acquired resistance to fractionated radiotherapy can be … · microenvironment and immunology...

Microenvironment and Immunology

Acquired Resistance to Fractionated Radiotherapy Can BeOvercome by Concurrent PD-L1 Blockade

Simon J. Dovedi1, Amy L. Adlard2, Grazyna Lipowska-Bhalla1, Conor McKenna1, Sherrie Jones1,Eleanor J. Cheadle1, Ian J. Stratford2, Edmund Poon3, Michelle Morrow3, Ross Stewart3, Hazel Jones3,Robert W. Wilkinson3, Jamie Honeychurch1, and Tim M. Illidge1

AbstractRadiotherapy is a major part in the treatment of most common cancers, but many patients experience local

recurrence with metastatic disease. In evaluating response biomarkers, we found that low doses of fractionatedradiotherapy led to PD-L1 upregulation on tumor cells in a variety of syngeneic mousemodels of cancer. Notably,fractionated radiotherapy delivered in combination with aPD-1 or aPD-L1 mAbs generated efficacious CD8þ

T-cell responses that improved local tumor control, long-term survival, and protection against tumor rechallenge.These favorable outcomes were associated with induction of a tumor antigen–specific memory immuneresponse. Mechanistic investigations showed that IFNg produced by CD8þ T cells was responsible for mediatingPD-L1 upregulation on tumor cells after delivery of fractionated radiotherapy. Scheduling of anti–PD-L1mAbwasimportant for therapeutic outcome, with concomitant but not sequential administration with fractionatedradiotherapy required to improve survival. Taken together, our results reveal themechanistic basis for an adaptiveresponse by tumor cells that mediates resistance to fractionated radiotherapy and its treatment failure. Withattention to scheduling, combination immunoradiotherapy with radiotherapy and PD-1/PD-L1 signalingblockade may offer an immediate strategy for clinical evaluation to improve treatment outcomes. Cancer Res;74(19); 5458–68. �2014 AACR.

IntroductionRadiotherapy remains themost important nonsurgical treat-

ment in the management of solid malignancies with around50% to 60% of all patients with cancer receiving this treatment.The inclusion of radiotherapy in treatment regimens reducesdisease recurrence and improves overall survival in most com-mon cancers (1–3). In addition to the direct cytoreductive effectof radiotherapy, emergingevidence suggests that the generationof antitumor immune responses may play an important rolein the effectiveness of this treatment (4, 5). Radiotherapy canlead to expression of ecto-calreticulin on tumor cells as wellas the release of several damage-associated molecular patterns

(DAMP), including High Mobility Group Box 1 (HMGB1) andATP, which can lead to recruitment and activation of antigen-presenting cells and priming of tumor antigen–specific T-cellresponses (6–10). Despite this, immune-escape frequentlyoccurs with tumor recurrence remaining the leading cause ofmortality in patients receiving radiotherapy (11). The identifi-cation and inhibition of key drivers of immunosuppressionmayaugment antitumor immune responses with the potential toimprove patient outcome.

The PD-1/PD-L1 axis is involved in the maintenance ofperipheral tolerance and modulation of acute inflammatoryresponses through inhibition of T-cell function such as loss of T-cell receptor signal transduction or through apoptosis of acti-vatedT cells (12, 13). In addition tobindingPD-1, PD-L1 canalsosuppress T-cell function through interaction with CD80 (14).ExpressionofPD-L1 is inducible and thought to respond to localinflammatory milieu, particularly type I and II IFN (12, 15, 16).Although barely detectable in most normal tissues, expressionof PD-L1 has beendescribed inmultiplemalignancies (reviewedin ref. 17). Importantly, recent clinical studies with either PD-1or PD-L1 targeting mAbs have demonstrated encouragingresponses in patients with advanced disease (18–21).

In this study, we demonstrate that radiotherapy leads to anadaptive upregulation of tumor cell PD-L1 expression that isdependent on CD8þ T-cell production of IFNg and may atten-uate the efficacy of the anticancer immune response. Usingsyngeneic models of melanoma, colorectal and triple-negativebreast cancer, we show that the efficacy of low doses of frac-tionated radiotherapy can be enhanced when delivered in

1Targeted Therapy Group, Institute of Cancer Sciences, Manchester Can-cer Research Centre, University of Manchester, Manchester AcademicHealth Sciences Centre, Manchester, United Kingdom. 2ExperimentalOncology Group, School of Pharmacy, University of Manchester, Man-chester Academic Health Sciences Centre, Manchester, United Kingdom.3MedImmune Ltd, Granta Park, Cambridge, United Kingdom.

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

R.W.Wilkinson, J.Honeychurch, andT.M. Illidge contributedequally to thisarticle.

Corresponding Author: Simon J. Dovedi, University of Manchester,Paterson Building, Wilmslow Road, Manchester M20 4BX, United King-dom. Phone: 161-9187024; Fax: 161-4463109; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-14-1258

�2014 American Association for Cancer Research.

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combinationwithmAb targeted against PD-1 andPD-L1. Impor-tantly, we show that dose scheduling is critical to the synergisticeffect of combination therapywithconcurrent butnot sequentialadministration (aPD-L1 mAb administered 7 days after com-pletion of radiotherapy) generating effective antitumor immu-nity and long-term tumor control. The results of this studyprovide evidence for thepotentialmechanisms thatmayunderlietreatment failure with low-dose fractionated radiotherapy andhave important implications for clinical translation demonstrat-ing concurrent but not sequential blockade of the PD-1/PD-L1axis with radiotherapy is required for long-term tumor control.

Materials and MethodsMice and cell linesBALB/c and C57Bl/6 mice were obtained from Harlan.

Animal experiments were approved by a local ethical commit-tee and performed under a United Kingdom Home Officelicense. CT26 murine colon carcinoma cells (ATCC) and4434 cells isolated from BRAFV600E p16�/� mice (RichardMarias, Cancer Research UK Manchester Institute, Manche-ster, United Kingdom) were maintained in DMEM, and 4T1triple-negative breast cancers (ATCC) maintained in RPMI-1640, supplemented with 10% FCS, 1% L-glutamine (Invitro-gen). All cell lines were cultured to limited passage beforeimplantation and were routinely screened to confirm theabsence of Mycoplasma contamination.

Tumor therapyMice were inoculated subcutaneously with either 5�105

CT26, 1�105 4T1, or 5�106 4434 cells. Irradiations were per-formed 7 to 10 days after inoculation (when tumors were atleast 100 mm3) as described previously (22). Administration ofaPD-1, aPD-L1, or isotype control mAb commenced on day 1of the fractionated radiotherapy cycle (unless otherwise stated)and was administered intraperitoneally (i.p.) 3qw for up to 3weeks at a dose of 10 mg/kg in a dose volume of 100 mL/10 g inPBS. Tumor volume (1,000 mm3 for CT26 and 4434models and500mm3 for 4T1)was the primary endpoint for efficacy studies.For cellular and cytokine depletion experiments, mice receivedeither aCD8 mAb, clone YTS169 (a gift from M. Glennie,Southampton University, Southampton, United Kingdom);aCD4 mAb, clone GK1.5 (Biolegend); aAsialo-GM1 (WakoChemicals); or aIFNg , clone XMG1.2 (BioXcell). Peripheralblood was sampled during therapy to confirm cellular deple-tion. For tumor rechallenge experiments, long-term surviving(LTS) mice were implanted contralaterally with tumor cells aminimum of 100 days after previous tumor implantation.Experimental groups contained at least 5 mice/group and arerepresentative of at least two independent experiments.

Measurement of cytokine production by CD8þ T cellsisolated from long-term surviving miceSplenocytes were isolated from either LTS or control mice

and cocultured with either irradiated tumor cells (50 Gy) or 1mmoL/mL of the H2-Ld restricted peptides SPSYVYHQF(AH1)/TPHPARIGL (b-galactosidase; Anaspec) as describedpreviously (22). Experimental groups contained 3 to 5mice andare representative of two independent experiments.

Tumor and immune cell phenotyping by flow cytomteryTo obtain single cell suspensions, tumors were processed

using a gentleMacs dissociator and a murine tumor dissoci-ation kit (Miltenyi Biotec). For analysis, nonspecific bindingwas blocked as described above and expression of CD4, CD8(BD Biosciences), CD45, CD11b, GR1, NKP46, PD-1, and PD-L1examined by multiparameter flow cytometry (all eBioscienceunless otherwise stated). For analysis, live cells were gated (byvital dye exclusion, Invitrogen) and populations phenotyped(as described above). An example of the gating strategy usedfor selection of either CD45� or CD45þ cells is provided inSupplementary Fig. S1.

In vitro coculturesTumor cells were cultured in the presence of 20 ng/mL IFNg

and/or TNFa for 24 hours before evaluation of PD-L1 expres-sion by flow cytometry as described above. For coculture assayeither resting or activated splenocytes (treated with PBS orphorbol 12-Myristate 13-Acetate and Ionomycin cell stimula-tion cocktail respectively, eBioscience) were cocultured at a 1:1ratio with tumor cells and tumor cell expression of PD-L1determined as described above. Silencing of IFNgR1 expres-sion was achieved by lentiviral transduction of cells withshRNA (cells were also transduced with nontargeting shRNAas controls; Thermo Scientific). Measurement of splenocytecytokine production (IFNg and TNFa) was measured by intra-cellular flow cytometry as described above.

ResultsBlockade of PD-1 or PD-L1 enhances the therapeuticefficacy of radiotherapy

Radiotherapy has been shown to modulate the immunoge-nicity of tumor cells but is rarely able to generate durabletherapeutic responses that lead to systemic antitumor immunityalone. We show that low doses of local fractionated-dose radio-therapy delivered as 10 Gy in 5 fractions lead to increased tumorcell expression of PD-L1 with elevated expression evident 1, 3,and 5 days after the last dose of radiotherapy when comparedwith time-matched, nontreated (NT) mice (Fig. 1A and B). Thisradiotherapy-mediated increase in tumor cell PD-L1 expressionpeaks at 72hours after the lastdose of radiotherapy andalthoughit declines significantly by 7 days after radiotherapy (whencompared with expression at days 1 and 3; P < 0.05 and P <0.01, respectively; Mann–Whitney test) remains elevated whencomparedwithNTmice (P< 0.05,Mann–Whitney test). A similarpattern of expression was also found on CD11bþ GR1Hi but noton CD11bþGR1Lo cells (Supplementary Fig. S2A and S2B).

Given these observations, we hypothesized that the immuneresponse generated following radiotherapy may be limitedthrough the PD-1/PD-L1 axis. Local radiotherapy delivered as10 Gy in 5 daily fractions was found to significantly improvesurvival in mice bearing established CT26 tumors when com-paredwithNTcontrols (Fig. 1C andD;P< 0.05 log-rank;Mantel–Cox test). Our data demonstrate that this radiotherapy-medi-ated local tumor control can be substantially improved throughcombination with either aPD-L1 mAb or aPD-1 mAb (Fig. 1Cand D; P < 0.001 log-rank; Mantel–Cox test). Combined therapywas curative in 66% and 80% of mice that received either

Efficacy of Fractionated Radiotherapy Enhanced by PD-1/PD-L1 Blockade

www.aacrjournals.org Cancer Res; 74(19) October 1, 2014 5459



radiotherapy and aPD-L1 mAb or aPD-1 mAb, respectively. Noadditional benefit was observed when mice received radiother-apy in combination with both aPD-1 and aPD-L1 mAb versuseither mAb alone (Supplementary Fig. S3). In contrast withcombination with radiotherapy, monotherapy with eitheraPD-L1 mAb or aPD-1 mAb did not significantly improvesurvival (Fig. 1C and D; P > 0.05 log-rank; Mantel–Cox test).

Blockade of PD-L1 also improved response to radiotherapy inmice bearing established 4T1 tumors where combined therapysignificantly reduced tumor burden by 38%, when comparedwith radiotherapy alone (10 days after start of therapy; 184.3 �13.5 mm2 vs. 292.8 � 14.3 mm2, respectively; P < 0.01 Mann–Whitney test) and significantly improved survival (P < 0.001 log-rank;Mantel–Cox test; datanot shown). Similar resultswerealso

Figure 1. Blockade of the PD-1/PD-L1 axis potentiates the efficacy offractionated radiotherapy. A and B,median fluorescence intensity (A)and representative histograms (B)of PD-L1 expression on CT26 cellsisolated from tumors 1, 3, or 7 daysafter treatment with 10 Gy in 5 dailyfractions of 2 Gy. C and D, CT26tumor-bearingmice received 10Gyradiotherapy delivered in 5 dailyfractions of 2 Gy either alone orin combination with either aPD-1(C) or aPD-L1 (D) mAb dosed at10 mg/kg 3qw for up to 3 weeks. E,tumor volumes 10 days after thestart of therapy in 4T1 tumor-bearing mice that received 20 Gyradiotherapy delivered in 5 dailyfractions of 4 Gy either alone or incombination with aPD-L1 mAb. F,4434 tumor-bearing mice received10 Gy radiotherapy delivered in 5daily fractions of 2 Gy either aloneor in combination with aPD-L1mAb. Experimental groupscontained at least 7 mice and arerepresentative of at least twoindependent experiments. A, E,and F show mean � SEM. �, P <0.05; ��, P < 0.01, Mann–Whitneytest. C and D, �, significance whencompared with control mice.þ, significance when comparedwith monotherapy. �� /þþþ, P <0.001, log-rank (Mantel–Cox) test.RT, radiotherapy.

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observed in mice bearing established 4434 melanomas (Fig. 1E).Combination therapy with local radiotherapy and mAb target-ing either PD-1 or PD-L1 was well tolerated in both BALB/c andC57Bl/6 mice (Supplementary Fig. S4A and S4B). These dataclearly demonstrate the potential to improve outcome followinglow-dose fractionated radiotherapy of established solid tumorsby blockade of the PD-1/PD-L1 axis.

NK cells contribute to local tumor control followingcombination therapy, but long-term survival isdependent on CD8þ T cellsWe next investigated the mechanisms underlying this long-

term tumor control observed following combination radio-therapy and aPD-L1 mAb therapy. Initially colony forming

assays were used to confirm thataPD-1 andaPD-L1mAbwerenot acting as radiation sensitizers through direct interactionwith the tumor cells (Supplementary Fig. S5A–S5C). Usingdepleting antibodies, we next explored the roles for effectorT cells and NK cells in mediating antitumor efficacy followingradiotherapy/aPD-L1 mAb combination therapy. Our datademonstrate that as early as 7 days after the start of a 5-dayfractionated radiotherapy cycle (with PD-L1 mAb therapycommencing on day 1 of the radiotherapy cycle), reducedtumor burden is evident in mice following combination ther-apy when compared with NT mice (207.5� 29.2 mm2 vs. 409.4� 86.88 mm2, respectively; P ¼ 0.067, Mann–Whitney Utest; Fig. 2A). However, this statistical trend to reduced volumewas lost following depletion of either CD8þ T cells or NK cells

Figure2. Therapeutic efficacyof fractionated radiotherapyandaPD-L1mAbcombination is dependenton the activity ofCD8þT lymphocytes. A, tumor volumeon day of therapy, 7 and 11 days after combination therapy with 5 fractions of 2 Gy and aPD-L1 mAb. Immune cell subsets (either CD8, CD4, or NK cell)were depleted 1 day before therapy with depletion maintained for 2 weeks. ��, P > 0.001; ��, P > 0.01; �, P > 0.05, Mann–Whitney test. B, survivalcurve. �� /þþþ,P <0.001, log-rank (Mantel–Cox) test. Data representative of 10mice per cohort. C, representative density plots of peripheral blood, confirmingdepletion of immune cell subsets. RT, radiotherapy.





where tumor volumes were not significantly different fromthose inNT cohorts (P¼ 0.52 and P¼ 0.70, respectively,Mann–WhitneyU test) but significantly larger thanmice that receivedcombination therapy without immune cell depletion (P < 0.01;combined therapy vs. CD8 depletion, and P < 0.05; combinedtherapy vs. NK cell depletion, Mann–Whitney U test). By day 11after treatment, combination therapy significantly reducedtumor burden when compared with NT controls (P < 0.001,Mann–Whitney U test). Although the depletion of either CD8þ

T cells or NK cells at this time point reduced the efficacy ofcombination therapy (P < 0.001 and P < 0.05, respectively,Mann–Whitney U test), the relative contribution of CD8þ Tcells and NK cells became more evident with significantlyreduced tumor control in CD8 versus NK cell-depleted mice(P < 0.05, Mann–Whitney U test). Our data also reveal that thedepletion of CD4þ T cells improved local tumor controlfollowing combination therapy (153.2 � 27.0 mm2 vs. 72.7 �17.3 mm2, respectively; P < 0.05, Mann–Whitney U test).

In contrast with early tumor control following combinationradiotherapy/aPD-L1 mAb therapy, LTS was not affected bythe depletion of NK cells (70% LTS mice following treatmentwith radiotherapy/aPD-L1 vs. 77.8% following combinationtherapy with NK cell depletion; Fig. 2B). The depletion of CD4þ

T lymphocytes increased the frequency of LTS mice from 70%to 87.5% (combination vs. combination þ CD4 depletion) butthis did not achieve significance (P >0.05 log-rank;Mantel–Cox

test). We show that the depletion of CD8þ T cells completelyabrogated the therapeutic efficacy of combination radiother-apy/aPD-L1 mAb therapy (P < 0.001 log-rank; Mantel–Coxtest). Depletion of immune cell populations was confirmed byflow cytometry on peripheral blood samples (Fig. 2C). Thesedata suggest that although NK cells may exert some localtumor control, this does not affect overall survival andalthough CD4þ T cells are dispensable or even capable ofsuppressing responses, CD8þ T cells are critical for medi-ating effective long-term tumor control following treatmentwith radiotherapy and aPD-L1 mAb.

Treatment with aPD-L1 mAb and radiotherapygenerates protective tumor antigen–specific memoryT-cell responses

We next investigated whether immunologic memory wasgenerated following treatment with radiotherapy and aPD-L1mAb. We show that LTS mice originally treated with radiother-apy and aPD-L1 mAb were able to completely reject tumorsfollowing contralateral rechallenge (Fig. 3A). To quantify thismemory response, splenocytes were harvested from LTS micethat had more than 100 days of disease-free survival and thecapacity of CD8þ T cells to produce IFNg following coculturewith either a CT26 tumor-associated antigen (AH1: SPSY-VYHQF), control peptide (b-galactosidase: TPHARIGL), or withirradiated CT26 cells was assessed (Fig. 3B and C). Our data

Figure 3. Fractionated radiotherapy and aPD-L1 mAb combination generates protective immunologic memory. A, survival curve of LTS mice followingcontralateral rechallenge with 5�105 CT26 cells. �, P < 0.05 compared with control mice (log-rank; Mantel–Cox test). Experimental groups contained atleast fourmice and are representative of at least two independent experiments. RT, radiotherapy. B, representative dot blot of IFNg production byCD8þT cellsisolated from either tumor naïve or LTS mice originally treated with radiotherapy and aPD-L1 mAb. C, frequency of IFNgþ CD8þ T cells isolated fromeither tumor naïve or LTS mice originally treated with radiotherapy and aPD-L1 mAb following coculture with either H2-Ld restricted peptides (AH1(SPSYVYHQF); a defined CT26 tumor-associated antigen or b-galactosidase (TPHPARIGL); control peptide of prokaryotic origin) or 50 Gy irradiated CT26cells for 5 days, followed by priming with 50 Gy irradiated CT26 cells. �, P < 0.05 (Mann–Whitney test). Data representative of two independent experiments.

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reveal that LTS mice were endowed with a significantly greaterfrequency of IFNg-producing CD8þ T lymphocytes followingcoculture with AH1 peptide than na€�vemice (6.6%� 0.8 vs. 2.3%� 0.2, respectively; P < 0.05, Mann–Whitney test). A similarresponse was observed following coculture of splenocytes withCT26 cells. Comparison of peptide and tumor cell cocultures,however, revealed that the frequency of memory CD8þ T cellswas approximately 3-fold lower in LTSmice following coculturewith tumor cells than with AH1 peptide and may reflect tumorcell-mediated suppression of T-cell activation. Taken together,these data demonstrate that radiotherapy when combined withblockade of the PD-1/PD-L1 axis can generate protective immu-nologic memory in long-term survivors.

Fractionated radiotherapy leads to CD8þ T-cell–dependent adaptive upregulation of tumor cell PD-L1expression

Initially we confirmed that treatment of tumor cells with arange of radiotherapy doses in vitro did not have any directimpact on expression of PD-L1 (Fig. 4A). To identify whichcellular populations within the tumormicroenvironment wereresponsible for modulating tumor cell expression of PD-L1,mice received a fractionated radiotherapy cycle in combina-tion with CD8, CD4, and NK cell depleting antibodies. Our datareveal that the depletion of CD8þ T cells but not NKcellscompletely abrogates the radiotherapy-mediated upregulationof PD-L1 on tumor cells (Fig. 4B and C). Interestingly, the

Figure 4. Fractionated radiotherapyincreases tumor cell PD-L1expression in vivo and isdependent on CD8þ T cells. A,expression of PD-L1 on CT26 cellsafter treatment with radiotherapy(2.5–10Gy) in vitro. B and C,representative contour plots (B)and median fluorescence intensity(C) of PD-L1 expression on CT26cells (gated as CD45� cells)isolated from tumors 3 days afterreceiving 10 Gy in 5 daily fractionsof 2Gy in combination with eitherCD8, CD4, or NK cell depletingantibodies. �, P < 0.05 (Mann–Whitney test). Experimental groupscontained at least fivemice and arerepresentative of at least twoindependent experiments. RT,radiotherapy; n/s, nonsignificant.





depletion of CD4þ T cells was found to further augmentradiotherapy-mediated upregulation of PD-L1 on tumor cells(� 2-fold compared with treatment with radiotherapy alone).

Adaptive upregulation of PD-L1 by tumor cells followingfractionated radiotherapy is IFNg dependent

Given the clinical correlation between IFNg in the tumormicroenvironment and PD-L1 expression (16) and the impactof TNFa on this response (23), we evaluated the impact of thesecytokines on PD-L1 in our cell lines. Coculture of tumor cellswith recombinant IFNg leads to a significant 20-fold increase incell surface expression of PD-L1 in vitro (Fig. 5A). Furthermore,

although addition of recombinant TNFa alone has no impacton tumor cell expression of PD-L1, a cocktail of both IFNg andTNFa can further augment tumor cell PD-L1 expression (�2-fold) when compared with expression with IFNg alone(Fig. 5A). We show that silencing of IFNg-receptor 1 (IFNgR1)on CT26 tumor cells using shRNA completely abrogates theupregulation of PD-L1 following coculture with either recom-binant IFNg or both IFNg and TNFa (Fig. 5A).

To establish whether activated immune cells could lead toan adaptive change in tumor cell expression of PD-L1 throughthe production of IFNg and TNFa, WT CT26 cells and thosetransduced with either nontargeting shRNA (NTC shRNA) and

Figure 5. CD8þ T-cell productionof IFNg is responsible for theupregulation of PD-L1 expressionon tumor cells followingfractionated radiotherapy. A,expression of PD-L1 on wild-typeCT26 tumor cells (WT) or cellstransduced with either anontargeting control (NTC) orIFNgR1 shRNA following coculturefor 24 hours with either 20 ng/mLIFNg , TNFa, or a combination ofboth cytokines. B, representativedensity plots showing expressionof IFNg and TNFa by CD8þ T cellsfollowing treatmentwith either PBSor phorbol 12-myristate 13-acetate(PMA) and ionomycin,C, frequencyof PD-L1–positive CT26 tumorcells (WT, NTC shRNA, or IFNgR1shRNA) following 24-hourcoculture with either PBS or PMA/ionomycin activated splenocytes.n/s, nonsignificant, P > 0.05; ��,P < 0.001; ��, P < 0.01 (two-tailedStudent t test). D and E,representative contour plots (D)and median fluorescence intensity(E) of PD-L1 expression on CT26tumor cells following treatmentwith radiotherapy (10 Gy in 5fractions) in the presence of anaIFNg blocking mAb (or isotypecontrol) in vivo. ��,P < 0.01, (Mann–Whitney test). Experimental groupscontained at least five mice. RT,radiotherapy.

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IFNgR1 shRNA were cocultured with isolated resting (PBStreated) and activated [phorbol 12-myristate 13-acetate (PMA)and ionomycin-treated] splenocytes (Fig. 5B and C). Impor-tantly, these experiments demonstrate that activated immunecells can elevate tumor cell PD-L1 expression at physiologicallyrelevant concentrations of IFNg and TNFa, in an IFNgR1-dependent manner.To confirm the role of IFNg on tumor cell expression of PD-

L1 in response to radiotherapy in vivo, mice received radio-therapy in combination with aIFNg-blocking antibody (orisotype control). IFNg blockade reduced tumor cell expressionof PD-L1 by 2.6-fold in NT mice to the level observed on CT26cells cultured in vitro, suggesting that adaptive upregulation ofPD-L1 occurs following implantation of tumor (Fig. 5D and E).However, the significant upregulation of PD-L1 observed fol-lowing radiotherapy (2.4-fold compared with NT control) wascompletely abrogated by IFNg blockade confirming this as thedriver of adaptive tumor cell expression of PD-L1.

Concomitant scheduling of aPD-L1 mAb andradiotherapy is critical for the generation of an effectiveantitumor immune responseThe optimal schedule for combining radiotherapy and

immune checkpoint inhibitors is currently unclear and maysignificantly affect the generation of a durable therapeuticantitumor immune response. We evaluated three distinctcombination schedules where mice bearing established CT26tumors received a fractionated radiotherapy cycle of 10 Gy in 5fractions with administration of aPD-L1 mAb commencingeither on day 1 of the fractionated radiotherapy cycle (scheduleA), day 5 of the cycle (schedule B), or 7 days after completion ofradiotherapy (schedule C; Fig. 6A). No significant difference inoverall survival was found between schedule A and B with LTSof 60% and 57%, respectively (P >0.05 log-rank; Mantel–Coxtest; Fig. 6B and Supplementary Fig. S6A). In contrast, sequen-tial treatment with radiotherapy followed 7 days later by aPD-L1 mAb (schedule C) was completely ineffective at enhancingoverall survival when compared with radiotherapy alone(median survival of 30 days vs. 35 days, respectively; P >0.05log-rank; Mantel–Cox test) despite similar tumor burdenacross groups (Supplementary Fig. S6B and S6C).Analysis of tumor-infiltratingCD4þ andCD8þTcells 24hours

after the last dose of radiotherapy revealed increased expressionof PD-1 when compared with time-matched NT controls (P <0.05, Mann–Whitney test; Fig. 6C). In contrast, by 7 days afterradiotherapy, no change in PD-1 expression was evident onCD4þ T cells and was found to be significantly decreased onCD8þ T cells when compared with time-matched NT controls(P < 0.05, Mann–Whitney test; Fig. 6D). Expression of PD-1 wasconsistently found to be higher on tumor-infiltrating CD8þ thanCD4þT-cells (Fig. 6C andD).Wenext compared efficacywith anacute versus extendedaPD-L1mAbdosingprotocol wheremicereceived either threedosesofmAbconcurrentwith radiotherapyor the same with dosing extended (3qw) for an additional 2weeks. No additional benefit was observed with extended aPD-L1 mAb administration (Supplementary Fig. S7).Taken together these data demonstrate that treatment with

low-dose fractionated radiotherapy leads to an acute increase

in PD-1 expression onT cells and that sequential therapywhereblockade of the PD-1/PD-L1 signaling axis is delayed untilcompletion of an radiotherapy cycle may be ineffective poten-tially due to deletion or anergy of tumor-reactive CD8þ T cells.

DiscussionIn this manuscript, we show that treatment with low doses

of fractionated radiotherapy leads to upregulation of PD-L1expression on tumor cells secondary to CD8þ T-cell produc-tion of IFNg . In models of melanoma, colorectal, and breastcancer, we demonstrate that the efficacy of radiotherapy canbe enhanced through combination with aPD-L1 mAb, leadingto the generation of memory immunity in LTS mice capable ofprotecting against tumor recurrence. Furthermore, our datareveal that dose scheduling may be critical for outcome withconcurrent but not sequential therapy effective at improvinglocal tumor control and survival.

Recent clinical trials have begun to evaluate blockade of thePD-1/PD-L1 axis with encouraging responses observed in mul-tiple disease settings with mAb targeted against both PD-1 andPD-L1 (18–20, 24, 25). Despite this, combination approachesmay be required to improve response rates and to generatedurable antitumor immunity.Weandothershavedemonstratedthe potential to improve systemic antitumor immune responsesthrough combination of immunotherapy with local radiother-apy, which has distinct advantages over chemotherapy in termsof highly focused tumor targeting and reduced systemic impacton the immune system (5, 22, 26, 27). Radiotherapy can lead totumor cell DAMP release, DC recruitment, and type I IFN-dependent cross-priming of tumor-specific CD8þ T-cellresponses (4, 6, 28). This is the first preclinical study to dem-onstrate that fractionated radiotherapy leads to increasedtumor cell expression of PD-L1 through CD8þ T-cell productionof IFNg . Here, tumor cell expression of PD-L1 may act as abiomarker of local antitumor response, suggesting that localradiotherapy may be sufficient to prime CD8þ T-cell responses.However, although treatment with radiotherapy alone wasunable to generate durable anticancer immunity, efficacy wasfound to be enhanced through blockade of either PD-1 or PD-L1,suggesting that signaling through this axis may limit theimmune response to radiotherapy suboptimally. We saw nosignificant differences in overall survival between radiotherapydelivered in combination with either aPD-1 or aPD-L1, or acombinationof bothmAb.Given this observation, it seems likelythat the efficacy of these mAb when delivered in combinationwith radiotherapy is through blockade of PD-1/PD-L1 signalingand notmediated through the PD-L1/CD80 or PD-1/PD-L2 axesalthough further experiments are needed to confirm this.

In addition togenerating adaptive immunity, radiotherapyhasbeen shown to enhance recognition andantitumor activity ofNKcells through increased tumor cell expressionofNKG2D(29). Ourdata reveal that the depletion of NK cells reduced local tumorcontrol at early time points (up to 11 days after completion ofradiotherapy) but did not affect overall survival. In addition, thedepletion of NK cells did not affect tumor cell expression of PD-L1 following radiotherapy, suggesting their limited contributionto local IFNg production. In contrast, although depletion of





CD4þ T cells did not affect survival following combinationtherapy, substantial increases in tumor cell expression of PD-L1 after radiotherapy was observed. The depletion of CD4þ Tcells affects both helper T-cell and Treg populations. Althoughfurther studies are needed to delineate the relative contributions

of these subpopulations of CD4þ T cells, we can speculate thatCD4þ helper T cells may be dispensable for the generation ofeffective CD8þ T-cell responses after radiotherapy/PD-L1 mAbtherapy. In addition, it is possible that tumor-infiltrating Tregsmay be actively suppressing IFNg production in the local tumor

Figure 6. Dosing schedule is critical to outcome with radiotherapy potentiation only observed with concurrent but not sequential aPD-L1 mAb therapy. A,schema for dose-scheduling studies. Mice received fractionated-dose radiotherapy (as 10 Gy in 5 daily fractions of 2 Gy) alone or in combination withaPD-L1 mAb starting either on day 1 of radiotherapy cycle (schedule A), day 5 of radiotherapy cycle (schedule B) or 7 days after the last dose of radiotherapy(schedule C). B, survival curves of therapy. þþ, P < 0.01 compared with monotherapy (log-rank; Mantel–Cox test). C and D, expression of PD-1 on CD4þ

(C) and CD8þ (D) T cells 24 hours and 7 days after the last dose of radiotherapy. �, P < 0.05 (Mann–Whitney test). Experimental groups contained at least fivemice and are representative of at least two independent experiments. RT, radiotherapy.

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milieu; however, further studies to assess the impact of Tregs ontumorcell PD-L1 expression following radiotherapy are required.PD-L1 expression can be modulated by a number of cyto-

kines, including type I and II IFNs, TNFa, and TGFb (16, 23, 30–32). We show that tumor cell expression of PD-L1 can beenhanced following coculture with IFNg and that addition ofTNFa can further augment this response. However, blockadeof IFNgR1 or in vivo depletion of IFNg demonstrates depen-dence on IFNg-mediated signaling for upregulation of PD-L1,with TNFa alone incapable of modulating tumor cell PD-L1expression. Interestingly, in vivo depletion of IFNg reduces PD-L1 expression on syngeneic tumor, matching the expressionprofile of tumor cells in vitro. This suggests that the local tumormicroenvironment in the absence of therapeutic interventionmay foster immunologically driven tumor adaptation, whichmay support tumor development. Similarly, in human mela-nocytic lesions, expression of PD-L1 has been shown to colo-calizewith areas of CD8þT-cell infiltration and is postulated torepresent an adaptive mechanism of immune escape (16).However, in our preclinical models, monotherapy with mAbtargeting PD-1/PD-L1 only provides modest efficacy, whichsuggests that targeting this axis alone may be unlikely to bringabout durable antitumor immune responses and underscoresthe requirement for a combinatorial approach.A recent study in the TUBO breast cancer model found that

treatment with a single dose of 12 Gy radiotherapy could alsoincrease both tumor cell andmonocyte-derived suppressor cellexpression of PD-L1 (30). In contrast with the use of a single 12-Gy dose, our results demonstrate that upregulation of PD-L1occurs at much lower biologic effective doses involving deliveryof fractionated radiotherapy using 10 Gy in 5 daily fractions.This is an important finding given that this dose per fraction iscommonly used in routine clinical practice. Several studies havedemonstrated the superiority of either single ablative doses orfractionated radiotherapy on the generation of antitumorimmune responses (4, 5, 22, 26, 33, 34). However, the varyingintrinsic radiosensitivity of the cell lines used, their inherentimmunogenicity, and the disparate phenotype of the tumormicroenvironments (which may be influenced both by thetumor cells and the background of the mouse) may contributeto the lack of concordance across these published studies.Whatis clear fromthese studies is that for a givenmodel, radiotherapydose fractionation does influence the phenotype and antitumoractivity of the immune response. Additional studies where theimmunogenicity of different radiotherapy dose fractionationregimens is directly compared are clearly required.To date, no studies have addressed the impact of sequencing

on the efficacy of radiotherapy and aPD-L1 mAb therapy. Thisis especially pertinent given the predisposition to adopting asequenced combination regimen in the clinic in an effort tolimit adverse reactions. We show that only blockade of PD-L1at the time of radiotherapy delivery can enhance therapeuticresponse with sequential therapy no better than treatmentwith radiotherapy alone. In addition, our data reveal that PD-L1 expression is elevated by 24 hours after completion of afractionated radiotherapy cycle and remains elevated for atleast 7 days after completion. Moreover, we found elevated PD-1 expression on tumor-infiltrating CD4þ and CD8þ T cells 24

hours after radiotherapy when compared with control cohorts.Expression of PD-1 was found to be consistently higher onCD8þ versus CD4þ T cells. Taken together, these data suggestthat local fractionated radiotherapy can prime antitumorCD8þ T-cell responses but these may be attenuated by signal-ing through the PD-1/PD-L1 axis and early inhibition of thisaxis may be critical for the generation of durable effectiveantitumor responses. Furthermore, the requirement for chron-ic versus acute blockade of the PD-1/PD-L1 axis when com-bined with radiotherapy, is unclear. We observed no differencein survival when aPD-L1 mAb was administered 3qw over 1week concurrent with radiotherapy delivery versus an extend-ed schedule of up to 3 weeks. These data suggest that com-bination approaches with radiotherapy may permit a reduc-tion in the duration of aPD-1/PD-L1 mAb therapy required toachieve therapeutic antitumor immune responses. Togetherthese data have important implications for clinical trial design.

In summary, this study demonstrates that treatment withfractionated radiotherapy leads to upregulation of tumor cellexpression of PD-L1 and that blockade of the PD-1/PD-L1 axiscan enhance the immune response to fractionated radiotherapyin multiple syngeneic models. Our data suggest that dose sched-uling of aPD-L1 mAb with radiotherapy may be critical to thegenerationof therapeutic immune responseswith the capacity toreduce tumor burden and improve survival. This therapeuticcombination may be a promising approach for the treatment ofmany solid malignancies where radiotherapy is commonly usedand translation to early-phase clinical trial is clearly warranted.

Disclosure of Potential Conflicts of InterestT.M. Illidge and J. Honeychurch received a commercial grant from Med-

Immune Ltd. S.J. Dovedi and I.J. Stratford received commercial research grant fromMedImmuneLtd. andAstraZeneca.R. Stewart isprincipal scientist inMedImmune.No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: S.J. Dovedi, I.J. Stratford, M. Morrow, R. Stewart,R.W. WilkinsonDevelopment of methodology: S.J. DovediAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.J. Dovedi, A.L. Adlard, G.L. Lipowska-Bhalla,C. McKenna, S. Jones, E.J. CheadleAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.J. Dovedi, G.L. Lipowska-Bhalla, E.J. Cheadle,R. Stewart, R.W. Wilkinson, J. Honeychurch, T.M. IllidgeWriting, review, and/or revision of the manuscript: S.J. Dovedi, E. Poon,M. Morrow, R. Stewart, R.W. Wilkinson, J. Honeychurch, T.M. IllidgeAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): R.W. WilkinsonStudy supervision: I.J. Stratford, E. Poon, M. Morrow, R.W. Wilkinson,J. Honeychurch, T.M. IllidgeOther (collaboration coordinator): E. Poon, M. Morrow

AcknowledgmentsThe authors thank the members of the Cancer Research UK Manchester

Institute BRU and Flow Cytometry Core Facilities for their help. They also thankall members of the Targeted Therapy Group for helpful discussion and criticalreview of this article.

Grant SupportThis work was funded with a grant fromMedImmune and Cancer Research UK.

The costs of publication of this article were defrayed in part 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 April 28, 2014; revised July 3, 2014; accepted July 9, 2014;published online October 1, 2014.





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2014;74:5458-5468. Cancer Res Simon J. Dovedi, Amy L. Adlard, Grazyna Lipowska-Bhalla, et al. Overcome by Concurrent PD-L1 BlockadeAcquired Resistance to Fractionated Radiotherapy Can Be

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