ctla-4 blockade expands inﬁltrating t cells and inhibits ... · 100 mci/ml of 51cr solution for 1...

Preclinical Development

CTLA-4 Blockade Expands Infiltrating T Cells and InhibitsCancer Cell Repopulation during the Intervals ofChemotherapy in Murine Mesothelioma

Licun Wu, Zhihong Yun, Tetsuzo Tagawa, Katrina Rey-McIntyre, and Marc de Perrot

AbstractCancer immunotherapy has shown promising results when combined with chemotherapy. Blocking

CTLA-4 signaling by monoclonal antibody between cycles of chemotherapy may inhibit cancer cell

repopulation and enhance the antitumoral immune reaction, thus improve the efficacy of chemotherapy

in mesothelioma. The impact of CTLA-4 blockade on the early stage of tumor development was

evaluated in a subcutaneous murine mesothelioma model. CTLA-4 blocking antibody was administered

following each cycle of chemotherapy, and monotherapy was included as controls. Antitumor effect

was evaluated by tumor growth delay and survival of the animals. Tumor cell repopulation was

quantified by bromodeoxyuridine incorporation and Ki67 by immunohistochemistry and/or flow

cytometry. In vitro cell killing was determined by classic chromium-released assay, and reverse

transcription PCR (RT-PCR) was carried out to determine the gene expression of associated cytokines.

Anti-CTLA-4 monoclonal antibody was able to inhibit tumor growth at early stage of tumor devel-

opment. Antitumor effect was achieved by administration of CTLA-4 blockade between cycles of

chemotherapy. Tumor cell repopulation during the intervals of cisplatin was inhibited by CTLA-4

blockade. Anti-CTLA-4 therapy gave rise to an increased number of CD4 and CD8 T cells infiltrating the

tumor. RT-PCR showed that the gene expression of interleukin IL-2, IFN-g, granzyme B, and perforin

increased in the tumor milieu. Blockade of CTLA-4 signaling showed effective anticancer effect,

correlating with inhibiting cancer cell repopulation between cycles of chemotherapy and upregulating

tumor-infiltrating T lymphocytes, cytokines, and cytolytic enzymes in a murine mesothelioma model.

Mol Cancer Ther; 11(8); 1809–19. �2012 AACR.

IntroductionImmunosuppressive components such as regulatory T

cells (Treg), cyototoxic T lymphocyte-associated antigen-4(CTLA-4), myeloid-derived suppressor cell (MDSC), andto some extent, tumor-infiltrating macrophages, play crit-ical roles in tumor tolerance through a variety of mechan-isms (1–5). Therefore, inhibitory checkpoints of immuneregulation provide potential targets for cancer immuno-therapy. CTLA-4, also known as CD152, is a member ofthe CD28/B7 immunoglobulin superfamily of immuneregulatory molecules. It shares its 2 ligands (B7-1 and B7-2) with its costimulatory counterpart CD28 (4, 6). CTLA-4

and CD28 and their ligands B7-1 (CD80) and B7-2 (CD86)are critically important for the initial activation of naive Tcells and regulation of the clonal composition of theresponding repertoire following migration of activateddendritic cells to lymphoid organs (7–9). Taken together,these 4 molecules are perhaps the most important cofac-tors functioning in an immunologic cascade, providingsignals that are crucial in T-cell activation and tolerance.Change in T-cell activation by blocking negative signalingreceptors such as CTLA-4 is one approach to overcomingtumor-induced immune tolerance. Therefore, the discov-ery of CTLA-4 as a key negative regulator in immuneactivity enabled to develop novel therapy to target thissignaling molecule (10, 11). Anti-human CTLA-4 antibo-dies ipilimumab and tremelimumab prolong antitumorimmune responses and lead to durable antitumor effects(12–14).

Current understanding of CTLA-4 function in T-cellresponses in vitro and in preclinical murine models madeit possible to initiate application of CTLA-4 blockade as anovel immunotherapy for cancer (15, 16). Other preclin-ical studies also show that CTLA-4 blockade can actsynergistically with other treatment modalities such asirradiation, cryotherapy, and chemotherapy (15–17). The

Authors' Affiliation: Latner Thoracic Surgery Research Laboratories andDivision of Thoracic Surgery, Toronto General Hospital, University HealthNetwork, University of Toronto, Toronto, Ontario, Canada

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author:Marc de Perrot, Toronto Mesothelioma ResearchProgram, Division of Thoracic Surgery, Toronto General Hospital, 9N-961,200 Elizabeth St, Toronto, ON M5G 2C4, Canada. Phone: 416-340-5549;Fax: 416-340-3478; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-1014

�2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

www.aacrjournals.org 1809

on September 11, 2020. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst May 14, 2012; DOI: 10.1158/1535-7163.MCT-11-1014

http://mct.aacrjournals.org/

initial clinical experience with antibody to CTLA-4 inhuman trials has been reported along with a perspectiveon adverse events observed with CTLA-4 blockade insome cancers (18–20).

One neglected area in cancer research that has recentlybeen highlighted is the importance of repopulation ofsurviving cancer cells between courses of chemotherapyand radiotherapy (21, 22). The rate of surviving cellrepopulationmay increase during fractionated radiother-apy and between cycles of chemotherapy, limiting theability to control tumors (23). Repopulation is likely to bemore important with chemotherapy than with radiationtherapy because of the longer intervals between cycles oftreatment and is a potential cause of clinical failure tochemotherapy.Accelerating repopulation after sequentialcourses of chemotherapy could lead to regrowth oftumors after initial shrinkage without any change in theintrinsic sensitivity of the cells to the drugs used (23).Thus, accelerating repopulation may be an importantcause of clinical drug resistance. Therefore, inhibition ofthis process may improve the efficacy of chemotherapy(24, 25).

Current evidence has shown that CTLA-4 blockade hassynergistic effect with chemotherapy in some animalstudies and clinical trials (11–14, 21). However, the opti-mal timing of such therapies has not been confirmed,especially in mesothelioma. To our best knowledge, theimpact of CTLA-4 blockade-based immunotherapy oncancer cell repopulation between cycles of chemotherapyhas not been reported. In this study, we administeredCTLA-4 blocking monoclonal antibody between cycles ofcisplatin treatments in murine mesothelioma models toevaluate the benefit of antitumor effect.

Materials and MethodsMurine mesothelioma cells and animal model

Murine malignant mesothelioma cell lines AB12 andAC29, derived from an asbestos-induced tumor in aBALB/c and CBA/J mouse, respectively, were kindlyprovided by Dr. Jay Kolls, University of Pittsburgh, Pitts-burgh, PA, in 2008 (2, 26). Both cell lines were revivedfrom the original stocks and cultured in RPMI-1640medi-um supplemented with 10% FBS and 1% penicillin andstreptomycin. No further authentication was done. Thecultures were maintained at 37�C in an atmosphere con-taining 5% CO2. AB12 and AC29 cells (2 � 106) wereinjected subcutaneously into the right flank of femaleBALB/c and CBA/J mice, which were provided by TheJackson Laboratory. All procedures followed the animalcare regulations of University Health Network afterapproval by the Research Ethic Board.

Treatment at the early stage of tumor developmentwith anti-CTLA-4 monoclonal antibody

Mice were treated with anti-mouse CTLA-4 monclonalantibody (clone: 9H10; eBioscience) 100 mg per dose by i.p.injection on days 1 and 3 after tumor cell injection (i.e.,

before the development of a palpable tumor). The impacton tumor growth was observed twice weekly. T-cellpopulation and function in tumor and spleen were quan-titatively evaluated by flow cytometry.

In vitro generation of tumor-specific CTLs bychromium-release assay

Splenocytes derived from naive or AB12-bearing miceat 7days after tumor challenge were pooled into 24-wellplates at a concentration of 2.5� 106 to 5� 106/mL.After 3days of culture, half of the medium was replaced withfresh medium containing interleukin (IL)-2 (final concen-tration of 10 U/mL). On day 5, cells were harvested andtested in a standard 4-hour 51Cr release assay (26). Briefly,target AB12 cells (106/100 mL of PBS) were labeled with100 mCi/mL of 51Cr solution for 1 hour and incubatedwith effector cells for 4 hours at different effector totarget (E:T) ratios in triplicate, and 51Cr release wasdetermined by analyzing the supernatants in a Micro-plate Scintillation Counter (Perkin Elmer). The per-centage of specific lysis was calculated according to theformula: 100 � [(experimental release � spontaneousrelease)/(maximal release� spontaneous release)]. Spon-taneous release and maximum release were obtainedfrom wells containing target cells incubated in mediumalone or in 2% acetic acid, respectively.

Combination treatment of tumor-bearing mice withCTLA-4 blocking antibody between cycles ofchemotherapy

Mice were randomly divided into 4 groups as followswhen tumor size reached 5 mm in diameter: (i) no treat-ment (NoRx); (ii) anti-CTLA-4monoclonal antibody alone(anti-CTLA-4), 100 mg per mouse was injected i.p. onceweekly for 3 doses, that is, on day 5, 12, and 19 after tumorcell injection; (iii) cisplatin alone (Cis), 5 mg/kg bodyweight was injected i.v. through the tail vein once weeklyfor 3 doses, that is, on day 4, 11, and 18 after tumor cellinjection; (iv) combination therapy, anti-CTLA-4 mono-clonal antibody (mAb) was given one day after each doseof cisplatin (Cis þ anti-CTLA-4), thus following the sameschedule as in groups 2 and 3.

Tumorsizewasmeasured twiceweeklybya caliper, andtumor volume was estimated by a formula: V ¼ ab2p/6,whereas a and b represent the longest and shortest max-imal perpendicular diameters, respectively. Mice wereeuthanized when they met a predetermined tumorvolume exceeded 350mm3 tominimize pain and sufferingand were scored as death. Survival time was evaluatedfrom the day of tumor challenge to euthanization.

CD8 T-cell depletionIn a separate experiment, the tumor-bearing mice were

treated with anti-CD8a mAb (clone: 53–6.7; eBioscience)100 mg per injection to deplete CD8 T cells before admin-istration of anti-CTLA-4 mAb to determine whether CD8T-cell depletion could reverse the effect induced byCTLA-4 blockade.

Wu et al.

Mol Cancer Ther; 11(8) August 2012 Molecular Cancer Therapeutics1810




Tumor cell repopulation was evaluated byimmunohistochemical staining and flow cytometryfor bromodeoxyuridine incorporationOn day 7 after the second dose of cisplatin, animals

were sacrificed about 3 hours after bromodeoxyuridine(BrdU; Roche) 100 mg/kg body weight was injected i.p.Tumor tissueswere removed at different time points aftertreatment and snap frozen immediately in liquidnitrogen,and then transferred to dry ice and kept at �80�C untilfrozen sectioning was conducted. Frozen sections werefixed in cold ethanol. Endogenous peroxidases, avidinand biotinwere blocked using 1%hydrogen peroxide andthe Avidin/Biotin Blocking Kit (Dako). Sections werestained with a primary monoclonal antibody (1:50)against Ki67 or BrdU (eBioscience), and secondary anti-body linked to streptavidin–horseradish peroxidase(Dako). After washing, one Sigma FAST (D-4168) DAB(3,30-diaminobenzidine) tablet and one urea hydrogentablet (Sigma) were added to ddH2O to serve as a perox-idase substrate (125 mL/section) and slides were counter-stained with hematoxylin to visualize nuclei. Sectionswere then dehydrated and mounted with DPX (Ultra-mount, Scot Scientific).Immunostained sections were quantified by using

Aperio ImageScope digital scanner and Aperio Image-Scope Viewer software version 9.0 (Vista, CA) under 200magnification. Tumor cell repopulation was quantified asthe proportion of Ki67 or BrdUrd-positive nuclear areasdivided by total nuclear areas (26).Single tumor cells were prepared by passing through

the cell strainer (�40 mm; BD Biosciences) and stainedsimilarly as for intracellular cytokine IFN-g staining.DNase I (Sigma) was applied after permeabilization.BrdUrd-FITC antibody 1:30 was added and cells wereexposed at room temperature in the dark for 30 minutes,then washed thrice for flow cytometry.

Immunohistochemistry and fluorescentimmunostaining of tumor-infiltrating T cellsTumor-infiltrating T cells were identified by primary

rabbit anti-mouse CD3 (clone: SP7), rat anti-mouse CD4(GK1.5), and CD8 (YTS169.4) monoclonal antibodies with1:100 dilution (Abcam). Antigen–antibody reactions werevisualized using DAB as the chromogen. For fluorescentimmunostaining, sections were incubated with goat anti-rat secondary antibodies, Alexa 488- or Alexa 568–labeledgoat anti-rat or rabbit IgG (1:400 dilution). Purified rat orrabbit IgGwas used as controls (Invitrogen). The detailedprocedures were followed the instructions provided bythe manufacturers.

Cell preparation and staining for flow cytometryT-cell subsets were determined by using flow cytome-

try. On day 7 after the second dose of cisplatin, spleensand draining lymph nodes were removed from tumor-bearingmice andplaced into ice-cold RPMI-1640mediumcontaining 1% FBS. The axillary lymph nodes from thetumor side were called draining lymph nodes. Peripheral

blood was drawn from the heart of mice that were imme-diately euthanized by inhalation of CO2. Homogenizedspleen and lymph node were passed through the cellstrainer to achieve single cells. ACK lysis buffer (Invitro-gen) was added and allowed to react for at least 15minutes at room temperature to lyse red blood cells. Afterwashing thricewith staining buffer, appropriate dilutions(1:50–1:100) of Abs or isotype controls were added to eachtube, 15minutes at room temperature in thedark. Stainingof surface markers including CD3, CD4, CD8, and ICOSwerewashed thricewith staining buffer and resuspendedin 1% paraformadehyde/PBS (v/v; Sigma). After fixationwith 1% paraformadehyde at 4�C overnight, cells werepermeabilized for intracellular staining of IFN-g , perforin,and granzyme B and fixedwith 200 mL Permeabilization/Fixation Solution (eBioscience) and then washed twicewith permeabilization buffer. Anti-mouse antibodiesagainst IFN-g (1:30), perforin (1:50), and granzyme B(1:50) were added and maintained at room temperaturefor 20 minutes in the dark.

Single-cell suspensions were stained with monoclo-nal antibodies conjugated with different fluorescent dyes,CD3 (clone: 17A2)-PerCP-Cy5.5,CD4 (clone:RM4-5)-FITC,CD8b (clone: H35-17.2)-APC, ICOS (clone: 7E.17G9)-PE,IFN-g (clone:XMG1.2)-PE,perforin (clone: eBioOMAK-D)-FITC, and granzyme B (clone:16G6)-PE. All antibodiesand isotypes were purchased from eBioscience or BioLe-gend. All cells from the same group were pooled togetherand considered to be as one sample for running flowcytometry. Becton Dickinson LSR II Flow Cytometer andFACSDiva software were used for data acquisition, andFlowJo software was used for analysis.

RNA extraction and real-time reverse transcriptionPCR

On day 7 after cisplatin treatment, spleens and tumortissues were collected and total RNA was extracted fromtumor tissues using TRizol Reagent (Invitrogen), andRNeasy MinElute Cleanup Kit (QIAGEN) enabled clean-up of RNA. cDNA was synthesized with High-CapacitycDNA Reverse Transcription Kits (ABI) on a PTC-100Programmable Thermal controller (MJ research Inc.) fol-lowing the manufacturer’s protocols. Regular PCR wascarried out to establish reverse transcription PCR (RT-PCR) standards of all target genes including CD3, CD4,CD8, ICOS, IL-2, IFN-g , granzyme B, and perforin andhousekeeping gene glyceraldehyde-3-phosphate dehy-drogenase (GAPDH).DNAfragmentswere obtained fromregular PCR on a PTC-100 Programmable Thermal. Reg-ular PCRwas carriedout by 10�HighFidelityPCRBuffer,Platinum Taq polymerase High Fidelity, 50 mmol/LMgSO4, 10 mmol/L dNTP Mix (Invitrogen). A SYBRGREEN real-time PCR was carried out on ABI PRISM7900HT system. PCR was composed of Power SYBRGREEN PCR 2� Master Mix (ABI), 200 nmol/L primer,and 2 mL 500 ng/mL cDNA�40 cycles. Primers of all targetgenes andhousekeepinggeneweredesignedbyusingABIPrism Primer Express software version 2.0.

Treatment of Mesothelioma with Chemoimmunotherapy

www.aacrjournals.org Mol Cancer Ther; 11(8) August 2012 1811




Statistical analysisAll data are presented as the mean � SEM. Statistical

analyses were conducted using GraphPad Prism 5 statis-tical software. For all statistical analyses, a 2-tailedP valueof less than 0.05 was considered to be statistically signif-icant. Single-group data were assessed using unpairedStudent t test. Tumor size, BrdUrd incorporation, andKi67 proportion of positive nuclear areas, the expressionof cytokine gene expression, T-cell subsets, among groupswere analyzed by using one-way repeated measuresANOVA Newman–Keuls test for multiple comparisons.A value of P < 0.05 was considered significantly different

for all comparisons. �, P < 0.05; ��, P < 0.01; ��, P < 0.001.Kaplan–Meier nonparametric regression analysis wasconducted to assess the survival time of tumor-bearinganimalswith significance determined by the log-rank test.

ResultsMurine mesothelioma AB12 is immunogenic

Specific cell lysis of cytotoxic T cells was observed intumor-bearing mice compared with naive mice in thepresence or absence of stimulation with tumor cell lysate(Fig. 1A). The production of representative cytotoxic

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Figure 1. Murine mesothelioma is immunogenic. The tumor responds to anti-CTLA-4 therapy at the early stage of tumor development in murinemesothelioma models and T-cell activation. A, on day 7 posttumor challenge, splenocytes were prepared from either tumor-bearing or naivemice and used for CTL assay as stated in Materials and Methods. Splenocytes derived from AB12-bearing mice with or without stimulation of tumor celllysate had more specific cell killing than those from naive mice. B, production of cytolytic cytokine IFN-g was greater in the CTLs from AB12-bearing mice than naive mice. C, on day 1 and 3 after tumor cell injection, mice were treated with anti-CTLA-4 mAb, as indicated by the arrows.Anti-mouse CTLA-4 mAb resulted in tumor growth delay in both AB12 and AC29 tumor models when compared with controls. D, on day 7 posttreatmentwith anti-CTLA-4 mAb, the absolute number of tumor-infiltrating CD4 and CD8 T cells increased.

Wu et al.





cytokine IFN-g was confirmed to increase in CD8 T cells,whereas theCD4T cells produced a fairly small amount ofIFN-g even though they were stimulated by tumor celllysate (Fig. 1B).

The impact of CTLA-4 blockade on tumor growth atthe early stage of tumor development in murinemesothelioma models and on T-cell activationAdministration of anti-CTLA-4 monoclonal antibody

on day 1 and 3 after tumor cell injection resulted indramatic growth delay in both AB12 and AC29 tumormodels (Fig. 1C). Some tumors completely disappearedafter treatment, and no tumor growth was observed inthese tumor-free mice when they were rechallenged withthe same tumor cell line. The absolute number of tumor-infiltrating CD4 and CD8 T cells after treatment increasedin both models, AC29 is not shown (Fig. 1D).

In tumor-bearing mice, CTLA-4 blockade duringchemotherapy delays tumor growth and improvedsurvival, but antitumor effect induced by CTLA-4blockade is reversed by CD8 T-cell depletionAdministration of anti-CTLA-4 mAbs during the inter-

vals of cisplatin treatments (Cis þ anti-CTLA-4) wassignificantly more effective than cisplatin alone (Cis),anti-CTLA-4 mAb alone (anti-CTLA-4), and untreatedcontrols (NoRx). Tumor growth curves indicated thatCisþ anti-CTLA-4 resulted in the best tumor growthdelayin AB12 tumor model, even though cisplatin alone (Cis)resulted in significant growth delay. In contrast to theeffect that was observed when CTLA-4 mAbwas injectedondays 1 and 3 after tumor cell injection, anti-CTLA-4 hadonly minor effect to delay the growth of AB12 tumorswhen it is given at 5 days after tumor challenge (Fig. 2A).The blockade of CTLA-4 signaling not only enhanced

antitumor activity, but also significantly prolonged sur-vival of tumor-bearing mice, as shown in Fig. 2B. Mediansurvival for the Cis þ anti-CTLA-4 is 38 days, comparedwith Cis 30 days [P ¼ 0.0139; HR, 14.62; 95% confidenceinterval (CI), 1.723–124.0], anti-CTLA-4 21 days (Cis þanti-CTLA-4 vs. anti-CTLA-4: P¼ 0.0477; HR, 0.5526; 95%CI, 0.3288–0.7764), and NoRx 18 days (P ¼ 0.0050; HR,0.4737; 95% CI, 0.2236–0.7238), respectively. Mice treatedwith Cis alone had longer survival thanNoRx, P¼ 0.0114;HR, 0.6000; 95% CI, 0.3499–0.8501. The body weight ofmice did not change significantly after completingtreatment.The antitumor effect induced by CTLA-4 blockade

could be reversed byCD8 T-cell depletion. Tumor growthwas similar with the untreated mice (Fig. 2C).

Inhibition of tumor cell repopulation byadministration of CTLA-4 blocking mAb during thecourses of chemotherapy in tumor-bearing miceRepresentative images of tumor sections stained with

hematoxylin and eosin (H&E), BrdUrd, and Ki67 areshown in Fig. 3A. In the Cis þ anti-CTLA-4–treatedtumor, it is common to see the condensed nuclei and

some areas replaced by infiltrating lymphocytes. Quan-titative analysis of the immunostaining sections indi-cated that the percentage of positive nuclear staining for

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Figure 2. The effect of CTLA-4 blockade between the intervals ofchemotherapy on tumor growth and survival. Treatment was initiatedwhen tumors grew to a certain size. Cisplatin was injected i.v. onceweekly for 3 doses, and anti-mouse CTLA-4 mAb was injected i.p. 1 dayafter each cycle of chemotherapy. Tumor size was recorded bymeasuring the 2maximal perpendicular diameters twiceweekly. A, tumorgrowth curves, n¼ 5 per group, and the experiment was repeated at leasttwice. Error bars indicate SEM. Cis þ anti-CTLA-4 (n ¼ 15) versus Cis(n ¼ 25), P ¼ 0.0351; versus anti-CTLA-4 (n ¼ 15), P < 0.0001; versusNoRx (n ¼ 25), P < 0.0001; Cis versus anti-CTLA-4, P ¼ 0.0043; versusNoRx, P < 0.0001; anti-CTLA-4 versus NoRx, P ¼ 0.0331. B, survivalcurves; mice were euthanized when tumor volume exceeded 350 mm3.Pooled results from 3 independent experiments are shown. Cis þ anti-CTLA-4 (n ¼ 15) versus Cis (n ¼ 25), P < 0.0001; versus anti-CTLA-4(n ¼ 12), P < 0.0001; versus NoRx (n ¼ 25), P < 0.0001; Cis versus anti-CTLA-4, P < 0.001; versus NoRx, P < 0.0001; anti-CTLA-4 versus NoRx,P < 0.05. C, anti-CTLA-4 resulted in some tumor growth delay whentreated at the early stage of tumor development, but the effect could bereversed by CD8 T-cell depletion with anti-CD8 mAb.






BrdUrd or Ki67 in tumor cells was significantly lowerafter treatment with cisplatin combined with anti-CTLA-4 mAb than with cisplatin alone, anti-CTLA-4alone, or untreated tumors (Fig. 3B and C). Flow cyto-metric data also showed that the BrdUrd-positive pro-portion of tumor cells dropped in the tumors treatedwith Cis þ anti-CTLA-4 when compared those treatedwith cisplatin alone, anti-CTLA-4 alone, or no treatment(Fig. 3D).

Infiltration of T cells in the tumormicroenvironmentafter treatment with CTLA-4 blocking antibodyfollowing chemotherapy in tumor-bearing mice

Tumor-infiltrating T cells were identified by CD3-specific immunostaining (Fig. 4A, top) and quantified bypositive area divided by total cellular areas including

T cells and tumor cells. The proportion of tumor-infiltrat-ing T cells increased significantly when CTLA-4 blockadewas combined to chemotherapy (Fig. 4B). Similar resultswere obtained from fluorescent immunostaining (Fig. 4A,middle and bottom). Flow cytometric results also showedthat the absolute number and proportion of total T cellsand CD4 and CD8 T cells rose after treatment with Cis þanti-CTLA-4 compared with other groups (Fig. 4C).

Gene expression of T-cell activation markers andassociated cytolytic cytokines in tumor-bearing mice

Thegene expression of T-cell activationmarkers such asICOS increased in the tumors after treatment with Cis þanti-CTLA-4, compared with Cis alone, anti-CTLA-4alone, or untreated tumors. More importantly, the genesof cytolytic cytokines and enzymes such as IL-2, IFN-g ,

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Figure 3. Cancer cell repopulationbetweencycles of chemotherapy isinhibited by anti-CTLA-4 therapy.A, tumor histology was shown inH&E staining, BrdUrdincorporation, and Ki67 staining.B and C, quantification ofBrdUrd incorporation and Ki67immunostaining was conductedusing Aperio ImageScope softwareto calculate the proportion ofpositive nuclear area occupied byBrdUrd or Ki67 divided by totalnuclear areas; �, P < 0.05;��, P < 0.01; ��, P < 0.001. D,BrdUrd incorporation in tumor cellswas shown as the percentage ofintracellular positive stainingdetermined by flow cytometry.Tumor cells from 5 tumors pergroup were pooled together. Theexperiment was repeated twice.

Wu et al.





granzyme B, and perforin had higher expression levels inthe Cisþ anti-CTLA-4–treated tumors, as shown in Fig. 5.However, once tumor grew to a certain size, at 50mm3 forexample, monotherapy with CTLA-4 blockade did notresult in significant growth delay of tumors or significantchange of the gene expression.

Perforin and granzyme B from T cells infiltrated intumor and draining lymph nodeOn day 7 after completing treatment, the expression of

perforin and granzyme B in CD8þ T cells infiltrated intumor and the draining lymph nodewas assessed by flowcytometry. Cis þ anti-CTLA-4 treatment resulted in anincrease of granzyme B on CD8þ T cells in both tumor(Fig. 6A) and draining lymph node (Fig. 6B), especially inthe draining lymph node, whereas perforin increasedlittle in tumor or draining lymph node.

DiscussionCTLA-4 blockade offered significant therapeutic bene-

fits to AB12 tumor-bearing mice when combined withchemotherapy. Administration of anti-CTLA-4 mAb dur-ing the intervals of cisplatin treatments slowed downtumor growth and prolonged survival of tumor-bearing

mice. This effectwas associatedwith significant inhibitionof tumor cell repopulation between cycles of cisplatintreatments and significant increased in the total numberof T cells and CD4þ, CD8þ T cells infiltrating into thetumor. Anti-CTLA-4 combined with chemotherapy alsoresulted in an increased gene expression of IL-2, IFN-g ,granzyme B, perforin, and ICOS in the tumor, suggestingthat the combination of chemotherapy and CTLA-4 inbetween cycles of chemotherapy enhanced the antitumorimmune responses.

Because it is difficult to evaluate the antitumor effect bymeasuring tumor size in orthotopicmodels of intrapleuralor intraperitoneal mesotelioma, we used a subcutaneoustumor model by injecting murine mesothelioma AB12cells into the right flank of BALB/c mice. This modelmade it easier to evaluate the effect on tumor growth andalso possible to study the impact of CTLA-4 blockade oncancer cell repopulation and the antitumoral immuneresponse during the intervals between treatments (26).Combination chemotherapy with cisplatin and peme-trexed is currently administered as the first-line chemo-therapy in the patients with advanced malignant pleuralmesothelioma (27, 28), this combination treatment wasable to prolong survival time by approximately 3 months,compared with cisplatin alone (29). Our in vivo studies in

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Figure 4. A, infiltration of T cells in tumor microenvironment after treatment with anti-CTLA-4 following chemotherapy was determined by immunohistochemicalstaining for CD3 (top,�200) and fluorescent immunostaining for CD3-green/CD4-red/DAPI-blue (middle,�400), CD3-green/CD8-red/DAPI-blue (bottom,�400),yellow color represents double positive of CD3/CD4 or CD3/CD8 T cells. B, quantitative analysis was conducted using an ImageScope software basedon completion of entire section scanning of CD3 immunostaining. Combination of cisplatin and anti-CTLA-4 mAb resulted in a significant increase ofCD3-positive proportion. Cisþ anti-CTLA-4 versus NoRx, P < 0.0001; Cisþ anti-CTLA-4 versus Cis, P¼ 0.0001, whereas anti-CTLA-4 versus NoRx, P¼ 0.144,and Cis versus NoRx, P ¼ 0.6369. C, the number of tumor-infiltrating CD3, CD4, and CD8 T cells at 7 days after the second dose of cisplatin was analyzedusing flow cytometry. DAPI, 40,6-diamidino-2-phenylindole.






this subcutaneous mouse model have indicated thatpemetrexed either alone or in combination with cisplatindid not result in significant benefit in controlling tumorgrowth (Supplementary Data, Supplementary Fig. 1S).Similar reports were also found in xenograft studies(30). Single-agent cisplatin was therefore selected as stan-dard chemotherapy in our mouse model. The tumor-bearing mice were treated once weekly to observe theeffect on tumor growth and survival. Our preliminaryresults indicated that tumor growth curves were clearlyseparated byweekly doses of chemotherapy (Supplemen-tary Data, Supplementary Fig. 2S). Three weekly dosesappeared to be most optimal for our mousemodel, as thismodality did not result in severe toxicity.

Therapies for mesothelioma are very limited so far(28, 29, 31). Therefore, immunotherapy such as targetingimmunosuppressive checkpoints has raised a lot of inter-est for the treatment of this disease. CTLA-4 blockingantibody has recently been approved to treat metastaticmelanoma by the U.S. Food and Drug Administration(32). This will open an avenue for treatment of othertypes of cancer. Even though limited studies have yetbeen carried out on mesothelioma, our current studyshows that administration of anti-CTLA-4 therapybetween cycles of chemotherapy results in tumorgrowth delay and improves survival, suggesting thatanti-CTLA-4–based immunotherapy may be a potentialtherapy for the treatment of patients with malignantpleural mesothelioma.

At the early stage of tumor development, treatmentwithCTLA-4 blockade resulted indramatic tumor growth

delay and even tumor disappearance. However, thisstrategy is not feasible clinically as diagnosis is usuallymade at relatively late stages in patients with meso-thelioma. Early treatment with CTLA-4 blockade led toan increase of tumor-infiltrating CD4þ and CD8þ T cellsin both AB12 and AC29 tumor models. As shown in thechromium-release assay, the splenocytes derived fromAB12 tumor-bearing mice had better cell killing thanthose from naive spleen through the production of morecytolytic cytokine IFN-g (Fig. 1). Early treatment resultedin complete tumor disappearance in some mice, andrechallenge with same tumor cells did not lead to tumorgrowth. All the above evidence suggests that both AB12andAC29mesotheliomas are immunogenic and accountsfor response to anti-CTLA-4 blockade-based immuno-therapy. We selected the AB12 model in the series ofexperiments carried out on tumor-bearing mice becausethe subcutaneous tumor growth was more homogenousin AB12 than in AC29 mice.

The mechanisms of CTLA-4 signaling have beenextensively studied (33). Other studies and ours showedthat blockade of CTLA-4 is able to enhance antitumorresponse (34). However, the mechanisms by whichCTLA-4 blockade enhances CD8 function in this contextis not yet fully understood. We believe that the para-digm between Treg and IL-17–producing CD4þ T cells(T-helper cell 17, TH17) and CD8þ T cells (Tc17) is ofimportance. Recent evidence suggests that the numbersof Treg and TH17 cells are inversely correlated in thesame tumor and that considerable functional plasticityexists between Treg and TH17 cells (35). CTLA-4 induces

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Figure 5. Gene expression of T-cell activation marker and associated cytolytic cytokines was determined by RT-PCR. Total RNA was extractedfrom the snap-frozen tumors of each group to quantify the expression level of CD3, CD4, CD8, IL-2, ICOS, IFN-g , granzyme B, and perforin in tumor. Thedetails are described in Materials and Methods.

Wu et al.





TH17 cells in peripheral blood of patients with meta-static melanoma and TH17 cells have been shown topromote antigen-specific antitumor immunity (36).Hence, the combination of chemotherapy and CTLA-4blockade could skew the Treg/TH17 balance toward a TH

17 phenotype. Although we have not measured thelevels of Treg and TH17 in this series of experiments,we had previously shown in a similar subcutaneousmodel of mesothelioma that Treg blockade in combina-tion with chemotherapy resulted in similar outcomewith improved survival and increased cytotoxicity byCD8þ T cells in the tumor microenvironment (26). Pre-liminary results in our laboratory have also shown anincreased level of IL-17 in the tumor microenvironmentin the group of mice receiving CTLA-4 blockade incombination with chemotherapy (data not shown).Considering the importance of CD8þ cells in mediatingthe CTLA-4 response, the recently described Tc17 cellscould also be of importance in generating the responseto CTLA-4 (37). Although Tc17 do not express granzymeB and perforin and are not able to mediate cell lysisin vitro, in vivo Tc17 can rapidly convert into an IFN-g–secreting phenotype producing CD8þ T cells andmediate tumor rejection (37). Although the potentialskewing of both CD8þ T cells and CD4þ T cells in thetumor microenvironment toward an IL-17–producingphenotype with the administration of CTLA-4 blocking

antibody remains to be shown, better understanding ofCTLA-4 in this context will likely help us refine cancertherapies.

CTLA-4 blockade is a promising cancer treatmentand its effect can be potentiated when it is combinedwith conventional therapies, such as chemotherapy,radiotherapy, or cryotherapy as recently showed in ourstudy and other preclinical work (38). In several phase Iclinical trials including melanoma, ovarian, and pros-tate cancers as well as lymphoma, blockade of CTLA-4resulted in tumor regression and was well tolerated,the main risk being the development of severe autoim-mune adverse effects (39–42). In our animal study, therewas no severe side effect using this treatment schema.Autoimmunity may be minimized by using sequentialadministration of the therapeutic antibody followingeach dose of cisplatin rather than concurrent adminis-tration. Surviving cancer cells may also be more effec-tively targeted by immunotherapy during the intervalsof chemotherapy. Further studies are required tooptimize the timing and scheduling of administrationof immunotherapy and chemotherapy to maximize thesynergistic effect.

Although the biology of mesothelioma is largely un-known, tumor progression is attributed to the balanceof cell proliferation and lack of apoptotic cell death.Some investigators have observed that a majority of

Figure 6. Cytolytic enzymes wereevaluated on day 7 after the seconddose of cisplatin by flow cytometry.Intracellular staining was carried outfor granzyme B and perforin fromT cells infiltrated in tumor (A) anddraining lymph node (B). The cellsfrom 5 mice per group were pooledtogether.

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malignant mesothelioma have high expression ofinhibitor apoptosis proteins and low expression of pro-liferation markers such as Ki67 (43). However, theexpression of these factors varied according to the ana-tomic site (peritoneum versus pleura) and the tumormicroenvironment (effusion vs. solid tumor; ref. 43).In addition, the expression of proliferation marker suchKi67, although present in a small proportion of thetumor, was always expressed and the level of expres-sion was more strongly associated with survival thaninhibiting apoptosis proteins. Hence, these findingssuggest that cell proliferation and dysregulation ofapoptotic cell death are both present in mesothelialtumors and that proliferation factors may be of greaterimportance for survival, thus supporting the role ofcell repopulation as a potential cause of clinical failureto chemotherapy in mesothelioma.

Anumber of studies, including our ownandotherworkonmesothelioma, have shown the effect of chemotherapyon the development of an antitumor immunity. Similarlyto our experience, Nowak and colleagues observed thatgemcitabine increased cellular antitumor immunitythrough a CD8 T-cell–dependent pathway in a mousemodel of mesothelioma (44). The resulting tumor-specificimmune response is critical for the eradication of tumorcells that may survive therapy. Recent evidence suggeststhat cancer stem cells survive chemotherapy and/orradiotherapy and repopulate the tumor leading to treat-

ment failure (45, 46). Hence, targeting stem cellsmay be ofprime importance for long-term remission of the tumor.Immunotherapy may be a promising therapeutic optionto target these cells and generate a long lasting memoryresponse (47).

In conclusion,we have shown that CTLA-4mayplay animportant role in mesothelioma tolerance, and blockadeof CTLA-4 signaling in combination with chemotherapyshowed promising results that may be of importance forthe development of new clinical trials.

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

AcknowledgmentsThe authors thank Dr. Yidan Zhao for his assistance in statistical

analysis.

Grant SupportThis work was partly supported by the Mesothelioma Applied

Research Foundation (MARF, USA) and the Mesothelioma Foundationat Princess Margaret Hospital (MFPMH, Canada). Dr. M. de Perrot is therecipient for both grants.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 11, 2012; revised May 9, 2012; accepted May 9, 2012;published OnlineFirst May 14, 2012.

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2012;11:1809-1819. Published OnlineFirst May 14, 2012.Mol Cancer Ther Licun Wu, Zhihong Yun, Tetsuzo Tagawa, et al. MesotheliomaCell Repopulation during the Intervals of Chemotherapy in Murine CTLA-4 Blockade Expands Infiltrating T Cells and Inhibits Cancer

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