Abscopal Effects of Radiotherapy Are Enhanced by Combined … · 2016-09-29 · Microenvironment and Immunology Abscopal Effects of Radiotherapy Are Enhanced by Combined Immunostimulatory

Microenvironment and Immunology

Abscopal Effects of Radiotherapy Are Enhancedby Combined Immunostimulatory mAbs and AreDependent on CD8 T Cells and CrossprimingMaría E. Rodriguez-Ruiz1,2, Inmaculada Rodriguez1, Saray Garasa1, Benigno Barbes1,Jose Luis Solorzano2, Jose Luis Perez-Gracia2, Sara Labiano1, Miguel F. Sanmamed1,3,Arantza Azpilikueta1, Elixabet Bola~nos1, Alfonso R. Sanchez-Paulete1, M. Angela Aznar1,Ana Rouzaut1, Kurt A. Schalper4,5, Maria Jure-Kunkel6, and Ignacio Melero1,2

Abstract

Preclinical and clinical evidence indicate that the proim-mune effects of radiotherapy can be synergistically augmentedwith immunostimulatory mAbs to act both on irradiatedtumor lesions and on distant, nonirradiated tumor sites. Thecombination of radiotherapy with immunostimulatory anti-PD1 and anti-CD137 mAbs was conducive to favorable effectson distant nonirradiated tumor lesions as observed in trans-planted MC38 (colorectal cancer), B16OVA (melanoma), and4T1 (breast cancer) models. The therapeutic activity was cru-cially performed by CD8 T cells, as found in selective depletionexperiments. Moreover, the integrities of BATF-3–dependentdendritic cells specialized in crosspresentation/crosspriming ofantigens to CD8þ T cells and of the type I IFN system wereabsolute requirements for the antitumor effects to occur. Theirradiation regimen induced immune infiltrate changes in the

irradiated and nonirradiated lesions featured by reductions inthe total content of effector T cells, Tregs, and myeloid-derivedsuppressor cells, while effector T cells expressed more intracel-lular IFNg in both the irradiated and contralateral tumors.Importantly, 48 hours after irradiation, CD8þ TILs showedbrighter expression of CD137 and PD1, thereby displayingmore target molecules for the corresponding mAbs. Likewise,PD1 and CD137 were induced on tumor-infiltrating lympho-cytes from surgically excised human carcinomas that wereirradiated ex vivo. These mechanisms involving crossprimingand CD8 T cells advocate clinical development of immuno-therapy combinations with anti-PD1 plus anti-CD137 mAbsthat can be synergistically accompanied by radiotherapy strat-egies, even if the disease is left outside the field of irradiation.Cancer Res; 76(20); 1–12. �2016 AACR.

IntroductionRadiotherapy is a solid pillar of cancer treatment used to treat

localized stages of a broad variety of malignant diseases and toalleviate local complications in advanced or metastatic cases as apalliative treatment. The mechanism of action of ionizing radio-therapy against cancer is thought to mainly rely on catastrophicdamage of genomic DNA, leading to apoptotic tumor cell death.Many cellular genetic and epigenetic factors affect the sensitivity of

each tumor to radiotherapy approaches. Recently, the tumorstroma component has been found to play a key role in theoutcome of irradiated tumors (1). When radiotherapy is pre-scribed to a patient, it is assumed that the normal nonmalignanttissue will also be irradiated giving rise to multifarious biologicaleffects including inflammation and scarring (1). Radiotherapycan be performed by applying an external beam of irradiation orby the temporal surgical insertion of radiation sources guided bycatheters into the cancer tissue using techniques collectivelyknown as brachytherapy.

Immunotherapy is emerging as another major pillar for thetreatment of cancer treatment. mAbs acting on immune systemreceptors to derepress or agonistically augment antitumorimmunity are being developed in the clinic (2). Antibodiesagainst the inhibitory (checkpoint) receptor CTLA-4 were thefirst to be clinically developed with ipilimumab receiving FDAand European Medicines Agency (EMA) approval for metastaticmelanoma (3). Among these checkpoint inhibitor monoclonalimmunostimulatory antibodies, agents blocking the PD1/PD-L1 receptor/ligand pair have already attained FDA and EMAapproval for metastatic melanoma (4), non–small cell lungcancer (5–7), and renal cell carcinoma (8) and other indica-tions are under regulatory evaluation. This achievement waspreceded by extensive and successful preclinical research inmouse models.

Agonist antibodies crosslinking CD137 (4-1BB) were alsoshown to enhance antitumor immunity in mice to the point of

1Division of Immunology and Immunotherapy, Center for AppliedMedical Research (CIMA), University of Navarra and Instituto deInvestigacion Sanitaria de Navarra (IdISNA), Pamplona, Spain. 2Uni-versity Clinic, University of Navarra and Instituto de InvestigacionSanitaria de Navarra (IdISNA), Pamplona, Spain. 3Department ofImmunobiology, Yale School of Medicine, New Haven, Connecticut.4Department of Pathology, Yale School of Medicine, New Haven,Connecticut. 5Department of Medicine (Medical Oncology), YaleSchool of Medicine, New Haven, Connecticut 6Bristol-Myers Squibb,Lawrenceville, New Jersey.

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

Corresponding Authors: Ignacio Melero, University of Navarra and Instituto deInvestigacion Sanitaria de Navarra (IdISNA), Av. Pio XII, 55, Pamplona, Navarra31008, Spain. Phone: 349-4819-4700; Fax: 349-4819-4717; E-mail:[email protected]; and Maria E. Rodríguez-Ruiz, [email protected]

doi: 10.1158/0008-5472.CAN-16-0549

�2016 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org OF1

Research. on October 9, 2020. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from

Published OnlineFirst August 22, 2016; DOI: 10.1158/0008-5472.CAN-16-0549

http://cancerres.aacrjournals.org/

causing the rejection of transplanted tumors (9). Two antibo-dies against CD137 are undergoing phase II clinical trialswith promising results (10, 11). Anti-PD1 and anti-CD137mAbs act on T cells that express these receptors on theirplasma membrane presumably as a consequence of an anti-gen-cognate activation process. Hence, the main mechanismof action is exerted on tumor-infiltrating lymphocytes thatexpress such receptors on their surface, thus becoming ame-nable to pharmacologic modulation with the correspondingmAb. In preclinical mouse models, anti-CD137 and anti-PD1mAbs exert powerful synergistic effects (12) that have givenrise to two ongoing clinical trials testing such a combination(NCT02253992, NCT02179918).

The interphase between radiotherapy and immunotherapy isan exciting emerging topic. Radiotherapy causes biologicaleffects known to both ignite (13, 14) and quench the cellularimmune response (13, 14). The type of cell death induced byradiotherapy is considered immunogenic (15, 16), because itsets in motion multiple alarmins (15, 16) and proinflamma-tory mechanisms (17). Radiotherapy-induced cell death is apotential source of tumor antigens to be uptaken, processed,and presented by dendritic cells to CD8þ T lymphocytes, aprocess that is collectively known as crosspresentation (18)and termed crosspriming if it results in CTL activation. Cross-presentation to CD8þ T cells is mainly mediated by a special-ized subset of dendritic cells, which are dependent for devel-opment on the Batf-3 transcription factor (19) and on sFLT-3Las a growth factor. We have published that this dendritic cell(DC) subset is critical for the therapeutic effects of anti-PD1and anti-CD137 mAbs by means of crosspresentation of tumorantigens (20). This DC subset is also known to be involvedin eliciting postradiotherapy CTL immune responses (21).However, other mechanisms such as irradiation-dependentTGFb production and myeloid cell recruitment are consideredimmunosuppressive.

Immunostimulatory mAbs have already been combinedwith radiotherapy in preclinical models. Anti-CTLA4 mAb(22), anti-PD1 mAb (23, 24), and anti-CD137 mAb(25–27), show evidence for synergistic effects with externalbeam irradiation. Furthermore, triple combinations of radio-therapy with anti CTLA-4 plus anti PD1 exert efficacious syn-ergistic effects against B16F10 melanoma tumors as seenagainst the directly irradiated tumor and a concomitant tumor,implanted outside the irradiation field (28), a phenomenonknown as the abscopal effect of radiotherapy (29).

Anecdotal evidence in the clinic suggests that in a patienttreated with anti CTLA-4 mAb (ipilimumab) and subsequentpalliative radiotherapy, there were objective responses outsidethe irradiation field, concurrent with increases in the titer ofantibodies against the shared tumor antigen NY-ESO1 (30). Ina phase II clinical trial testing the ipilimumab plus radiotherapycombination, there was a trend toward better overall survival inmetastatic melanoma patients (28).

In this study, we use different mouse models to demonstratethat external beam radiotherapy synergizes with immunostimu-latory anti-PD1 and anti-CD137mAbs as single agents and whenused in combination. The therapeutic effects were attributed toCD8 T cells by depletion experiments and involved profoundchanges in the tumor microenvironment that include and aug-ment the expression of the receptors to be targeted by the immu-nomodulatory mAb.

Materials and MethodsCell lines

Tumor cells lines, MC38, a colon adenocarcinoma cell line ofC57BL/6 origin whose identity (Case 6592-2012) was providedto us by Dr. Karl E. Hellstr€om (University of Washington,Seattle, WA). The 4T1 breast carcinoma cells of BALB/c originwere originally provided by Dr. Claude Lecrec, Institute Pas-teur, Paris, France, and verifed in the master cell bank at Insti-tute Pasteur (Paris, France). B16F10-OVA melanoma–derivedcells that are transfected to express chicken ovalbumin (OVA)were verified by Idexx Radil in 2012 and kept in a master cellbank as vials thawn every 3–6 months and were cultured inRPMI1640 supplemented with 10% FBS, 2 mmol/L L-gluta-mine, 0.05 mmol/L 2-mercaptoethanol, HEPES, penicillin, andstreptomycin at 37�C in a humidified atmosphere containing5% CO2. All these cells lines were certified as being free ofcontamination by Mycoplasma using the Mycoplasma detec-tion kit (MycoAlert Mycoplasma Detection Kit from Lonza).

In vivo tumor experimentsC57BL/6 female mice were injected subcutaneously with

5 � 105 MC38 and 5 � 105 B16OVA cells, respectively, inthe right flank (primary tumor) and with 3 � 105 MC38 and3 � 105 B16OVA cells in the left flank (secondary tumor). Asimilar scheme was used to subcutaneously engraft 4T1 cells infemale BALB/c mice. Perpendicular tumor diameters were mea-sured with a Vernier calipers every 2–3 days, and tumorvolumes were calculated. On day 11, when both tumors werepalpable, animals were randomly assigned to 8 groups receiv-ing or not receiving radiotherapy (8 Gy � 3 fractions), to onlyone of the two tumors, in combination or not in combinationwith intraperitoneal immunostimulatory mAbs (anti-PD1,anti-CD137, or both). Anti-PD1, anti-CD137, the combina-tion, or anti-rat IgG control antibody were administered intra-peritoneally at the dose of 200 mg/mouse (10mg/kg) or 100 mg/mouse (5 mg/kg) on days 13, 15, and 17. In some experiments,monoclonal immunostimulatory antibodies were adminis-tered on days 17, 19, 20. Tumor size was monitored every2–3 days and mice were sacrificed when tumor size reached4,000 mm3. Tumor radiotherapy procedures are detailed inSupplementary Materials section.

Flow cytometry and ELISA assaysTumor tissue was processed to obtain single-cell suspension for

flow cytometry analysis (see Supplementary Methods). To esti-mate absolute numbers in cell suspension, perfect count micro-spheres were used as an internal standard according to themanufacturer's instructions (Cytognos).

Levels of human IFNg in mouse plasma samples were mea-sured by a commercial ELISA (Human IFNg Elisa Set, BDOptEIA, BD Biosciences), following the manufacturer's instruc-tions. All samples were measured in duplicate. The detectioncut-off levels of the assay were 4.7 pg/mL for IFNg . Thecoefficient of variation was <15%. For tumor antigen–specificCD8 T-cell assessment, a H-2Kb KSPWFTTL tetramer labeledwith PE (manufactured by Biolegend) was used. For gatingand costaining, the following mAbs were used: CD45.2 PerCP/Cy5.5 (clone 104 from Biolegend), CD4 BV421 (clone RM4-5from Biolegend), CD8 BV510 (clone 53-6.7 from Biolegend),CD137 biotin (clone 17B5 from Biolegend), and PD-1 FITC(clone 29F.1A12 from Biolegend).

Rodriguez-Ruiz et al.

Cancer Res; 76(20) October 15, 2016 Cancer ResearchOF2




Statistical analysisStatistical differences between survival curves were analyzed

with the Mantel–Cox, log-rank test, nonlinear regression anddifferences between other groups were analyzed with theMann–Whitney U test using GraphPad Prism (GraphPad Soft-ware Inc.)

ResultsAbscopal effects of radiotherapy are synergized by anti-CD137and anti-PD1 immunomodulatory mAb

Mice bearing bilateral tumors derived from subcutaneousengraftment of MC38 colorectal carcinoma cells were used as amodel to monitor the abscopal effects of radiotherapy in com-bination with immunostimulatory mAbs. Eight Gy fractionateddoses of external beam radiotherapy were selectively applied onlyto one of the tumor lesions, while a contralateral tumor was setoutside the irradiation field (see representative dosimetry inSupplementary Fig. S1A). Contralateral concomitant tumors wereinoculated the same day with 10-fold fewer tumor cells. Radio-therapy given every other day was followed on alternate days bythree doses of anti-CD137or/and anti-PD1mAbs. ThemAbsweregiven as single agents or in combination as detailed in Supple-mentary Fig. S1B. Supplementary Table S1A individually showsthe statistical comparisons of the evolution of irradiated andnonirradiated tumor lesions. Results collectively indicate thatboth anti-PD1 and anti-CD137 mAb contributed to controlcontralateral tumor growth when in conjunction with unilateralradiotherapy. Strikingly, the mice receiving radiotherapy and thecombination of the two immunostimulatory mAbs were thegroup that achieved faster and almost constant completeresponses (Supplementary Fig. S1C), translated in 100% long-term overall survival (Supplementary Fig. S1D). Of note, curedmice were immune 3 months later to MC38 tumor cell rechal-lenge, while able to engraft B16OVA melanoma cells as anantigenically unrelated control (Supplementary Fig. S1E).

Of note, combined treatment was well tolerated by the mice interms of safety. Given the fact that CD137 mAb can cause liverinflammation (31), we assessed ALT serum levels and checkedliver pathology specimens that ruled out increased toxicity due tothe addition of local radiotherapy to the immunostimulatoryantibody combination (unpublished observations).

Similar experiments were carried out with bilateral B16OVAmelanoma (Fig. 1A), known to be of difficult treatment byimmunotherapy (32, 33). In this case, mice bearing tumors for11 days showed a radiotherapy-dependent control of contralat-eral tumors, when distant radiotherapy was combinedwith eitheranti-PD1 or anti-CD137 mAb. When both antibodies were com-bined together, all the tumors regressed bilaterally, even thoughcombined immunotherapy without irradiation also induced theregression of most tumors (Fig. 1B; Supplementary Table S1A)achieving long-term survival (Fig. 1C). When mice cured by theradiotherapy plusmAb combinationwere rechallenged 3monthslater, 3 of 5 mice were protected from B16OVA, while growingMC38 as a contralateral antigenically unrelated control tumor(unpublished observations). Remarkably, measurements of theconcentration of IFNg in sera from mice undergoing triple com-bined treatment (radiotherapy plus anti-PD1 plus anti-CD137)showed much higher levels than any other treatment regimen ondayþ18 (Fig. 1D). This fact strongly indicated an ongoing cellularimmune response of far greater intensity.

4T1 breast cancer is an exceedingly difficult tumor model totreat with immunotherapy (34) that causes spontaneous lungmetastases. In this setting, we again performed experimentswith bilateral tumors, irradiating only one of the lesions(Fig. 2A). Tumor growth analyses indicated better local anddistant tumor control when radiotherapy was combined withimmunotherapy but without achieving complete responses(Fig. 2B; Supplementary Table S1A), although this treatmentdid lead to longer survival (unpublished observations). Fur-thermore, spontaneous metastases to the lung were followed byCT scans and by surgical inspection upon sacrifice with thequantification of the number and size of metastases (Fig. 2Cand Supplementary Fig. S2). As can be seen in Fig. 2C, overallnumbers of spontaneous lung metastases were reduced in theradiotherapy plus combined mAb immunotherapy group. InSupplementary Fig. S2, individual CT scan sections and repre-sentative excised lungs are shown.

In the case of bilateral MC38 tumors, experiments were alsoperformed starting treatment as late as day þ14 (Fig. 3A) aftertumor cell engraftment to ascertain the limits of the strategyand demonstrate radiotherapy synergy with the anti-CD137plus anti-PD1 combination regimen. In this case, the treat-ments were not curative in any case (Fig. 3B and C), but thedelay in tumor progression induced by the triple combination(radiotherapy plus anti-PD1 plus anti-CD137) was readily seenin comparison when monitoring the contralateral tumor. Nonoticeable effects were exerted by each of the mAb when usedseparately in this regimen, or when the immunotherapy com-bination was employed without radiotherapy (Fig. 3B and C;Supplementary Table S1B).

CD8 T cells, BATF-3–dependent dendritic cells, and the type IIFN system are necessary for the radiotherapy abscopal effectspotentiated by anti-CD137 and anti-PD1 mAbs

Using treatment conditions comparable with those in Fig. 1and Supplementary Fig. S1, on bilateral MC38-derived tumors,we repeatedly depleted CD8b

þ T cells, CD4þ T cells, and NK1.1þ lymphocytes with specific mAbs (Fig. 4A). As can be seenin Fig. 4B and C, in mice treated with the combinatorialregimen of radiotherapy plus anti-CD137 and anti-PD1 mAbs,we observed that CD8 T cells were absolutely required for thecontralateral antitumor effects. In contrast, CD4 T-cell deple-tion resulted in a more pronounced therapeutic effect with allanimals achieving complete bilateral regressions and showing astriking effect on overall survival. NK1.1 depletion had littleeffect on the outcome of the contralateral tumors (Fig. 4B andC; Supplementary Table S1C). These results show the involve-ment of cytolytic T lymphocytes in the beneficial effect of thecombinatorial regimen. CD4 depletion also eliminates regula-tory T cells likely explaining the better outcome upon depletionwith anti-CD4 mAb. Induction of CTLs against tumor antigensis mainly mediated by BATF-3–dependent DC (20). Accord-ingly, we performed similar experiments in BATF-3–deficientmice, which showed no abscopal effects and had weaker localtumor control by radiotherapy (Fig. 5A and B). In line with this,BATF-3�/� mice showed no increases in serum IFNg followingcombined treatment (Fig. 5C)

As both CTLs (35) and crosspriming (36) are known to bedependent on type I IFNs, we next studied treatment in thesame experimental setting (Fig. 5A) of mice devoid or notof type I IFN receptor (IFNAR�/�). As can be observed

Radioimmunotherapy and Abscopal Effects

www.aacrjournals.org Cancer Res; 76(20) October 15, 2016 OF3




in Fig. 5D, the abscopal effects exerted by combined radio-immunotherapy were completely abrogated in IFNAR-deficientmice. Importantly, the local effects on the directly irradiatedtumors were also decreased to same extent, suggesting animportant role of the IFNa/b system on the therapeutic effectsexerted by radiotherapy.

Radiotherapy changes the immune tumor microenvironmentin nonirradiated tumor lesions

Observations of therapeutic effects on tumor lesions outsidethe irradiation fields prompted us carry out experiments toinvestigate changes in the immune contexture of the tumormicroenvironment due to irradiation. Our 8 Gy fractionateddoses on alternate days (scheme in Supplementary Fig. S3A) wereapplied tomice bearing bilateral MC38-derived tumors. Absolutenumbers and density of T lymphocyte subsets were quantitated atthe end of the regimen on day þ17. We observed that CD4 T-celland CD8 T-cell numbers were clearly reduced both at the tumor

site receiving radiation and, importantly, at the tumor lesionoutside the irradiation field. FOXP3þ CD4þ Treg cells were alsoreduced at the contralateral site and less clearly so at the irradiatedsite (Supplementary Fig. S3B and S3C). Myeloid-derived suppres-sor cells (MDSC) were also evaluated as CD11b (Ly6C or Ly6G)positive cells in the tumors. Our results indicate a trend toward adecrease in G-MDSC in the irradiated tumor, whereas in thenonirradiated sites M-MDSCs were decreased to some extent(Supplementary Fig. S3DandS3E).However, our repeated experi-ments did not reach statistical significance. The results on radio-therapy-dependent reduction of tumor-infiltrating T lymphocyteswere confirmed in mice bearing MC38 tumors in which abscopaleffects were noted (Supplementary Fig. S4A–S4C). However, inthis case, combined radiotherapy plus combined immunotherapygave rise to dramatic increases of CD4 and CD8 T cells infiltratingthe irradiated and distant tumors (Supplementary Fig. S4C).

Moreover, analyses with a MHC tetramer that detects specificCD8 T cells recognizing the gp70 immunodominant antigen in

Figure 1.

Combined radiotherapy and immunotherapy with anti-CD137 and/or anti-PD1 mAbs against B16OVA-derived tumors. A, scheme of tumor engraftment andcombined treatments.B, bilateral tumor size follow-up (mean) of the indicated treatment regimens (only the primary tumors received radiotherapywhen indicated)Supplementary Table S1A shows statistical comparisons. C, overall survival in the same groups of mice. D, serum concentrations of IFNg pretreatment and 48 hoursafter completing the regimen of the indicated mAbs with or without prior radiotherapy (RT) dose of 8 Gy � 3 fractions.






MC38 tumor cells showed a clear tendency to higher numbers ofsuch tumor-reactive CD8 T lymphocytes in the tumor microen-vironment observed in mice undergoing combined treatment(Supplementary Fig. S4D and S4E).

It was also important to assess functionality in terms of theability of tumor-infiltrating T lymphocytes to produce IFNg . Tothis end, we treated mice as shown in Supplementary Fig. S1(Fig. 6A) and mice were sacrificed on day þ16 to monitortumor-infiltrating T cells. In this setting, experimental groupsundergoing combined treatment showed smaller tumor lesionson both sides (Fig. 6B). Intratumoral CD4 and CD8-gated Tcells were assessed for the intensity of intracellular IFNg stain-ing with further stimulation ex vivo with PMA and ionomycin.

As seen in Fig. 6C and D, lymphocytes from mice undergoingcombined radiotherapy plus immunotherapy attained moreintense IFNg production both in lymphocytes from the irradi-ated and nonirradiated lesions. Of note, these differences werealso observed if the lymphocytes were not stimulated with PMAplus ION (unpublished observations).

Radiotherapy enhances the expression of CD137 and PD1 ontumor-infiltrating lymphocytes

One possible explanation of the synergy between radio-therapy and immunostimulatory mAbs was that radiotherapyresulted in a more intense expression of CD137 and/orPD1 on tumor-infiltrating T lymphocytes. Using multicolor

Figure 2.

Combined radiotherapy andimmunotherapy with anti-CD137and/or anti-PD1 mAb mediatecombined effects against 4T1-derived breast carcinomas andreduction of spontaneous lungmetastases. A and B, treatmentscheme (A) and follow-upsubcutaneous tumor growth follow-up (mean) of the primary andcontralateral tumors (B). Only theprimary tumor received radiotherapywhen indicated (SupplementaryTable S1A shows statisticalcomparisons). C, number of lungmetastasis identified by CT-SCAN onday þ36 (mean � SD) in theindicated treatment groups.Representative data are shown inSupplementary Fig. S2 includingphotographs with the spontaneousmetastases seen in mice whose lungswere excised upon necropsy.






immunofluorescence and flow cytometry, an increase of CD137expression levels was observed among TILs 48 hours after a 20-Gysingle dose (Supplementary Fig. S5A). Such an effect was moreconspicuous on CD8þ TILs, while PD1 expression was pre-served at a similar bright level as in the case of the nonirradiatedtumor. PD-L1 was expressed on TILs with slight increasesrelated to radiotherapy. On TILs in the contralateral nonirra-diated lesion, CD137 levels also increased on CD8 T cells butnot on CD4 T cells. Increases in PD1 were also noted but onlyon CD8 T cells in the contralateral side (Supplementary Fig.S5A and S5B). This was not observed when only a single dose ofradiotherapy was given, as after three fractionated 8 Gy doses,an increase in the expression of PD-1 and CD137 was alsodocumented (Supplementary Fig. S5C and S5D).

To study these effects on human tumor samples, we irradiatedfreshly surgically explanted adenocarcinomas (two gastric carci-nomas, five colon cancers, and one chondrosarcoma). Fragmentsof the excised tumor received 20 Gy, while the other fragments

were leftwithout irradiation (mock irradiated). Tumors fragmentswere subsequently maintained in culture medium and 48 hourslater, samples were formalin-fixed and paraffin-embedded forimmunohistochemical analysis. As seen in SupplementaryFig. S6A and S6B, there was a clear increase in the percentageof TILs with CD137 and PD1-detectable surface expression, whilethe total number of T cells remained without noticeablechanges. Supplementary Figure S6B shows microphotographsof representative IHC fields of one of the cases. To study theseincreased expression of CD137 and PD-1 at the single-cell levelin a more quantitative fashion, flow cytometry analyses wereperformed in two cases of colon cancer as freshly surgicallyexcised tumors. Figure 7A shows clear increases in the immu-nofluorescence intensity for PD-1 and CD137 on viable CD8 orCD4 T cells that was contingent upon irradiation. In Fig. 7B, dotplots of these cases show that CD8 and CD4 T cells frequentlycoexpressed CD137 and PD-1 after irradiation. Furthermore,multiplex tissue immunofluorescence on a surgical specimen of

Figure 3.

Delayed treatment of bilateral MC38-derived tumors shows synergistic effects combining immunotherapy with anti-CD137 plus anti-PD1 mAbswith radiotherapy.A,scheme of treatment as in Supplementary Fig. S1 but delaying treatment onset until day þ14. As in previous figures, only the primary tumors receivedradiotherapy when indicated. B, average bilateral tumor progression in the indicated treatment groups. C, follow-up of individual bilateral subcutaneous tumorsfrom B. Statistical comparisons by nonlinear regression are shown in Supplementary Table S1B.






gastric cancer showed augmented expression of CD137 and PD-L1 upon irradiation, and representative images are shownin Fig. 7C.

All in all, radiotherapy-induced increases in expression of themAb-targeted receptors PD1 and CD137 are thus likely toaccount, at least in part, for the combinatorial synergistic effect

Figure 4.

CD8þ T cells are necessary for immune-mediated abscopal effects of radiotherapy potentiated with the combination of anti-CD137 and anti-PD1 mAbs. A, schemeof treatments and depletions of CD4, CD8, and NK lymphocytes with specific antibodies. B, follow-up of the growth of subcutaneous MC38-derived tumorsas bilaterally implanted (again only the primary tumors received the doses of radiotherapy when indicated). C, overall survival. Statistical comparisons areshown in Supplementary Table S1C.






of radiotherapy and infusion of immunomodulatory mAb tar-geted to such receptors.

DiscussionCancer therapeutics are likely to benefit from the combination

of radiotherapy and immunotherapy strategies (13). Our studyalso strongly indicates that radiotherapy can become a treatmentwith systemic beneficial antitumor effects (abscopal effects), inaddition to the well-known local effects of irradiation. In ourhands, radiotherapy modifies the immune microenvironment ofdistant nonirradiated tumors, but only the addition of immu-nostimulatory mAbs was able to elicit a meaningful therapeuticeffect against nonirradiated tumors and consolidate or enhancethe response against the directly irradiated malignant lesions.

To explain the abscopal effects of radiotherapy, manymechan-isms have been invoked. Radiotherapy causes vascular inflam-mation (13) and activation of antigen-presenting DCs (37).Recently, the key contribution of sensing tumor-released DNAby the cytoplasmic pattern recognition receptor STINGwas foundto be crucially important. This mechanism critically causes a localrelease of type I IFN involving DCs (36). Irradiation is also

reported to kill tumor cells showing the hallmarks of immuno-genic cell death, as definedbyKroemer and colleagues (38). In thisstudy, we found that abscopal effects are contingent on a DCsubset specialized in antigen crosspriming to induce CTLs (20). Itis tempting to speculate that such antigen-presenting cell subset isthe main mediator of productive tumor antigen presentation toCD8 T cells.

CTL responses and crosspriming are known to be dependenton type I IFN in mice (36, 38). This cytokine system has evolvedto raise the alarm upon acute viral infection and is involvedin setting in action an optimal immune response for viralclearance. Our findings demonstrate that IFNa/b is criticallyinvolved in the abscopal effects of radiotherapy. DNA releasedfrom dying tumor cells is probably involved in eliciting IFNa/bvia STING (39) and, in turn, IFNa/b may act both on cross-priming DCs (36) and of CD8 T cells (35) to favor, as a neces-sary factor, the CTL immune response. Strategies aiming at localenhancement of IFNa/b could render radiotherapy-inducedtumor cell death more immunogenic as recently shown forchemotherapy (40).

However, immunogenic cell death as induced by radiother-apy only exceptionally offers systemic control of the spread of

Figure 5.

BATF-3�/� and IFNAR�/� mice lose the abscopal effects of radiotherapy upon combination treatment with anti-PD1 and anti-CD137 mAbs. A, control or combinedimmunotherapy plus radiotherapy treatment regimens were given as in Supplementary Fig. S1 to C57Bl6 WT mice or to syngeneic BATF3�/� or IFNAR�/�

mice. B, the average tumor growth in the directly irradiated tumor lesion and the contralateral tumor in BATF-3�/� mice in comparison with WT mice.C, concentrations of IFNg in the serumsamples from the indicatedgroups ofmice taken48hours after the last treatmentdose.D, similar experiment as inB comparingIFNAR�/� mice with WT mice.






disease. This could be due to the relative weakness of theimmunizing effects or because of concomitantly elicited immu-nosuppressing factors and mechanisms such as those mediatedby TGFb (41).

Strategies to enhance the antitumor immune effects of radio-therapy have been explored in preclinical models, and resultsfrom pioneering clinical research have also been reported (28).For instance, mouse tumor lesions were treated with the TLR7agonist imiquimod cream (42, 43), or injected with TLR9 CpGagonist nucleotides showing evidence for stronger immunitywiththe ability to partially tackle distant disease (44, 45). In the clinic,strategies based on combinations of radiotherapy with imiqui-mod (42) or subcutaneous GM-CSF (46) have been reported withpromising proof-of-concept results.

Regarding the optimal combinations of radiotherapy andimmunotherapy, several parameters are to be optimized includ-ing dose, fractionation, and interval between doses. We chosethree fractions of 8 Gy based on published evidence (47) suggest-ing that this regimen attains better results from the immunologicpoint of view at least when combining radiotherapy with anti-CTLA-4 mAb (22). However, this issue remains open to debate.

mAbs that tamper with immunoinhibitory receptors(checkpoints; ref. 2) or agonist antibodies to lymphocytecostimulatory receptors (48) have taken the centerstage ofoncology drug development. A plethora of clinical trials areexploring their efficacy against multiple malignant diseases,first when used as single agents and then in combinations(49). To date, very little clinical experience exists with com-bining radiotherapy with immunostimulatory mAbs. Onlyresults from two clinical trials combining radiotherapy andipilimumab are available for metastatic melanoma (28) andprostate cancer (50). These showed limited efficacy that mightbe patient subset–specific. As for abscopal effects, evidence iseven more scanty although there is reported anecdotal evi-dence (30).

Systemic effects of anti-PD1mAb have not yet been reported topotentiate abscopal effects of radiotherapy in patients. In immu-nogenicmousemodels, anti-CD137 (25) and anti-PD1mAb (21)have been reported to enhance the antitumor effects of radio-therapy. In our case, we report that these antibodies, and espe-cially their combination, can unleash a very potent therapeuticeffect against the contralateral tumors (abscopal effects), when

Figure 6.

Combined immunostimulatory mAbs and unilateral radiotherapy induce more intense expression of IFNg in CD8þ and CD4þ tumor-infiltrating T cells. A, scheme oftreatment and tumor surgical excision in mice bearing bilateral MC38-derived tumors. B, tumor weight at sacrifice (day þ16). C, mean fluorescence intensity(MFI) of intracellular IFNg immunostaining in the gated CD8þ or CD4þ tumor-infiltrating T lymphocytes as indicated after a 4-hour restimulation with PMA þionomycin. D, percentage of lymphocytes expressing intracellular IFNg among CD8þ and CD4þ tumor-infiltrating lymphocytes.






both irradiated and nonirradiated tumor lesions were very wellestablished for longer than one week.

In our experiments, potentiation of abscopal effects resulted inlong-term survival, comparablewith recently reported effectswiththe combination of anti-PD1 and anti-CTLA-4 mAb in B16F10-bearingmice (28). In this case, the combinedmechanism resultedfrom a reinvigoration of antitumor CTLs that were not repressedby the PD1/PD-L1 axis if combined treatment was given. Ourselective depletion experiments point in the same direction.

Our results on abscopal effects contrast with the fact thatradiotherapy by itself reduced in our hands the content of Tcells in the tumors, although it also slightly reduced the numberof MDSCs both in the irradiated tumor and in the contralateralsite. However, radiotherapy enhanced IFNg production on a percell basis and the level of CD137 and PD1 expression on T cells,in this way making them more amenable to pharmacologic

therapeutic costimulation. This was also observed in humantumor fragments irradiated ex vivo. Interestingly, our depletionexperiments reveal that only the function of CD8þ T cells is anabsolute requirement. Moreover, at least a subset of CD4þ cellsseems to be operating in detriment of efficacy. These are likelyto be Treg cells. Importantly, at the single-cell level there aretumor-infiltrating lymphocytes that coexpress the CD137 andPD-1 receptors, arguing in favor of a double hit by the immu-nostimulatory mAbs on single T-cell basis.

In our hands, radiotherapy plus immunostimulatory mAbsdramatically enhance the T-cell infiltrate after 8 days of combinedtreatment with evidence for more CD8 T cells recognizing thegp70 tumor antigen in the MC38 tumor model. These tetramer-positive cells are CD137þ and PD-1þ in keeping with previousreports showing that CD137þ cells in humanmelanomas tend tobe specific for tumor neoantigens (51).

Figure 7.

Ex vivo irradiation inducesCD137, PD-L1, andPD1 expression in human carcinoma tissue samples.A, two primary human colon carcinomaswere surgically excised andfollowing pathology assessment, tumor fragments were minced to 5 �5 mm pieces and kept in tissue culture. Samples were irradiated or nonirradiated(mock-irradiated) with a single dose of 20 Gy and 48 hours later cell suspensions were immunostained for flow cytometry and fluorescence intensity ofimmunostainings for surface CD137 and PD-1 on gated CD4 and CD8 T lymphocytes are shown.B, dot plots show the percentages of double positive lymphocytes forCD137 and PD-1 on CD4 and CD8 T cells in the tumor-derived cell suspensions corresponding to irradiated and mock-irradiated tissue samples as indicated.C, multiplexed immunofluorescence microphotographs of a representative gastric carcinoma explant whose fragments were either irradiated or mockirradiated, showing stainings for tumor cells (cytokeratin-positive, green channel), CD3 (green channel), CD137 or PD-L1 (red channels). Nuclei were highlightedwith DAPI (blue fluorescence). Scale bar, 100 mm.






Overall, our data strongly support initiation of clinical trialstesting anti-CD137 mAb in combination with PD1/PD-L1 block-ade together with concomitant irradiation of some of the tumormetastatic sites, in search of a way powerful to make the most ofthese novel immunotherapies.

Disclosure of Potential Conflicts of InterestM. Jure-Kunkel has ownership interest (including patents) in Bristol-Myers

Squibb. I. Melero reports receiving a commercial research grant from Pfizer andis a consultant/advisory board member for Bristol-Myers Squibb, BoehringerIngelheim, Roche-Genentech, Incyte, andAstra Zeneca. Nopotential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: M.E. Rodriguez-Ruiz, I. Rodriguez, M.A. Aznar,I. MeleroDevelopment of methodology: M.E. Rodriguez-Ruiz, I. Rodriguez, B. Barbes,S. Labiano, A. Azpilikueta, E. Bola~nos, A. RouzautAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.E. Rodriguez-Ruiz, I. Rodriguez, J.L. Solorzano,J.L. Perez-Gracia, A. Rouzaut, K.A. SchalperAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.E. Rodriguez-Ruiz, I. Rodriguez, J.L. Perez-Gracia,A. Azpilikueta, A.R. Sanchez-Paulete, A. Rouzaut, K.A. Schalper, I. MeleroWriting, review, and/or revision of the manuscript: M.E. Rodriguez-Ruiz,I. Rodriguez, B. Barbes, J.L. Perez-Gracia, S. Labiano, M.F. Sanmamed,A. Azpilikueta, E. Bola~nos, A.R. Sanchez-Paulete, M. Jure-Kunkel, I. Melero

Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M.E. Rodriguez-Ruiz, S. Garasa, B. Barbes,J.L. Solorzano, M.F. SanmamedStudy supervision: M.E. Rodriguez-Ruiz, I. Melero

AcknowledgmentsWe acknowledge generous help by Drs. Martinez-Monge, Aristu, and

Gil-Bazo from the Department of Oncology at CUN. We are also grateful forthe advice from Drs. Lozano, Echeveste, and Idoate from the pathologydepartment at CUN. We are grateful for Sciencific discussion with Drs. MarianoPonz, David Sancho, Nicola Tinari, and Antonio Rull�an. Excellent dosimetry byArantza Zubiria and dedicated animal care by Eneko Elizalde are alsoacknowledged.

Grant SupportThis work was financially supported by grants from MICINN (SAF2011-

22831 and SAF2014-52361-R). I. Melero was also funded by the Departamentode Salud del Gobierno deNavarra, Redes tem�aticas de investigaci�on cooperativaRETICC, European Commission VII Framework and Horizon 2020 programs(AICR and PROCROP), SUDOE-IMMUNONET, Fundaci�on de la Asociaci�onEspa~nola Contra el C�ancer (AECC), Fundaci�on BBVA and Fundaci�on CajaNavarra. M.E. Rodriguez-Ruiz receives a Rio Hortega contract from ISCIII.S. Labiano is recipient of predoctoral scholarship from MICINN.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 25, 2016; revised July 15, 2016; accepted July 27, 2016;published OnlineFirst August 22, 2016.

References1. Shalgunov V, van Wieringen JP, Janssen HM, Fransen PM, Dierckx RA,

Michel MC, et al. Synthesis and evaluation in rats of homologous seriesof [(18)F]-labeled dopamine D 2/3 receptor agonists based on the 2-aminomethylchroman scaffold as potential PET tracers. EJNMMI Res2015;5:119.

2. Sharma P, Allison JP. The future of immune checkpoint therapy. Science2015;348:56–61.

3. Lipson EJ, Drake CG. Ipilimumab: an anti-CTLA-4 antibody for metastaticmelanoma. Clin Cancer Res 2011;17:6958–62.

4. Ugurel S, Rohmel J, Ascierto PA, Flaherty KT, Grob JJ, Hauschild A, et al.Survival of patients with advanced metastatic melanoma: the impact ofnovel therapies. Eur J Cancer 2016;53:125–34.

5. Borghaei H, Paz-Ares L, Horn L, Spigel DR, Steins M, Ready NE, et al.Nivolumab versus docetaxel in advanced nonsquamous non-small-celllung cancer. N Engl J Med 2015;373:1627–39.

6. Brahmer J, Reckamp KL, Baas P, Crino L, Eberhardt WE, Poddubskaya E,et al. Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer. N Engl J Med 2015;373:123–35.

7. Garon EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP, et al.Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl JMed 2015;372:2018–28.

8. Motzer RJ, Escudier B, McDermott DF, George S, Hammers HJ, Srinivas S,et al. Nivolumab versus everolimus in advanced renal-cell carcinoma.N Engl J Med 2015;373:1803–13.

9. Melero I, Shuford WW, Newby SA, Aruffo A, Ledbetter JA, Hellstrom KE,et al. Monoclonal antibodies against the 4-1BB T-cell activation moleculeeradicate established tumors. Nat Med 1997;3:682–5.

10. Fisher TS, Kamperschroer C, Oliphant T, Love VA, Lira PD, Doyonnas R,et al. Targeting of 4-1BB by monoclonal antibody PF-05082566 enhancesT-cell function and promotes anti-tumor activity. Cancer ImmunolImmunother 2012;61:1721–33.

11. Ascierto PA, Simeone E, SznolM, Fu YX,Melero I. Clinical experiences withanti-CD137 and anti-PD1 therapeutic antibodies. Semin Oncol 2010;37:508–16.

12. Wei H, Zhao L, Li W, Fan K, Qian W, Hou S, et al. Combinatorial PD-1blockade and CD137 activation has therapeutic efficacy in murinecancer models and synergizes with cisplatin. PLoS One 2013;8:e84927.

13. Barker HE, Paget JT, Khan AA, Harrington KJ. The tumour microenviron-ment after radiotherapy:mechanisms of resistance and recurrence. Nat RevCancer 2015;15:409–25.

14. Demaria S, Golden EB, Formenti SC. Role of local radiation therapy incancer immunotherapy. JAMA Oncol 2015;1:1325–32.

15. Bloy N, Pol J, Manic G, Vitale I, Eggermont A, Galon J, et al. Trial Watch:radioimmunotherapy for oncological indications. Oncoimmunology2014;3:e954929.

16. Stone HB, Peters LJ, Milas L. Effect of host immune capability on radio-curability and subsequent transplantability of a murine fibrosarcoma.J Natl Cancer Inst 1979;63:1229–35.

17. Kepp O, Senovilla L, Vitale I, Vacchelli E, Adjemian S, Agostinis P, et al.Consensus guidelines for the detection of immunogenic cell death.Oncoimmunology 2014;3:e955691.

18. Segura E, Amigorena S. Cross-presentation in mouse and human dendriticcells. Adv Immunol 2015;127:1–31.

19. Hildner K, Edelson BT, Purtha WE, Diamond M, Matsushita H,Kohyama M, et al. Batf3 deficiency reveals a critical role forCD8alphaþ dendritic cells in cytotoxic T cell immunity. Science2008;322:1097–100.

20. Sanchez-Paulete AR, Cueto FJ, Martinez-Lopez M, Labiano S, Morales-Kastresana A, Rodriguez-Ruiz ME, et al. Cancer immunotherapy withimmunomodulatory anti-CD137 and anti-PD-1 monoclonal antibo-dies requires BATF3-dependent dendritic cells. Cancer Discov 2016;6:71–9.

21. Deng L, Liang H, Burnette B, Beckett M, Darga T, Weichselbaum RR, et al.Irradiation and anti-PD-L1 treatment synergistically promote antitumorimmunity in mice. J Clin Invest 2014;124:687–95.

22. RuoccoMG, Pilones KA, KawashimaN, CammerM,Huang J, Babb JS, et al.Suppressing T cell motility induced by anti-CTLA-4 monotherapyimproves antitumor effects. J Clin Invest 2012;122:3718–30.

23. Verbrugge I, Hagekyriakou J, Sharp LL, Galli M, West A, McLaughlin NM,et al. Radiotherapy increases the permissiveness of established mammarytumors to rejection by immunomodulatory antibodies. Cancer Res2012;72:3163–74.

24. Park SS, Dong H, Liu X, Harrington SM, Krco CJ, Grams MP, et al. PD-1restrains radiotherapy-induced abscopal effect. Cancer Immunol Res2015;3:610–9.






25. Shi W, Siemann DW. Augmented antitumor effects of radiation therapyby 4-1BB antibody (BMS-469492) treatment. Anticancer Res 2006;26:3445–53.

26. Belcaid Z, Phallen JA, Zeng J, See AP, Mathios D, Gottschalk C, et al. Focalradiation therapy combined with 4-1BB activation and CTLA-4 blockadeyields long-term survival and a protective antigen-specific memoryresponse in a murine glioma model. PLoS One 2014;9:e101764.

27. Newcomb EW, Lukyanov Y, Kawashima N, Alonso-Basanta M, Wang SC,Liu M, et al. Radiotherapy enhances antitumor effect of anti-CD137therapy in a mouse Glioma model. Radiat Res 2010;173:426–32.

28. Twyman-Saint\VictorC, RechAJ,MaityA, RenganR,PaukenKE, Stelekati E,et al. Radiation and dual checkpoint blockade activate non-redundantimmune mechanisms in cancer. Nature 2015;520:373–7.

29. Nobler MP. The abscopal effect in malignant lymphoma and its relation-ship to lymphocyte circulation. Radiology 1969;93:410–2.

30. Postow MA, Callahan MK, Barker CA, Yamada Y, Yuan J, Kitano S, et al.Immunologic correlates of the abscopal effect in a patient withmelanoma.N Engl J Med 2012;366:925–31.

31. Dubrot J, Milheiro F, Alfaro C, Palazon A, Martinez-Forero I, Perez-GraciaJL, et al. Treatmentwith anti-CD137mAbs causes intense accumulations ofliver T cells without selective antitumor immunotherapeutic effects in thisorgan. Cancer Immunol Immunother 2010;59:1223–33.

32. Quetglas JI, Dubrot J, Bezunartea J, Sanmamed MF, Hervas-Stubbs S,Smerdou C, et al. Immunotherapeutic synergy between anti-CD137 mAband intratumoral administration of a cytopathic Semliki Forest virusencoding IL-12. Mol Ther 2012;20:1664–75.

33. Quetglas JI, Labiano S, Aznar MA, Bola~nos E, Azpilikueta A, Rodriguez I,et al. Virotherapy with a semliki forest virus-based vector encoding IL12synergizes with PD-1/PD-L1 blockade. Cancer Immunol Res 2015;3:449–54.

34. Demaria S, Kawashima N, Yang AM, Devitt ML, Babb JS, Allison JP, et al.Immune-mediated inhibition of metastases after treatment with localradiation and CTLA-4 blockade in a mouse model of breast cancer. ClinCancer Res 2005;11:728–34.

35. KolumamGA, Thomas S, Thompson LJ, Sprent J, Murali-Krishna K. Type Iinterferons act directly on CD8 T cells to allow clonal expansion andmemory formation in response to viral infection. J Exp Med 2005;202:637–50.

36. Le Bon A, Etchart N, Rossmann C, AshtonM, Hou S, Gewert D, et al. Cross-priming of CD8þ T cells stimulated by virus-induced type I interferon. NatImmunol 2003;4:1009–15.

37. Gupta A, Probst HC, Vuong V, Landshammer A, Muth S, Yagita H, et al.Radiotherapy promotes tumor-specific effector CD8þ T cells via dendriticcell activation. J Immunol 2012;189:558–66.

38. Kroemer G, Galluzzi L, Kepp O, Zitvogel L. Immunogenic cell death incancer therapy. Annu Rev Immunol 2013;31:51–72.

39. Deng L, LiangH, XuM, Yang X, Burnette B, Arina A, et al. STING-dependentcytosolic DNA sensing promotes radiation-induced type I interferon-

dependent antitumor immunity in immunogenic tumors. Immunity2014;41:843–52.

40. Sistigu A, Yamazaki T, Vacchelli E, Chaba K, Enot DP, Adam J, et al. Cancercell-autonomous contribution of type I interferon signaling to the efficacyof chemotherapy. Nat Med 2014;20:1301–9.

41. Vanpouille-Box C, Diamond JM, Pilones KA, Zavadil J, Babb JS, FormentiSC, et al. TGFbeta is a master regulator of radiation therapy-inducedantitumor immunity. Cancer Res 2015;75:2232–42.

42. Adams S, Kozhaya L, Martiniuk F, Meng TC, Chiriboga L, Liebes L, et al.Topical TLR7 agonist imiquimod can induce immune-mediated rejectionof skin metastases in patients with breast cancer. Clin Cancer Res2012;18:6748–57.

43. Dewan MZ, Vanpouille-Box C, Kawashima N, DiNapoli S, Babb JS, For-menti SC, et al. Synergy of topical toll-like receptor 7 agonist with radiationand low-dose cyclophosphamide in a mouse model of cutaneous breastcancer. Clin Cancer Res 2012;18:6668–78.

44. 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.

45. 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.

46. Golden EB, Chhabra A, Chachoua A, Adams S, Donach M, Fenton-Kerimian M, et al. Local radiotherapy and granulocyte-macrophagecolony-stimulating factor to generate abscopal responses in patientswith metastatic solid tumours: a proof-of-principle trial. Lancet Oncol2015;16:795–803.

47. Dewan MZ, Galloway AE, Kawashima N, Dewyngaert JK, BabbJS, Formenti SC, et al. Fractionated but not single-dose radio-therapy induces an immune-mediated abscopal effect whencombined with anti-CTLA-4 antibody. Clin Cancer Res 2009;15:5379–88.

48. Melero I,Hirschhorn-CymermanD,Morales-KastresanaA, SanmamedMF,Wolchok JD. Agonist antibodies to TNFRmolecules that costimulate T andNK cells. Clin Cancer Res 2013;19:1044–53.

49. Melero I, Berman DM, Aznar MA, Korman AJ, Perez Gracia JL, Haanen J.Evolving synergistic combinations of targeted immunotherapies to combatcancer. Nat Rev Cancer 2015;15:457–72.

50. Kwon ED, Drake CG, Scher HI, Fizazi K, Bossi A, van den EertweghAJ, et al. Ipilimumab versus placebo after radiotherapy in patientswith metastatic castration-resistant prostate cancer that had pro-gressed after docetaxel chemotherapy (CA184-043): a multicentre,randomised, double-blind, phase 3 trial. Lancet Oncol 2014;15:700–12.

51. Gros A, Robbins PF, YaoX, Li YF, Turcotte S, Tran E, et al. PD-1 identifies thepatient-specific CD8(þ) tumor-reactive repertoire infiltrating humantumors. J Clin Invest 2014;124:2246–59.






Published OnlineFirst August 22, 2016.Cancer Res María E. Rodriguez-Ruiz, Inmaculada Rodriguez, Saray Garasa, et al. CD8 T Cells and CrossprimingCombined Immunostimulatory mAbs and Are Dependent on Abscopal Effects of Radiotherapy Are Enhanced by

Updated version

10.1158/0008-5472.CAN-16-0549doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2016/08/20/0008-5472.CAN-16-0549.DC1

Access the most recent supplemental material at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://cancerres.aacrjournals.org/content/early/2016/09/29/0008-5472.CAN-16-0549To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-16-0549

http://cancerres.aacrjournals.org/content/suppl/2016/08/20/0008-5472.CAN-16-0549.DC1

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/early/2016/09/29/0008-5472.CAN-16-0549


Documents

Abscopal Effects of Radiotherapy Are Enhanced by Combined … · 2016-09-29 · Microenvironment and Immunology Abscopal Effects of Radiotherapy Are Enhanced by Combined Immunostimulatory