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Radiotherapy Complements Immune Checkpoint BlockadeShin Foong Ngiow,1,2Grant A.McArthur,3,4and Mark J.Smyth1,2,*1Immunology in Cancer and Infection Laboratory,QIMR Berghofer Medical Research Institute,Herston,QLD4006,Australia2School of Medicine,University of Queensland,Herston,QLD4006,Australia3Cancer Therapeutics Programs,Trescowthick Laboratories,Peter MacCallum Cancer Centre,St.Andrews Place,East Melbourne,VIC3002, Australia4Sir Peter MacCallum Department of Oncology,University of Melbourne,Parkville,VIC3010,Australia*Correspondence:mark.smyth@.au/10.1016/ell.2015.03.015Adaptive immune resistance ablates effective anti-tumor immune responses.In a recent issue of Nature, Victor and colleagues describe that anti-PD-L1combats adaptive immune resistance upon localized radiation plus anti-CTLA-4therapy.The superior activity of radiation and dual immune checkpoint blockade is mediated by non-redundant immune mechanisms in cancer.Combinations of two or more immuno-therapies with additive or synergistic benefit in preclinical models(Uno et al., 2006)have set the framework for combi-nation successes in the clinic(Wolchok et al.,2013).With the recent success of immune checkpoint inhibitors and other immunomodulators,there has been re-newed interest in evaluating the combina-tion of such agents with radiation therapy (RT)in clinical trials(Verbrugge et al., 2012;Formenti and Demaria,2013).The biologic premise behind such a strategy is that the tumor-antigen release and change in the tumor microenvironment achieved by localized RT will promote specific tumor targeting by the adap-tive immune system,which can be augmented further by systemic immune-stimulating agents(Tang et al.,2014).In this manner,clinicians hope to induce a phenomenon known as the abscopal effect,whereby localized RT results in im-mune-mediated tumor regression in dis-ease sites well outside of the radiation field.RT also induces DNA damage and tumor cell death by promoting tumor-cell expression of Fas and MHC class I.Dying tumor cells release not only tumor anti-gens but also ATP and danger signals such as HMGB1and calreticulin.RT also has some potentially deleterious effects by increasing the tumor cell expression of PD-L1,secretion of TGF b,and induc-tion of Treg.A small pilot phase I/II study in prostate cancer(Slovin et al.,2013) and case reports in melanoma(Postow et al.,2012;Hiniker et al.,2012;Golden et al.,2013)combining RT with ipilimu-mab show the possible clinical activity ofthis approach.Now,Victor et al.(2015)report a phase Iclinical trial of22patients with advancedmelanoma treated with RT and anti-CTLA-4.In the trial,a single lesion wasirradiated with hypofractionated stereo-tactic body radiation(6–8Gy deliveredover two or three fractions),followed byfour cycles of ipilimumab beginning3–5days after the last fraction of RT.Assess-ment of unirradiated lesions using RECISTcriteria demonstrated18%patients hada partial response as the best response,18%had stable disease,and64%hadprogressive disease.So,although partialresponses were observed,like mono-therapy with anti-CTLA-4,the majority ofthe patients did not respond.The authors then describe the use ofthe B16-F10melanoma mouse model,where RT plus anti-CTLA-4was moreeffective than either treatment alone,pro-moting the regression of both irradiatedand unirradiated tumors(Victor et al.,2015).RT given before,or concurrentlywith,anti-CTLA-4yielded similar results.Complete responses in these combina-tion-treated mice were CD8+T cell-dependent,and memory to rechallengewas demonstrated.However,not allmice responded,and melanoma celllines from relapsing mice were derived.In these cell lines,resistance to the com-bination was confirmed,but the lesionswere not RT resistant.Random forest ma-chine learning analysis for sub-types ofTIL isolated from the resistant melanomasdemonstrated that the top predictor forresistance was the CD8+CD44+to Tregratio,which failed to increase after RTplus anti-CTLA-4(as it did in sensitivetumors).Transcriptomic profiling revealed thatPD-L1was among the top0.2%ofupregulated genes in melanoma(RT plusanti-CTLA-4resistance signature).Ge-netic elimination of PD-L1on the ther-apy-resistant Res499melanoma cells byCRISPR dramatically restored responseto RT plus anti-CTLA-4(0%to60%sur-vival),suggesting upregulation of PD-L1was one major mechanism,but notthe only mechanism of resistance to RTplus anti-CTLA-4.In the original parentalB16-F10,RT plus anti-CTLA-4increasedthe proportion of PD-1+Eomes+CD8T cells and the proportion that wereKi67+and granzyme(Grz)B+.But in theresistant sublines,the Ki67+GrzB+CD8T cells were not increased.The frequencyof PD-1+CD8+T cells that were Eomes+was a striking modifier of the likelihoodof a complete response(CR),becausenearly all the CRs occurred when thefrequency of Ki67+GrzB+in PD-1+CD8+was high,but the relative size of the PD-1+Eomes+-exhausted population wassmall.Critically,adding anti-PD-L1improved responses to the naive B16-F10and resistant sublines after RT plusanti-CTLA-4.Similar results were ob-tained in the mouse TSA mammary tumorand a mouse pancreatic tumor model.Random forest modeling showed thatanti-CTLA-4predominantly caused adecrease in Treg cells,anti-PD-L1strongly increased CD8TILfrequency,Cancer Cell27,April13,2015ª2015Elsevier Inc.437 Cancer CellPreviewsand blockade of both increased the CD8/Treg ratio.RT only caused a modest increase in CD8TILs;however,TCR sequencing revealed that there was an increased diversity of TCR clonotypes,which was also observed with RT plus anti-CTLA-4.This effect was also seen in the spleen and peripheral blood.Some clones reached 20%post-treatment in the blood with triple treatment.By contrast,peripheral T cell expansion was modest with RT and anti-CTLA-4alone.So the concept gleaned from the mouse studies was that favorable immune changes in TILs after immune check-point blockade promoted their peripheral clonal expansion.When combined with increased TCR repertoire diversity af-forded by RT,selection and oligoclonal peripheral expansion of clones with distinct TCR traits were favored (Figure 1).From the clinical trial of RT +ipilimu-mab,12patients had pre-treatment tumor biopsies,and pre-and post-treatment blood was available on 10patients.From these low numbers,it was revealed that PD-L1lo intensity on melanoma cells was associated with re-invigoration of PD-1+Eomes +CD8+T cells after RT plus anti-CTLA-4,while PD-L1hi status was associated with persistent exhaustion of the CD8+T cells.PD-L1hi on melanoma biopsies collected from therapy-resistant patients is potentially reflecting the failure of CTL anti-tumor cytotoxicity,consistent with the lower frequencies of Ki67+GrzB +PD1+CD8+T cells.As dis-cussed above,it is also worth empha-sizing that a proportion of the mice with PD-L1knockout Res499cells still suc-cumbed to disease after RT plus anti-CTLA-4treatment,suggesting a role for non-tumor PD-L1in promoting resistance to RT/anti-CTLA-4.Intriguingly,despite other recent reports (Herbst et al.,2014)PD-L1status on the macrophages was neither associated with reinvigoration nor independently predictive of progres-sion-free survival.Although there may be activation of other T cell checkpoint path-ways,the hierarchy of PD-L1expression on tumor cells and non-tumor cells in driving T cell exhaustion warrants further study in larger patient cohorts.Nonethe-less,the mechanism of radiation to diversify TCR clonotypes remains to be discerned.Radiotherapy is a major component of cancer treatment,with over 50%of can-cer patients receiving radiation during the course of their disease.Radiation is used for both palliation of local symptoms and curative therapy in some malig-nancies,like lung,prostate,or head and neck cancer.However,although radiation offers clear benefits in terms of both local control and survival,disease recurrence outside the radiation field remains all toocommon,and most patients eventually die from progressive metastatic disease.A considerable amount of work remains to be done to create successful com-binations of immunotherapeutics and radiation,which includes identifying the optimal radiation dose,fractionation,and sequence for use in combination with im-mune checkpoint inhibitors.ACKNOWLEDGMENTSWe wish to acknowledge funding support from the National Health and Medical Research Council of Australia,The Susan Komen for the Cure,and the Cancer Council of Queensland.REFERENCESFormenti,S.C.,and Demaria,S.(2013).J.Natl.Cancer Inst.105,256–265.Golden,E.B.,Demaria,S.,Schiff,P.B.,Chachoua,A.,and Formenti,S.C.(2013).Cancer Immunol Res 1,365–372.Herbst,R.S.,Soria,J.C.,Kowanetz,M.,Fine,G.D.,Hamid,O.,Gordon,M.S.,Sosman,J.A.,McDermott, D.F.,Powderly,J.D.,Gettinger,S.N.,et al.(2014).Nature 515,563–567.Hiniker,S.M.,Chen,D.S.,Reddy,S.,Chang,D.T.,Jones,J.C.,Mollick,J.A.,Swetter,S.M.,and Knox,S.J.(2012).Transl Oncol 5,404–407.Postow,M.A.,Callahan,M.K.,Barker,C.A.,Ya-mada,Y.,Yuan,J.,Kitano,S.,Mu,Z.,Rasalan,T.,Adamow,M.,Ritter,E.,et al.(2012).N.Engl.J.Med.366,925–931.Slovin,S.F.,Higano,C.S.,Hamid,O.,Tejwani,S.,Harzstark,A.,Alumkal,J.J.,Scher,H.I.,Chin,K.,Gagnier,P.,McHenry,M.B.,and Beer,T.M.(2013).Ann.Oncol.24,1813–1821.Tang,C.,Wang,X.,Soh,H.,Seyedin,S.,Cortez,M.A.,Krishnan,S.,Massarelli,E.,Hong,D.,Naing,A.,Diab,A.,et al.(2014).Cancer Immunol Res 2,831–838.Uno,T.,Takeda,K.,Kojima,Y.,Yoshizawa,H.,Akiba,H.,Mittler,R.S.,Gejyo,F.,Okumura,K.,Ya-gita,H.,and Smyth,M.J.(2006).Nat.Med.12,693–698.Verbrugge,I.,Hagekyriakou,J.,Sharp,L.L.,Galli,M.,West,A.,McLaughlin,N.M.,Duret,H.,Yagita,H.,Johnstone,R.W.,Smyth,M.J.,and Haynes,N.M.(2012).Cancer Res.72,3163–3174.Victor, C.T.,Rech, A.J.,Maity,A.,Rengan,R.,Pauken,K.E.,Stelekati,E.,Benci,J.L.,Xu, B.,Dada,H.,Odorizzi,P.M.,et al.(2015).Nature Pub-lished online March 9,2015./10.1038/nature14292.Wolchok,J.D.,Kluger,H.,Callahan,M.K.,Postow,M.A.,Rizvi,N.A.,Lesokhin, A.M.,Segal,N.H.,Ariyan,C.E.,Gordon,R.A.,Reed,K.,et al.(2013).N.Engl.J.Med.369,122–133.Figure 1.Schematic Model for Non-redundant Mechanisms of RT/Anti-CTLA-4/Anti-PD-L1Therapy to Combat Immune Resistance in MelanomaAdapted from Victor et al.(2015)Extended Data Figure 6.438Cancer Cell 27,April 13,2015ª2015Elsevier Inc.Cancer CellPreviews。