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Human Cancer BiologySee commentary by Choi et al., p. 6086

IDOExpression inBrain Tumors Increases theRecruitment ofRegulatory T Cells and Negatively Impacts Survival

Derek A. Wainwright, Irina V. Balyasnikova, Alan L. Chang, Atique U. Ahmed, Kyung-Sub Moon,Brenda Auffinger, Alex L. Tobias, Yu Han, and Maciej S. Lesniak

AbstractPurpose:Glioblastomamultiforme (GBM) is an aggressive adult brain tumorwith a poor prognosis.One

hallmark of GBM is the accumulation of immunosuppressive and tumor-promoting CD4þFoxP3þGITRþ

regulatory T cells (Tregs). Here, we investigated the role of indoleamine 2,3 dioxygenase (IDO) in brain

tumors and the impact on Treg recruitment.

Experimental Design: To determine the clinical relevance of IDO expression in brain tumors, we first

correlated patient survival to the level of IDO expression from resected glioma specimens. We also used

novel orthotopic and transgenic models of glioma to study how IDO affects Tregs. The impact of tumor-

derived and peripheral IDO expression on Treg recruitment, GITR expression, and long-term survival was

determined.

Results:Downregulated IDO expression in glioma predicted a significantly better prognosis in patients.

Coincidently, both IDO-competent and deficient mice showed a survival advantage bearing IDO-deficient

brain tumors,when comparedwith IDO-competent brain tumors.Moreover, IDOdeficiencywas associated

with a significant decrease in brain-resident Tregs, both in orthotopic and transgenicmouse gliomamodels.

IDO deficiency was also associated with lower GITR expression levels on Tregs. Interestingly, the long-term

survival advantage conferred by IDO deficiency was lost in T-cell–deficient mice.

Conclusions:These clinical andpreclinical data confirm that IDOexpression increases the recruitment of

immunosuppressive Tregs that lead to tumor outgrowth. In contrast, IDO deficiency decreases Treg

recruitment and enhances T-cell–mediated tumor rejection. Thus, the data suggest a critical role for

IDO-mediated immunosuppression in glioma and support the continued investigation of IDO–Treg

interactions in the context of brain tumors. Clin Cancer Res; 18(22); 6110–21. �2012 AACR.

IntroductionGlioblastoma multiforme (GBM) is the most common

and aggressive form of primary brain cancer. Even aftersurgical resection, irradiation, and chemotherapy, themedi-an survival for patients with GBM remains at only 14.6months, with 26%of patients alive after 2 years (1). Becauseof the invasiveness of GBM, it remains a significant chal-lenge to resect all of the tumor cells during surgery. Theincomplete surgical removal likely contributes to the highrate of relapse in patients. An additional factor that con-tributes to the poor prognosis in GBM is the potentlyimmunosuppressive tumor environment. The induction

and/or maintenance of immunosuppression in GBM isdue in part to the infiltration and accumulation of thehighly immunosuppressive regulatory T cells (Tregs; CD4þ

FoxP3þCD25þrefs. 2–6).Thepathogenic roleofTregs inbrain tumorshaspreviously

been reported by several independent groups (3, 5, 7–9).Tregs constitutively express high levels of the receptor, glu-cocorticoid-induced tumor necrosis factor receptor–relatedprotein (GITR; ref. 10), suppress the functions of antigen-presenting cells (APC) via the expression of immunosup-pressive cytokines, interleukin-10 (IL-10), and transforminggrowth factor-beta (11) and arise from thymus-derived (nat-ural; nTreg) or induced (iTreg; converted from a CD4þ

FoxP3� conventional T cell) progenitors (12). Specific toGBM, Tregs accumulate at high levels (2, 7), universallyexpress GITR (4), and are predominantly thymus-derived(5). Several factors have been implicated to be critical forrecruiting Tregs to glioma including the Treg-recruitingchemokines,CCL2andCCL22(13), although there areotheragents that may be involved (14). Moreover, while selectchemokines control Treg recruitment, a "master" centralregulator orchestrating Treg infiltration and/or accumulationhas yet to be implicated in the context of CNS malignancy.

Authors' Affiliation: The Brain Tumor Center, The University of Chicago,Chicago, Illinois

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

Corresponding Author: Maciej S. Lesniak, The Brain Tumor Center, ThePritzker School of Medicine, The University of Chicago, 5841 S. MarylandAve., Chicago, IL 60637. Phone: 773-834-4757; Fax: 773-834-2608;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-2130

�2012 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 18(22) November 15, 20126110

on February 9, 2021. © 2012 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst August 29, 2012; DOI: 10.1158/1078-0432.CCR-12-2130

http://clincancerres.aacrjournals.org/

Indoleamine 2,3 dioxygenase (IDO) is a cytoplasmicenzyme that catabolizes the essential amino acid, trypto-phan, to the metabolite, kynurenine (15). In vitro, previouswork has shown that GBM cells can be induced to expressIDO (16) and that this induction may be associated withtumorigenesis (17), although this had not been shown invivo before this study. Moreover, IDO has been shown topotently inhibit T-cell activation and/or proliferation (18),while also regulating the recruitment (19), expansion (20),and activation of Tregs (21) through an APC-dependentmechanism. On the basis of these observations, it is nosurprise that dendritic cell-expressed IDO has been posi-tively associated with promoting tumorigenesis in tumorsthat localize outside of the central nervous system (CNS;ref. 22). However, given the immunospecialization of APCsin the brain (23), as well as previous evidence indicatingthat GBM cells express IDO (24), we hypothesized that IDOexpression by glioma cells, rather than peripheral cells,would promote Treg accumulation and tumor outgrowth.Herein, we present our comprehensive in vivo-based

investigation showing the role of IDO in brain tumors. Weshow that the downregulation of IDO in human glioma isassociated with a significant survival advantage in patients.Using the GL261 orthotopic and/or RasB8 transgenicmouse glioma models, we also show the impact of IDOcompetency on tumor-infiltrating Tregs in wild-type (WT)and IDO-deficient (IDO�/�) mice bearing IDO-competentand -deficient brain tumors, in addition to the analysis ofRasB8mice with variable IDO competency. Orthotopically,we show that IDO deficiency, specifically in tumor cells, isassociatedwith a significant long-term survival advantage inimmunocompetent mice, regardless of IDO expression inthe periphery. This survival benefit is associated with a

significant decrease in the level of brain tumor infiltratingTregs. Similarly, the lack of IDO expression in RasB8mice isalso associatedwithdecreased Treg recruitment to the brain,confirming that the effects of IDO expression on Tregrecruitment are not exclusive to an orthotopic model.Furthermore, there is an association between the expressionof tumor-derived IDO andGITR on infiltrating Tregs. Final-ly, we identify a contextual role for the effects of IDO on Tcells as CD4-, CD8-, and Rag1-deficientmousemodels withIDO-deficient brain tumors lose the long-term survivaladvantage. This work advances our understanding of therole of IDO in human glioma and provides further under-standing into the interaction between IDO and Tregs in thecontext of brain tumors.

Materials and MethodsMice, procedures, and murine glioma cell line

C57BL/6 (WT; Cat# 000664), IDO�/� (Cat# 005897),CD4�/� (Cat# 002663), CD8�/� (Cat# 002665), andRag1�/� (Cat# 002216) mice were obtained from JacksonLaboratories, maintained in the University of ChicagoCarlson Barrier Facility and intracranially injected betweenthe ages of 6 and 8 weeks. Hemizygous transgenic RasB8mice were obtained from Dr. Abjihit Guha (Hospital forSick Children, Toronto, Canada), endogenously expressinga single allele of oncogenic V12HA-ras and maintained inour laboratory. The RasB8 mice were backcrossed to a WT(IDOþ/þ) or IDO-deficient (IDO�/�) background. RasB8mice were analyzed at 5 months postnatal and/or whensymptomatic onset occurred including abnormal fur coat,hunched posture, lack of normal motion/activity andweight loss. All mouse strains used in the included studieswere on the C57BL/6 background and were provided auto-claved food pellets and water ad libitum. All surgical proce-dures were completed in accordance with NIH guidelineson the care and use of laboratory animals for researchpurposes. Mice were euthanized by cervical dislocation.GL261 cells were obtained from NCI Frederick NationalTumor Repository Lab and cultured inDulbecco’s ModifiedEagle’s Media supplemented with 10% fetal calf serum aswell as streptomycin (100 mg/mL) and penicillin (100 U/mL) at 37�C in a humidified atmosphere of 95% air/5%CO2. No authentication of the cell line was conducted.Details about the genetic modifications of the GL261 cellline are enclosed in the supplementary methods and Sup-plementary Fig. S1. All cell culture products were purchasedfrom Gibco Invitrogen. Intracranial injection proceduresare described further in the supplementary methods.

Flow cytometryT-cell isolation and stainingwere conducted as previously

described (5, 6; and detailed in the supplementarymethods).

Immunofluorescence and histologyIDO�/� mice were intracranially injected (ic.) with 4 �

105 Vc or IDOkd GL261 cells, euthanized at 2 weeks post-injection, followed bybrain extraction andflash freezing [in

Translational RelevanceEffective immunotherapy of glioblastomamultiforme

(GBM) remains a significant challenge because of thestrong immunosuppression induced by the tumor. Oneof the immunosuppressive barriers in GBM is the resultof a significant recruitment and accumulation of CD4þ

FoxP3þ regulatory T cells (Tregs). Moreover, as indolea-mine 2,3 dioxygenase (IDO) has been implicated in theregulation of Treg function and expansion, we sought todetermine its impact in the context of glioma, given theprogressive accumulation of Tregs during brain tumorprogression. Here, we show that IDO expression is aprognostic factor in glioma patients. Likewise, tumor-derived, rather than peripheral IDO, increases Tregrecruitment and decreases survival. Reciprocally, IDOdeficiency in glioma leads to long-term survival associ-ated with a significant decrease in Treg infiltration. Thiswork provides both clinical and preclinical rationale forthe therapeutic inhibition of IDO in brain tumors as afuture treatment for patients with incurable forms ofbrain cancer.

IDO Regulates Treg Infiltration in Brain Tumors

www.aacrjournals.org Clin Cancer Res; 18(22) November 15, 2012 6111




62.5% n-Butyl Bromide (Fisher Scientific) þ 37.5% 2-methylbutane (Fisher Scientific) surrounded by crusheddry ice]. Frozen brains were sectioned on the Micromcryostat with vacutome system (Thermo Scientific) with atemperature of�24�C and immersed into Tissue Teck O.C.T. Compound (Sakura Finetek USA, Inc.) at 8 mm intervalsand thaw-mountedontoprecleaned SuperFrost slides (Fish-er Scientific). Sections were postfixed with 4% paraformal-dehyde, blocked for nonspecific staining with 10% bovineserum albumin (BSA; A4503; Sigma-Aldrich) in PBS for 1hour. Sections were incubated overnight with mouse anti-GFAP alexa fluor 488 (131-17719; Invitrogen) and rat anti-CD3-AF647 (145-2C11; Ebioscience) in PBS with 1% BSAat 4�C. Sections were washed extensively (PBS), 1 drop ofglycerolwas added and the slideswere cover slipped. Imagesof antibody-stained sections were captured using the Sp5Tandem Scanner 2-photon confocal microscope (LeicaMicrosystems) running LAS AF software (Leica Microsys-tems) using the argon and red NeHe laser lines. Fluorescentimages were captured using the �20 objective. For detailson the method of tumor quantification, see the Supple-mentary methods.

Patient datasets and data analysisAll glioma patient data was publicly available in deiden-

tified form and obtained from the NCI Repository forMolecular Brain Neoplasia Data (REMBRANDT) data-base (https://caintegrator.nci.nih.gov/rembrandt/; ref. 25),using the data available on April 18, 2012. Thus, noInstitutional Review Board approval was needed. Therewere 343 glioma patient samples that were correlatedbetween IDO expression and overall survival. Althoughall of the patient samples are represented in the Kaplan–Meier Survival Plot, not all of the patients in the databasewere identified for glioma subtype. Thus, only thosepatients that were positively identified as astrocytoma orGBM were used in the analysis for calculating the per-centage of patients with upregulated, intermediate ordownregulated IDO expression. The differences betweengroups were analyzed by log-rank P value. The graphswere created using Rembrandt data for Affymetrix probes210029 using the Lowest Geometric Mean Intensity andassociated survival data. Up- or down-regulation amongglioma specimens refers to a twofold (or greater) changein IDO expression, when compared with specimens fromnonglioma patients.

Statistical analysisSurvival curves were calculated according to the method

of Kaplan–Meier. Overall, survival is defined as the timefrom injection of GL261 cells to day 150 of the time course.The P valuewas obtained by log-rank statistical analysis andwas considered significant when P � 0.05. For nonsurvivalcurves, data are presented as� SEMandwere analyzed by 2-way ANOVA, 1-way ANOVA, or the 2-tailed Student’s t testand a P value � 0.05 were considered significant. Post-hocanalyses were conducted using the Newman–Keuls methodfor multicomparison procedures. All analyses were con-

ducted using GraphPad Prism version 4.00 (GraphPadSoftware, Inc.).

ResultsUpregulationof IDO inglioma is associatedwithapoorprognosis

Previous work has shown that the upregulation of glio-ma-expressed tryptophan dioxygenase, an enzyme associ-ated with mediating tryptophan catabolism, is positivelycorrelated to an earlier time of death in patients (26).However, whether other enzymes that mediate tryptophancatabolism, such as IDO, have a similar correlation betweenpatient survival and expression pattern has not been deter-mined. Using the REpository ofMolecular BRAinNeoplasiaDaTa (REMBRANDT) and on the basis of a sample size ofdata taken from 343 glioma patients, we analyzed themolecular expression levels for IDO in glioma specimensand correlated those data to patient survival (Fig. 1A). IDOexpression was upregulated (triangles; n ¼ 75), intermedi-ately expressed (squares; n ¼ 215) and downregulated(circles; n ¼ 53) in 75, 215, and 53 patients, respectively.The overall survival of patients with upregulated, interme-diate, and downregulated expression in glioma specimenswas 24.9 � 2.76, 34 � 2.71, and 44.3 � 6.21 months,respectively. Moreover, glioma upregulated for IDO wassignificantly correlated to an earlier average time ofdeath (postdiagnosis), when compared with patients withintermediate (P < 0.05)- and downregulated (P < 0.005)-expressing glioma. To better understand whether theincreased IDO expression in glioma was associated witha particular grade of astrocytoma, we also investigated thepercentage of grades II, III, and IV astrocytoma associatedwith upregulated-, intermediate-, and downregulated-IDOexpression. Figure 1B shows that of the IDO-upregulatedspecimens, 17%, 10%, and 73% of specimens wereassociated with astrocytoma grades II, III, and IV,respectively. Figure 1C shows that of the IDO-intermedi-ately expressed specimens, 22%, 16%, and 62% of speci-menswere associatedwith astrocytoma grades II, III, and IV,respectively. Figure 1D shows that of the IDO-downregu-lated specimens, 29%, 20%, and 50% were associated withastrocytoma grades II, III, and IV, respectively. Furtheranalysis correlating the absolute expression level of IDOwith survival, age of diagnosis and Karnofsky score, amonggrade(s) II, III, and IV astrocytoma, is presented in Supple-mentary Fig. S2. Collectively, the data show that upregu-lated IDO expression in glioma predicts a poor prognosis inpatients and that this expression level tends to predominatein high-grade glioma; although it is not an absolute indi-cator of a GBM diagnosis.

Peripheral IDO expression neither affects survival norT-cell levels in brain tumors

Previous work has shown that IDO expression by den-dritic cells (DC) plays a critical role in regulating the anti-tumor immune response (22). Coincidentally, it has alsobeen shown that CNS-resident glia express IDO under

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Clin Cancer Res; 18(22) November 15, 2012 Clinical Cancer Research6112




neuroinflammatory conditions (27–29). However, noinvestigation has determined whether peripheral IDO is acritical source of immune regulation in the context of braintumors. To investigate whether non-tumor–derived IDOplays a critical role in regulating T-cell levels in braintumors, we ic. GL261 cells into the brain of WT or IDO-deficient (IDO�/�) mice. Figure 2A and B indicates thatperipheral (non–tumor-derived) IDO did not significantlyaffect the frequency nor absolute number of CD4þ-, CD8þ-,or CD4þFoxP3þ regulatory T cells in the brain or drainingcervical lymph nodes (dLN) at 3 weeks post-ic. To deter-mine whether peripheral IDO expression is required toaffect overall survival from brain tumors, we ic. injectedGL261 cells into the brain ofWTor IDO-deficient (IDO�/�)

mice and found an average survival time of 30 and 28 days,respectively (Fig. 2C). Collectively, these data suggest thatthe peripheral expression of IDO neither affects the T-cellresponse in the brain tumor and draining lymph nodes nordoes it affect the overall survival.

Tumor-derived IDO decreases survival and increasesTreg recruitment in brain tumors

As our previous data show that peripheral IDO is unim-portant to the level of T-cell infiltration in brain tumors, wenext determined whether tumor-derived IDOwas involved.We comparedWTmice ic. with IDO-competentGL621 cells[transduced with a scrambled shRNA (vector control; Vc)]or with IDO-deficient GL261 cells [transduced with IDO-

Figure 1. The upregulation ofIDO in glioma is associated with adecreased overall lifespan inpatients. Data was isolated from theREMBRANDT and represents theanalysis of 343 different gliomapatients. A, the Kaplan–Meiersurvival curve for IDO1 was plottedfor upregulated (triangle),intermediate (square), anddownregulated (circle) expressionover time. Upregulated (B),intermediate (C), and downregulated(D) IDO expression in glioma wascorrelated to the percentage ofgrade II (Astro II), grade III (Astro III),or GBM (grade IV astrocytoma).�, P < 0.05; ��, P < 0.005.

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specific shRNA (IDOkd)]. As shown in Fig. 3A and B, theinhibition of IDO had profound effects on the levels ofbrain tumor-infiltrating T-cell levels. As the ratio of Tregs:effector T cells has been shown to reflect the overall likeli-hood of an effective antitumor response, we analyzed boththe frequency of, as well as the absolute numbers of T cellsin thebrain. In thefirstweekpost-ic. injection, therewerenosignificant changes in the frequency and absolute cell num-ber of T cells in the brain. However, by 2 weeks post-ic.injection, the level of Tregs were significantly increased inthe IDO competent, when compared with IDO-deficientbrain tumors (P < 0.05). This difference was furtherincreased at 3 weeks post-ic. injection (P < 0.01). However,this effect was highly localized as there were no significantdifferences in Treg levels in the tumor-draining lymphnodes or spleen (Supplementary Fig. S3). Also, in contrastto the increased level of Tregs in the brain, the frequency ofCD8þ T cells decreased at 2 and 3weeks post-ic. injection inIDO competent, when compared with the IDO-deficientbrain tumors. Collectively, these data suggest that theexpression of IDO by brain tumors increases the ratio ofimmunosuppressive Tregs to cytotoxic CD8þ T cells.

To understand the functional importance of the T-cellinfiltration, we also studied the interaction between IDOand T cells using WT, CD4-deficient (CD4�/�; CD4þ T cell-deficient), CD8-deficient (CD8�/�; CD8þ T-cell–deficient),and recombinase activating gene 1-deficient (Rag1�/�; defi-cient for functional T andB cells)micewith IDO-competentand IDO-deficient brain tumors. Figure 3C shows that WT(immunocompetent) mice with IDO-competent braintumors have an average survival of 30 days post-ic. injec-tion, whereas most WTmice ic. injected with IDO-deficienttumors survive until 150 days (when the study was termi-nated; P < 0.001). In contrast, T-cell–deficient mice wereincapable of mediating long-term survival. CD4�/� micewith IDO-competent and IDO-deficient brain tumors sur-vive an average of 20 and 34.5 days post-ic. injection,respectively. Similarly, CD8�/� mice with IDO-competentand IDO-deficient brain tumors survive an average of 21.5and 31 days post-ic. injection, respectively. Finally, Rag1�/�

mice with IDO-competent and IDO-deficient brain tumorssurvive an average of 28 and 33 days post-ic. injection,respectively. Taken together, these data show that braintumor-derived IDO increases Tregs while simultaneously

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Figure2. Peripheral IDOdeficiencyhas noeffect onbrain tumor-infiltrating Tcell levels andsurvival.WT (white bars) or indoleamine 2,3dioxygenase1-deficient(IDO�/�; black bars) mice were ic. injected with 4� 105 normal GL261 cells. The frequency and absolute numbers of (A) total CD4þ T cells, CD8þ T cells, andCD4þFoxP3þ regulatory T cells isolated from thebrain andbilateral deep and superficial cervical dLNof tumor-bearingmicewere analyzed at 3wp-ic. All T cellpopulationswere initially identifiedby the expressionofCD3.B, representative flowcytometric plots show thegating strategyused for the identificationof totalCD3þCD4þ, CD3þCD8þ, and CD3þCD4þFoxP3þ T cells. Bar graphs in A are shown as mean � SEM and are representative of 2 independent experiments(n ¼ 3–5 mice/group). C, the Kaplan–Meier curve represents mouse survival times over a time course of 50 days (n ¼ 7–9 mice/group).

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decreasing the frequency of CD8þ T cells that suppressestumor rejection and leads to mortality. Conversely, theinhibition of IDO is associated with a reduction of Tregsand an increased frequency of CD8þ T cells leading toeffective tumor rejection and long-term tumor immunity.Moreover, this protective immune-mediated response iscritically dependent on the coordinated response by bothCD4þ and CD8þ T cells as it is abrogated by both types ofT-cell deficiency.

The global loss of IDO significantly decreases Tregrecruitment to the brainAlthough we established that tumor-derived IDO expres-

sion significantly affects the tumor-resident immuneresponse, we sought to further determine the effects ofglobal IDO deficiency on Tregs, given the previous workshowing the critical impact of peripheral IDOexpression on

immune-mediated tumor immunity (22). Similarly towhatwas observed in WT mice, IDO�/� mice had significantlyfewer Tregs in IDO-deficient brain tumors, when comparedwith IDO-competent tumors (P < 0.05; Fig. 4A). Interest-ingly, we also observed that Tregs were also decreased in thecervical dLNs of IDO�/� with IDO-deficient brain tumors,which had not been observed in WT mice (SupplementaryFig. 3; P < 0.05) or in the spleens of tumor-bearing mice.This specific interaction suggests that IDOexpressing cells intumor draining lymph nodes, as has previously beenascribed to APCs, play a critical role in Treg expansionoutside of the tumormicroenvironment (21), yet have littleimpact on Treg recruitment to the actual malignancy.Accordingly, IDO�/� mice with IDO-competent braintumors survive an average of 26.5 days post-ic. injection,whereas most IDO�/� mice with IDO-deficient braintumors survive until 150 days post-ic. injection (when the

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Figure 3. The absence of brain tumor-derived IDO regulates T cell infiltration and long-term survival in T-cell–competent, but not deficient mice. WT micewere ic. injectedwith 4� 105GL261 cells transducedwith scrambled shRNA (vector control, Vc; white bars) or shRNA specific to IDO (IDO knockdown, IDOkd;black bars). Naïve (control; gray bars)micewere usedas abaseline of normal T cell levels in the brain. The (A) frequency and (B) absolute numbers of total CD4þ

T cells, CD8þ T cells, and CD4þFoxP3þ regulatory T cells isolated from the brain of tumor-bearing mice were analyzed from naïve, or at 1, 2, and 3 weekspostinjection mice. All T cell populations were initially identified by the expression of CD3. Bar graphs in Fig. 3A and B are shown as mean � SEM and arerepresentative of 2 independent experiments (n¼ 4–5mice/group). Kaplan–Meier survival curves for (C)WT, CD4-deficient (CD4�/�; lack CD4þ T cells), CD8-deficient (CD8�/�; lack CD8þ T cells), and recombinase activating gene 1-deficient (Rag1�/�; lack all functional T or B cells) mice ic. injectedwith 4� 105 Vc orIDOkd GL261 cells. Mouse survival was analyzed for up to 150 days (n ¼ 7–16 mice/group) and reflects the pooled data resulting from 2 to 3 independentexperiments. �, P < 0.05; ��, P < 0.01; ��, P < 0.001.






study was terminated; P < 0.002; Fig. 4B). Importantly, invitro analysis of apoptosis and proliferation attributesshowed no differences between the Vc and IDOkd GL261cell lines (Supplementary Fig. S4). To assess whether theeffects of global IDO deficiency were restricted to an ortho-topic tumor model, we also analyzed the RasB8 transgenicglioma model that was on an IDO-competent or IDO-deficient background. IDO-competent RasB8 mice showedan increased Treg frequency (P < 0.05) and absolute cellnumber (P < 0.01) in the brain, when compared with IDO-deficient counterparts (Fig. 4C). Also in accordance withwhatwas observed in the orthotopicmodel, Treg levelsweredecreased in the draining lymph nodes of IDO�/� RasB8mice, confirming the critical role of IDO in this anatomicallocation; although this effect was not as exaggerated in theRasB8 transgenic model, when compared with the GL261

orthotopic model. Collectively, these data show a series oflocal interactions between IDO expression and Treg accu-mulationwhen comparing the brain tumorwith the periph-ery. However, it is important to note that brain tumorexpression of IDO overrides the peripheral contribution ofIDO in so far as determining the level of brain-residentTregs, as well as overall impact on survival.

IDO deficiency in brain tumors is associated withdecreased T cell infiltration

Our data show that T cell levels increase in IDO-compe-tent brain tumors over time. However, it was not clearwhether T-cell infiltration was simply due to a function ofincreasing tumor size or if IDO expression endowedthe tumor with special T-cell–recruiting capabilities. Todetermine this, we chose to quantify the average size of

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Figure 4. Global IDO-deficiencyaffects Treg levels in brain tumorsand draining lymph nodes withouteffects in the spleen. IndoleamineIDO�/� mice were ic. injected with4 � 105 GL261 cells transducedwith scrambled shRNA (vectorcontrol, Vc; white bars) or shRNAspecific to IDO (IDO knockdown,IDOkd; black bars). The (A)frequency and absolute numbersof CD4þFoxP3þ regulatory T cellsisolated from the brains, ipsilateraldeep and superficial cervical dLNand spleen of tumor-bearing micewere analyzed at 3wp-ic. injection.Representative flow cytometricplots show the gating strategyused for the identification of totalCD3þCD4þFoxP3þ Tregs in theGL261-cell based orthotopicglioma mouse model. B, theKaplan–Meier curve representsmouse survival times over a timecourse of 150 days(n ¼ 9–18 mice/group) andrepresents the combined datafrom 3 independent experiments.C, RasB8 mice that were IDO-competent (white bars) or deficientwere analyzed for the frequencyand (E) absolute numbers of Tregs.Representative flow cytometricplots show the gating strategyused for the identification of totalCD3þCD4þFoxP3þ Tregs in theRasB8 transgenic glioma mousemodel. For all experiments, Tregswere initially first identified for thecoexpression of CD3 and CD4,before FoxP3. Bar graphs in A andC are shown as mean � SEM andare representative of 2independent experiments(n ¼ 3–5 mice/group). �, P < 0.05;��, P < 0.01; ��, P < 0.001.

Wainwright et al.





IDO-competent and IDO-deficient brain tumors at 1, 2, and3 weeks post-ic. injection. As shown in Fig. 5A, tumor sizeis significantly larger in IDO-competent, when comparedwith IDO-deficient brain tumors throughout the durationof the time course. Moreover, the IDO-competent tumor issignificantly larger at 3wp-ic. injection, when comparedwith all other groups and time points (P < 0.001). However,when the tumor size was taken into consideration, thenumber of infiltrating T cells was still significantly increasedin IDO-competent brain tumors, when compared withIDO-deficient tumors (P < 0.01). To confirm this observa-tion, IDO-competent and deficient brain tumors were ana-lyzed for infiltrating T cells at 2 weeks post-ic. injection inIDO�/�mice; a time point where the brain tumors were themost similar in size throughout the time course. As shownin Fig. 5A and B, there was a robust level of T-cell infiltrationin IDO competent, when compared with IDO-deficientbrain tumors. Finally, to determine whether increasing thelevel of IDO expression would negatively impact survivalfrom brain tumors, we compared the survival of mice ic.injected with (pEF6) control and IDO-overexpressing braintumors cells and found no differences between overall

lifespans (Fig. 5C). Collectively, these data suggest that IDOexpressionbybrain tumors significantly impacts factors thatrecruit T cells. In addition, these data imply that althoughIDO promotes tumorigenesis, increasing IDO expressionbeyond a certain threshold does not impact survival.

Brain tumor–derived IDO upregulates GITR oninfiltrating T cells

Wehave previously shown that GITR-expressing Tregs arepreferentially increased in brain tumors, when comparedwith Tregs of the spleen (5). To determine whether IDOplays a role in this regulation, we determined the relativelevel of expression for GITR on CD4þFoxP3�-, CD8þ- andCD4þFoxP3þ regulatory- T cells in the brain, dLNs andspleen ofWTmice at 1, 2, and 3wp-ic. with IDO-competentor IDO-deficient brain tumors. As shown in Fig. 6A–C,micebearing IDO-competent tumors were associated with upre-gulated GITR expression on all types of infiltrating T cells inthe brain, when compared withmice bearing IDO-deficienttumors at 2 and 3 wp-ic. injection. However, the actuallevel of expression for GITR varied widely and depended onthe T-cell–subtype. Figure 6A shows that for CD4þFoxP3�

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Figure 5. The expression of IDO in brain tumors is associated with increased T cell level infiltration. A, indoleamine IDO�/� mice were ic. injected with 4� 105

GL261 cells transduced with scrambled shRNA (vector control, Vc; white bars) or shRNA specific to IDO (IDO knockdown, IDOkd; black bars). Mice wereeuthanized at 1, 2, or 3 wp-ic., brains were flash frozen and systematically sectioned throughout the tumor injection site. Brain sections were stained withhematoxylin and eosin and scanned by the CRi Pannoramic Whole Slide Scanner. In each section, tumors (black arrows) were traced using thePannoramic Viewer software and the areawas digitally calculated. Scale bar, 1mm. Bar graphs representing theVc (white bars) and IDOkd (black bars)mediantumor volume are represented for the 1, 2, and 3 wp-ic. time points. The 2 wp-ic. tumor volumes were then divided by the absolute amount of CD3þ

T cells based on flow cytometric quantification. Bar graphs are shown as mean � SEM (n ¼ 4 mice/group). B, fluorescent microscopy for CD3 and GFAPexpression, as well as nuclear 40, 6-diamidino-2-phenylindole staining, was conducted in IDO�/� mice ic. Vc and IDOkd GL261 cells at 2 wp-ic.Photomicrographs of the deep tumor parenchyma (top) or at the tumor/brain border (bottom) were imaged at �20 magnification. Bars, 50 mm. C, IDO�/�

mice were ic. 4 � 105 GL261 cells transfected the pEF6 vector alone (control) or the pEF6 vector with an inserted IDO cassette (IDO overexpressing,IDOover). �, P < 0.05; ��, P < 0.01.






effector T cells, GITR expression was 2,088 � 103 in thebrainof IDO-competent tumor-bearingmice at 1wp-ic. andsignificantly upregulated to4,142�126and3,003�42at 2and 3 wp-ic., respectively (P < 0.01). In contrast, the level ofGITR expression by CD4þFoxP3� effector T cells in thebrains of IDO-deficient tumor-bearing mice was 1,899 �95, 2,425 � 223, and 1,678 � 128 at 1, 2 and 3 wp-ic.injection, respectively, and not significantly different fromone another. A similar trend in the regulation of GITR onCD8þ T cells was observed, albeit, at a lower overall level ofrelative expression (Fig. 6B). Specifically, GITR expressionwas 817.8 � 41 in the brain of IDO-competent tumor-bearing mice at 1 wp-ic. injection and significantly upre-gulated to 1,536.2 � 116 and 2,169 � 22 at 2 and 3 wp-ic.injection, respectively (P < 0.01), whereas the level of GITRexpression by CD8þ cytotoxic T cells in the brain of IDO-deficient tumor-bearing mice was 781 � 18, 1,079 � 45,and 810 � 90 at 1, 2, and 3 wp-ic. injection, respectively,

and not significantly different from one another. In accor-dance with previous reports, Tregs expressed amuch higherlevel ofGITRwhen comparedwithCD4þFoxP3� andCD8þ

T cells (10). Specifically, Fig. 6C shows that GITR expressionwas 9,108 � 584 in the brain of IDO-competent tumor-bearingmice at 1wp-ic. injection andupregulated to16,360� 747 at 2 wp-ic. injection (P < 0.01) and further upregu-lated to 21,133 � 504 at 3 wp-ic. injection (p < 0.01). Incontrast, the level of GITR expression by Tregs in the brainsof IDO-deficient tumor-bearing mice was 8,692 � 420,10,132 � 573, and 6,869 � 1,974 at 1, 2, and 3 wp-ic.injection, respectively, and not significantly different fromone another. Notably, GITR levels were not significantlychanged in tumor-draining lymph nodes or the spleenthroughout the time course. Collectively, these data suggestthat IDO expression plays a critical role in the regulation ofGITR on tumor-infiltrating T cells and particularly withregard to Tregs.

Figure 6. Brain tumor–expressed IDO upregulates GITR levels on tumor-infiltrating T cells. WTmice were ic. injectedwith 4� 105 GL261 cells transduced withscrambled shRNA (vector control, Vc; white bars) or shRNA specific to IDO (IDO knockdown, IDOkd; black bars). The mean fluorescence intensity of GITRexpressed by (A) CD4þFoxP3� T cells, (B) CD8þ T cells, and (C) CD4þFoxP3þ regulatory T cells isolated from the brains, ipsilateral cervical dLN and spleen oftumor-bearingmicewere analyzed at 1, 2, and 3weeks postinjection. All T cell populationswere initially identifiedby the expression ofCD3. Bar graphs in A–Care shownasmean�SEMandare representative of 2 independent experiments (n¼5mice/group).D,when IDO is absent frombrain tumors,GITRexpressionremains low on infiltrating T cells. Moreover, while Treg levels remain decreased, CD4þ non-Tregs and CD8þ cytotoxic T cells are increased, resulting ineffective tumor rejection. Reciprocally,when IDO is expressed,GITRexpression is upregulatedon infiltratingTcells; this is particularly evident onTregs. This iscoincident with a significant increase in the ratio of Tregs, when compared with CD4þ non-Tregs and CD8þ cytotoxic T cells. The increased fraction ofimmunosuppressive Tregs is then associated with decreased tumor rejection and increased outgrowth. �, P < 0.05; ��, P < 0.01.

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DiscussionThe potent immunosuppression induced by GBM is

one of the primary obstacles to finding effective immu-notherapies (30, 31). This immunosuppression is asso-ciated with a significant accumulation of Tregs within thetumor microenvironment (2–4, 32, 33) and is consideredto be one of the primary obstacles inhibiting the tumorrejection functions of CD8þ cytotoxic T cells. It is, there-fore, important that future immunotherapies simulta-neously arm effector CD8þ cytotoxic T cells, while simul-taneously inhibiting immunosuppressive mechanisms.Relevant to this requirement, IDO is a molecule that hasbeen shown to serve as a central regulator of both immu-nosuppression, as well as T-cell function (18, 34). How-ever, before this investigation, a comprehensive analysisof IDO in tumors of the CNS had not been conducted.Here, we elucidate the role of IDO in brain tumors andthe overall impact on tumor immunity.In the CNS, the expression of IDO is normally main-

tained at low to undetectable levels. However, inflamma-tory processes activated by bacterial byproducts or theproinflammatory cytokine, IFN-g , rapidly upregulate IDOexpression by glia (35). Moreover, IDO activity contributesto the suppression of proinflammatory T-cell responses inthe CNS, based on studies showing the exacerbation ofpathology related to the experimental autoimmune enceph-alomyelitis model (mouse model of multiple sclerosis) inthe context of pharmacologic IDO inhibition (28, 36).Although this effect would be undesirable in an autoim-mune disorder of the CNS, it would be greatly appreciatedin the context of CNS tumors, whereby IDO inhibitionwould lead to decreased immunosuppression and subse-quently more effective immune-mediated tumor rejection.However, before this investigation, the in vivo role of IDO inthe context of CNS malignancy had not been investigatedcomprehensively.Here, we have shown that IDO expression by brain

tumors, alone, promotes tumor outgrowth in an orthotopicmouse model. This was based on the analysis of the ortho-topic GL261 cell–based tumormodel that was deficient for,normal for, or overexpressed IDO in WT and/or IDO�/�

mice. Using these models, we found a reciprocal relation-ship whereby IDO-competent brain tumors were infiltratedby an increased frequency of Tregs, coincident with adecreased frequency of CD8þ Tc cells, when compared withIDO-deficient brain tumors. These changes were coinciden-tal with a significant reduction of overall T-cell levels inIDO-deficient, when compared with IDO-competent braintumors, even when taking into account the difference inoverall tumor size. These data were further supported by theanalysis in the spontaneously developing RasB8 mouseglioma model whereby global IDO competency led to asignificant increase in the overall level of Tregs found in thebrain, suggesting that this IDO-regulatedTreg accumulationis a generalizable phenomenon and not exclusive to theGL261-based brain tumor model. More importantly, ourdata show that IDO-deficient brain tumors lead to a signif-icant and long-term survival advantage with clinically

relevant benefits. It is also important to understand thatthis survival benefit is maintained both in the presenceand absence of peripheral IDO competency. This suggeststhat the tumor, rather than tumor-draining lymphnode APCs, plays an overarching role in regulating theantitumor immune response. Although determining if thiseffect is specific to CNS malignancies requires furtherinvestigation.

Our data further elucidate IDO T-cell–mediatedmechan-isms through the use of T-cell–deficient mice wherebyCD4þ and/or CD8þ T-cell deficiency abrogates the long-term survival mediated by IDO deficiency in immunocom-petent mice. Finally, we reveal the impact of IDO on GITRexpression levels by demonstrating a strong upregulationon IDO-competent tumor-infiltrating T cells, potentiallyproviding additional future therapeutic value as it hasrecently been shown that targeting Treg-expressed GITR canpositively impact tumor immunity (37, 38). It was inter-esting to find that all T-cell subtypes upregulated GITRexpression, although Tregs showed the highest level ofexpression (5–10� higher than that of CD4þFoxP3� andCD8þ T cells). Collectively, our data are summarizedschematically in Fig. 6D.

Our results from animal models parallel the gliomapatient data showing that the upregulation of IDO expres-sion by brain tumors is associated with a significantly worseprognosis in patients. It is interesting that the upregulationof IDO was more common among GBM patients, whencompared with lower tumor grades that were more associ-atedwith downregulated IDOexpression. This suggests thatoneof the selective pressures brain tumorsmayuse to transitfrom earlier to later stages of astrocytoma may be throughthe upregulation of IDO. Furthermore, these data alsoimplicate IDOas a future biomarker for theuse inpredictingthe course of treatment (i.e.,whether ornot to administer anIDO inhibitor), as well as predicting the overall survival inpatients. However, it is not yet knownwhether patients withupregulated and downregulated IDO expression will pos-sess similar responses to pharmacologic IDO inhibitors,such as 1-methyl tryptophan (1-MT).Clinical trials aimed atIDO inhibition are currently recruiting patients with solidtumors. Perhaps an interesting retrospective follow-up tothis trial will be whether patients with upregulated IDOexpression do better when administered a pharmacologicIDO inhibitor compared with those patients with down-regulated IDO expression.

It has been established that IDO-expressing tumors sys-temically alter the accumulation of tryptophan catabolites(39). The first catabolic byproduct of IDO catabolism,kynurenine, was recently highlighted as a critical cataboliteinvolved inmediating immunosuppression in glioma (26).Coincidentally, kynurenine has been associated with theexpansionof Tregs via interactionwith the aryl hydrocarbonreceptor (Ahr) in CD4þ T cells (40). It was, therefore,interesting to find that Treg levels were simultaneouslydecreased in brain tumors and dLN, but not in the spleen,in the context of global IDO deficiency. This may reflect acombinationof factors including the lackof locally draining






tumor-produced kynurenine, the limitedmigration patternof brain tumorAPCs, aswell as the involvement of primarilythymus-derived, rather than tumor-induced Tregs. Notably,the effects of kynurenine on Ahr-expressing thymus-derivedTregs are currently unknown and are thus a future target ofinvestigation.

The Treg accumulation in IDO-competent brain tumorswas associated with a significant and time-dependent upre-gulation of GITR. The selective increase in GITR expressionon tumor-infiltrating, but not dLN- and spleen-residentTregs, may predispose those tumor-resident Tregs to low-dose GITRmAb–mediated therapy; potentially minimizingthe immunotoxic effects created by systemic administra-tion. In support of this hypothesis are data from a mousemelanoma model showing that GITR mAb decreases thestability and intratumoral accumulation of Tregs, coinci-dent with the regression of small-established tumors (38).More recently, the phosphorylation status of c-jun N-ter-minal kinase was identified to be a critical regulator ofimmunosuppressiveness in Tregs, as well as a downstreamtarget of activated GITR; potentially providing additionaltherapeutic value (41).

Collectively, our data provide the foundation and ratio-nale for further investigation into determining the role ofIDO in brain tumors. Future directions include the thera-peutic targeting of IDO with the pharmacologic inhibitor,1-MT, as well as other agents identified to have the potentialfor IDO modulation such as Imatinib (42) and Acyclovir(43).Downstream catabolite production (i.e., kynurenine),Treg levels and T-cell–expressed GITR will be among theoutcome measures analyzed. Finally, to provide betterpreclinical tools for future therapeutic testing, we are cur-rently developing transgenic models that faithfully recapit-ulate high-grade glioma and are conditionally deficient forIDO.

Inhibiting immunosuppression in glioma is one of themost critical barriers to effective and long-lasting antitumorimmunity (30, 31). Here, we have shown that the upregula-tion of IDO expression in patient glioma strongly predictsa poor prognosis. This association from glioma patients

was then applied to mouse models confirming that tumor-derived IDO expression contributes to a decreased lifespanin a T-cell–dependent manner. Moreover, the IDO expres-sion in brain tumors was coincident with increased Tregrecruitment and upregulated GITR expression levels. Col-lectively, these data implicate IDO as central player inregulating brain tumor immunity, independently of periph-eral sources, as well as providing the rationale for increasedclinical and preclinical efforts for understanding its fullimpact in brain tumors.

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

Authors' ContributionsConception and design: D.A. Wainwright, A.U. Ahmed, M.S. LesniakDevelopment of methodology: D.A. Wainwright, I.V. Balyasnikova, M.S.LesniakAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.):D.A.Wainwright, A.L. Chang, K.-S. Moon, Y. HanAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): D.A. Wainwright, A.L. Chang, A.U.Ahmed, K.-S. Moon, B. Auffinger, M.S. LesniakWriting, review, and/or revision of the manuscript:D.A. Wainwright, B.Auffinger, M.S. LesniakAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): D.A. Wainwright, B. AuffingerStudy supervision: D.A. WainwrightHelped with experimentation: A.L. Tobias

AcknowledgmentsThe authors thank Ms. Lingjiao Zhang, M.S. for her expertise in the

statistical analysis of our studies. The authors also thank Prof. RobertDantzer, D.V.M./Ph.D. (MD Anderson Cancer Center) for his suggestionsand critical comments during the preparation of this work.

Grant SupportThis work was supported by the NIH grants R01 CA138587 (M.S.

Lesniak), NIH R01 CA122930 (M.S. Lesniak), NIH U01 NS069997 (M.S.Lesniak), and NIH F32 NS073366 (D.A. Wainwright).

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 June 26, 2012; revised August 1, 2012; accepted August 18, 2012;published OnlineFirst August 29, 2012.

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2012;18:6110-6121. Published OnlineFirst August 29, 2012.Clin Cancer Res Derek A. Wainwright, Irina V. Balyasnikova, Alan L. Chang, et al. Regulatory T Cells and Negatively Impacts SurvivalIDO Expression in Brain Tumors Increases the Recruitment of

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