent on CD4þ T cells, anddid not require CD8þ T cells (11). Similarly, CD4þ T cellscould mediate full immunity in response to irradiated B16melanoma cells administered intraperitoneally with freea-GalCer (10). In contrast, the antitumor activity of ana-GalCer–pulsed live B16 tumor cell vaccine was abrogatedin both MHC class I�/� and MHC class II�/� animals (12).These contradictory findings suggest the type of responseinduced is a consequence of the tumor antigens presentedand/or the timing and manner in which the infused tumorcells die. Shimizuand colleagues’ analysis of their live tumorcell vaccine showed that the injected a-GalCer–loadedtumor cells were killed by iNKT cells and NK cells, therebyproviding a reservoir of antigens for uptake by residentAPCs,withCD8aþdendritic cells in spleen being importantfor cross-presentation (12, 44). In contrast, in the gliomastudy presented here, the tumor cells in the Glioma/Galvaccine were CD1d-negative and, although alive at the timeof injection, had been lethally irradiated. These differentpathways to cell death are likely to result in differences inthe timing of cell death and in the nature of the danger-associated molecular patterns and ligands for phagocytosisreceptors expressed by the dying tumor cells. This in turnmay determine the location, subset and maturation stateof the APCs that take up the tumor cells and shape theimmune response.

Because the Glioma/Gal vaccine was infused into thecirculation, we anticipated a role for APCs in the spleen,and for CD8aþ dendritic cell in particular, as these APCshave been shown to have a selective capacity to take up andpresent antigen from circulating dying cells (12, 45). Sur-prisingly, in our model splenic dendritic cell were redun-dant. Instead we found evidence that the MdLNs was animportant site of T-cell priming. Two major CD11cþ den-dritic cell subsets have been described in the lung withrecent studies suggesting the 2 populations have distinctfunctional roles (46). CD11blo CD103þ dendritic cells,which are langerinþ, present antigen to CD8þ T cells. Incontrast, CD11bhi CD103� dendritic cells preferentiallyinduce proliferation of CD4þ T cells (46, 47). Given thatantitumor responses to Glioma/Gal were not abrogatedwhen langerinþ cells were depleted, we speculate thatinfused tumor cells lodging in the pulmonary capillary bed

Hunn et al.





might be preferentially taken up by CD11bhi CD103�

dendritic cells that in turn generate the predominantlyCD4þ T-cell–mediated response observed. However, a rolefor other cell types of the monocyte/macrophage/dendriticcell lineage, either circulating in the blood or resident in theMdLNs, cannot be excluded. It has recently been reportedthat CD169þ macrophages in the subcapsular sinus oflymph nodes are able to take up a-GalCer and activateiNKT cells (48), phagocytose dead tumor cells arriving viaafferent lymphatics, and act as APCs to initiate antitumorimmune responses (49).Tregs progressively accumulate within GL261 tumors in

vivo (50). Not surprisingly, in this study established intra-cranial tumors were resistant to therapeutic vaccinationunless mice were also treated with an anti-CD25 depletingantibody. Although it has also been reported that activatediNKT cells can unexpectedly expand Tregs (51), it is unlikelythat the Glioma/Gal vaccine induced Treg activity becauseTreg depletion was not required for successful prophylacticvaccination. In addition, we have previously shown that ana-GalCer–pulsed dendritic cell vaccine did not alter Tregnumber or function (13).Tregs are just one of several immuno-suppressive and

immuno-evasion mechanisms hindering immunotherapyfor high-grade glioma (52). In this study, 1 of 5 a-GalCer–pulsed GBM cell lines failed to stimulate iNKT cell expan-sion or elicit an alloresponse. Studies in progress suggestthis might have been due to a tumor-derived soluble factorthat causes a general inhibition of T-cell proliferation (datanot shown). Attenuation of immuno-suppressive mechan-isms will be a vital component of successful glioma immu-notherapies in the future.There is still a lack of identified glioma-associated anti-

gens that are consistently expressed and/or capable ofeliciting strong antitumor responses. For this reason, per-sonalized vaccines incorporating autologous tumor extractshave been the most common immunotherapeutic strategytested inpatientswith glioma.Other benefits of usingwholetumor-based vaccines include applicability to all HLA types,the provision of MHC classes I and II epitopes, and thestimulation of a broad polyclonal response that reduces therisk of immune escape. Most studies of autologous tumor-based vaccines in patients with glioma have involved load-ing antigen onto autologous monocyte-derived dendriticcells. However, generating dendritic cells frompatients withadvanced disease can be problematic and there are manyuncertainties surrounding the optimum phenotype of theinfused dendritic cells (53). Thus, a vaccine that deliverswhole tumor-derived antigens to endogenous dendriticcells may be a better approach than classical dendritic celltherapy, and intact autologous tumor cells constitute aready-made vehicle for this purpose.The use of irradiated whole tumor cells in anticancer

vaccines raises a concern about possible induction of auto-immunity, particularly if the treatment also includes delib-erate attenuation of peripheral tolerance mechanisms suchas Treg.However, we sawnohistologic evidence of immunecell infiltration in the normal brain parenchyma of treated

mice distant from the tumor site (data not shown). Anotherconcern is the risk of acute toxicity associated with theintravenous infusion of irradiated tumor cells. Toxicityencountered in this study at higher doses of the vaccinewas effectively abrogated by pretreating mice with intrave-nous heparin, suggesting that tissue factor or some otherpro-coagulant factor was responsible (54, 55).

Reduced numbers of iNKT cells and functional iNKT celldefects that could impact on the use of a-GalCer as animmune adjuvant have been reported in patients withadvanced solid cancers (14, 15, 56). However, GBM seemsto be an exception in this regard. A small study of iNKT cellsin the blood of 9 patients with gliomas of various grades(57) reported nodifference in iNKT cell function or number(expressed as percentage of total lymphocytes) comparedwith control subjects. Here a much larger and more homo-geneous group of patients was examined, with iNKT cellnumber and function compared with age-matched healthycontrols. All patients had a histologic diagnosis of GBM,and almost all were taking moderate to high doses oflymphotoxic corticosteroid. Importantly, we found no dif-ference in the absolute iNKT cell count in peripheral bloodin patients compared with controls, even though thepatients were relatively lymphopenic. The relative sparingof iNKT cells in patients might reflect a greater resistance ofthis cell type to corticosteroid (58). We did find somedeficiencies in the iNKT/DC axis in patients. A mildimpairment of iNKT cell expansion might be explained bycorticosteroid treatment (59). We also observed reducednumbers of circulating myeloid dendritic cells but circulat-ing CD14þ monocytes, known to be capable of differenti-ating into mature dendritic cells (60), were present innormal quantities and expressed normal levels of CD1d.Taken together, these data suggest there are nomajor defectsin the iNKT/DC axis in patients with high-grade glioma andthat a-GalCer–pulsed irradiated autologous glioma cellscould be an effective vaccine in human patients.

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

Authors' ContributionsConception and design: M.K. Hunn, K.W.R. Broadley, T. Petersen, M.J.McConnell, I. HermansDevelopment of methodology: M.K. Hunn, K.W.R. Broadley, P.M. Fergu-son, T. Petersen, M.J. McConnell, I. HermansAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): M.K. Hunn, K.J. Farrand, K.W.R. Broadley, P.M.Ferguson, R.J. Miller, C.S. Field, I. HermansAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): M.K. Hunn, K.J. Farrand, K.W.R. Broad-ley, P.M. Ferguson, C.S. Field, M.J. McConnell, I. HermansWriting, review, and/or revision of the manuscript: M.K. Hunn, K.W.R.Broadley, P.M. Ferguson, I. HermansAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M.K. Hunn, I. HermansStudy supervision: M.K. Hunn, I. Hermans

AcknowledgmentsThe authors wish to acknowledge that the Cd1d monomer was obtained

from the NIH Core Tetramer Facility. We also thank the personnel of theBiomedical Research Unit of the Malaghan Institute of Medical Research foranimal husbandry, G.F. Painter and C. Hayman for providing the a-GalCer,

Glioma/Gal Vaccine





and A. Slocombe and the Radiology Department of Wellington Hospital fortheir assistance with obtaining the MRI scans.

Grant SupportThis work was supported by the Cancer Society of New Zealand

(grant 08/10), the Neurological Foundation of New Zealand, and theWellington Medical Research Foundation Inc. M.K. Hunn was supportedby a New Zealand Health Research Council Clinical Fellowship, the

Royal Australasian College of Surgeons, and the Surgical ResearchTrust.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 29, 2012; revised August 31, 2012; accepted September16, 2012; published OnlineFirst November 12, 2012.

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2012;18:6446-6459. Published OnlineFirst November 12, 2012.Clin Cancer Res Martin K. Hunn, Kathryn J. Farrand, Kate W.R. Broadley, et al. That Stimulates NKT Cells Is an Effective Treatment for GliomaVaccination with Irradiated Tumor Cells Pulsed with an Adjuvant

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