Combinatorial Immunotherapy of Polyinosinic …clincancerres.aacrjournals.org/content/clincanres/20/5/...via intraperitonial injections: anti-CD8, 500 mg/injection; anti-CD4, 200 mg/injection;

Cancer Therapy: Preclinical

Combinatorial Immunotherapy of Polyinosinic–PolycytidylicAcid and Blockade of Programmed Death-Ligand 1 InduceEffective CD8 T-cell Responses against Established Tumors

Toshihiro Nagato1,4, Young-Ran Lee1, Yasuaki Harabuchi4, and Esteban Celis1,2,3

AbstractPurpose: Epitope-based cancer vaccines capable of inducing CD8 T-cell responses to tumor-associated

antigens (TAA) expressed by tumor cells have been considered as attractive alternatives for the treatment of

some types of cancer. However, reliable TAAs have not been identified formostmalignant diseases, limiting

the development of epitope-based vaccines. Herein, we report that the combinatorial therapy of poly-

inosinic–polycytidylic acid (poly-IC) and antiprogrammed death-ligand 1 (PD-L1) monoclonal antibody

(mAb) can be implemented with good results for tumors where no known TAAs have been identified.

Experimental Design: Three cancer mouse models (melanoma, lung, and colon) were used to evaluate

therapeutic efficacy and examine the immunologic mechanisms of the poly-IC/anti–PD-L1 mAb therapy.

Results: The combined administration of poly-IC and anti–PD-L1 mAb into tumor-bearing mice

generated potent immune responses resulting in the complete eradication or remarkable reduction of

tumor growth. In some instances, the poly-IC/anti–PD-L1 mAb therapy induced long-lasting protection

against tumor rechallenges. The results indicate that CD8T cells but not CD4T cells orNK cellsmediated the

therapeutic efficacy of this combinatorial therapy. Experiments using genetically deficientmice indicate that

the therapeutic efficacy of this combinatorial therapy depended in part by the participation of type-I IFN,

whereas IFN-g did not seem to play a major role.

Conclusions: The overall results suggest that immunotherapy consisting of the combination of poly-

IC/anti–PD-L1 mAb could be a promising new approach for treating patients with cancer, especially

those instances where no reliable TAAs are available as a therapeutic vaccine. Clin Cancer Res; 20(5);

1223–34. �2014 AACR.

IntroductionConventional treatments for cancer such as surgery, radio-

therapy, and chemotherapy are commonly associated withsuboptimal therapeutic efficacy and detrimental side effects.Therefore, different treatment modes such as immunother-apy using therapeutic vaccines or monoclonal antibodies(mAb) that enhance ongoing antitumor immune responsesare being explored as alternatives or adjunct treatments (1,2). Especially, the use of cancer vaccines that induce tumor-reactive CD8 T cells is being considered as a strategy to treatestablished tumors and prevent recurrences (3–5). Never-theless, todevelop such therapeutic vaccines it is necessary toidentify tumor-associated antigens (TAA) containing pep-

tide epitopes for tumor-reactive CD8 T cells (6, 7). Severalinvestigators including us have considered using syntheticpeptides representing defined CD8 T-cell epitopes derivedfrom TAAs such asmelanosomal differentiation proteins, asvaccines for treating melanoma (8–11). Being cognizantthat significant challenges exist regarding the use of peptidevaccines such as those related to MHC restriction, whichlimit the use of a peptide to a subset of patients expressing aparticularMHC class I (MHC-I) allele. In addition, formanytumor types such as lung cancer, no reliable TAAs capable ofeliciting effective antitumor T-cell responses have beenidentified. In viewof this, alternative approaches to generateantitumor CD8 T-cell responses should be explored. Onestrategy would be to generate or enhance existing tumor-specific CD8 T-cell responses via the combinatorial use ofstrong immune adjuvants such as Toll-like receptor (TLR)ligands and mAbs that block immune regulatory pathwaysthat suppress CD8 T cells. While studying the therapeuticeffects of peptide vaccines administered in combination ofpolyinosinic–polycytidylic acid (poly-IC) and antipro-grammed death-ligand 1 (PD-L1) mAb in a mouse modelof melanoma, we observed significant therapeutic effects inmice that received an irrelevant control peptide (11). Theseresults suggested that the combined administration of these

Authors' Affiliations: 1Immunology Program, Moffitt Cancer Center;Departments of 2Oncologic Sciences and 3Molecular Medicine, Universityof South Florida, Tampa, Florida; and 4Department of Otolaryngology-Head and Neck Surgery, AsahikawaMedical University, Asahikawa, Japan

Corresponding Author:EstebanCelis, Cancer Immunology, Inflammationand Tolerance Program, Georgia Regents University Cancer Center, 1410LaneyWalker Blvd., CN-4121, Augusta, GA 30912. Phone: 706-721-5668;Fax: 706-721-1670; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-2781

�2014 American Association for Cancer Research.

ClinicalCancer

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on July 30, 2019. © 2014 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst January 3, 2014; DOI: 10.1158/1078-0432.CCR-13-2781

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immune modulating agents could provide a therapeuticbenefit against established tumors. Here, we report thatnon–antigen-specific immunotherapy consisting of repeat-ed co-administration of poly-IC and anti–PD-L1 mAbresulted in dramatic antitumor responses in several cancermouse models, which were mediated by CD8 T cells.

Materials and MethodsMice and cell lines

Six- to 8-week-old female C57BL/6 (B6) mice were fromthe National Cancer Institute/Charles River Program (Wil-mington, MA). IFN-g–deficient (IFN-g�/�) mice (B6 back-ground) were from Jackson Laboratories. IFN-ab receptor–deficient (IFN-abR�/�) mice (B6 background) wereobtained fromDr. PhilippaMarrack (National JewishMed-ical and Research Center, Denver, CO). All animal care andexperiments were conducted according to our institutionalanimal care and use committee (IACUC) guidelines. Lewislung carcinoma (LLC)-A9F1 cells are a subclone of LLC (12)provided by Lea Eisenbach (Weizmann Institute of Science,Rehovot, Israel). Mouse colorectal adenocarcinoma MC38cells were provided by Dmitry Gabrilovich (Moffitt CancerCenter, Tampa, FL). Mouse melanoma B16F10 cells wereprovided by Alan Houghton (Memorial Sloan KetteringCancer Center, New York, NY). Mouse thymoma EL4 cellswere from the American Type Culture Collection. All of thecell lines were cultured as recommended by the providersand were not authenticated by the authors.

Reagents and antibodiesPoly-IC was provided by Andres Salazar [poly-lysine and

carboxymethylcellulose (poly-ICLC)/Hiltonol; Oncovir,Inc.] or purchased from InvivoGen (Poly-IC HMW).CpG-1826 was prepared by the Mayo Clinic Molecular

Core Facility. Anti-mouse PD-L1 (10F.9G2), anti-NK 1.1(PK136), anti-CD4 (GK1.5), anti-CD8 (2.43), and anti-mouse interleukin-2 (IL-2; JES6-5H4) mAbs were fromBioXCell. Anti-mouse–programmed death-1 (PD-1;RMP1–14) mAb was provided by Hideo Yagita (JuntendoUniversity School of Medicine, Tokyo, Japan). Recombi-nant mouse IL-2 and IFN-g were from PeproTech. Fluo-rescence labeled Abs were from eBioscience and BDBiosciences.

Flow cytometric analysisThe expression of MHC molecules and PD-L1 on LLC-

A9F1, MC38, and B16F10 cells pretreated or not for 24, 40,or 48 hours with 100 ng/mL IFN-g was evaluated by flowcytometry using FITC-conjugated anti–H-2Db, PE-conju-gated anti–H-2Kb, APC-conjugated anti-MHC class II(MHC-II), and PE-conjugated anti–PD-L1 Abs. Cell linesalso were stained by 7-AAD to exclude nonviable cells.Fluorescence was measured using a FACSCalibur flowcytometer (BD Biosciences) and analyzed using FlowJosoftware.

Therapeutic protocols and evaluation of antitumoreffects

Mice were injected subcutaneously with 5 � 105 LLC-A9F1, 5 � 105 MC38, or 4 � 105 B16F10 cells in a shavedrear flank. Seven (B16F10 tumor model), 8 (MC38 tumormodel), or 9days (LLC-A9F1 tumormodel) later, poly-ICorCpG-1826 was administered intravenously at 50 mg/dose.The administration of poly-IC or CpG-1826 was repeated 3times, 5 days apart. Anti–PD-L1 or PD-1 mAb was admin-istered intraperitoneally on days 1 and 3 after each poly-ICor CpG-1826 administration at a 200 mg/dose. IL-2/anti–IL-2 mAb complexes (IL-2C�) were prepared by incubating 2mg recombinant mouse IL-2 with 10 mg anti-mouse IL-2mAb per dose for 18 hours at 4�C. IL-2C� were adminis-tered intraperitoneally on 2 and 1 days before the first poly-IC administration. Survivor mice were rechallenged subcu-taneously with the same number of tumor cells (in theiropposite flanks). For in vivo cell depletions (CD8 T cells,CD4 T cells, or NK cells), mice received the following mAbsvia intraperitonial injections: anti-CD8, 500 mg/injection;anti-CD4, 200 mg/injection; or anti-NK1.1, 300 mg/injec-tion on days �3, �1, and þ4 of the first poly-IC adminis-tration. Depletions were confirmed by flow cytometry anal-ysis of blood samples (data not shown). Tumor growth wasmonitored every 2 to 3 days in individually tagged mice bymeasuring 2 opposing diameters with a set of calipers. Micewere euthanatized when the tumor area reached 400 mm2.Results are presented as the mean tumor size (area in mm2)� SD for every treatment group at various time points untilthe termination of the experiment.

Measurement of immune responsesFor detection of CD8 T cells, secreting IFN-g EliSpot

assays were performed as described (13), using purifiedspleen CD8 T cells (Miltenyi Biotec). CD8 T cells wereincubated at 1 � 105 together with 1 � 105 stimulator cells

Translational RelevanceFor many malignant diseases, few if any reliable

tumor-associated antigens (TAA) capable of elicitingeffective antitumor CD8 T-cell responses have beenidentified, limiting the development of epitope-basedvaccines. Thus, there is a clear need to explore alternativeand novel immunotherapeutic approaches to induceand amplify tumor-reactive CD8 T cells without depend-ing on the use of defined TAAs. Here we describe a non–antigen-specific immunotherapy consisting of repeatedco-administration of polyinosinic–polycytidylic acid(poly-IC) and antiprogrammed death-ligand 1 (PD-L1) antibodies that results in dramatic antitumor effectsin several cancermousemodels, whichweremediatedbyCD8 T cells. Because both poly-IC and anti–PD-L1monoclonal antibody are available for clinical use, webelieve that our preclinical studies can readily translateinto the treatment for patients with cancer, especially inthose instances where reliable TAAs have not beenidentified.

Nagato et al.

Clin Cancer Res; 20(5) March 1, 2014 Clinical Cancer Research1224




(EL4, LLC-A9F1, andMC38 cells pretreated or not for 24 or48 hours with 100 ng/mL IFN-g). Cultures were incubatedat 37�C for 20 hours and spots (IFN-g producing cells) weredeveloped as described by the EliSpot kit manufacturer(Mabtech, Inc.). Spot counting was done with an AIDEliSpot Reader System (Autoimmun Diagnostika GmbH).

Statistical analysesStatistical significance to assess the numbers of tumor-

specific CD8 T cells (EliSpot) was determined by unpairedStudent t tests. As required by our IACUC guidelines, thenumbers of mice included in each treatment group wereselected based on the expected outcomes and variabilitybetween mice in each group (observed in previous experi-ments), which were taken into account to assess statisticalsignificance of the therapy. Tumor sizes between 2 popu-lations throughout time were analyzed for significanceusing 2-way ANOVA. All analyses and graphics were doneusing GraphPad Prism 6.02 (GraphPad Software). Allexperiments were repeated at least 2 times with similarresults.

ResultsTherapeutic effects of the combinatorialimmunotherapy with poly-IC and anti–PD-L1 mAbagainst established B16 melanomaIn a recent study, we observed a significant antitumor

effect in a control group of mice that received an irrelevantpeptide vaccine combined with poly-IC [TLR3 ligand andmelanoma differentiation-associated protein 5 (MDA5)agonist] and anti–PD-L1 mAb against established subcuta-neous B16 tumors (11). In view of this interesting observa-tion, we first explored the therapeutic efficacy and examinedthe immunologic mechanisms involved of the combinedadministration of poly-IC and anti–PD-L1 mAb in the B16mouse melanoma model. Mice were inoculated subcutane-

ously with B16F10 cells and 7 days later they received poly-IC alone, anti–PD-L1 mAb alone, or poly-IC plus anti–PD-L1 mAb. As shown in Fig. 1, tumors grew at a somewhatlower rate inmice that received poly-IC or anti–PD-L1 mAbas compared with the untreated group. In contrast, thecombined administration of poly-IC/anti–PD-L1 mAbresulted in a remarkable synergistic therapeutic effect. Nota-bly, depletion of CD4 T cells or NK cells did not reduce theeffectiveness of the combination therapy. However, deple-tion of CD8 T cells abrogated the antitumor effect. AlthoughB16 tumor growth was slowed down by the poly-IC/anti–PD-L1mAb combination therapy, none of themice rejectedtheir tumors.

Therapeutic effects of poly-IC/anti–PD-L1 mAbcombinatorial immunotherapy against establishedlung and colon tumors

Next, we determined whether the therapeutic efficacy ofpoly-IC/anti–PD-L1 mAb combinatorial therapy wouldextend to other tumor types. For these studies, we selectedthe transplantable LLC and the MC38 colon carcinoma.Because the tumor recognition of T cells and the efficacy ofanti–PD-L1 mAb may depend on the expression levels ofMHC and PD-L1 molecules, we evaluated the expression ofMHC-I, MHC-II, and PD-L1 on LLC, clone A9F1 (LLC-A9F1), and MC38 cells that were pretreated or not withIFN-g . B16F10 melanoma cells were also included in theseevaluations. Both LLC-A9F1 and MC38 expressed highlevels ofMHC-I (H-2Db andH-2Kb), which were somewhatincreased by IFN-g treatment (Fig. 2). The B16F10 cellsexpressed low levels of MHC-I, but these were dramaticallyincreased by IFN-g . All 3 tumors didnot expressMHC-II andIFN-g treatment was able to upregulate its expression onlyon B16F10. Expression of PD-L1was found expressed on all3 tumors and treatment with IFN-g enhanced its expressionby approximately 10-fold.

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Figure 1. Therapeutic effects inducedby the combinatorial therapy of poly-IC/anti–PD-L1mAbagainst establishedB16F10 tumors. B6mice (5 per group) wereinoculated subcutaneously on day 0 with 4 � 105 B16F10 cells and later treated on days 7, 12, and 17 with poly-IC at 50 mg/dose given intravenously.Anti–PD-L1 mAb (200 mg/dose) was administered intraperitoneally 1 and 3 days after each poly-IC treatment. Various subsets of immune cells(CD8Tcells, CD4Tcells, orNKcells) weredepletedusingmAb3and1daysbefore and4days after receiving the first poly-IC treatment. Nontreatedmicewereincluded as controls. As noted, some mice received poly-IC alone or anti–PD-L1 mAb alone. Points, mean for each group of mice; bars, SD. P valueswere compared with no treatment group and calculated using 2-way ANOVA tests (�, P < 0.05; ��, P < 0.001; ��, P < 0.0001).

Combinatorial Immunotherapy of Poly-IC and Anti–PD-L1 mAb

www.aacrjournals.org Clin Cancer Res; 20(5) March 1, 2014 1225




The effectiveness of poly-IC/anti–PD-L1 mAb therapywas evaluated against 9-day established subcutaneousLLC-A9F1 tumors. Tumor growth was effectively controlledin mice receiving the combination of poly-IC/anti–PD-L1mAb (Fig. 3A). Interestingly, administration of poly-ICalone resulted in an equally significant antitumor effect.However, anti–PD-L1mAb alone had a substantially lower,but statistically significant antitumor effect. The use of adifferent TLR ligand (CpG, a TLR9 agonist) with anti–PD-L1mAb did not increase the effectiveness of the therapy ascompared with the use of anti–PD-L1 mAb alone. Admin-istration of IL-2/anti–IL-2 mAb complexes (IL-2C�) hasbeen shown to potentiate in vivo CD8 T-cell expansion (14,15), and increase the antitumor effects of the T cells (10, 16).The addition of IL-2C� did not further improve the effec-tiveness of the combination therapy. Most remarkably, theadministration of poly-IC alone and the combination ofpoly-IC/anti–PD-L1 mAb (with or without IL-2C�)resulted in complete tumor eradications in 80% (4/5) ofmice. To assess the generation of long-term systemic immu-nity, the mice that rejected their tumors in the 3 groupsmentioned above were rechallenged on day 39 with theLLC-A9F1 tumor. One half of mice that received the com-bination of poly-IC/anti–PD-L1mAb or poly-IC alone wereable to reject the second tumor challenge (Fig. 3B). Surpris-ingly, in the case of mice treated with the combinatorialtherapy plus IL-2C�, no rejections were observed aftertumor rechallenge.

To assess whether tumor-reactive CD8 T cells wereinduced in this tumor model, CD8 T cells were isolatedfrom spleens of LLC-A9F1–bearing mice that were treatedwith poly-IC/anti–PD-L1mAb on day 12, when the averagetumor sizehaddecreasedby approximately 50%. TheCD8Tcells were effective in recognizing LLC-A9F1 cells and thisrecognition was increased by IFN-g pretreatment of thetumor cells (Fig. 3C). Interestingly, although the CD8 T

cells did not produce IFN-g spots when cultured alone, theydid recognize an irrelevant tumor (EL4 thymoma). Theseresults suggest that the CD8 T cells induced by this therapyin this tumor model may recognize shared antigensexpressed by LLC-A9F1 and EL4 tumor cells. The presenceof antitumor CD8 T cells was also evaluated in spleens ofmice treatedwith poly-IC/anti–PD-L1mAbwith or withoutIL-2C� on day 32, after their tumors had been completelyrejected. CD8 T cells from mice treated with poly-IC/anti–PD-L1mAb significantly recognized the LLC-A9F1 cells andthe response was increased by IFN-g pretreatment of thetumor cells (Fig. 3D, left). However, CD8 T cells from themice that received the therapy plus IL-2Cx did not show asignificant response to the LLC-A9F1 tumor (Fig. 3D, right).A comparison of the levels of antigen-reactive CD8 T cellsobserved during tumor rejection (day 12, Fig. 3C) and afterrejection (day 32, Fig. 3D) indicates that during the courseof tumor rejection a marked reduction (approximately 10-fold) in tumor-reactive T cells occurred in this modelsystem, which would explain the lack in the ability of somemice to resist a tumor rechallenge (Fig. 3B). These resultsalso suggest that the administration of IL-2C� before theinjection of the combinatorial therapy may be detrimentalfor the acquisition of long-term immunity in this tumormodel.

Next, the effectiveness of the poly-IC/anti–PD-L1 mAbcombination therapy was examined in the MC38 coloncancermodel. Here, tumor growth inmice that were treatedwith poly-IC/anti–PD-L1 mAb (with or without IL-2C�)was inhibited significantly as comparedwith the nontreatedor poly-IC alone-treated mice (Fig. 4A). In this tumormodel, 60% (3/5) of mice that received the poly-IC/anti–PD-L1mAb treatment and 80% (4/5) of those receiving thecombinatorial therapy plus IL-2C� completely rejectedtheir tumors. Mice that failed to reject their tumors hadrelatively small tumors (<100 mm2) at the end of the

Figure 2. Expression levels ofMHC-I (H-2Db and H-2Kb),MHC-II, and PD-L1 on LLC-A9F1,MC38, and B16F10 cells.LLC-A9F1, MC38, and B16F10cells were incubated or not with100 ng/mL of IFN-g for 24, 40,or 48 hours, and stained withspecific Abs as indicated, followedby flow cytometric analysis.

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experiment. However, in mice treated with poly-IC alone,although tumors significantly grew at a slow rate as com-pared with no treatment, only 1 of 5mice rejected its tumor(generating large error bars in Fig. 4A). Notably, in thismodel none of survivor mice developed tumors after atumor rechallenge on day 39, regardless of their initialtreatments (data not shown).To assess the antitumor CD8 T-cell responses, EliSpot

was performed using spleen CD8 T cells from survivormice on day 55, after the rechallenge tumors wererejected. The CD8 T cells from the poly-IC/anti–PD-L1mAb treated mice exhibited high responses against MC38

cells regardless of whether the tumor cells were treated ornot with IFN-g (Fig. 4B, middle). In contrast, the CD8 T-cell responses from the single mouse that was treated withpoly-IC alone, that was able to reject the initial tumor andthe tumor rechallenge, showed approximately 5-foldlower reactivity (Fig. 4B, left), as compared with micethat received the poly-IC/anti–PD-L1 mAb combinatorialtherapy. Interestingly, the tumor recognition of CD8 Tcells was somewhat decreased by the addition of IL-2C�to the combinatorial therapy (Fig. 4B, right), suggestingthat the use of IL-2C� before administrating poly-IC andanti–PD-L1 mAb may be unfavorable for the generation

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Figure 3. Therapeutic effects induced by the combinatorial therapy of poly-IC/anti–PD-L1 mAb against established lung carcinoma tumors. A, B6 mice (5 pergroup) were inoculated subcutaneously on day 0 with 5� 105 LLC-A9F1 cells and injected intravenously on days 9, 14, and 19 with poly-IC or CpG at 50 mg/dose. Anti–PD-L1 mAb was administered intraperitoneally 1 and 3 days after each poly-IC or CpG administration at 200 mg/dose. IL-2C� wasadministered intraperitoneally 2 and1daysbefore the first poly-IC administration. Points,mean for eachgroup ofmice; bars, SD.P valueswere comparedwithno treatment group and calculated using 2-way ANOVA test (��, P < 0.01; ��, P < 0.001; ��, P < 0.0001). Mice with complete tumor rejections:poly-IC¼4/5; poly-IC/aPD-L1¼4/5; Poly-IC/aPD-L1þIL-2C�¼ 4/5. B, at the termination of the experiment presented in A, on day 39,mice (4 per group) thatwere originally treated with poly-IC alone or poly-IC/anti–PD-L1 mAb with or without IL-2C� and had successfully rejected the tumors wererechallenged subcutaneouslywith 5�105 LLC-A9F1cells on their flanks contralateral to the initial tumor challenge. Each line corresponds to the tumor size ofeach individual mouse. C, CD8 T cells were purified from pooled splenocytes of mice treated with the combination of poly-IC/anti–PD-L1 mAb onday 12, and tumor cell recognition was evaluated using an IFN-g EliSpot assay. Stimulator cells: LLC-A9F1 cells previously treated or not with IFN-g(100 ng/mL, 24 hours) and EL4 cells. Photos in left panel represent examples of wells obtained using 1 � 105 CD8 T cells and 1 � 105 tumor cellsper well. D, on day 32, IFN-g EliSpot assay using purified CD8 T cells from mice treated with the combination of poly-IC/anti–PD-L1 mAb with orwithout IL-2C�were performed using EL4 cells and IFN-g–treated (100 ng/mL, 24 or 48 hours) or nontreated LLC-A9F1 cells as stimulator cells. Results of C(right) and D represent the average number of spots from triplicate wells with SD (error bars) of the mean. P values of D were compared with CD8 alone andcalculated using unpaired Student t test (�, P < 0.05; ��, P < 0.01).






or persistence of the tumor-reactive CD8 T cells. It shouldbe noted that in the colon cancer model, the CD8 T cellsspecifically recognized the MC38 cells because almost noreactivity was observed toward EL4 and LLC-A9F1 cells(Fig. 4B).

The antitumor effect of anti–PD-L1 mAb is most likelybecause of its blocking effect on the PD-1 inhibitory path-way. However, it is possible that the anti–PD-L1mAb couldhave a direct cytolytic effect on the tumor cells, for examplevia ADCC. Because PD-1 blockade can also be achievedusing antibodies specific for the PD-1 receptor (expressedon T cells), we compared the efficacy of anti–PD-1 and anti–PD-L1 mAbs in combination with poly-IC against estab-lished MC38 tumors. As shown in Fig. 4C, both anti–PD-1and anti–PD-L1 mAbs were equally effective in controllingtumor growth when administered in combination withpoly-IC.

Effector mechanism of poly-IC/anti–PD-L1 mAbtherapy against lung and colon tumors

To assess the contribution of various lymphocyte subsetsin the rejection of LLC-A9F1 and MC38 tumors, the anti-tumor efficacy of the poly-IC/anti–PD-L1 mAb combina-torial therapywas evaluated inmice depleted of CD8T cells,CD4 T cells, or NK cells. In both the LLC-A9F1 and MC38tumor models, the therapeutic effects of the combinationtherapy disappeared when CD8 T cells were depleted (Fig.5A and B). Conversely, the elimination of CD4 T cells andNK cells had no significant deleterious effect. It has beenreported that CD8 T cells require help of CD4 T cells tobecome functional long-term memory cells (17, 18). Thus,the depletion of CD4 T cells may impair the acquisition oflong-term immunity in mice treated with the combinationof poly-IC/anti–PD-L1 mAb. To examine whether CD4 T-cell depletion during the treatment for the primary MC38

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Figure 4. Therapeutic effects induced by the combinatorial therapy of poly-IC/anti–PD-L1mAb against established colon carcinoma tumors. A, B6mice (5 pergroup) were inoculated subcutaneously on day 0 with 5 � 105 MC38 cells and injected intravenously on days 8, 13, and 18 with poly-IC at 50 mg/dose.Anti–PD-L1 mAb and IL-2C� were administered as described in Fig. 3. Points, mean for each group of mice; bars, SD. P values were compared with notreatment group and calculated using 2-way ANOVA test (�,P < 0.05; ��,P < 0.0001). Mice with complete tumor rejections: poly-IC¼ 1/5; poly-IC/aPD-L1¼3/5; poly-IC/aPD-L1þIL-2C� ¼ 4/5. B, CD8 T cells were purified from pooled splenocytes of each group on day 55 (after a successfully rejectingas tumor rechallenge), and tumor cell recognition was evaluated using IFN-g EliSpot assays. Stimulator cells were as follows: MC38 and LLC-A9F1 cellspreviously treated or not with IFN-g (100 ng/mL, 24 or 48 hours) and EL4 cells. Results represent the average number of spots from triplicate wellswith SD (error bars) of the mean. Photos represent examples of wells obtained using 1 � 105 CD8 T cells and 1 � 105 tumor cells per well. C, B6 mice(4–5 per group) were inoculated with MC38 cells and injected with poly-IC as described above in A. Anti–PD-L1 or PD-1 mAbs were administeredintraperitoneally 1 and 3 days after each poly-IC administration at 200 mg/dose. Points, mean for each group of mice; bars, SD. Mice with complete tumorrejections: poly-IC/aPD-L1 ¼ 3/4; poly-IC/aPD-1 ¼ 4/5.

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tumor challenge affected the generation of long-termimmunity, the surviving mice from the experiment shownin Fig. 5B were rechallenged on day 39 with fresh MC38cells. Notably, 80% (4/5) of the CD4 T-cell depleted miceand 100% (5/5) of nondepleted animals rejected the sec-ondary tumor challenge (Fig. 5C), suggesting that long-termantitumor immunity in this tumor model could be gener-ated in the absence of CD4 T cells. Nevertheless, when CD8T cells were isolated from the spleens of the 2 groups ofmice(CD4 depleted and untreated) that rejected the MC38rechallenges and were analyzed for their ability to reactwith tumor cells, it was evident that the responses of CD8 Tcells from the CD4-depleted mice were approximately 40%lower as comparedwith the responses of the untreatedmice(Fig. 5D).It is known that CD8 T cells can exert their antitumor

function through the secretion of cytostatic lymphokinessuch as IFN-g (19, 20). Furthermore, stimulation of TLR3

and RIG-I–like receptors by poly-IC induces the activationof antigen-presenting cells (APCs) and the generation ofhigh amounts of type-I IFN (21, 22), which is implicated inthe potentiation of CD8 T-cell responses (23, 24). Thus, theefficacy of poly-IC/anti–PD-L1 mAb therapy against LLC-A9F1 was evaluated in mice deficient for IFN-g (IFN-g�/�)or type-I IFN receptors (IFN-abR�/�). Surprisingly, IFN-g�/� mice treated with the combination of poly-IC/anti–PD-L1 mAb with IL-2C� completely rejected their tumors(Fig. 6A). On day 35, the IFN-g�/� mice were rechallengedwith live LLC-A9F1 cells and although the tumors startedgrowing, they were all rejected (data not shown). Theseresults indicate that IFN-g is not required for tumor erad-ication and long-term protection in the LLC-A9F1 tumormodel. A different outcome was observed in IFN-abR�/�

mice, where only 33% (2/6) animals treated with poly-IC/anti–PD-L1 mAb with IL-2C� rejected their tumors (Fig.6B). These results indicate that type-I IFN plays an

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MC38 cells on their flankscontralateral to the initialchallenge. Points, mean for eachgroup of mice; bars, SD. Rightportion of C, tumor growth curvesare shown for individual mice fromthe CD4-depleted group. D, at thetermination of the experimentpresented in C, on day 17 aftertumor rechallenge (day 56 afterinitial tumor challenge), CD8 T cellswere purified from pooledsplenocytes of mice thatcompletely rejected theirrechallenged tumors, and tumorcell recognition was evaluatedusing IFN-g EliSpot assays.Stimulator cells were as follows:MC38 cells previously treated ornot with IFN-g (100 ng/mL, 24hours) and EL4 cells. Resultsrepresent the average number ofspots from triplicate wells with SD(error bars) of the mean. Photosrepresent examples of wellsobtained using 1� 105 CD8 T cellsand 1 � 105 tumor cells per well.






important role in generating immunity necessary to achieveeffective therapeutic responses against established LLC-A9F1 tumors.

DiscussionNumerous groups including ours are involved in devel-

oping T-cell epitope-based vaccination strategies for malig-nant diseases such asmelanoma, cervical cancer, and breastcarcinoma. Thesemalignancies were selected because of theexistence of defined TAAs that can be used to stimulateantigen-specific, tumor-reactive CD8 T-cell responses(8, 13, 25). However, formany other tumor types includinglung and colon carcinomas, which are the leading world-wide causes of cancer death (26), few if any reliable TAAs fortriggering tumor-specific CD8 T-cell responses have beenidentified, limiting the development of epitope-based vac-cines. Thus, we explored an alternative and novel immu-notherapeutic approach to induce and efficiently amplifytumor-reactive CD8 T cells without depending on the use ofdefined TAAs. To achieve this goal, we took advantage of arecent unexpected observation where immunization with acontrol-irrelevant peptide in combinationwith poly-IC andPD-1 blockade substantially decreased the rate of tumorgrowth in the B16 mouse melanoma model (11). Wehypothesize that the CD8 T-cell responses that in manyinstances are naturally generated against TAAs throughoutthe course of the disease are in general ineffective and thatthe administration of poly-IC and anti–PD-L1 mAb some-

how improves these responses, or alternatively generatesnew antitumor T-cell responses that result in therapeuticeffectiveness. On one hand, poly-IC, a TLR3 and RIG-I–likereceptor agonist is known to stimulate various immune cellsincluding professional APCs such as dendritic cells, enhanc-ing tumor antigen cross-presentation to CD8 T cells. Onecould easily envision that tumor-infiltrating dendritic cellsthat capture TAAs (either in the form of shed antigens ordead tumor cells) after exposure to poly-IC would becomepotent APCs capable of priming a new CD8 T-cell response(or alternatively of expanding and reactivating an existingsuboptimal response) capable of delaying tumor growthand even in some instances eradicating disease. In the caseof the LLC-A9F1 tumor, poly-IC by itself was effective ineliciting outstanding antitumor effects that were not furtherenhanced by PD-1 blockade (Fig. 3A). On the other hand,co-administration of poly-IC and anti–PD-L1 mAb wasrequired to obtain similar remarkable antitumor effects inthe B16 melanoma and MC38 colon carcinoma models(Figs. 1 and 4A). The additive effect of anti–PD-L1 mAb inthe combination therapy could be because of variousreasons depending on the specific tumor model and stageof disease. For example, it is possible that TAA-reactiveCD8 T cells naturally generated before therapy express theinhibitory PD-1 receptor, which is a marker of exhaustedT cells (27) and that PD-1 blockade during their inter-actions with dendritic cells (which express PD-L1 and PD-L2) rescues the T cells to expand and become more potenteffector cells (28, 29). Because the tumor cells themselves

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Figure 6. Role of IFN-g and type-IIFN in the therapeutic antitumoreffects of poly-IC/anti–PD-L1mAb. IFN-g�/� mice (A) andIFNabR�/� mice (B) wereinoculated subcutaneously on day0 with 5 � 105 LLC-A9F1 cells andtreated with the combination ofpoly-IC/anti–PD-L1 mAb with IL-2C� in the same manner asdescribed in Fig. 3. Nontreatedmice were included as controls.Tumor sizes were determined inindividual mice by measuring 2opposing diameters and arepresented as tumor areas in squaremillimeters. For A, points, mean foreach group of mice; bars, SD. ForB, lines, tumor size in area of eachindividual mouse.

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express PD-L1 (30), which is enhanced by IFN-g (Fig. 2),it is also likely that PD-1 blockade enhances the effectorphase of the CD8 T-cell response increasing tumor killingand perhaps promoting T-cell survival and proliferationat the tumor site (31). Previous studies in mouse modelsof immunotherapy have shown remarkable therapeuticeffects of PD-1 blockade (9–11, 32, 33). Poly-IC is wellknown to induce high levels of type-I IFN, which has beenshown to induce the expression of PD-1 on T cellslimiting their function (34, 35). In addition, poly-IC hasbeen reported to stimulate the production of IFN-g by NKcells (36), which will contribute to enhance the expres-sion of PD-L1 on the tumor cells. Thus, it should be of nosurprise that in most instances PD-1 blockade wouldsynergize with the antitumor effects of poly-IC.Our results indicate that the antitumor effects of the poly-

IC/anti–PD-L1 mAb combination therapy were mediatedprincipally by CD8 T cells and that CD4 T cells andNK cellsplayed a minimal role, if any (Figs. 1 and 5A and B). Inaddition, the EliSpot assays clearly showed that tumor-reactive CD8 T cells were induced by this combinatorialtherapy (Figs. 3C and 4B). Although in the LLC-A9F1 lungcancermodel, themajority of theCD8T cells inducedby thecombination therapy seemed to recognize a shared antigenpresent in another completely different tumor (EL4thymoma; Fig. 3C), the CD8 T cells generated by thecombination therapy in theMC38 colon carcinoma seemedto recognize antigen(s) not present inother tumor cells (EL4and LLC-A9F1). At present, we do not know the nature ofTAAs recognized by the tumor-reactive CD8 T cells inducedby poly-IC/anti–PD-L1mAb therapy. InMC38 tumormod-el, we examined whether spleen CD8 T cells from micetreated with the combination therapy could recognize EL4cells pulsed with the p15E604–611 (KSPWFTTL) peptide, animmunodominant H-2Kb restricted CD8 T-cell epitopederived from an endogenous murine leukemia virusexpressed by numerous tumors including MC38 (37, 38).Nevertheless, in EliSpot assays EL4 cells pulsed withp15E604–611 were barely recognized by tumor-reactive CD8T cells (comprising only approximately 3% of the responseobserved with MC38 tumor cells, data not shown). Futureand complex studies will be required to identify the TAAsrecognized by the CD8 T cells.The overall effectiveness of tumor immunotherapy will

not only depend in achieving an initial antitumorresponse, hopefully capable of reducing tumor massesto an undetectable level, but one would also hope that theimmune response would persist for long-time periods toprevent tumor recurrences and metastatic spread. Usingtumor rechallenges in mice that rejected their initialtumors as a way to evaluate long-term immunity allowedus to evaluate the establishment of CD8 T-cell memory bythe poly-IC/anti–PD-L1 mAb combination therapy. How-ever, the 2 tumor models where complete rejections wereachieved by this therapy gave somewhat divergent results.In the MC38 colon carcinoma model, all the mice thatrejected their original tumors resisted the tumor rechal-lenges, indicating the establishment of effective CD8

memory T cells. However, in the LLC-A9F1 lung cancermodel, only one half of the mice that rejected theirprimary tumors resisted a tumor rechallenge. However,100% of IFN-g�/� mice that rejected their LLC-A9F1primary tumors were able to resist a subsequent tumorrechallenge (data not shown), indicating that IFN-gdecreases the generation of long-lived (memory) CD8 Tcells as previously noted in a microbial infection model(39). Numerous additional factors could determine thegeneration of long-term CD8 T-cell memory in this modeof immunotherapy, such as the nature of the TAAs rec-ognized by the T cells and the immune suppressive effectof the tumor microenvironment that may facilitate theestablishment of exhausted CD8 T cells incapable ofreacting to a subsequent tumor encounter. It is wellknown that helper CD4 T cells play a role in the estab-lishment of memory CD8 T cells (17, 18). Our results inthe MC38 tumor model showed that the majority (80%)of the mice that were depleted of CD4 T cells whilereceiving poly-IC/anti–PD-L1 mAb therapy and rejectedthe original tumor were able to resist a tumor rechallenge(Fig. 5C). However, the level of CD8 T-cell responses wasreduced by approximately 40% as compared with thenondepleted mice (Fig. 5D). However, in the LLC-A9F1tumor model, removal of CD4 T cells reduced the level ofprotection against tumor rechallenge from 50% (Fig. 3B)to 0% (data not shown). Thus, our results suggest thatindeed, CD4 T cells may play a role in promoting long-term survival of tumor reactive CD8 T cells, which insome instances such as with the LLC-A9F1 tumor, deter-mines the ability to resist a tumor rechallenge. The mech-anism(s) by which CD4 T cells may facilitate the gener-ation of long-lived CD8 T cells could be numerous,including the production of IL-2 and enhancing thefunction of dendritic cells via CD40 ligand/CD40 inter-actions. Nevertheless, our results indicate that adminis-tration of IL-2 (as IL-2C�) decreased the ability of mice toresist a tumor rechallenge (Fig. 3B) and decreased thelevels of tumor-reactive CD8 T cells (Figs. 3D and 4B). Itshould be noted that the type of IL-2C� we used has beenreported to enhance proliferation and survival of memoryCD8 T cells and NK cells but does not result in stimula-tion of CD4 T regulatory cells (14, 15). An importantissue that remains to be determined is whether the poly-IC/anti–PD-L1 mAb therapy generates CD4 T cells reac-tive with TAAs, which could be somehow involved inCD8 long-term immunity, or whether the role of the CD4T cells in this process is independent of their antigenspecificity.

IFN-g has been considered to be a essential cytokine forthe antitumor effects of CD8 T cells (19). Specifically, IFN-gincreases the expression of MHC-I molecules on tumorcells, which in many instances enhances their recognitionby CD8 T cells. In addition, IFN-g has direct antitumoractivity, limiting cell proliferation (40, 41). In fact, as shownhere, B16F10, LLC-A9F1, andMC38 cells treatedwith IFN-gincreased their levels of MHC-I molecules (Fig. 2) andsignificantly increased recognition by CD8 T cells from






mice treated with the combinatorial therapy in the case ofLLC-A9F1 (Fig. 3C). Although IFN-g clearly has a positiveantitumor effect, this cytokine can also exhibit immuno-suppressive activities (20). Specifically, many tumorsincluding the ones used in this study when exposed toIFN-g increase their expression of PD-L1 (Fig. 2), whichinhibits the function of T cells (42). Furthermore,although IFN-g increases MHC-I expression, in someinstances it may decrease CD8 T-cell recognition byeither, decreasing the generation of some peptide epi-topes, through the induction of immunoproteasomes(43), or through the production of excessive noncognatepeptide/MHC-I complexes that limit antigen-specific T-cell recognition (9). In addition, IFN-g may exhibit directinhibitory/toxic effects on T cells, limiting clonal expan-sion (39). Irrespective of all these issues, our results withLLC-A9F1 indicate that IFN-g did not play an essentialrole in limiting the tumor growth produced by the poly-IC/anti–PD-L1 mAb combination therapy (Fig. 6A). Infact, whereas 80% of wild-type B6 mice receiving thistherapy rejected their tumors (Fig. 3A), 100% of the IFN-g�/� mice eliminated the tumors (Fig. 6A). Similar to thefindings presented here, we have recently described thatpeptide vaccination with poly-IC (with and without anti-CD40 mAb) generated remarkable antitumor effects inIFN-g�/� mice against B16 melanoma and a humanpapilloma virus mouse tumor model (8, 9, 25). In theseinstances, the antitumor effect of the CD8 T cells wasmediated by perforin-mediated cytolysis, but not IFN-g .

Poly-IC is recognized by TLR3 and cytoplasmic RIG-I–like receptors, such as the MDA5, resulting in the activa-tion of APCs and the generation of high levels of type-IIFN as well as other proinflammatory cytokines such asTNF-a, IL-6, and IL-12 (21, 22). Because type-I IFN hasimportant roles for activating and expanding CD8 T cells(23, 24), we predicted that the poly-IC/anti–PD-L1 mAbtherapy would be ineffective in IFN-abR�/� mice. Indeed,the antitumor effects of the poly-IC/anti–PD-L1 mAbcombination therapy in the LLC-A9F1 model werereduced in IFN-abR�/� mice (Fig. 6B) as compared withthe wild-type B6 mice (Fig. 3A). Nevertheless, this therapystill elicited significant antitumor effects in 4 of 6 mice,where 2 animals rejected their tumors and 2 had asubstantial decrease in tumor growth rate as comparedwith the untreated controls. These results suggest thattype-I IFN signals are important but not absolutelyrequired for inducing the antitumor effects. The antitu-mor effects of the combinatorial therapy in the absence oftype-I IFN signals may be because of the participation ofother T-cell stimulatory cytokines such as IL-12 generated

by poly-IC–stimulated APCs or could be the result oftype-I IFN signals directly on the tumor cells.

Finally, it should be mentioned that the combinatorialpoly-IC/anti–PD-L1mAbcancer immunotherapydescribedhere could be expediently taken into the clinic. Currently,there is a formulation of poly-IC being developed as atherapeutic. Hiltonol that was used in this study is a highmolecular weight poly-IC formulation stabilized withpoly-lysine and carboxymethylcellulose (poly-ICLC) thathas already used in humans as a monotherapy or as animmune adjuvant for cancer vaccines (44–49). Further-more, several humanized mAbs for the purpose of imple-menting PD-1 blockade (anti–PD-L1 or anti–PD-1) arebeing developed and are currently undergoing clinicaltesting (50, 51). Our results in the MC38 tumor modelsuggest that effective antitumor effects using immuno-therapy with poly-IC and PD-1 blockade can be achievedwith either mAb specific for the PD-1 receptor or its ligandPD-L1 (Fig. 4C). Because both poly-IC and various Abs toinduce PD-1 blockade are being developed for clinicaluse, we believe that our preclinical studies could readilybe translated into the treatment for patients with cancer,especially in those instances where no reliable TAAs havebeen identified.

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

Authors' ContributionsConception and design: T. Nagato, Y.-R. Lee, Y. Harabuchi, E. CelisDevelopment of methodology: T. Nagato, Y.-R. Lee, Y. Harabuchi, E. CelisAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): T. Nagato, Y.-R. LeeAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): T. Nagato, Y.-R. Lee, Y. Harabuchi, E.CelisWriting, review, and/or revision of the manuscript: T. Nagato, Y.-R. Lee,E. CelisStudy supervision: E. Celis

AcknowledgmentsThe authors thank Dr. A. Salazar (Oncovir, Inc.) for kindly providing

Poly-ICLC (Hiltonol).

Grant SupportThis work was supported by NIH grants R01CA136828 and

R01CA157303.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedOctober 21, 2013; revisedDecember 2, 2013; acceptedDecember4, 2013; published OnlineFirst January 3, 2014.

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Nagato et al.





2014;20:1223-1234. Published OnlineFirst January 3, 2014.Clin Cancer Res Toshihiro Nagato, Young-Ran Lee, Yasuaki Harabuchi, et al. T-cell Responses against Established Tumorsand Blockade of Programmed Death-Ligand 1 Induce Effective CD8

Polycytidylic Acid−Combinatorial Immunotherapy of Polyinosinic

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