Blocking Indolamine-2,3-Dioxygenase Rebound Immune ......Cancer Therapy: Clinical Blocking...

Cancer Therapy: Clinical

Blocking Indolamine-2,3-Dioxygenase ReboundImmune Suppression Boosts Antitumor Effects ofRadio-Immunotherapy in Murine Models andSpontaneous Canine MalignanciesArta M. Monjazeb1, Michael S. Kent2, Steven K. Grossenbacher3, Christine Mall3,Anthony E. Zamora3, Annie Mirsoian3, Mingyi Chen4, Amir Kol5, Stephen L. Shiao6,Abhinav Reddy1, Julian R. Perks1,William T.N. Culp2, Ellen E. Sparger2, Robert J. Canter7,Gail D. Sckisel3, and William J. Murphy3,8

Abstract

Purpose: Previous studies demonstrate that intratumoral CpGimmunotherapy in combination with radiotherapy acts as anin-situ vaccine inducing antitumor immune responses capable oferadicating systemic disease. Unfortunately, most patients fail torespond. We hypothesized that immunotherapy can paradoxi-cally upregulate immunosuppressive pathways, a phenomenonwe term "rebound immune suppression," limiting clinicalresponses. We further hypothesized that the immunosuppressiveenzyme indolamine-2,3-dioxygenase (IDO) is a mechanism ofrebound immune suppression and that IDO blockade wouldimprove immunotherapy efficacy.

Experimental Design: We examined the efficacy and immu-nologic effects of a novel triple therapy consisting of local radio-therapy, intratumoral CpG, and systemic IDOblockade inmurinemodels and a pilot canine clinical trial.

Results: In murine models, we observed marked increase inintratumoral IDO expression after treatment with radiotherapy,

CpG,or other immunotherapies. The additionof IDOblockade toradiotherapy þ CpG decreased IDO activity, reduced tumorgrowth, and reduced immunosuppressive factors, such as regu-latory T cells in the tumor microenvironment. This triple combi-nation induced systemic antitumor effects, decreasingmetastases,and improving survival in a CD8þ T-cell–dependent manner. Weevaluated this novel triple therapy in a canine clinical trial,because spontaneous canine malignancies closely reflect humancancer. Mirroring our mouse studies, the therapy was well toler-ated, reduced intratumoral immunosuppression, and inducedrobust systemic antitumor effects.

Conclusions: These results suggest that IDO maintainsimmune suppression in the tumor after therapy, and IDOblockade promotes a local antitumor immune response withsystemic consequences. The efficacy and limited toxicity ofthis strategy are attractive for clinical translation. Clin CancerRes; 22(17); 4328–40. �2016 AACR.

IntroductionImmunotherapy is revolutionizing metastatic cancer therapy.

Patients responding to these therapies can achieve durable long-

term remissions. However, most patients fail to respond, andsome can experience significant immune-mediated toxicities (1–3). There is growing interest in understanding mechanisms ofresistance to immunotherapy and finding combinatorial strate-gies to further increase efficacy while minimizing toxicity.

Local radiotherapy (RT) is an ideal candidate for combinedmodality immunotherapy strategies. In addition to debulkingtumor and releasing tumor antigens, RT has well-establishedimmunomodulatory effects (4). Preclinical and clinical reportsconfirm the safety and efficacy of multimodality strategiesemploying RT and immunotherapy. One particularly promisingstrategy is RT in combination with the immune-stimulatory toll-like receptor 9 (TLR9) agonist CpG oligodeoxynucleotide (CpG),which has demonstrated significant synergy in preclinical models(5) and the ability to induce regression of systemic disease inclinical trials (6, 7). Systemic response rates were about 20%withdisease stability in another 20% of patients with refractory sys-temic (6) or cutaneous lymphoma (7). However, patients whosetumors induced regulatory T cells (Tregs) responded poorly. Theeffects of CpG on Treg-mediated immune suppression can beparadoxical. CpG can reduce Tregs by converting them to a T-helper phenotype via IL6 production (8) and can also directlyreverse Treg function (9). Conversely, CpG can directly induce

1Department of RadiationOncology, UCDavis ComprehensiveCancerCenter, Sacramento, California. 2Department of Surgical and Radio-logical Sciences, UC Davis School of Veterinary Medicine, Davis, Cali-fornia. 3DepartmentofDermatology,UCDavisHealth Sciences, Sacra-mento, California. 4Department of Pathology, UC Davis HealthSciences, Sacramento, California. 5Department of Pathology, Micro-biology, and Immunology, UC Davis School of Veterinary Medicine,Davis, California. 6Departments of Radiation Oncology and Biomed-ical Sciences, Cedars-Sinai Medical Center, Los Angeles, California.7Division of Surgical Oncology, Department of Surgery, UC DavisComprehensive Cancer Center, Sacramento, California. 8Division ofHematology and Oncology, Department of Internal Medicine, UCDavis Comprehensive Cancer Center, Sacramento, California.

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

Corresponding Author: Arta M. Monjazeb, School of Medicine, University ofCalifornia, Davis, 4501 X St., G-140, Sacramento, CA95817. Phone: 916-734-8252;Fax: 916-703-5069; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-3026

�2016 American Association for Cancer Research.

ClinicalCancerResearch

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Published OnlineFirst March 15, 2016; DOI: 10.1158/1078-0432.CCR-15-3026

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Foxp3 expression (10) and upregulate indolamine-2,3-dioxygen-ase (IDO; ref. 8) which is known to induce and maintain Tregs.

IDO is an immunosuppressive enzyme that catalyzes the rate-limiting step in the catabolismof tryptophan to kynurenine and isexpressed by numerous human malignancies (11, 12). IDOexpression can induce immune tolerance to malignancies (13)via complex mechanisms (14). In dendritic cells (DC), IDO canactivate Treg-suppressive function in a CTLA-4–dependent man-ner (15). It can also prevent DC IL6 production, thereby prevent-ing Treg conversion and maintaining elevated Treg levels despiteinflammatory signals such as CpG (16). Furthermore, IDO candirectly induce Tregs from na€�ve CD4þ T cells via 3-hydroxyan-thranillic acid, a downstream catabolite of IDO tryptophanmetabolism (17, 18). IDO can, through similarly complexmechanisms, inhibit natural killer cells and prevent effector T-cell activation and proliferation (14, 19). IDO expression can beparadoxically upregulated after inflammatory signals (20, 21)presumably as a mechanism to limit inflammation andmaintainimmune homeostasis. 1-Methyl-Tryptophan (1MT) is a pharma-cologic inhibitor of IDOwithdemonstrated antitumorproperties,which is being tested alone or in combinationwith chemotherapyin human trials (22).

Using a novel immunotherapy strategy combining local RT,intratumoral CpG, and IDO blockade, we tested the hypothesisthat IDO upregulation after immunostimulatory therapies,such as RT þ CpG, maintains tumor microenvironmentimmune suppression and limits treatment efficacy. We showthat immunostimulatory therapies paradoxically upregulatedIDO expression, which we termed "rebound immune suppres-sion." The addition of 1MT decreased IDO activity, decreasedTregs and other immune-suppressive factors within the tumor,and significantly improved the antitumor effects of RT þ CpGin a CD8þ T-cell–dependent manner. Although the immuneeffects were primarily limited to the microenvironment of thetreated tumor, systemic antitumor responses were observed. Weconfirmed these results in a veterinary clinical trial of compan-ion canines since canine cancers closely represent humanmalignancy. We observed significant responses at the RT-trea-ted primary tumor as well as at untreated sites of metastaticdisease in most dogs. There were also significant changes in the

immunosuppressive tumor microenvironment, corroboratingour mechanistic mouse data. In marked contrast with immunecheckpoint inhibitors, little toxicity was observed in mouse orcanine studies. These results confirm the potency of combina-torial immunotherapy strategies and suggest that IDOmay playa critical role in maintaining the immunosuppressive tumormicroenvironment after immunotherapy. The addition of IDOblockade may safely improve the efficacy of immunotherapy bypreventing rebound immune suppression.

Materials and MethodsIn vivo reagents

CpG ODN 1826 (mouse studies) and 2006 (canine studies)were purchased from Invivogen. 1-methyl-D,L-tyrptophan waspurchased from Sigma-Aldrich. CD8-depleting antibody (clo-neYTS169.4) was administered prior to the start of therapy andonce weekly thereafter. The agonistic anti-mouse CD40 antibody(FGK115.B3) was generated as previously described (23). Rat IgG(Jackson ImmunoResearch Laboratories) was used as a control foranti-CD40. Recombinant human interleukin-2 (rhIL2; Teceleu-kin; Roche) was provided by the NCI.

Mouse tumor studiesAll studieswere approved by theUCDavis Institutional Animal

Care and Use Committee (IACUC), and humane endpoints wereused. Female 8- to 12-week-old C57BL/6 or BALB/c mice werepurchased from the animal production area at the NCI. All micewere housed at the UC Davis animal facilities under specificpathogen-free conditions. Mouse tumor studies were performedas outlined (Fig. 1D). 4T1 breast adenocarcinoma tumors weregrown orthotopically in the mammary fat pad of BALB/c mice,and B16 melanoma tumors were implanted into the flank ofC57BL/6 mice. Mouse tumors were irradiated with 8 Gy perfraction delivered with 9 MeV electrons on an Elekta synergyclinical accelerator using a 2-cm diameter electron field and 0.5-cm bolus. Treatment accuracy was confirmed using anthropo-morphic phantoms as previously described (24). Mice weresacrificed and tissues harvested at 7 or 14 days after treatmentcompletion for mechanistic studies.

Canine clinical trialThis clinical trial was approved by the UC Davis School of

Veterinary Medicine Clinical Trials Review Board and IACUC.Five canines with histologically confirmed metastatic melano-ma or sarcoma were enrolled with informed consent of theirowners. The trial schema is outlined in Supplementary Fig. S1.Treatment consisted of weekly 8 Gy fractions of RT repeated fora total of four treatments over 4 weeks. RT was delivered to theprimary tumor only using a clinical grade linear accelerator atthe UC Davis Center for Companion Animal Health. After eachRT treatment, canines received a 2-mg intratumoral injection ofCpG. Patients also received oral 1MT at 1,200 mg daily for the28-day treatment period. Radiologic scans, blood, and tissuesamples were obtained before treatment and at follow-up visitsat weeks 5, 8, and 20. Radiographic response rates were deter-mined using irRECIST criteria (25). Patients were evaluated by aveterinary radiation oncologist for treatment toxicity weeklyduring therapy and at the 5, 8, and 20-week follow-up visitsusing clinical exam and blood laboratories. Toxicity was gradedaccording to the VRTOG criteria (26).

Translational Relevance

Cancer immunotherapy consisting of radiotherapy in com-bination with intratumoral CpG has proven highly effective insome patients, and additional trials are ongoing. Unfortunate-ly, most patients fail to respond to this therapy. In this report,we demonstrate that the effectiveness of this therapy may belimited by upregulation of IDO in the tumor microenviron-ment in response to the therapy itself. IDO upregulationmaintains immune suppression within the tumor and limitsan effective antitumor immune response. Addition of IDOblockade to this therapy reverses intratumoral immune sup-pression and substantially improves local and systemic effi-cacy without any apparent increase in toxicity. Given the use ofIDO inhibitors and radiotherapy þ CpG in clinical trials, andthat this triple therapy has now been tested in large animalmodels, this therapy is ready for clinical translation.

Blocking IDO Rebound Immune Suppression Boosts Immunotherapy

www.aacrjournals.org Clin Cancer Res; 22(17) September 1, 2016 4329




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Flow cytometryStaining procedures and antibodies are detailed in Supplemen-

tary Methods. All data were collected using a BD Fortessa flowcytometer equipped with BD FACSDiva software. Data wereanalyzed using FlowJo v10 (TreeStar).

Quantitative real-time PCRQuantitative real-timePCR(qRT-PCR)wasperformedusingAB

Step-ONE Plus (Applied Biosystems) in the presence of SYBRGreen Supermix (Applied Biosystems). Details of RNA isolation,cDNA synthesis, and qRT-PCR can be found in SupplementaryMethods.

Kynurenine/tryptophan ratioKynurenine to tryptophan ratio was determined using mass

spectrometry at the UC Davis Metabolomics Center as detailed inSupplementary Methods.

Statistical analysisStatistical analysis was performed with Prism software (Graph-

Pad). Data were expressed asmean� SEM. For analysis of three ormore groups, the one-way ANOVA was performed with theHolm–Sidak correction for multiple comparisons. Analysis ofdifferences between two normally distributed test groups wasperformed with the Student t test. Welch correction was appliedto Student t test datasets with significant differences in variance. �,P < 0.05; ��, P < 0.01; ��, P < 0.001; and ��, P < 0.0001.

Immunofluorescence and Immunohistochemistry methodsare detailed in Supplementary Methods.

ResultsIDO upregulation in the tumor microenvironment afterimmunostimulatory therapies

To test our hypothesis that IDO-mediated "rebound immunesuppression" maintains immune suppression and limits treat-ment efficacy, we employed the poorly immunogenic and highlymetastatic mouse 4T1 orthotopic breast tumor model (27). Wefirst evaluated IDO expression within the tumor microenviron-ment after immunostimulatory therapies and observed a signif-icant increase in IDO-expressing cells compared with untreatedcontrols in the glandular nests of malignant cells (Fig. 1A and B; P< 0.0001). IDO can be upregulated by tumor-infiltrating immunecells as well as tumor cells. In our model, IDO upregulationappears to be predominately in the neoplastic epithelial cells.IDO upregulation within 4T1 cells was verified in vitro as well(data not shown). We likewise observed a parallel statisticallysignificant 3- to 5-fold increase in IDO1 mRNA expression (Fig.1C). Importantly, this upregulation of IDOwas not limited to RTand CpG but was also observed with other immunotherapy

strategies (Fig. 1A–C). These results indicate that immunotherapycan paradoxically upregulate immunosuppressive pathways, suchas IDO, which may limit efficacy.

Systemic 1MT significantly decreases IDO activity andaugments the antitumor efficacy of local RT þ CpG

To investigate if IDO-mediated "rebound immune suppres-sion" after RTþ CpG limits treatment efficacy, we next tested theeffects of adding systemic IDO blockade. Our treatment schema(Fig. 1D) consisted of two 8 Gy fractions of local RT administeredto the primary tumor over a 7-day period. Each fraction of RT wasaccompanied by intratumoral injection of 20 mg of CpG based ondose response data. CpG was administered locally to mirrorhuman clinical trials and minimize systemic toxicity (6, 7). ForIDOblockade, 1MTwas administered by daily 2mg i.p. injectionsthroughout the study period. The triple combination decreasedIDO enzymatic activity, as measured by serum kynurenine/tryp-tophan ratio, below the level of untreated controls (Fig. 1E; P ¼0.03). We next evaluated the antitumor effects of 1MT, RT, andCpG, alone or in combination. CpG and 1MT alone or incombination had no significant effect on tumor growth (Fig.1F). Local RT alone significantly reduced tumor growth, but byday 34, there was accelerated tumor outgrowth, and mean tumorsize was no longer statistically different than controls (Fig. 1F andG). Although CpG and 1MT had no antitumor effects alone or incombination, when either one was combined with RT, theysignificantly inhibited tumor growth (Fig. 1F and G). Notably,the triple combination was significantly better than either RT þCpG or RT þ 1MT decreasing tumor growth by an additional 3-fold (day 34mean tumor size: 377mm3, 1,060mm3, 1,057mm3,respectively; Fig. 1F). The triple therapy also significantlyimproved the survival of 4T1-bearing mice (Fig. 1H, P <0.001). To ensure these results were not tumor or mouse strainspecific, we tested this regimen in C57BL/6 mice bearing B16melanoma tumors. Again, mice treated with the triple combina-tion had significantly smaller tumors thanmice treated with RTþCpG (Fig. 1I, P ¼ 0.042). Importantly, in both models, there wasno evidence of autoimmune or other toxicities induced by thetherapy. Thus, as hypothesized, the addition of 1MT significantlyimproved the antitumor effects of RT þ CpG in 4T1 and B16tumors (Fig. 1G and I).

We next examined the systemic antitumor effects of this tripletherapy in 4T1-bearing mice. At day 40, heavy burdens of pul-monary metastatic disease could be grossly identified in controlbut not treated mice (Fig. 1J). The primary tumors (located in thedistalmammary fat pad)were treatedwith 2-cm radiation portals,and the lungs were well outside of the radiation fields. Similarresults were also observed by CT imaging (Fig. 1K) and furthercorroborated in quantifiable fashion using in-vitro lung tumor

Figure 1.1MT limits radiationþ CpG–induced IDO upregulation and improves therapeutic efficacy. Expression of IDO in control or treated 4T1 tumors by immunofluorescence(A and B) or qPCR (C). IDO þ cells stain bright pink, and nuclei are counterstained by DAPI; white arrows indicate examples of positive staining cells.Balb/cmice bearing orthotopic 4T1 breast tumors or C57/BL6mice bearing B16melanoma tumorswere treated as outlined in the schema (D). IDO enzymatic activityin 4T1 tumor–bearing mice as measured by serum kynurenine to tryptophan ratio (E). 4T1 tumor growth (F, G) and tumor-bearing mouse survival (H). B16melanoma tumor growth (I). Lung metastases in orthotopic 4T1-bearing mice as assessed by gross examination (J), CT (K), and lung colony-forming assay (L). Redarrows indicate examples of lung metastases. n ¼ 3 to 4 mice per group for correlative studies and n ¼ 6 to 10 mice per group for tumor growth studies andsurvival studies. Bar graphs represent mean � SEM. Results analyzed by one-way ANOVA, Student t test, or Kaplan–Meier analysis between the indicatedgroups (� , P < 0.05; �� , P < 0.01; �� , P < 0.001).






colony-forming assays with a 6-fold reduction in lung colony-forming units in the triple combination group compared withcontrols at day 28 (Fig. 1L, P ¼ 0.001). Crucially, lungs from themajority of the triple combination–treated mice grew no tumorcolonies.

Taken together, our findings indicate that RTþCpG resulted inantitumor effects but also upregulated IDO expression. Theaddition of IDO blockade with 1MT decreased IDO activity andsignificantly improved the antitumor effects. This triple therapywas well tolerated, increased survival, and markedly reducedsystemic metastases.

RT þ CpG þ 1MT induces systemic antitumor effects inspontaneous canine malignancies

Based on our murine studies, we initiated a pilot clinical trialfor outbred companion canines with spontaneous metastaticmelanomas and sarcomas. Due to the aggressive nature andpoor prognosis of these cancers, the standard of care for thesecanine patients is palliative RT to the primary tumor consistingof 4 weekly fractions of 8 Gy. Our treatment protocol (Sup-plementary Fig. S1A) built on this palliative regimen butmirrored our mouse studies with an intratumoral injection ofCpG at the time of RT and daily administration of 1MT for thetreatment course. CpG was administered at 2 mg/dose to mirrorhuman clinical studies. The activity of CpG ODN 2006 used inhuman clinical trials was verified in canines with an in-vitroperipheral blood mononuclear cell proliferation assay (Sup-plementary Fig. S1B). 1MT was administered orally at 1,200 mgper day based on pharmacokinetic studies in canines (28). Weverified the ability of 1MT to reduce IDO enzymatic activity incanines by measuring serum kynurenine/tryptophan ratio insamples collected from trial canines (Supplementary Fig. S1C; P¼ 0.02). We enrolled 5 canines with rapidly progressing met-astatic disease to this pilot trial (Supplementary Table S1). Fourhad mucosal melanomas with pulmonary metastases, and onehad soft tissues sarcoma with nodal and pulmonary metastases(Supplementary Table S1). Canines with metastatic melanomashave an extremely poor prognosis (29). The survival of caninesin this study (5.8 months, 95% confidence interval: 3.2–9.2months) exceeded published historic controls (29). All 5canines responded at the primary site of disease which waswithin the radiation portals (Supplementary Table S1; Fig. 2A–D). The local response to therapy was robust with a 50% to100% reduction in tumor mass (Fig. 2A–D). Photographic andCT imaging examples documenting local responses are shownin Fig. 2B–D. Crucially, when examining best systemicresponses by immune-related response criteria (25) at distantmetastatic sites outside of the radiation portals, we observedone subject with a complete response, two with partialresponses, one with disease stabilization, and one with pro-gressive disease (Supplementary Table S1; Fig. 2E–H). CTimaging examples of partial (Fig. 2F–G) and completeresponses (Fig. 2H) of index lesions are depicted. Mirroringhuman cancer immunotherapy trials (1, 3), responses wererapid and robust with >80 to 100% reduction in the volume ofindex lesions (Fig. 2E) and regression of even large bulkytumors (Fig. 2F and H). Some of the partial responses consistedof a mixed response with regression of some lesions and growthor stability of others (Fig. 2G). This type of mixed response hasalso been observed in human immunotherapy trials and isassociated with a good prognosis (25). Thus, in canines with

previously rapidly progressing disease, the local response ratewas 100% and the systemic response rate was 60%, withanother 20% disease stabilization. Upon cessation of treatmentin this trial, all dogs eventually had CT documented or clinical/symptomatic progression. No further treatment was providedbeyond the brief treatment course outlined in the schema, andassessment of longer administration, as is used in human trials,is needed.

Canines were monitored closely for toxicity with regularphysical examinations and lab work. Importantly, the onlyadverse effects observed were mild mucositis and skin toxicitywithin the radiation portals that did not exceed what would beexpected from palliative RT alone (Supplementary Table S1).These results demonstrate that this triple combination therapycan be safely administered resulting in significant antitumoreffects in metastatic disease and validate the findings of ourmouse studies in a model more representative of humancancer.

Additionof IDOblockade toRTþCpG transforms the immune-suppressive tumor microenvironment

The primary rationale for adding 1MT to RT þ CpG was ourhypothesis that IDO-mediated rebound immune suppressionlimited efficacy bymaintaining Tregs and an immunosuppressivetumor microenvironment despite inflammatory signals. Clinicalefficacy has been observed with RT þ CpG, but patients whosetumors induce Tregs are unlikely to respond (6).We examined thefate of immunosuppressive Tregs after the completion of therapyin mice and canines. In mice, Treg levels were maintained withinthe tumor after RT þ CpG (Fig. 3). The addition of 1MT signif-icantly and substantially reduced the Treg subset of CD4þ T cellswithin the tumormicroenvironment but not in the periphery (Fig.3; Supplementary Figs. S2 and S3). The addition of 1MT resultedin a greater than 4-fold decrease in intratumoral Tregs comparedwith RT þ CpG alone 14 days after therapy (Fig. 3A and B; P ¼0.02). This triple combination, but not immunotherapy alone(CpGþ 1MT), RT alone, or RTþCpG, significantly reduced Tregscompared with control mice (Fig. 3A and B; control: 24.4% �2.1% vs. RTþCpGþ 1MT 6.2%� 2.2%; P < 0.01). Similar resultswere seen if Tregswere analyzed as a percentage of total cells in thetumor (control: 0.093% � 0.072% vs. RTþ CpGþ 1MT 0.018%� 0.003%; P ¼ 0.07, data not shown). IL6 induction afterimmunostimulatory therapies contributes to the reduction ofTregs. IDO can maintain Tregs in tumors by downregulating IL6(8). We observed a 9-fold increase in IL6 expression within thetumor microenvironment after triple combination therapy (Sup-plementary Fig. S4; P¼ 0.03). We also observed a significant 3- to4-fold increase in theCD4þ/Treg ratio, which is a known indicatorof immune status and prognosis (11, 30, 31), in RT þ CpG þ1MT–treated mice compared with tumor-bearing control mice (P< 0.01), CpGþ 1MT–treatedmice (P < 0.05), RT-treatedmice (P <0.01), and RT þ CpG–treated mice (P < 0.01; Fig. 3C). Theaddition of 1MT provided no added benefit in terms of Tregreduction and improvement in the conventional CD4/Treg ratioin the dLN (Supplementary Fig. S2A–S2C), spleens (Supplemen-tary Fig. S2D–S2F), or nondraining lymph nodes (data notshown). This is in contrast with the tumor, where the greatestTreg reductions were seen in the 1MT-containing groups (CpG þ1MT and RT þ CpG þ 1MT; Fig. 3A–C). Similar results were alsoseen at earlier time points (Supplementary Fig. S3). Thus,although 1MT was administered systemically, its primary action

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is within the tumor microenvironment where IDO is alsoupregulated.

Informed by our murine studies, we examined the fate of Tregsin the tumors, dLN, and systemic circulation of canines receivingRTþCpGþ 1MT (Fig. 3; Supplementary Fig. S2). When possible,tumor biopsy, tumor-draining lymph node biopsy, and periph-eral blood samples were obtained before and after therapy.Peripheral blood was obtained from all dogs. A posttherapytumor biopsy could not be obtained from one dog due to acomplete response. In addition, some dogs displayed massive

tissue death in the posttreatment tumor biopsy samples limitinganalyses. Lymph node biopsies could not be obtained from twodogs in which dLNswere not accessible with aminimally invasiveprocedure. Mirroring the results of our mouse studies, the per-centage of tumor-infiltrating Tregs was significantly reduced in alldogs after therapy with virtually no Tregs remaining in the tumormicroenvironment after therapy (Fig. 3D and E; P ¼ 0.014).Conversely, in the three dLN samples, we did not observe astatistically significant change in Tregs across the cohort withTregs dropping substantially (3–4-fold) after therapy in some

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Figure 2.Efficacy of radiationþ CpGþ 1MT ina canine clinical trial. Therapeuticresponse of local irradiated tumors(A–D) and untreated metastaticlesions (E–H) in a canine clinical trialare depicted. Waterfall plot of bestresponse at the primary-treatedtumor (A). Photographs (B) and CT(C) depicting response of amelanoma of the buccalmucosa. CTdepicting response of an abdominalwall sarcoma (D). Waterfall plot ofbest response at untreatedmetastatic index lesions (E). CTdemonstrating a partial response(F), mixed response (G), andcomplete response (H) ofmetastatic pulmonary index lesions.






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dogs but increasing in others (Supplementary Fig. S2G). Alsomirroring our mouse results, no significant change in peripheralTregs was observed (Supplementary Fig. S2H). To confirm ourresults from flow cytometric analysis, immunofluorescence stain-ing for FoxP3 expression in canines that had sufficient remainingbiopsy samples was performed. Representative fields and quan-tification from a canine with Treg decreases in the tumor aredepicted in Fig. 3F andG confirmingmarked reduction in Foxp3þ

cells following triple combination therapy.In the mouse tumor models, we also observed significant

reductions in other immune-suppressive factors in the tumormicroenvironment. Tumor associated macrophages (TAM) areknown to play a critical suppressive role in themicroenvironmentof 4T1 tumors (32).Mirroring the reduction seen inTregs only, the

RT þ CpG þ 1MT triple combination resulted in a statisticallysignificant reduction of TAMs by nearly 5-fold compared withcontrol (Fig. 4A andB; 18.4%�10.3%vs. 3.8%�1.5%;P<0.05).At baseline, intratumoral TAMs were M2 polarized as assessed byarginase to inducible nitric oxide ratio (data not shown). We nextexamined the expression of the TGF-beta gene (TGFB)which bothinduces and is produced by Tregs and TAMs (reviewed in 33, 34),is produced by IDO-expressing DCs (35), and is also known toblock lymphocyte activation (36). All of the therapies reducedTGFB expression relative to control, but this was most pro-nounced and most significant in the triple combination (Fig.4C; P < 0.0001). Taken together, these studies indicate that thistherapy reduces multiple immune-suppressive factors andrestores IL6 signaling within the tumor microenvironment.

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Figure 4.Radiation þ CpG þ 1MT reduces tumor-associated macrophages. Levels of intratumoral CD45þCD11bþF4/80þ macrophages as assessed by flow cytometry in4T1-bearing mice (A and B). Representative flow cytometry contour plots demonstrating staining of intratumoral CD45þ cells for F4/80 and CD11b (A). Flowcytometry data represented as a bar graph expressed as% tumor-associatedmacrophages of all CD45þ cells (B). Bar graph representation of intratumoral TGF-betamRNA as assessed by qPCR (C). n ¼ 3 to 4 mice per group. Bar graphs represent mean � SEM. Results analyzed by one-way ANOVA between the indicatedgroups (� , P < 0.05; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001).

Figure 3.Radiationþ CpGþ 1MT reduces intratumoral regulatory CD4þ T cells in mice and canines. Day-28 levels of tumor-infiltrating regulatory CD4þ T cells as assessed byflow cytometry and immunofluorescence in 4T1-bearing mice (A–C) or canine patients (D–G) treated with RT þ CpG þ 1MT. Representative flow cytometrycontour plots demonstrating staining of intratumoral CD4þ cells for FoxP3 and CD25 (A). Flow cytometry data represented as a bar graph expressed as%Treg (CD4þ, CD25þ, FoxP3þ) of CD4þ cells (B). Bar graph representation of CD4þ to Treg ratio asmeasured by flow cytometry (C). Representative flow cytometryplots demonstrating staining of canine intratumoral CD4þ cells for FoxP3 pre- and post-RTþCpGþ 1MT therapy (D). Bar graph representation of intratumoral Tregspre- and posttherapy expressed as a percentage of CD3þ cells in 4 canine patients as assessed by flow cytometry (E). Line graph demonstrates changes inTreg levels in individual patients as assessedbyflowcytometry (E). Immunofluorescent stainingof canine tumor samples for FoxP3 (F). FoxP3þ cells stain bright pink,and nuclei are counterstained by DAPI; white arrows point out examples of positive staining cells. Bar graph quantification of intratumoral FoxP3-positivecells (G). n ¼ 3 to 4 mice per group and 4 canine patients. Bar graphs represent mean � SEM. Results analyzed by one-way ANOVA or Student t test between theindicated groups (� , P < 0.05; �� , P < 0.01).






RT þ CpG þ 1-MT triple therapy antitumor effects are CD8þ T-cell dependent

We next examined the effects of this triple therapy on CD8þ

T cells andDCs. The percentage of tumor-infiltrating CD8þ T cells(expressed as a percentage of total viable cells within the tumor)significantly increased from 0.68% in control mice to 2.71% 7days after RTþ CpGþ 1MT (Fig. 5A and B; P ¼ 0.02). The CD8þ

T-cell/Treg ratio in the tumor was also increased by therapy from6.3 to 21.7 (Fig. 5C; P ¼ 0.0006). There were no changes inactivation/functional/exhaustionmarkers such as PD-1, CD69, orIFN-gamma in the CD8þ T cells after therapy, but the majority of

intratumoral CD8þ T cells expressed these markers at baseline(data not shown).We also evaluated changes in tumor-infiltratingCD8þ T cells in treated canines. By flow cytometric analysis, therewas a posttherapy increase in intratumoral CD8þ T cells (as apercentage of all CD3þ intratumoral cells) in all three evaluablecanines (Fig. 5D and E). Overall, the frequency of intratumoralCD8þ T cells doubled after therapy, which trended toward sta-tistical significance (Fig. 5D and E; 24% � 8.5% vs. 47.6% �16.4%; P¼ 0.06). IHC staining likewise demonstrated an increasein intratumoral CD8þ T cells after therapy (30 � 33 vs. 50 � 39cells/HPF; Fig. 5F and G; P ¼ 0.09). Mirroring our mouse studies

ControlA B C

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Figure 5.RadiationþCpGþ 1MT increases tumor-infiltrating CD8þ T cells inmice and canines. Representative flow cytometry contour plots demonstrating staining of tumor-infiltrating CD45þCD3þ cells for CD8 and CD4 (A). Flow cytometry data of tumor-infiltrating CD8þ T cells represented as a bar graph expressed as % CD8þ

cells of all CD45þ cells (B). CD8þ T cell to Treg ratio as determined by flow cytometry (C). Levels of tumor-infiltrating CD8þ T cells as assessed by flow cytometry incanine tumors (D and E). Representative flow cytometry contour plots demonstrating staining of canine intratumoral CD8þ and CD4þ T cells pre- andpost-RTþ CpGþ 1MT therapy (D). Bar graph representation of intratumoral CD8þ T cells expressed as a percentage of CD3þ cells in 3 canine patients as assessedby flow cytometry (E). Line graph demonstrates changes in Treg levels in individual patients as assessed by flow cytometry (E). Immunohistochemicalstaining with tumor-infiltrating CD8þ T cells stained in red, white arrows point out examples of positive staining cells (F). Bar graph quantification of intratumoralCD8-positive cells (G). CD8þ T cell to Treg ratio as determined by flow cytometry (H). Line graph demonstrates changes in CD8þ T cell to Treg ratio inindividual patients before to after treatment (H). n ¼ 3 to 4 mice per group and 3 canine patients. Bar graphs represent mean � SEM. Results analyzed by Studentt test (� , P < 0.05; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001).

Monjazeb et al.





therapy induced a significant increase in the CD8þ/Treg ratio (Fig.5H; P ¼ 0.014).

We tested the CD8 dependence of this therapy in mousemodels. Depletion of CD8þ T cells by intraperitoneal adminis-tration of anti-CD8 reduced the number of circulating CD8þ

T cells by >99% (Supplementary Fig. S5A and S5B). In micedepleted of CD8þ T cells, we observed that the antitumor effectsof RT þ CpG þ 1MT were significantly diminished. After treat-ment with the triple combination, tumors were more than tripledin size in CD8-depleted mice compared with those treated withcontrol IgG (Supplementary Fig. S5C; 2,066 mm3 vs. 684mm3; P< 0.0001). The survival benefit of the triple therapy was alsosignificantly diminished in CD8-depleted mice (SupplementaryFig. S5D; P ¼ 0.03), indicating the critical role of CD8þ T cells inthe antitumor effects.

In addition to the effects of CD8þ T cells, we also evaluated DCactivation and phenotypes following triple therapy. IDO expres-sion has been linked to a tolerogenic DC phenotype, associatedwith decreased expression of CD80 and MHC II and increasedTGF-beta production (35). No change in DC numbers or activa-tion within dLNs was observed (data not shown). Recent datademonstrate that intratumoral DCs, although a minor popula-

tion, play a critical role in antitumor T-cell responses (37, 38).Given that the major effects of this therapy occur in the tumormicroenvironment, we also examined DCs in the tumor micro-environment. There was an increased number of activated DCswithin treated tumors, with a 4-fold increase in CD80-expressingDCs (Supplementary Fig. S6A–S6C; P ¼ 0.02) and a near dou-bling of DCMHCII mean fluorescence intensity (MFI) comparedwith control mice (Supplementary Fig. S6D and S6E; P ¼ 0.01).

Canine immunologic biomarkersFinally, in three of the canine patients for which sufficient pre-

and posttreatment tumor tissue was available, we evaluated genesignatures by qPCR (Fig. 6). Based on ourmouse data, we focusedon expressionof three immunosuppressivemarkers (IDO1,TGFB,and FOXP3). Two of these patients demonstrated systemic partialresponses, and one had systemic progressive disease. Interesting-ly, in the two responders, expression of all three immunosup-pressive genes was markedly decreased after therapy, whereas allthree were increased in the setting of progressive disease. Thesedata suggest that this gene signature in the local tumor mayrepresent a predictive biomarker for systemic response to thistherapy and requires further evaluation in future studies. Overall,

Figure 6.Intratumoral expression of immunosuppressive molecules as a gene signature of systemic antitumor immune response in canines treated with radiationþ CpG þ 1MT. Sufficient pre- and posttherapy tumor tissue was available for further analysis in 3 canines: two responders and one with progressive disease.Expression of the immunosuppressive molecules IDO, TGF beta, and FoxP3 was assessed by qPCR, and results are expressed relative mRNA expressionof technical triplicates. Bar graphs represent mean � SEM.






althoughhypothesis-generating innature, these canine correlativeimmune analyses corroborate our mechanistic mouse data andprovide rationale for further exploration of these immunologicendpoints in future studies.

DiscussionThis is the first report of a novel immunotherapy strategy

combining local RT þ CpG with systemic IDO blockade. Theprimary purpose of this study was to examine whether IDO-mediated rebound immune suppression after RT þ CpG immu-notherapy maintains an immunosuppressive microenvironmentand limits efficacy. As hypothesized, RT þ CpG paradoxicallyupregulated IDO expression, which we termed "rebound immunesuppression." Importantly, this immunotherapy-induced increasein IDOexpressionwas alsoobservedwithother immunotherapies,suggesting that thesemechanismsmay have broader implications.Other studies also demonstrate the upregulation of IDO afterinflammatory signals (20, 21). This rebound immune suppressionsuggests a physiologic regulatory mechanism, whereby theimmune system tempers responses in inflammatory settings tomaintain homeostasis. The addition of IDO blockade decreasedIDO activity and substantially increased local and systemic anti-tumor effects. Mechanistically, this therapy reduced multipleimmune-suppressive factors, includingTregs, TAMs, andTGF-beta;increased intratumoral CD8þ T cells; activated intratumoral DCs;and restored IL6 signaling within the tumor microenvironment.

Although IDO blockade was administered systemically, theimmunologic effects of IDO were most pronounced in the tumormicroenvironment but limited in the tumor-draining lymphnodes. This suggests that the immune reactivation is likely occur-ring directly within the tumor microenvironment and that this issufficient to induce a systemic antitumor immune response. Arecent report by Levy and colleagues indicates that reversal ofimmune suppression triggering an active immune response at asingle site is sufficient to induce an effective systemic antitumorimmune response (39). Our data are also corroborated by find-ings demonstrating that after checkpoint inhibition or IDOblockade, little effect is seen in the dLNs, but there are markedeffects on CD8þ T cells in the tumor (40). Likewise, recent datademonstrate that intratumoral DCs, although a minor popula-tion, play a critical role in antitumor T-cell responses (37, 38).Overall, our findings are in line with the published literature andsupport an emerging concept that augmenting a pre-existent butpreviously ineffective antitumor immune response directlywithinthe local tumor microenvironment can serve as the nidus for asystemic response.

A principal finding of this study is the utility of RT in combi-nationwith immunotherapy. We observed no effect of the immu-notherapies alone or in combination but robust effects in com-bination with RT (Fig. 1). This synergy was seen in both theantitumor effects and the mechanistic immune changes inducedby therapy. The reduction in tumor-infiltrating Tregs and TAMswas only seen when RT was combined with immunotherapy (Fig.3). Likewise, the increase in tumor-infiltrating CD8þ T cellsinduced by RT þ CpG þ 1MT (Fig. 5) was not seen with RT orimmunotherapy alone (data not shown). These results add to thegrowing bodyof literature demonstrating the potent synergy of RTand immunotherapy.

The utility of mice to model human disease has recently beencalled into question (41). Mouse models provide an excellent

platform for exploratory studies, but often fail to adequatelyrecapitulate treatment efficacy or toxicity in human clinicaltrials (42). The introduction of pretranslational confirmatorystudies in a more robust model of human disease can helpimprove the efficiency of the translational pipeline by limitinghuman testing of therapies that are unlikely to be effective ortolerated. We tested our therapy in such a model by initiating apilot veterinary clinical trial in companion canines with late-stage metastatic spontaneous melanomas or sarcomas. Thesecancers arise, grow, and metastasize in the setting of an intactimmune system and with patterns akin to human tumors (43).Canine immune systems are genetically and developmentallymuch more similar to humans than rodent models (44–47). Inaddition, companion dogs share many environmental riskfactors with their human owners. In dogs with rapidly progres-sive systemic disease, our results demonstrated a significantresponse at the local RT-treated primary tumor as well asresponse or disease stabilization in 80% of dogs at untreatedsites of metastatic disease. This included substantial reductionsof bulky pulmonary metastases which were well outside of theirradiated fields. We also observed significant changes in theimmunosuppressive tumor microenvironment confirming themechanistic data from our mouse models. Overall, the antitu-mor effects and mechanistic immunologic findings from ourmouse models were paralleled in the canine clinical trial.

Immunotherapy can provide durable responses in a number ofmetastatic cancers (1–3). Unfortunately, most patients will fail torespond to these therapies, and these treatments can inducesignificant toxicities. To increase response rates, combinatorialstrategies are being actively investigated. Recent clinical trialsdemonstrate that combining PD-1 and CTLA-4 checkpoint inhi-bitors can increase response rates in metastatic melanoma but arealso accompanied by an equally marked increase in toxicity with40%of patients responding but also 53%of patients experiencinggrade 3 or 4 toxicities (48). These toxicities were attributed to theimmune dysregulation induced bymultiple checkpoint blockade.RT þ CpG þ 1MT employs immune stimulation in combinationwith blockade of immune suppression limiting overlapping tox-icity profiles and leavingmultiple checkpoint pathways intact.Weobserved minimal toxicity with this regimen.

An area for future study is the assessment of longer courses oftherapy. Treatment only lasted 2 weeks in mice and 4 weeks incanines. All of our canines eventually recurred after treatmentdiscontinuation. Although robust responses were observed to thisshort treatment course, most published immunotherapy strate-gies employ prolonged courses of therapy, and it is likely thatprolonging the course of therapy in our models would furtherincrease the magnitude and duration of response.

In summary, this study substantiates the power of combina-torial immunotherapy strategies and confirms that RT can be apotent partner for immunotherapy. Furthermore, it demonstratesthat combinatorial strategies designed to minimize overlappingtoxicity can be safe and effective. The correlative studies describedhere demonstrate the importance of posttreatment tumor biop-sies, which are rarely employed in human trials, to evaluate theimmunologic changes induced by therapy and to further ourunderstanding of the mechanistic underpinnings of therapyresponse and failure. In addition, they suggest that IDO-mediatedrebound immune suppression may be a general mechanism ofresistance to some immunotherapy strategies and that additionof IDO blockade may improve the effectiveness of such


Monjazeb et al.




strategies. They demonstrate the feasibility of preclinical testing ofimmunotherapy in spontaneous canine malignancies as a pre-translational step in a model more reflective of human disease.Overall, these studies provide a strong rationale for clinicaltranslation of this immunotherapy strategy to substantiallyimprove the already documented efficacy of RT þ CpG.

Disclosure of Potential Conflicts of InterestM. Chen reports receiving a commercial research grant from Bayer. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: A.M. Monjazeb, M.S. Kent, M. Chen, G.D. SckiselDevelopment ofmethodology: A.M.Monjazeb,M.S. Kent, S.K. Grossenbacher,A. Mirsoian, M. Chen, A. Kol, E.E. SpargerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.M. Monjazeb, M.S. Kent, S.K. Grossenbacher,A. Mirsoian, M. Chen, A. Kol, S.L. Shiao, A. Reddy, J.R. Perks, W.T.N. CulpAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.M. Monjazeb, S.K. Grossenbacher, C. Mall, A.E.Zamora, M. Chen, A. Kol, S.L. Shiao, A. Reddy, W.T.N. Culp, E.E. Sparger,R.J. CanterWriting, review, and/or revision of themanuscript:A.M.Monjazeb,M.S. Kent,S.K. Grossenbacher, C. Mall, A.E. Zamora, A. Mirsoian, M. Chen, A. Kol,S.L. Shiao, W.T.N. Culp, R.J. Canter, G.D. Sckisel, W.J. Murphy

Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.M. Monjazeb, S.K. Grossenbacher, C. Mall,A.E. Zamora, A. Mirsoian, A. ReddyStudy supervision: A.M. Monjazeb, W.J. Murphy

AcknowledgmentsThe authors acknowledge Weihong Ma, Shuaib Juma, Hong Chang, Hung

Kieu, and Monja Metcalf for their technical support and Teri Guerrero forveterinary clinical trials support.

Grant SupportThis study was supported by UC Davis CTSC K12 Scholar Program, NIH 2

KL2 RRO24144-06; American Cancer Society Institutional Research Grant,IRG-95-125-07; Amador Cancer Research Foundation; Christine and HelenLandgraf Award; and NIH R01 CA 095572, NIH R01 CA 072669, and NCIP30CA093373.

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 December 18, 2015; revised February 25, 2016; accepted February28, 2016; published OnlineFirst March 15, 2016.

References1. Brahmer JR, Tykodi SS, ChowLQ,HwuWJ, Topalian SL,HwuP, et al. Safety

and activity of anti-PD-L1 antibody in patients with advanced cancer.N Engl J Med 2012;366:2455–65.

2. Hodi FS,O'Day SJ,McDermott DF,Weber RW, Sosman JA,Haanen JB, et al.Improved survivalwith ipilimumab in patients withmetastaticmelanoma.N Engl J Med 2010;363:711–23.

3. Topalian SL,Hodi FS, Brahmer JR,Gettinger SN, SmithDC,McDermottDF,et al. Safety, activity, and immune correlates of anti-PD-1 antibody incancer. N Engl J Med 2012;366:2443–54.

4. Formenti SC, Demaria S. Systemic effects of local radiotherapy. LancetOncol 2009;10:718–26.

5. Milas L, Mason KA, Ariga H, Hunter N, Neal R, Valdecanas D, et al. CpGoligodeoxynucleotide enhances tumor response to radiation. Cancer Res2004;64:5074–7.

6. Brody JD, Ai WZ, Czerwinski DK, Torchia JA, Levy M, Advani RH, et al. Insitu vaccination with a TLR9 agonist induces systemic lymphoma regres-sion: A phase I/II study. J Clin Oncol 2010;28:4324–32.

7. Kim YH, Gratzinger D, Harrison C, Brody JD, Czerwinski DK, Ai WZ, et al.In situ vaccination againstmycosis fungoides by intratumoral injection of aTLR9 agonist combined with radiation: A phase 1/2 study. Blood2012;119:355–63.

8. Baban B, Chandler PR, Sharma MD, Pihkala J, Koni PA, Munn DH, et al.IDOactivates regulatory T cells andblocks their conversion into Th17-like Tcells. J Immunol 2009;183:2475–83.

9. Peng G, Guo Z, Kiniwa Y, Voo KS, Peng W, Fu T, et al. Toll-like receptor 8-mediated reversal of CD4þ regulatory T cell function. Science 2005;309:1380–4.

10. Wang Z, Zheng Y,HouC, Yang L, Li X, Lin J, et al. DNAmethylation impairsTLR9 induced Foxp3 expression by attenuating IRF-7 binding activity infulminant type 1 diabetes. J Autoimmun 2013;41:50–9.

11. Mitsuka K, Kawataki T, Satoh E, Asahara T, Horikoshi T, Kinouchi H.Expression of indoleamine 2,3-dioxygenase and correlation with patho-logical malignancy in gliomas. Neurosurgery 2013;72:1031–8; discussion1038–9.

12. Ye J, LiuH,HuY, Li P, ZhangG, Li Y. Tumoral indoleamine 2,3-dioxygenaseexpression predicts poor outcome in laryngeal squamous cell carcinoma.Virchows Arch 2013;462:73–81.

13. UyttenhoveC, Pilotte L, Theate I, Stroobant V, ColauD, Parmentier N, et al.Evidence for a tumoral immune resistance mechanism based on trypto-phan degradation by indoleamine 2,3-dioxygenase. Nat Med 2003;9:1269–74.

14. Munn DH, Mellor AL. Indoleamine 2,3-dioxygenase and tumor-inducedtolerance. J Clin Invest 2007;117:1147–54.

15. Sharma MD, Baban B, Chandler P, Hou DY, Singh N, Yagita H, et al.Plasmacytoid dendritic cells from mouse tumor-draining lymph nodesdirectly activate mature Tregs via indoleamine 2,3-dioxygenase. J ClinInvest 2007;117:2570–82.

16. Sharma MD, Hou DY, Liu Y, Koni PA, Metz R, Chandler P, et al.Indoleamine 2,3-dioxygenase controls conversion of Foxp3þ Tregs toTH17-like cells in tumor-draining lymph nodes. Blood 2009;113:6102–11.

17. ChenW, Liang X, Peterson AJ, Munn DH, Blazar BR. The indoleamine 2,3-dioxygenase pathway is essential for human plasmacytoid dendritic cell-induced adaptive T regulatory cell generation. J Immunol 2008;181:5396–404.

18. Yan Y, Zhang GX, Gran B, Fallarino F, Yu S, Li H, et al. IDO upregulatesregulatory T cells via tryptophan catabolite and suppresses encephalito-genic T cell responses in experimental autoimmune encephalomyelitis.J Immunol 2010;185:5953–61.

19. Mellor AL, Munn DH. IDO expression by dendritic cells: Tolerance andtryptophan catabolism. Nat Rev Immunol 2004;4:762–74.

20. Mellor AL, Baban B, Chandler PR, Manlapat A, Kahler DJ, Munn DH.Cutting edge: CpG oligonucleotides induce splenic CD19þ dendriticcells to acquire potent indoleamine 2,3-dioxygenase-dependent T cellregulatory functions via IFN Type 1 signaling. J Immunol 2005;175:5601–5.

21. Von Bubnoff D, Scheler M,Wilms H, Fimmers R, Bieber T. Identification ofIDO-positive and IDO-negative human dendritic cells after activation byvarious proinflammatory stimuli. J Immunol 2011;186:6701–9.

22. Hou DY, Muller AJ, Sharma MD, DuHadaway J, Banerjee T, Johnson M,et al. Inhibition of indoleamine 2,3-dioxygenase in dendritic cells bystereoisomers of 1-methyl-tryptophan correlates with antitumorresponses. Cancer Res 2007;67:792–801.

23. MurphyWJ, Welniak L, Back T, Hixon J, Subleski J, Seki N, et al. Synergisticanti-tumor responses after administration of agonistic antibodies to CD40and IL-2: coordination of dendritic and CD8þ cell responses. J Immunol2003;170:2727–33.

24. Perks JR, Lucero S, Monjazeb AM, Li JJ. Anthropomorphic phantoms forconfirmation of linear accelerator-based small animal irradiation. Cureus2015;7:e254.

25. Wolchok JD, Hoos A, O'Day S, Weber JS, Hamid O, Lebbe C, et al.Guidelines for the evaluation of immune therapy activity in solid






tumors: Immune-related response criteria. Clin Cancer Res 2009;15:7412–20.

26. Ladue T, Klein MK, Veterinary Radiation Therapy Oncology G. Toxicitycriteria of the veterinary radiation therapy oncology group. Vet RadiolUltrasound 2001;42:475–6.

27. Aslakson CJ,Miller FR. Selective events in themetastatic process defined byanalysis of the sequential dissemination of subpopulations of a mousemammary tumor. Cancer Res 1992;52:1399–405.

28. Jia L, Schweikart K, Tomaszewski J, Page JG, Noker PE, Buhrow SA, et al.Toxicology and pharmacokinetics of 1-methyl-d-tryptophan: Absenceof toxicity due to saturating absorption. Food Chem Toxicol 2008;46:203–11.

29. Proulx DR, Ruslander DM, Dodge RK, Hauck ML, Williams LE, HornB, et al. A retrospective analysis of 140 dogs with oral melanomatreated with external beam radiation. Vet Radiol Ultrasound 2003;44:352–9.

30. Koyama K, Kagamu H, Miura S, Hiura T, Miyabayashi T, Itoh R, et al.Reciprocal CD4þ T-cell balance of effector CD62Llow CD4þ andCD62LhighCD25þ CD4þ regulatory T cells in small cell lung cancerreflects disease stage. Clin Cancer Res 2008;14:6770–9.

31. Sinicrope FA, Rego RL, Ansell SM, Knutson KL, Foster NR, Sargent DJ.Intraepithelial effector (CD3þ)/regulatory (FoxP3þ) T-cell ratio predicts aclinical outcome of human colon carcinoma. Gastroenterology 2009;137:1270–9.

32. Luo Y, Zhou H, Krueger J, Kaplan C, Lee SH, Dolman C, et al. Targetingtumor-associated macrophages as a novel strategy against breast cancer. JClin Invest 2006;116:2132–41.

33. Chen W, Konkel JE. TGF-beta and 'adaptive' Foxp3(þ) regulatory T cells. JMol Cell Biol 2010;2:30–6.

34. Teicher BA.Transforming growth factor-beta and the immune response tomalignant disease. Clin Cancer Res 2007;13:6247–51.

35. Yang J, Yang Y, Fan H, Zou H. Tolerogenic splenic IDO (þ) dendritic cellsfrom the mice treated with induced-Treg cells suppress collagen-inducedarthritis. J Immunol Res 2014;2014:831054.

36. Stephen TL, Rutkowski MR, Allegrezza MJ, Perales-Puchalt A, Tesone AJ,Svoronos N, et al. Transforming growth factor beta-mediated suppressionof antitumor T cells requires FoxP1 transcription factor expression. Immu-nity 2014;41:427–39.

37. Broz ML, Binnewies M, Boldajipour B, Nelson AE, Pollack JL, Erle DJ, et al.Dissecting the tumormyeloid compartment reveals rare activating antigen-presenting cells critical for T cell immunity. Cancer Cell 2014;26:638–52.

38. Ruffell B, Chang-StrachanD, Chan V, Rosenbusch A, Ho CM, Pryer N, et al.Macrophage IL-10 blocks CD8þ T cell-dependent responses to chemo-therapy by suppressing IL-12 expression in intratumoral dendritic cells.Cancer Cell 2014;26:623–37.

39. Marabelle A, Kohrt H, Sagiv-Barfi I, Ajami B, Axtell RC, Zhou G, et al.Depleting tumor-specific Tregs at a single site eradicates disseminatedtumors. J Clin Invest 2013;123:2447–63.

40. Spranger S, Koblish HK, Horton B, Scherle PA, Newton R, Gajewski TF.Mechanism of tumor rejection with doublets of CTLA-4, PD-1/PD-L1, orIDO blockade involves restored IL-2 production and proliferation of CD8(þ) T cells directly within the tumor microenvironment. J ImmunotherCancer 2014;2:3.

41. Seok J, Warren HS, Cuenca AG, Mindrinos MN, Baker HV, Xu W, et al.Genomic responses in mouse models poorly mimic human inflammatorydiseases. Proc Natl Acad Sci U S A 2013;110:3507–12.

42. Voskoglou-Nomikos T, Pater JL, Seymour L. Clinical predictive value of thein vitro cell line, human xenograft, and mouse allograft preclinical cancermodels. Clin Cancer Res 2003;9:4227–39.

43. Felsburg PJ. Overview of immune system development in the dog: Com-parison with humans. Hum Exp Toxicol 2002;21:487–92.

44. Gu YC, Bauer TRJr, Ackermann MR, Smith CW, Kehrli MEJr, Starost MF,et al. The genetic immunodeficiency disease, leukocyte adhesion deficien-cy, in humans, dogs, cattle, and mice. Comp Med 2004;54:363–72.

45. Holsapple MP, West LJ, Landreth KS. Species comparison of anatomicaland functional immune system development. Birth Defects Res B DevReprod Toxicol 2003;68:321–34.

46. Govindaraj RG, Manavalan B, Basith S, Choi S. Comparative analysis ofspecies-specific ligand recognition in Toll-like receptor 8 signaling: ahypothesis. PLoS One 2011;6:e25118.

47. Liu J, Xu C, Hsu LC, Luo Y, Xiang R, Chuang TH. A five-amino-acidmotif inthe undefined region of the TLR8 ectodomain is required for species-specific ligand recognition. Mol Immunol 2010;47:1083–90.

48. Wolchok JD, Kluger H, CallahanMK, PostowMA, Rizvi NA, Lesokhin AM,et al. Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med2013;369:122–33.


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2016;22:4328-4340. Published OnlineFirst March 15, 2016.Clin Cancer Res Arta M. Monjazeb, Michael S. Kent, Steven K. Grossenbacher, et al. Murine Models and Spontaneous Canine MalignanciesSuppression Boosts Antitumor Effects of Radio-Immunotherapy in Blocking Indolamine-2,3-Dioxygenase Rebound Immune

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