pd-1/pd-l1blockadeenhancest-cellactivityand antitumor ......dec 08, 2016 · published onlinefirst...

Cancer Therapy: Preclinical

PD-1/PD-L1 Blockade Enhances T-cell Activity andAntitumor Efficacy of Imatinib in GastrointestinalStromal TumorsAdrian M. Seifert1, Shan Zeng1, Jennifer Q. Zhang1, Teresa S. Kim1, Noah A. Cohen1,Michael J. Beckman1, Benjamin D. Medina1, Joanna H. Maltbaek1, Jennifer K. Loo1,Megan H. Crawley1, Ferdinand Rossi1,2, Peter Besmer2, Cristina R. Antonescu3, andRonald P. DeMatteo1

Abstract

Purpose: Tyrosine kinase inhibitors are effective in gastroin-testinal stromal tumors (GISTs) but often are of transient benefitas resistance commonly develops. Immunotherapy, particularlyblockade of the inhibitory receptor programmed death 1 (PD-1)or the ligand programmed death ligand 1 (PD-L1), has showneffectiveness in a variety of cancers. The functional effects of PD-1/PD-L1 blockade are unknown in GISTs.

Experimental Design:We analyzed tumor andmatched bloodsamples from 85 patients with GISTs and determined the expres-sion of immune checkpoint molecules using flow cytometry. Weinvestigated the combination of imatinib with PD-1/PD-L1blockade in KitV558D/þ mice that develop GISTs.

Results: The inhibitory receptors PD-1, lymphocyte activationgene 3, and T-cell immunoglobulinmucin-3 were upregulated on

tumor-infiltrating T cells compared with T cells from matchedblood. PD-1 expression on T cells was highest in imatinib-treatedhuman GISTs. Meanwhile, intratumoral PD-L1 expression wasvariable. In human GIST cell lines, treatment with imatinibabrogated the IFNg-induced upregulation of PD-L1 via STAT1inhibition. In KitV558D/þ mice, imatinib downregulated IFNg-related genes and reduced PD-L1 expression on tumor cells.PD-1 and PD-L1 blockade in vivo each had no efficacy alone butenhanced the antitumor effects of imatinib by increasing T-celleffector function in the presence of KIT and IDO inhibition.

Conclusions: PD-1/PD-L1 blockade is a promising strategy toimprove the effects of targeted therapy in GISTs. Collectively, ourresults provide the rationale to combine these agents in humanGISTs. Clin Cancer Res; 1–12. �2016 AACR.

IntroductionThe advent of targeted molecular therapy has revolutionized

the treatment of many cancers, including gastrointestinal stromaltumors (GISTs). The majority of GISTs contain an activatingmutation in either the KIT or PDGFRA oncogene (1, 2). Imatinibmesylate (Gleevec) is a tyrosine kinase inhibitor that specificallytargets KIT and PDGFRA (3). The tumor response to imatinib inGISTs is impressive, but most often transient. Resistance com-monly develops within 2 years, often due to a secondary KITmutation (4, 5). Imatinib acts primarily via direct effects on tumorcells. However, we previously showed that imatinib also inhibitstumor cell production of the immunosuppressive enzyme indo-leamine 2,3-dioxygenase (IDO; ref. 6).

Many tumors express ligands that engage inhibitory receptorson T cells and decrease T-cell activation and function within thetumor microenvironment. There is growing evidence that tumorcells commonly exploit the programmed death 1 (PD-1, PDCD1)and programmed death ligand 1 (PD-L1, PDCD1LG1) axis toevade the immune system (7). PD-1 is an inhibitory receptor thatis upregulated after T-cell activation and remains elevated withantigen persistence and therefore is often increased on tumor-infiltrating T cells (8–10). The ligands for PD-1 are PD-L1 (B7-H1,CD274) and PD-L2 (B7-DC, CD273, PDCD1LG2). PD-L1 isexpressed widely on immune cells and can be upregulated byproinflammatory stimuli, such as interferons, but is alsoexpressed by tumor cells in a variety of cancers (11). PD-L1expression on tumors can lead to impaired T-cell proliferationand effector function, leading to apoptosis of tumor-specificT cells (12). PD-L2 expression is restricted mostly to hematopoi-etic cells. PD-1/PD-L1 blockade has demonstrated encouragingantitumor effects in several solid tumors, including kidney, blad-der, and lung cancer, as well as melanoma (13–19).

Despite the efficacy of tyrosine kinase inhibition, nearly allpatients with a metastatic GIST develop tumor progression andeventually succumb to their disease. There has not been anyimprovement in the first-line therapy for GISTs since imatinibwas approved in 2002. In this study, we analyzed freshly isolatedT cells from the tumor andperipheral bloodof patientswithGISTsfor the expression of inhibitory receptors. We determined theeffects of imatinib on IFNg-related genes and the PD-1/PD-L1 axis

1Department of Surgery, Memorial Sloan Kettering Cancer Center, New York,New York. 2Department of Developmental Biology, Memorial Sloan KetteringCancer Center, New York, NewYork. 3Department of Pathology, Memorial SloanKettering Cancer Center, New York, New York.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Ronald P. DeMatteo, Memorial Sloan Kettering CancerCenter, 1275 York Avenue, New York, NY 10065. Phone: 212-639-3976; Fax: 212-639-4031; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-16-1163

�2016 American Association for Cancer Research.

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in GISTs. Furthermore, in a genetically engineered mouse modelofGISTs, we tested the combination of imatinibwith PD-1/PD-L1blockade.

Materials and MethodsPatient samples

Tumor specimens and matched peripheral blood wereobtained from 85 patients with GISTs who underwent surgeryat our institution and were consented to a protocol approved bythe Institutional Review Board. Blood was drawn before surgicalincision, andperipheral bloodmononuclear cellswere isolated bydensity centrifugation over Ficoll–Plaque PLUS (GE Healthcare).Tumor tissue was subjected to mechanical dissociation to obtainsingle-cell suspensions, as described previously (6). After pro-curement, all specimens were processed, and cells were immedi-ately analyzed with flow cytometry. Tumor (KITþ) and stromalcells (KIT�) were isolated using human CD117 microbeads(Miltenyi Biotec). The purity of isolated cells was greater than90% by flow cytometry.

Cell lines and treatmentsThe human GIST cell lines GIST-T1 (KIT exon 11 mutant;

ref. 20), HG129 (also KIT exon 11 mutant; ref. 21), and GIST882(KIT exon13mutant; provided by Jonathan Fletcher [Departmentof Pathology, Brigham and Women's Hospital, Harvard MedicalSchool, Boston, MA]) were maintained at 37�C in 5% CO2 inRPMI1640 medium supplemented with 10% \FBS, 2 mmol/L L-glutamine, 50 U/mL penicillin–streptomycin, 0.1% 2-mercap-toethanol, and 10 mmol/L Hepes. Cells were incubated withrecombinant human IFNg (100 ng/mL; R&D Systems), imatinib(100 nmol/L; Novartis), or the pan-JAK inhibitor tetracyclicpyridine 6 [P6; 1 mmol/L; provided by Jacqueline Bromberg fromthe Department of Medicine, Memorial Sloan Kettering CancerCenter (MSKCC), New York, NY (22)].

Mice and treatmentsKitV558D/þ mice (23) that were 6 to 12 weeks old were main-

tained in a specific pathogen-free animal facility, age- and sex-matched for experiments, and used in accordance with an insti-tution-approved protocol. Tumors were minced, incubated in 5mg/mL collagenase IV (Sigma-Aldrich) plus 0.5 mg/mL DNAse I(Roche Diagnostics) in HBSS for 30 minutes at 37�C, then

quenched with FBS, and washed through 100- and 40-mm nyloncell strainers (Falcon; BD Biosciences) in PBS with 1% FBS.Tumor-draining lymph nodes and spleens were mechanicallydissociated as described previously (6). Cells were immediatelyanalyzed with flow cytometry. Imatinib was administered at90 mg/kg per day in the drinking water. 1-Methyl-D-tryptophan(1-MT; SigmaAldrich) was prepared and administered twice dailyby oral gavage at 400 mg/kg per day as before (6). Anti-PD-1(RMP1-14; Bio X cell) or anti-PD-L1 (10F.9G2; Bio X cell) block-ing antibodies or isotypes (rat IgG2a or rat IgG2b) were admin-istered intraperitoneally at a dose of 250 and 200 mg per mouse,respectively, at the indicated time points. Murine T cells werepurified from mesenteric lymph nodes from C57BL/6J mice(B6, The Jackson Laboratory) and tumors from KitV558D/þ miceusing CD3-biotin and anti-biotin MicroBeads (Miltenyi Bio-tec). Positive selection was performed using two sequentialMACS columns. CD3þ T cells were then cultured in anti-CD3–coated 96-well plates (BD Biosciences) with 10 mg/mL of eitheranti-PD-1 antibody or isotype, and supernatant was harvestedat 48 hours.

Flow cytometry and cytokine detectionHuman-specific antibodies were purchased from BD Bios-

ciences (CD45, 2D1; CD3, SK7; CD4, RPA-T4; PD-1, MIH4),eBioscience (CD8, RPA-T8), BioLegend (PD-L1, 29E.2A3), R&DSystems (TIM-3, 344823), and Enzo Life Sciences (LAG-3, 17B4).Mouse-specific antibodies were purchased from BD Biosciences(CD3, 125-2C11; CD4, GK1.5; CD44, IM7; CD62L, MEL-14; PD-L1, MIH5; CD69, H1.2F3; IFN-g , XMG1.2; TNF, MP6-XT22),eBioscience (CD8, 53-6.7; PD-1, J43; PD-L2, 122; Foxp3, FJK-16s; Ki67, SolA15; Granzyme B, NGZB), and BioLegend (CD45,30-F11; PD-L1, 10F.9G2; CD25, PC61). Appropriate isotypecontrols were used where applicable. A viability dye was typicallyused to exclude dead cells. Intracellular staining was performedusing the eBioscience Fixation and Permeabilization Buffer Kit.For intracellular cytokine staining, cells were stimulated withphorbol 12-myristate 13-acetate (PMA, 50 ng/mL) and ionomy-cin (750 ng/mL) for 4 hours at 37�C, 5%CO2 in the presence of 1mg/mL brefeldin A (BD Biosciences). Surface staining was per-formed and cells were fixed and permeabilized with the BDCytofix/Cytoperm Kit and stained for IFNg and TNFa. Superna-tant cytokines were measured by cytometric bead array accordingto the manufacturer's instructions (Mouse Inflammation Kit;BD Biosciences). Data were acquired using a BD FACSAria orFortessa LSR flow cytometer and analyzed using FlowJo software(Tree Star).

Immunofluorescence and IHCFormalin-fixed and paraffin-embedded specimens were sec-

tioned at 5-mm thickness and mounted on glass slides. The PD-L1 (clone 5H1) antibody was obtained from Lieping Chen(Department of Immunobiology, Yale University School of Med-icine, New Haven, CT) (12). In brief, antigen retrieval wasachieved with citrate buffer. Anti-PD-L1/B7-H1 murine IgG(1:1,000) was applied on tissue sections overnight at 4�C. Afterwashing with TBS (0.05 mol/L Tris base, 0.9% NaCl, pH 8.4),tissue sections were incubated with biotinylated anti-mouse IgG(1:100; BA-2001, Vector Laboratories), followed by incubationwith Elite ABC Kit for 30minutes at room temperature. Antibodybinding was detected with the TSA Biotin System(NEL700A001KT, PerkinElmer) and DAB (DAKO, K3468).

Translational Relevance

Although GISTs are often initially sensitive to imatinib orother tyrosine kinase inhibitors, resistance generally develops,necessitating additional therapeutic strategies. There has notbeen any improvement in the first-line therapy for GISTs sinceimatinib was approved in 2002. Immunotherapy is beingtested in a variety of cancers.Ourfindings provide new insightsinto the combination of tyrosine kinase inhibition and immu-notherapy andprovide a strong incentive to clinically combineimatinib and anti-PD-1/PD-L1 in GISTs. Furthermore, ourdata challenge the current notion that immunotherapy iseffective only in tumors with a high number of mutations, asthere is only onemutation in ourmousemodel, as is typical inhuman GISTs.

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Murine PD-L1 staining was performed using the polyclonalantibody against mouse B7-H1/PD-L1 antibody (1:100, R&DSystems). Terminal deoxynucleotidyl transferase–mediated dUTPnick end labeling (TUNEL) and KIT immunostaining (1:200,D13A2; Cell Signaling Technology) were performed as describedpreviously (24). Slides were analyzed and imaged on the AxioImager 2 wide-field microscope (Zeiss).

qRT-PCRTotal RNA was extracted from each human GIST specimen,

cell line, or murine GIST specimen, reverse transcribed, andamplifiedwith PCR TaqMan probes for human PDCD1LG1 (i.e.,PD-L1, Hs01125301_m1), IFNGR1 (Hs00988303_m1), IRF1(Hs00971960_m1), GAPDH (Hs02758991_g1), murine Pdl1(Mm00452054_m1), Ido1 (Mm00492590_m1), and Gapdh(Mm99999915_g1, all Applied Biosystems). Quantitative PCRwas performed using a ViiATM7 real-time PCR system (AppliedBiosystems). Relative expression was calculated by the 2�DDCt

method according to the manufacturer's protocol and expressedas the fold increase over the indicated control.

STAT1 knockdownFor transient STAT1 knockdown, GIST-T1 cells were transfected

with 30 nmol/L of ON-TARGET plus SMARTpool siRNA specificfor human STAT1 (L-003543-00) or a nontarget control siRNA(D-001810-10-05; Thermo Scientific) using Lipofectamine RNAi-MAX (Invitrogen) for 48 hours. GIST-T1 cells were treated withPBS or IFNg (100 ng/mL) for 6 hours.

Western blot and cytokine arrayWestern blot analysis of whole-protein lysates from frozen

tumor tissues or cells was performed as described previously (6).Antibodies for PD-L1 (E1L3N), phosphorylated SRC (Tyr416),phosphorylated STAT1 (Tyr701), phosphorylated STAT3 (Ser727or Tyr705), total and phosphorylated KIT (Tyr719), phosphory-lated ERK1/2 (Thr202/Tyr204), phosphorylated SMAD2 (Ser465/467), TGFb, and GAPDH were purchased from Cell SignalingTechnology. Anti-IDO antibody (clone 10.1) was purchased fromEMD Millipore. Whole protein from frozen tumor tissues waslysed, and total protein was tested for cytokine/chemokine expres-sion using a Proteome Profiler Array (R&DSystems). Densitometrywas conducted on blots using ImageJ (NIH, Bethesda, MD).

Statistical analysisData are expressed as mean � SEM or median. Unpaired two-

tailed Student t test or one-way ANOVA comparisons were per-formed as applicable using GraphPad Prism 6.0 (GraphPadSoftware). P < 0.05 was considered significant.

ResultsT cells infiltrating human GISTs express high levels ofinhibitory receptors compared with blood

The inhibitory receptors PD-1, LAG-3, and TIM-3 (HAVCR2)are typically expressed on antigen-specific and dysfunctional Tcells within tumors and during chronic viral infection (10, 25). Toidentify the inhibitory receptors that are relevant in GISTs, weperformed flow cytometry on T cells from 106 tumor specimensandmatched blood, freshly obtained from85 patients with GISTsundergoing surgery (Supplementary Table S1). The percentages ofCD4þ and CD8þ T cells among leukocytes were lower in tumors

than in matched blood. CD8þ T cells were less frequent thanCD4þ T cells in both compartments (Fig. 1A). Intratumoral T cellsdisplayed greater cell-surface expression of PD-1, LAG-3, andTIM-3 compared with T cells from matched blood (Fig. 1B–D). PD-1was expressed at the highest levels on both intratumoral CD4þ

and CD8þ T cells (48% and 39%, respectively). Moreover, thepercentage of PD-1 on CD4þ and CD8þ T cells correlated withinthe same tumor specimen (Fig. 1E).

Patterns of PD-1 expression in T cells in human GISTsThe expression of inhibitory receptors on tumor-infiltrating

T cells suggested that T-cell immune evasion occurs in humanGISTs. PD-1þ T cells tended to also express LAG-3 and lessfrequently TIM-3 (Fig. 2A). In contrast, PD-1� T cells had littleexpression of LAG-3 or TIM-3. The majority of T cells frommatched blood did not express any inhibitory receptors (Fig.2B). Previously, we showed that tumors sensitive to tyrosinekinase inhibition (based on serial radiologic assessment prior tosurgery) contained more CD3þ and CD8þ T cells, but a lowerpercentage of CD4þ T cells and T regulatory cells (Treg) comparedwith untreated and resistant tumors (6). Here, we found thatcompared with untreated tumors, there was higher PD-1 expres-sion on CD4þ T cells in sensitive tumors and CD8þ T cells intumorswith acquired resistance to imatinib (i.e., increasing tumorsize or vascularity after a period of stable or shrinking disease byserial radiologic assessment; Fig. 2C). There was no associationbetween PD-1 expression and type of oncogene mutation (KIT,PDGFRA, or wild-type), tumor location, size, or mitotic rate (datanot shown). In 10 patients who had multiple tumors removedduring surgery, we found a similar expression pattern of inhib-itory receptors within individuals, despite differences in tumorlocation, size, and mitotic rate (Fig. 2D).

PD-L1 is expressed in a subset of human GISTsTo assess the expression of PD-L1 in human GISTs, we evalu-

ated humanGIST specimens by IHC. PD-L1 expressionwas low inmost cases and tended to be focal (Fig. 3A). PD-L1 was present ontumor cells, but also on some intratumoral leukocytes. To ascer-tain the relative location of PD-L1 within the tumor, wemeasuredPD-L1 mRNA levels in freshly isolated tumor (CD45�KITþ) andstroma (CD45�KIT�) cells from two resistant human GISTs. PD-L1 mRNA was equally distributed between these two compart-ments (Fig. 3B). Furthermore, we performed real-time PCR on 41bulk human GIST samples. The PD-L1 mRNA level was variablebut didnot appear to correlatewith treatment response to tyrosinekinase inhibition (Fig. 3C),mutation (Fig. 3D), or tumor size (Fig.3E). There was a weak inverse relationship between mitotic rateand PD-L1 expression (Fig. 3F). Overall, intratumoral PD-L1expression was variable and heterogeneous.

Imatinib abrogates IFNg-induced upregulation of PD-L1 onhuman GIST cell lines

Next, we found that PD-L1 expression was low on threeimatinib-sensitive human GIST cell lines (Fig. 4A). Because IFNgis known to induce PD-L1 expression (12), we tested its effect onGIST cell lines. IFNg upregulated PD-L1 mRNA in GIST-T1 cellsover time as measured by real-time PCR (Fig. 4B). Similar resultswere observed in GIST882 and HG129 cells (data not shown).IFNg-induced upregulation of PD-L1 transcripts was abolishedwith either the JAK inhibitor pyridine 6 or the KIT inhibitor

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imatinib. IFNg treatment also induced PD-L1 protein expression,but imatinib abrogated the effect as measured by flow cytometry(data not shown) and Western blot analysis (Fig. 4C). Themechanism appeared to be partly through the inhibition ofSTAT-1 activation, as JAK inhibition by pyridine 6 also reducedPD-L1 upregulation and, like imatinib, was associated withdecreased phosphorylated STAT1 (Fig. 4C) and STAT1 mRNAlevels (data not shown). Furthermore, STAT1 knockdown bysiRNA in GIST-T1 cells blocked IFNg-induced upregulation ofPD-L1 protein andmRNA (Fig. 4D andE). STAT1 knockdownwasconfirmed by Western blot analysis (Fig. 4D). We next measuredthe mRNA levels of IFNg receptor (IFNGR1) and its major down-stream signaling component IFN response factor 1 (IRF1). Nota-bly, imatinib reduced the IFNg-mediated increase in IFNGR1 andIRF1 mRNA (Fig. 4F). Collectively, IFNg played a significant rolein the regulation of PD-L1 expression in humanGIST cells, but itseffect was inhibited by imatinib.

Imatinib modulates IFNg-related genes and PD-L1 expressionin GISTs

To determine whether imatinib also alters tumor IFNg signal-ing in KitV558D/þ mice, we assessed IFNg-related genes in ourpreviously published mRNA microarray data (6). After 1 weekof imatinib therapy, the expression ofmultiple IFNg-related geneswas reduced in bulk tumor (Fig. 5A). Pdl1 was not present on thearray. Notably, PD-L1 expression was markedly reduced after 4

weeks of imatinib by IHC (Fig. 5B). To further investigate theextent to which imatinib treatment modulates PD-1 and PD-L1expression in GISTs, we performed flow cytometry on intratu-moral T cells and tumor cells (Fig. 5C). PD-L1 was present ontumor cells and decreased with 1 week of imatinib treatment. PD-L2 expression was minimal (data not shown). T-cell subsetswithin the tumor expressed both PD-1 and PD-L1 at baseline,but imatinib had little to no effect on expression levels. CD8þ Tcells from the tumor-draining lymph node and spleen generallyhad very low PD-1 expression (Fig. 5D). Compared with intra-tumoral PD-1�CD8þ T cells, PD-1þCD8þ T cells from the tumorproduced only minimal amounts of IFNg after stimulation withPMA and ionomycin, suggesting that these cells are dysfunctional(Fig. 5D). However, in vitro treatment of bulk intratumoral T cellsfromuntreatedKitV558D/þmicewith anti-PD-1 antibody increasedtheir production of both TNF and IFNg (Fig. 5E). In contrast, Tcells from themesenteric lymph node of wild-type mice had littlecytokine production at baseline or after PD-1 blockade. Thus, PD-1 blockade preferentially affected tumor-infiltrating T cells com-pared with T cells from the mesenteric lymph node, whichessentially lacked PD-1 expression.

PD-1/PD-L1 blockade enhances the antitumor effects ofimatinib in murine GISTs

Given the presence of PD-1 and PD-L1 in both human andmurine GISTs, we hypothesized that blockade of the PD-1/PD-L1

Figure 1.

T cells infiltrating human GISTs express high levels of inhibitory receptors compared with blood. A, CD4þ and CD8þ T cells as a percentage of leukocytes(CD45þ cells) inmatchedblood and tumor specimens (n¼ 106) from85humanpatientswithGISTs..B,Histogramsof PD-1, LAG-3, and TIM-3 expression onCD4þ andCD8þ T cells in blood (gray) and tumor (black) from a representative sample. PD-1þ, LAG-3þ, and TIM-3þ cells as percentage of CD4þ (C) and CD8þ T cells(D) from matched peripheral blood and tumor samples. E, Linear regression analysis between PD-1 expression on CD4þ and CD8þ T cells in 106 human GISTspecimens. Data, mean � SEM. � , P < 0.05.

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axis would have an antitumor effect. We treated KitV558D/þ micewith imatinib or vehicle combined with PD-1 or PD-L1 antibo-dies or isotype control for 4 weeks. The combination of imatinib

and PD-1/PD-L1 blockade was not associated with noticeabletoxicity in our model, and mice did not show signs of autoim-munity. Notably, anti-PD-1 and anti-PD-L1 altered tumor weight

Figure 2.

Patterns of PD-1 expression in T cells in human GISTs. A, Expression of individual or the combination of inhibitory receptors (PD-1, LAG-3, and TIM-3) onCD4þ and CD8þ T cells in 63 human GIST specimens by Boolean gate analysis. Data, mean � SEM. � , P < 0.05. B, Total number of inhibitory receptors expressedby CD4þ (top) and CD8þ T cells (bottom) from the blood (left) and tumor (right). C, Percentage of PD-1þ cells among CD4þ (top) and CD8þ T cells(bottom) in blood and tumor from untreated (n ¼ 36), imatinib-sensitive (n ¼ 38), and imatinib-resistant (n ¼ 32) GIST specimens. Each dot represents aseparate specimen. Horizontal red line, median values. � , P < 0.05. D, Expression of inhibitory receptors on tumor-infiltrating T cells from patients with GISTs withmultiple metastases (n ¼ 10). Each dot represents a separate tumor specimen.

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only when combined with imatinib (Fig. 6A). The combinationtreatment decreased phosphorylated KIT, phosphorylated STAT1,IDO, and TGFb and its upstream mediator phosphorylatedSMAD2 (Fig. 6B). Furthermore, Pdl1 and Ido1 mRNA levels weredownregulated after 4 weeks of treatment (Fig. 6C). Importantly,the enhanced antitumor effect of imatinib andanti-PD-1persisted

at 3months of treatment (Fig. 6D) andwas detectable as early as 1week (Fig. 6E). After 1 week of combined treatment, we noticeddecreased KIT staining, indicating a reduction of tumor cells,especially in tumors from mice treated with imatinib plus anti-PD-L1, and increased TUNEL staining, indicating increased tumorcell apoptosis (Fig. 6F). There was no change in the frequency of

Figure 3.

PD-L1 is expressed in a subset of human GISTs. A, Representative IHC with anti-PD-L1 (clone 5H1) shows membranous and cytoplasmic expression in humanGISTs (black arrow; scale bar, 50 mm). B, Freshly isolated KIT� (stroma) and KITþ (tumor) cells from two human GISTs were analyzed for PD-L1mRNA by real-timePCR. Bars, mean � SEM. C, RNA was isolated from fresh-frozen untreated (n ¼ 14), imatinib-sensitive (n ¼ 9), and imatinib-resistant (n ¼ 18) human GISTs andanalyzed for PD-L1 mRNA using real-time PCR. Bars, means. D, PD-L1 mRNA relative expression in 25 GIST samples with confirmed mutation. Horizontal red line,median values. E, Scatter plots to analyze the correlation of PD-L1mRNA with tumor size and (F) mitotic count. HPF, high-power field. Values are log transformed.

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CD4þ or CD8þ T cells or Tregs after 1 or 4 weeks of combinationtreatment (data not shown). Remarkably, intratumoral CD8þ Tcells showed increased proliferation and inflammatory cytokineproduction upon combination treatment for 1 week compared

with imatinib alone (Fig. 6G). In accordance with our observa-tions from the mRNA microarray, IFNg protein and two of thechemokines it induces, CXCL9 and CXCL10, were downregulatedafter 1 week of treatment (Fig. 6H). Furthermore, PD-1 blockade

Figure 4.

Imatinib abrogates IFNg-induced upregulation of PD-L1 on human GIST cell lines. A, PD-L1 expression in human GIST cell lines as determined by flowcytometry. Gray histograms, isotype controls. B,GIST-T1 cells were treated with either PBS, IFNg (100 ng/mL), IFNg plus the JAK inhibitor tetracyclic pyridine 6 (P6;1 mmol/L), or IFNg plus imatinib (Im; 100 nmol/L). PD-L1 mRNA relative expression was determined at the indicated time points by real-time PCR. C, Westernblot analysis of GIST-T1 treated as described above for 20 hours. D, GIST-T1 cells were transfected with nontarget control siRNA or ON-TARGET plusSMARTpool siRNA (STAT1 siRNA) and then treated with PBS or IFNg (100 ng/mL) for 6 hours. Western blot analysis demonstrating efficiency of STAT1 knockdownand PD-L1 expression. E, PD-L1 expression in GIST-T1 cells was measured by PCR. F, GIST-T1 cells were analyzed for IFNGR1 and IRF1 by real-time PCR after6 hours of the indicated treatment. Data are normalized to control. Data, mean � SEM. � , P < 0.05.

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Figure 5.

Imatinib modulates IFNg-related genes and PD-L1 expression in GISTs. A, Bar graph showing mRNA levels of IFNg-related genes determined by microarray analysis oftumors from KitV558D/þ mice treated with imatinib for 1 week. Data are shown as log2 fold change compared with vehicle-treated KitV558D/þ mice (3/group). Bars,mean. B, Representative IHC with anti-PD-L1 of tumors from KitV558D/þ mice treated with vehicle or imatinib for 4 weeks (scale bar, 100 mm). C, Histograms of PD-1 andPD-L1 expression on tumor cells (CD45�Kitþ), tumor-associated macrophages (TAMs, CD45þF4/80þCD11bþ), and T-cell subsets (CD4þ Tconv cells: CD45þ

CD3þCD4þFoxp3�, Tregs: CD45þCD3þCD4þCD25þFoxp3þ, and CD8þ T cells: CD45þCD3þCD8þ) in GIST-bearing KitV558D/þ mice treated with vehicle or imatinib for 1week (3–5/group). Representative plots are depicted with isotype control (gray), vehicle (blue), and imatinib (red). D, Expression of IFNg in stimulated CD8þ T cellsfrom spleens, tumor-draining lymph nodes (TdLN), and tumors from untreated KitV558D/þ mice. CD8þ T cells from the indicated tissues were isolated, stimulatedin vitro with PMA and ionomycin, and then analyzed by flow cytometry. E, Effect of PD-1 blockade on cytokine production in T cells. T cells were harvested from themesenteric lymphnode (mLN) fromB6mice [wild-type (WT)] or the tumor fromuntreatedKitV558D/þmiceand cultured in vitro in the presenceof anti-PD-1 (10mg/mL)orisotype control. After 48 hours, culture supernatant was collected, and cytokines were measured by cytometric bead array. Data, mean � SEM. � , P < 0.05.

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combined with the IDO inhibitor 1-MT was more effective after 1week of treatment in our GISTmousemodel than IDO inhibitionalone (Fig. 6I). Thus, IDO inhibition (which also occurs afterimatinib therapy alone) is sufficient to enable the antitumoreffect of anti-PD-1. Taken together, we showed that KIT inhi-bition combined with PD-1/PD-L1 blockade reduced tumorweight in KitV558D/þ mice, which was associated with increasedtumor cell apoptosis and enhanced frequency of cytokine-producing CD8þ T cells.

DiscussionIn this study, we found that the PD-1/PD-L1 axis contributes to

tumor immune evasion in GISTs. Among the inhibitory receptorsanalyzed on intratumoral T cells in human GISTs, PD-1 wasexpressed at the highest frequency. Notably, the PD-1 expressionon CD4þ and CD8þ T cells within sensitive and resistant humanGISTs had a bimodal distribution, with a distinct populationhaving very high expression, suggesting that a subset of patientsmight particularly benefit from PD-1/PD-L1 blockade. The levelof PD-1 expression on intratumoral CD4þ T cells correlated withthat on CD8þ T cells within a patient. Furthermore, in patientswith multiple tumors, the different tumors had similaramounts of inhibitory receptors on intratumoral T cells, despitedifferences in tumor location, size, and mitotic rate. Thisobservation does not resolve whether the antitumor immuneresponse is either tumor driven, despite the known heteroge-neity among tumor subclones, or patient driven, as systemichost factors may be responsible for a consistent T-cell response.PD-1 expression has been demonstrated to identify T cells thatrecognize tumor-specific proteins (26). PD-1þCD8þ T cellsaccumulated in murine GISTs but produced less IFNg upon invitro stimulation compared with PD-1�CD8þ T cells, suggestingthat PD-1 marks functionally impaired intratumoral T cells inour model. Nevertheless, bulk intratumoral T cells madeinflammatory cytokines after treatment with anti-PD-1 in vitro,as did intratumoral CD8þ T cells from KitV558D/þ mice that hadbeen treated with anti-PD-1 in vivo.

PD-L1 expression in human GISTs was variable and did notcorrelate with treatment status, tumor mutation, and tumorsize. Tumors with a lower mitotic rate had a very modestcorrelation with higher PD-L1 expression, which has beensuggested by prior data (27). We showed that PD-L1 expressionin human GISTs was similar between tumor and stromal cells.It is unclear whether PD-L1 expression by either tumor orimmune cells is required for response to anti-PD-1/PD-L1therapy. In a previous report, a subset of melanoma patientswith PD-L1� tumors responded to PD-1 blockade, suggestingthat PD-L1 expression may not be necessary for response (28).

Investigation of this question is hampered by the suboptimalantibodies for PD-L1 IHC. In fact, in several human GISTs, wefound only focal immunostaining despite relatively high PD-L1mRNA levels.

Multiple mechanisms have been shown to drive PD-L1expression. First, PD-L1 can be induced by IFNg (12, 29).Although human GIST cell lines had low PD-L1 expression,IFNg treatment markedly increased it, likely through STAT1.IFNg generally enhances the inflammatory response but mayalso promote immunosuppression by reducing tumor recog-nition and T cell–mediated lysis (30). PD-L1 expression hasalso been linked to EGFR mutations, PI3K/AKT signaling, and,in BRAF-resistant melanoma cell lines, reactivation of theMAPK pathway (31–33). In GISTs, PD-L1 expression mayconceivably be induced by other cytokines, signaling pathways,and immune cells within the tumor microenvironment, as wellas by IFNg . Oncogene inhibition downregulates PD-L1 expres-sion in melanoma cell lines (33, 34). Similarly, in our mousemodel, imatinib decreased PD-L1 mRNA, protein, and cell-surface expression. In addition, imatinib treatment of GIST celllines abrogated the effect of IFNg on PD-L1 expression. Theeffects of imatinib were likely mediated by suppression of JAK/STAT and PI3K/AKT, which are both downstream of KIT, and byrepression of IFNg and IFNg-related genes. The reduction inIFNg is consistent with the previous finding that imatinibreduced MHC class I expression in human GISTs (35) and ourdemonstration that imatinib lowered class II in murine andhuman tumor-associated macrophages in GISTs (36).

Tumor cells treated with targeted therapy were recently shownto induce a reactive secretome that promotes the survival ofsensitive cells, and the expansion and dissemination of drug-resistant clones (37). This therapeutic obstaclemight beovercomeby combining targeted therapy with mobilization of the immunesystem. Indeed, our data demonstrated that anti-PD-1 and anti-PD-L1 in KitV558D/þ mice with established tumors increased theeffects of imatinib by enhancingCD8þT-cell function, resulting insubstantial tumor cell apoptosis. Administered alone, anti-PD-1and anti-PD-L1 were ineffective. It seems unlikely that pretreat-ment with the blocking antibodies would further improve thecombination of PD-1/PD-L1 blockade and imatinib. Imatinib-induced tumor cell death could sensitize GISTs to PD-1/PD-L1blockade through the release of endogenous tumor antigens thatsubsequently activate the immune system. However, the rapidtumor response by 1 week makes it more likely that a preexistingimmune response was amplified. It does not seem that PD-1inhibition in our model reduced tumor growth independentof the adaptive immune system, a mechanism recentlyshown inhumanmelanoma tumor cells,which frequently expressPD-1 (38).

Figure 6.PD-1/PD-L1 blockade enhances the antitumor effects of imatinib in murine GISTs. A, Tumor weights from KitV558D/þ mice (3–5/group) treated with imatinib (Im)or vehicle (Veh) for 4 weeks. Anti-PD-1 or anti-PD-L1 antibodies or their isotypes (Iso) were given on days 0, 4, 8, and 12. Mice were sacrificed at day 28. B,Western blot analysis of tumor samples from KitV558D/þmice treated as inA. C, Bulk tumors were analyzed for expression of Pdl1 and Ido1 by real-time PCR. Data arenormalized to control. Data, mean of at least three samples � SEM. � , P < 0.05. D, Tumor weights after 3 months of treatment (3–5 mice/group). Antibodies orisotypes were given as in A. Mice were sacrificed at day 90. E, Tumor weights from KitV558D/þ mice (3–5/group) treated with imatinib for 1 week. Anti-PD-1 oranti-PD-L1 antibodies were given on days 0, 2, 4, and 6. Mice were sacrificed at day 7. F, Tumor KIT and TUNEL staining after 1 week of treatment. Scale bars, 50 mm.G, Percentages of intratumoral CD8þ T cells that were Ki67þ or IFNg/TNF–producing intratumoral CD8þ T cells after in vitro stimulation with PMA andionomycin. Data,means of three to four samples� SEM. � ,P <0.05.H,Cytokine array densitometry of tumors fromKitV558D/þmice after 1 week of treatment. I, Tumorweights from KitV558D/þ mice treated with 1-MT for 1 week (3–5/group). Anti-PD-1 antibody was given as in E. Mice were sacrificed at day 7. All tumorweights are shown normalized to vehicle control.


Seifert et al.




The other contributing factor that explains the efficacy ofcombining imatinib with anti-PD-1 or anti-PD-L1 is the inhibi-tion of IDO. IDO is an enzyme that catalyzes the degradation ofthe essential amino acid tryptophan to kynurenine, whose meta-bolites suppress T cells. Previously, we showed that imatinibreduces tumor cell production of IDO in ourmodel by decreasingthe levels of the transcription factor ETV4, which regulates IDOtranscription (6). Recent studies have suggested that elevatedexpression of metabolic enzymes (e.g., IDO) and inhibitorymolecules (e.g., PD-1) is induced by IFNg as an adaptive responseby the tumor (29, 39). Our data imply that IDO inhibition byimatinib partially accounts for the antitumor efficacy of concom-itant PD-1/PD-L1 blockade. This supposition is consistent with aprevious report showing that IDO inhibition augmented theefficacy of T-cell immunotherapy in B16 melanoma (40). Fur-thermore, tumor regression after therapeutic PD-1 blockade hasbeen demonstrated to require preexisting CD8þ T cells that arenegatively regulated by PD-1/PD-L1–mediated adaptive immuneresistance. Response to pembrolizumab (anti-PD-1) was associ-ated with higher numbers of CD8þ, PD-1þ, and PD-L1þ cells inthe tumor microenvironment before treatment (41). The combi-nation of imatinib and PD-1/PD-L1 blockade may be mosteffective in treatment-na€�ve tumors, which are the most sensitiveto oncogene inhibition. Melanomas with high levels of somaticmutations have been shown to be more likely to respond tocheckpoint immune blockade (42). However, our data show thatmurine GISTs, which have only one mutation, respond to PD-1/PD-L1 blockade in the setting of tyrosine kinase inhibition.

In conclusion, PD-1 was expressed at high levels on tumor-infiltrating T cells in human GISTs, while PD-L1 levels werevariable. In a mouse model of GIST, anti-PD-1 and anti-PD-L1had no antitumor effect when used alone but did increase theefficacy of imatinib. The mechanism involved a reduction of IDOlevels and an increase in CD8þ T-cell function. PD-1/PD-L1blockade is a promising strategy to improve the effects of targetedtherapy in GISTs.

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

Authors' ContributionsConception and design: A.M. Seifert, S. Zeng, T.S. Kim, N.A. Cohen, R.P.DeMatteoDevelopment of methodology: A.M. Seifert, S. Zeng, T.S. Kim, P. Besmer,R.P. DeMatteoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.M. Seifert, S. Zeng, T.S. Kim, M.J. Beckman,J.H. Maltbaek, J.K. Loo, M.H. Crawley, C.R. Antonescu, R.P. DeMatteoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.M. Seifert, S. Zeng, T.S. Kim, N.A. Cohen,M.J. Beckman,M.H.Crawley, F. Rossi, P. Besmer, C.R. Antonescu, R.P.DeMatteoWriting, review, and/or revision of the manuscript: A.M. Seifert, S. Zeng,J.Q. Zhang, N.A. Cohen, M.J. Beckman, B.D. Medina, J.H. Maltbaek, J.K. Loo,F. Rossi, C.R. Antonescu, R.P. DeMatteoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.M. Seifert, S. Zeng, T.S. Kim, R.P. DeMatteoStudy supervision: A.M. Seifert, R.P. DeMatteo

AcknowledgmentsWe are grateful to the Tissue Procurement Service for assistance in the

acquisition of human tumor specimens. We thank members of the SloanKettering Institute Laboratory of Comparative Pathology, Molecular Cytology,Flow Cytometry, and Pathology Core Facilities, Colony Management Group,and Research Animal Resource Center. We thank Lieping Chen for giving us themurine anti-human PD-L1 monoclonal antibody (clone 5H1) and JacquelineBromberg for providing the JAK inhibitor pyridine 6. We thank Russell Holmesfor logistical and administrative support.

Grant SupportThe investigators were supported by NIH grants R01 CA102613 and T32

CA09501, Cycle for Survival, SwimAcross America, Stephanie and Fred Shumanthrough the Windmill Lane Foundation, and David and Monica Gorin (to R.P.DeMatteo), GISTCancer Research Fund (to R.P.DeMatteo andC.R. Antonescu),F32 CA162721 and the Claude E. Welch Fellowship from the MassachusettsGeneral Hospital (to T.S. Kim), F32 CA186534 (to J.Q. Zhang), P01 CA47179(to C.R. Antonescu), and P50 CA140146-01 (to C.R. Antonescu and P. Besmer).The Molecular Cytology and Flow Cytometry Core Facilities were supported byCancer Center Support Grant P30 CA008748.

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

Received May 7, 2016; revised June 28, 2016; accepted July 14, 2016;published OnlineFirst July 14, 2016.

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Published OnlineFirst July 28, 2016.Clin Cancer Res Adrian M. Seifert, Shan Zeng, Jennifer Q. Zhang, et al. Efficacy of Imatinib in Gastrointestinal Stromal TumorsPD-1/PD-L1 Blockade Enhances T-cell Activity and Antitumor

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