Microenvironment and Immunology

IL10 Release upon PD-1 Blockade SustainsImmunosuppression in Ovarian CancerPurushottam Lamichhane1,2,3, Lavakumar Karyampudi1,3, Barath Shreeder2,3,James Krempski1, Deborah Bahr2, Joshua Daum2, Kimberly R. Kalli4,Ellen L. Goode5, Matthew S. Block4, Martin J. Cannon6, and Keith L. Knutson1,2,3

Abstract

Ligation of programmed cell death-1 (PD-1) in the tumormicroenvironment is known to inhibit effective adaptive antitumorimmunity. Blockade of PD-1 in humans has resulted in impressive,durable regression responses in select tumor types. However,durable responses have been elusive in ovarian cancer patients.PD-1was recently shown tobe expressedonand thereby impair thefunctions of tumor-infiltrating murine and human myeloid den-dritic cells (TIDC) in ovarian cancer. In the present work, wecharacterize the regulation of PD-1 expression and the effects ofPD-1 blockade on TIDC. Treatment of TIDC and bone marrow–derived dendritic cells (DC) with IL10 led to increased PD-1expression. Both groups of DCs also responded to PD-1 blockade

by increasing production of IL10. Similarly, treatment of ovariantumor–bearing mice with PD-1 blocking antibody resulted in anincrease in IL10 levels in both serum and ascites. While PD-1blockade or IL10 neutralization as monotherapies were inefficient,combination of these two led to improved survival and delayedtumor growth; this was accompanied by augmented antitumorT- and B-cell responses and decreased infiltration of immunosup-pressive MDSC. Taken together, our findings implicate compensa-tory release of IL10 as one of the adaptive resistance mechanismsthat undermine the efficacy of anti–PD-1 (or anti–PD-L1) mono-therapies and prompt further studies aimed at identifying suchresistance mechanisms. Cancer Res; 77(23); 6667–78. �2017 AACR.

IntroductionProgrammed cell death-1 (PD-1) andprogram cell death ligand

1 (PD-L1) constitute a major immune regulatory axis that blocksantitumor effector responses in the tumor microenvironment.PD-1 is an inhibitory receptor expressed on several adaptiveand innate immune cells, including T cells, B cells, monocytes,macrophages, natural killer (NK) cells, and dendritic cells (DC;refs. 1–4). KnownPD-1 ligands include PD-L1 (B7-H1or CD274)and PD-L2 (B7-DC or CD273; ref. 1). PD-L1 is constitutivelyexpressed on many cells types like DCs and macrophages. PD-L1is inducible on T cells, B cells, tumor cells, epithelial cells, andendothelial cells. Similarly, PD-L2 is primarily induced, depend-ing on the cytokine milieu, on DCs, B cells, macrophages, andmonocytes (1, 5–7). The ligation of PD-1 by its ligands, during T-

lymphocyte activation, has been shown to result in the tyrosinephosphorylation of its cytoplasmic tail, leading to recruitment ofSrc homology domain-containing phosphatase-2 (SHP-2). Acti-vated SHP-2 subsequently dephosphorylates T cell receptor (TCR)proximal signaling complexes such as ZAP70/CD3z and PI3K andinhibits Akt phosphorylation culminating in the inhibition of Tcell proliferation, cytotoxic functions, and cytokine release, andpromotion of apoptosis (8, 9).

PD-1 expression on lymphocytes is dynamic and is upregulatedfollowing TCR or B cell receptor (BCR) ligation. Studies also haveshown that T cells are programmed epigenetically upon persistentantigenic stimulation for rapid PD-1 expression upon restimula-tion (10, 11). One crucial observation that is made frequently isthat the expression of PD-1 on T cells infiltrating in tumors is veryhigh compared with their peripheral or normal tissue counter-parts (12, 13). This high expression of PD-1 on tumor-infiltratingT cells not only identifies them as tumor antigen-specific butalso correlates with blunted responsiveness to further stimulation(13, 14). Furthermore, infiltration by PD-1þ lymphocytes oftencorrelates with distant metastatic relapse and poor prognosis inmany tumor types (12, 14, 15).

Inmanymalignant tissues, it has been observed that up to 40%of the infiltrating immune cells are tumor-infiltrating murine andhuman myeloid dendritic cells (TIDC) and disease progressioncorrelates with the phenotypic switch of TIDCs from immunestimulatory to immune suppressive (4, 16). We have shown in amurine model of ovarian carcinoma that TIDCs also becomeincreasingly PD-1þ as disease progresses rendering them ineffec-tive as immune stimulators by inhibiting the cytokine release, co-stimulatory molecules expression, and antigen presentationcapacity (4). It is now clear from many recent studies thatthe TME is adept at inducing PD-1 expression on the infiltrating

1Department of Immunology, Mayo Clinic Rochester, Minnesota. 2Department ofImmunology, Mayo Clinic Florida, Jacksonville, Florida. 3The Cancer Vaccinesand Immune Therapies Program, Vaccine and Gene Therapy Institute, Port St.Lucie, Florida. 4Division of Oncology, Mayo Clinic Rochester, Minnesota.5Department of Health Sciences Research, Mayo Clinic Rochester, Minnesota.6Department of Microbiology and Immunology, University of Arkansas forMedical Sciences, Little Rock, Arkansas.

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

Current address for L. Karyampudi: Iovance Biotherapeutics, Tampa, Florida.

Corresponding Author: Keith L. Knutson, Mayo Clinic, 4500 San Pablo Road,Jacksonville, FL 32224. Phone: 507-250-2935; Fax: 904-953-6233; E-mail:knutson.keith@mayo.edu.

doi: 10.1158/0008-5472.CAN-17-0740

�2017 American Association for Cancer Research.

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immune effector cells, rendering them ineffective. The identifica-tion of TME-associated factors and mechanisms responsible forthe unique regulation of PD-1 expression on infiltrating immunecells remains underdeveloped.

As we and others have shown, ovarian cancer creates a pro-nounced immune suppressive TME (17) and of many factors, thecytokine IL10 has been correlated with volume of ascites, poorsurvival, and relapse (18–20). In the ID8murinemodel of ovariancancer, we have seen that IL10 is present at high levels in both theblood serum and ascites. Additionally, the ascites and tumor-infiltrating immune cells, including the TIDCs, express very highlevels of PD-1 compared with their peripheral counterparts (4).Given the immune regulatory role of IL10 and its correlation withdisease severity and poor prognosis in ovarian cancer, we soughtto explore if IL10 induces PD-1 expression on the TIDCs. Ourresults show that IL10 treatment of TIDCs results in increasedexpression of PD-1 in a STAT3-dependent manner. Antibody-mediated blockade of PD-1 on TIDCs subsequently leads toincreased release of IL10 into theTMEof treatedmice.Coblockadeof both PD-1 and IL10 results in significant enhancement insurvival of tumor-bearing mice, reduced tumor burden, andaugmented adaptive antitumor immunity.

Materials and MethodsAnimals

Four- to 12-week-old female C57BL/6J (B/6J) mice, obtainedfrom an in-house breeding program or from The Jackson Labo-ratory (Bar Harbor, ME), were used for experimentation. Animalcare and use was in accordance with the Institutional Animal Careand Use Committee at Mayo Clinic.

Cell linesLeukocytes were cultured in modified RPMI 1640 media

as previously described (4). ID8 tumor cells, obtained fromDr. K. Roby, University of Kansas (Lawrence, KS) in 2005, werederived from immortalized ovarian epithelial cells generated byrepeated passage in culture and were grown in complete DMEMmedia (4). Theywere last authenticated asmouse origin by IDEXXBioResearch in 2014. ID8 cells used in the described experimentswere used within 12 passages upon receipt.

Tumor implantationID8 tumor cells (5� 106 cells) were injected i.p. at a volume of

500 mL in PBS. Tumor and ascites were harvested from the micewhen moribund, usually 50 to 100 days following challenge. Forcomparison of tumor burden, the weight of omentum was mea-sured from tumor-bearing mice as a surrogate. This was done toreduce variability associated with harvesting diffuse nodulesscattered throughout the peritoneal cavity. We found in prelim-inary studies that the weight of the tumor bearing greater omen-tum is linearly proportional to the total tumor burden (R2¼ 0.81,P < 0.0001, n ¼ 56). In order to more accurately represent tumorburden, theweight of omentumof age-matched healthymice wassubtracted from the omentum of the tumor-bearing mice.

In vivo PD-1 and IL10 blockadeMice were inoculated with ID8 cells and on day 25 received

either 200 mg hamster IgG (Jackson ImmunoResearch Laborato-ries) or 200mgG4 clone PD-1–blockingmonoclonal antibody i.p.as described (21, 22). Mice were treated until moribund up to 11

treatments (8–11 times up to 7 weeks). IL10-neutralizingantibody (BioLegend) and IL10R antagonist antibody (BD Phar-mingen)were injected i.p. alternating between the two antibodiesat a concentration of 100 mg and 200 mg per mouse, respectively.The same amount of isotype matched antibodies (Rat IgG1k orRat IgG1c) from the respective suppliers was injected in thecontrol groups. Tumor and ascites were harvested as necessarywhen mice were moribund.

Leukocyte isolation and cultureLeukocytes fromascites of tumor-bearingmice were isolated by

discontinuous Ficoll gradient as described previously (23).Tumor-infiltrating leukocytes (TIL) were harvested by mincingthe tumor through a 70-mm nylon cell strainer and separationwith a discontinuous Ficoll gradient. From single-cell suspen-sions,DCsweremagnetically isolated basedon the cells of interestusing CD11c microbeads obtained from Miltenyi Biotec. Na€�vemouse leukocytes were obtained from BL/6J mice spleens bygrinding the spleen through a 70-mm nylon cell strainer. Thesplenocytes were centrifuged at 3,000 rpm for 10 minutes andresuspended in 4 mL (ammonium–chloride–potassium) ACKbuffer to lyse red blood cells. The remaining cells were thenwashed and resuspended in culturemedia. Bonemarrow–deriveddendritic cells (BMDC) were obtained from BL/6J by flushingfemurs and tibias with media and centrifuging cells at 1,500 rpmfor 10 minutes, followed by ACK treatment. Cells wereresuspended with media containing 10 ng/mL mGM-CSF and1 ng/mL mIL4 and plated into 6-well plates. Culture media waschanged with fresh media containing 10 ng/mL mGM-CSF and1 ng/mL mIL4 at 48 and 96 hours. The cells were matured with40 ng/mL TNFa on day 5 of cell culture. After 2 additional days,the cells were washed with PBS, resuspended in fresh media andcytokines (VEGF-A, Genway Biotech; IL4, Peprotech); IL10,Peprotech; IL12, Peprotech) or control vehicle were added torespective wells.

Flow cytometryCell-surface molecule staining and flow cytometry were

done essentially as previously described on whole populationsand/or enriched populations (21, 22). For flow cytometricanalysis, a similar number of events, usually 20,000 to200,000 were collected for all groups. Antibodies against CD3,CD8, CD11c, F4/80, PD-1, CD45, PD-L1, PD-L2, CD40, CD27,CD69, CD80, and CD86 were purchased from eBioscience.Antibodies against CD4 and CD25 were purchased from BDPharmingen. Appropriate isotype-matched nonspecific antibo-dies were used as controls.

STAT3 inhibition and siRNA knockdownFor STAT3 inhibition, STAT3 Inhibitor VI (InSolution Calbio-

chem) was used at 150 mmol/L. IL10 was added to the respectivewells 1 hour following the addition of the STAT3 inhibitor. STAT3siRNAs (siRNA I and siRNA II) and the control siRNA werepurchased from Cell Signaling Technologies and were used at aconcentration of 150 nmol/L. Mission siRNA transfection reagent(Sigma Aldrich) was used as transfection agent. SiRNA-mediatedreduction in STAT3 protein was confirmed using Western blotanalysis after 48 or 72 hours. Forty-eight hours after additionof siRNA, cells were either left untreated or treated with IL10

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(10 ng/mL) for an additional 48 hours. Cells were then stained forflow cytometric analysis.

Western blotBMDCs in respective experiments were lysed in lysis buffer A

(150mmol/LNaCl, 20mmol/L Tris Cl (pH7.4), 5mmol/L EDTA,1 mmol/L CaCl2, 10 mmol/L NaF, 1% Triton X 100, 5% glycerol,HALT protease/phosphatase inhibitor (Thermo Scientific). Pro-tein (10mg)was loaded and separated by SDS-PAGE and analyzedby immunoblotting for PD-1 (Novus Biologicals, AF1021),STAT3 (79D7) and p-STAT3 (D3A7; Cell Signaling Technology),and b-actin (LI-COR). Corresponding anti-goat, anti-mouse, andanti-rabbit secondary antibodies conjugated to IRDyes were pur-chased from LI-COR. Signal was detected using LI-COR OdysseyImaging System.

RT-PCRRNA was isolated from purified TIDCs using Qiagen RNeasy

Plus Mini Kit (Qiagen). First-strand cDNA was synthesized usingSuperscript First-Strand Synthesis System III (Invitrogen). PD-1–specific quantitative PCR was performed using the PD-1(NM_008798.2) primer/probe set (Mm01285676_m1) pur-chased from ThermoFisher. GAPDH was used as internal controlusing the primer/probe set (Mm99999915_g1) from Thermo-Fischer. IL10 (NM_010548.2) specific PCR amplification wasperformed using the following primers: forward primer 50-ATGCCTGGCTCAGCACTGCT-30, reverse primer 50-CATT-CATGGCCTTGTAGACACCTTGG-30. b-Actin (NM_007393.5)was used as a control. The following primers were used for b-actinspecific PCR amplification: Murine forward primer 50-GTCCCTCACCCTCCCAAAAG-30, murine reverse primer 50-GCTGCCTCAACACCTCAAC CC-30. Bands were resolved by run-ning PCR products on a 1% agarose gel.

Cytokine ELISATIDCs or BMDCs were treated with 10 mg/mL anti–PD-1

antibody (G4 clone), 10 mg/mL isotype control IgG, or leftuntreated for 24 or 48 hours, after which the supernatants werecollected for IL10 and soluble PD-L1 assessments. TIDCs, serum,and ascites samples were also collected for IL10 and PD-L1assessments. To measure mouse IL10, a Ready-Set-Go ELISA kitfrom eBioscience was used according to the manufacturer's pro-tocol. Soluble PD-L1 ELISA was performed on the cell culturesupernatants according to the manufacturer's protocol (MyBioSource).

FRa antibody ELISAELISA plates were coated in bicarbonate coating buffer with

recombinant mouse FRa (R&D Systems) at a concentration of 1mg/mL. Wells that did not receive any serum were used to deter-mine the background. Serial dilutions of purifiedmouse IgGwereused as a standard curve to convert the O.D. reading to a con-centration of antigen-specific IgG. After overnight incubation at4C, the plates were washed with 0.05% Tween in PBS, blockedwith 1% BSA in PBS for 1 hour at room temperature, washedagain, and then serum samples were added. Plates were thenincubated overnight at 4�C followed by wash with PBS/Tween.Anti-mouse HRP secondary Ab was then added to each well andincubated for 1 hour at room temperature before washing anddeveloping with TMB substrate. OD was read using an ELISAreader after reaction was stopped by reducing the pH of thereactions with acid.

Statistical analysisStatistical analysis was performed using GraphPad Prism ver-

sion 4.00 (GraphPad; http://www.graphpad.com). The Studentt test, Mann–Whitney U test, or two-way ANOVA test was per-formed to determine statistically significant difference. A P value�0.05 was considered statistically significant.

ResultsIL10 increases the expression of PD-1 and its ligand PD-Ll onovarian TIDCs

Our recent studies have shown that PD-1, which has beenprimarily studied in T cells, is also expressed on the TIDCs (4).In the present study, we wanted to determine what factors (e.g.,cytokines), commonly found in tumor microenvironment, leadto the increased expression of PD-1 on the TIDCs. BecauseTIDCs already have upregulated PD-1 and in general aredifficult to access, we developed an in vitro model usingBMDCs, which consistently have a much lower level expressionof PD-1 relative to TIDCs (4). TNFa-activated BMDCs weretreated with a variety of well-described immunologic agonists(IL10, 10 ng/mL; IL12, 20 ng/mL; VEGF-A, 25 ng/mL; poly:IC, 50 mg/mL; CpG, 50 mg/mL; or IL4, 5 ng/mL) for 48 hoursafter maturation, followed by flow cytometric analysis of PD-1expression. As shown in Fig. 1A, of the agents tested, only IL10was able to induce significantly elevated expression of PD-1 onBMDCs. IL10 dose assessments (Fig. 1B) showed dose depen-dency with maximal effective concentration of 5.1 � 1.3 ng/mL(n ¼ 18). Fig. 1C shows a flow cytometry histogram of PD-1expression on BMDCs upon treatment with 10 ng/mL Il10.Time course experiments showed PD-1 was maximally upre-gulated at 48 hours following exposure to IL10 (10 ng/mL;Fig. 1D). To determine the validity of the BMDC, we also observedthe effects of IL10 on expression of PD-1 on the surface of murineovarian TIDCs derived from ascites. As shown in Fig. 1E and F,while TIDCs express high levels of PD-1, treatment with IL10 for48 hours led to a further increase in PD-1 expression by approx-imately double. Increased expression of PD-1 induced by IL10wasdue to augmented pdcd1 gene expression, which resulted inincreased total cellular levels of PD-1 protein (Fig. 1G).

We previously showed that PD-1 maintains an immune sup-pressive phenotype of TIDCs and that a primarymediator of T cellsuppression is TIDC-expressed PD-L1 (4, 24). Thus, as a readoutfor immune suppressive potential, we also analyzed PD-L1expression following IL10 treatment of BMDCs. As shownin Fig. 2H and I, IL10 also led to an increased expression ofPD-L1 on the surface. We also observed that IL10 treatment ofBMDCs induced the release of soluble PD-L1 (sPD-L1) by thesecells (Fig. 1J). In contrast, DCs express only low levels of PD-L2,which was not increased following treatment with IL10 (Fig. 1Kand L). The effects of IL6 were also examined in comparison withIL10 because both cytokines have common proximal signalingelements (e.g., JAK-STAT3; ref. 25). IL6 was able to induce PD-1expression in BMDCs but was not nearly as effective as IL10(Supplementary Fig. S1A). In fact, the effect of IL6 was biphasic,peaking at 30ng/mLand then ineffective at higher concentrations.In contrast, IL6 appeared to be more efficient at driving PD-1expression on CD4 T cells during T-cell activation, whereas IL10had no effect (Supplementary Fig. S1B). Similar effects wereobserved with CD8 T cells, although much less pronounced(Supplementary Fig. S1C). Thus, IL10, which is typically at

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high levels in the ovarian cancer TME, is a key driver of PD-1expression on TIDCs.

IL10-mediated increase in PD-1 on the surface of DCs is STAT3dependent

Various transcription factors have been implicated in eitherpromoting or blocking PD-1 expression primarily in T cells(26, 27). We found that pSTAT3 and STAT3 levels were increasedin the DCs isolated from ascites of ID8 ovarian tumor–bearingmice and correlated with increased PD-1 expression (Fig. 2A).Because signaling emanating from IL10 receptor involves theactivation of STAT3 molecules via tyrosine phosphorylation,subsequent dimerization, and nuclear translocation, resulting in

binding to the DNA and gene transcription, we next asked ifSTAT3 activity has a role in IL10-mediated upregulation ofPD-1 expression on DCs. BMDCs in this experiment weretreated with STAT3 inhibitor (which prevents the dimerizationand hence the nuclear translocation of STAT3) 1 hour prior toaddition of IL10. As shown in Fig. 2B and C, STAT3 inhibitioncompletely reversed the IL10-mediated increase in expressionof PD-1 on the BMDCs. In a separate approach, we suppressedSTAT3 in the BMDCs with siRNA (Fig. 2D) prior to the additionof IL10, which resulted in a decrease in PD-1 expression (Fig. 2Eand F). These results are consistent with the recent findings thatimplicate activated STAT3 as an important transcription factorin PD-1 expression (28).

Figure 1.

IL10 increases the expression of PD-1 and its ligand PD-Ll on ovarian DCs. A, Mean fluorescence intensity (MFI) of PD-1 staining on BMDCs following 48 hoursof exposure to cytokines and TLR agonists. Each bar represents mean � SEM (n ¼ 3). B, Line graph of mean � SEM (n ¼ 3) relative MFI (rMFI) values forPD-1 expression on BMDCs treated for 48 hourswith increasing concentrations of IL10.C,A representative histogram of PD-1 expression on BMDCs treated with IL10.D, A line graph of the MFI values for PD-1 expression on BMDCs treated over a time course with 10 ng/ml IL10, control vehicle (NT), or anti-IL10–neutralizingantibody. E,Themean� SEM (n¼ 3) rMFI values for PD-1 on ID8-derived TIDCs, obtained fromascites, treated or untreatedwith IL10 for 48 hours.F,A representativehistogram of data from E. G, Photograph of representative Western blot (top) and PCR (bottom) of PD-1 protein and mRNA levels, respectively, in BMDCS.H, The mean � SEM (n ¼ 3) rMFI values of surface-bound PD-L1 expression on BMDCs treated as above. I, A representative histogram for H. J, Mean � SEM(n ¼ 3) levels of soluble PD-L1 (sPD-L1) in supernatants of BMDCs either untreated or treated with IL10. K, Mean � SEM (n ¼ 3) rMFI values of surface-boundPD-L2 expression on BMDCs treated as above. L, A representative histogram for K. Filled histogram, isotype stain; solid line, untreated (NT) DCs; dashed lines,IL10-treated DCs. P values were calculated with a Student paired t test. All data are representative of three individual experiments with similar results.

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PD-1blockade results in compensatory release of IL10byTIDCsPD-1 blockade has been shown to be therapeutic in mouse

models as well as in humans for certain types of cancers, yet amajority of patients among various tumor types do not benefit(29). Although the response of T and B cells to PD-1 therapycontinues to be extensively explored, the effects of PD-1 blockadeon DCs are not well established (21). We have shown that TIDCsrespond to PD-1 blockade by increasing costimulatory moleculeand MHC class I expression as well as increasing antigen presen-tation, the effects that were primarily dependent on release ofinhibition on the NFkB pathway (21). Here, we show thatdisruption of the PD-1–PD-L1 axis on TIDCs using PD-1 blockingor PD-L1 blocking antibody leads to increased release of theimmune regulatory cytokine IL10 (Fig. 3A). We also show thatPD-1 blockade on TIDCs leads to an increase of IL10 mRNA,suggesting that the increased production of IL10 upon PD-1blockade is not merely due to enhanced release of preexistingintracellular stores of IL10 (Fig. 3B). The effect of different con-centrations of PD-1 blocking antibody on IL10 release by TIDCswas also measured. As shown in Fig. 3C, progressive increases inthe concentration of the blocking antibody beyond 10 mg/mL didnot progressively increase the production of IL10. In all theexperiments onward, 10 mg/mL antibody was used in the in vitroexperiments. From time course experiments, a significant differ-ence in IL10 release was detected as early as 7 hours (Fig. 3D). Inthe ID8 tumor–bearing mice, a significant increase in the IL10

levels was detected both in the blood serum and ascites of micethat were treatedwith PD-1 blocking antibody comparedwith theuntreated ones (Fig. 3E).

The effect of PD-1 blockade on BMDCswas also evaluated. PD-1þBMDCs (BMDCspost treatmentwith IL10 as shown in Fig. 1C)were treated with PD-1 blocking Ab for 24 hours. PD-1 blockadeon these cells also led to significantly increased release of IL10 (Fig.3F). In order to check if IL10, released upon PD-1 blockade, can inturn lead to increased expressionof PD-1on surface of BMDCs,wecollected the cell culture supernatants from the BMDCs treated asin Fig. 3F. Cell culture supernatants were then either left untreatedor pretreated with IL10-neutralizing Ab before adding to freshBMDC cultures. As shown in Fig. 3G and H, IL10 preneutraliza-tion prevented the increase in PD-1 expression on BMDCs treatedwith conditioned media from BMDCs treated with anti–PD-1.This shows that PD-1 and IL10 form a feedback loop that ampli-fies upon PD-1 blockade. These results identify a mechanism inDCs whereby the blockade of PD-1 on these cells leads to acompensatory release of IL10, hence potentially maintaining andfostering the suppressive environment despite PD-1 blockade.

Combination treatment with PD-1 blocking Ab and IL10neutralization/IL10R blockade reduces tumor burden andenhances survival in tumor-bearing mice

Given the complex interdependent interaction between IL10and PD-1 that we observed in the above studies and their role in

Figure 2.

IL10-mediated increase in PD-1expression on DCs is STAT3dependent. A, RepresentativeWestern blot of phospho-STAT3(p-stat3), STAT3, PD-1, and b-actinon lysates from CD11cþ DCs isolatedfrom spleen and ascites of ID8tumor–bearingmice.BandC,Thebargraph (� SEM, N ¼ 3; B) andrepresentative histograms (C) ofmean fluorescence intensity (MFI)values of PD-1 on BMDCs eithertreated with IL10 alone, STAT3inhibitor alone, or STAT3 inhibitorfollowed by IL10 for 48 hours. Filledhistogram, isotype stain; solid darkline, untreated; solid gray line, STAT3inhibitor treated; dotted line, IL10 þSTAT3 inhibitor treated; dashed line,IL10-treated BMDCs. D, Therepresentative Western blot (left) ofSTAT3 on lysates from STAT3 siRNAtreated BMDCs. E, The bar graph(� SEM, N ¼ 2–5) and F, therepresentative histogram of PD-1expression (MFI) on BMDCs eithertreated or untreated with IL10 postsiRNA transfection. Filled histogram,isotype stain; solid dark line, CtrlsiRNA treated; dotted dark line, CtrlsiRNA þ IL10 treated; solid gray line,STAT3 siRNA treated; dotted grayline, STAT3 siRNA þ IL10–treatedBMDCs. The P valueswere calculatedwith a Student t test. All data arerepresentative of three individualexperiments with similar results. n.s.,nonsignificant.

PD-1 Feedback Loop

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immune suppression in tumor microenvironment, we looked tosee if combining these two therapies (PD-1 and IL10/IL10Rblockade) has any effect in the generationof antitumor immunity.As shown in Fig. 4A, anti–PD-1 or anti-IL10(R) as monotherapyhad minimal effect on the overall survival or tumor burden; butthe combination of these significantly delayed tumor growth andenhanced the survival of the tumor-bearing mice. Combinationtreatment also significantly reduced the tumor burden in themice(Fig. 4B) compared with the control group.

Combination treatment enhances the infiltration of immuneeffectors in the ascites of tumor-bearing mice

In order to determine if the combination treatment modulatedthe quantity and quality of infiltrating immune cells, we lookedfor T- and B-cell infiltrates in the ascites of tumor-bearingmice. Asshown in Fig. 5A, there was a significant increase in the totalnumber of T cells in the ascites of tumor-bearing mice that weretreated with the PD-1 blocking antibody alone or the combina-tion regimen of PD-1 blocking and IL10(R)-neutralizing antibo-dies. A slight increase in the fraction of CD8þ T cells and a slightdecrease in the frequency of CD4þ T cells among the total CD3þ Tcells compared with other treatment groups were observed in the

ascites of mice from the combination treatment group (Fig. 5Band C). Importantly and consistent with an inhibitory role ofIL10, we observed a robust increase in both the numbers ofactivated CD4þ and CD8þ T cells in the combination treatmentgroup (Figs. 5D and E), whereas those levels were not differentfrom controls in the monotherapy groups.

Similar to the changes in T-cell numbers in the ascites, asignificant increase in the frequency of B cells (CD19þB220þ)was observed in the ascites of tumor-bearing mice that weretreated with PD-1 blocking antibodies alone or the combina-tion regimen (Fig. 6A and B). Thus, we sought to determine iftumor antigen-specific antibody response was enhanced in thecombination treatment group. Folate receptor alpha (FRa) is awell-known tumor antigen that has been shown to be over-expressed in various epithelial derived cancer cells, includingovarian cancer, and deemed capable of driving oncogenictransformation (30). In order to determine if an antigen-spe-cific antibody response was generated, the sera from differenttreatment groups were assayed for FRa-specific antibodies. Asshown in Fig. 6C, there were significantly increased levels ofcirculating FRa-specific antibodies in the serum of mice fromcombination treatment group. Also, increased levels of FRa-

Figure 3.

PD-1 blockade on DCs enhances the release of IL10, creating a feedback loop. A, Bar graph of mean (� SEM; N ¼ 3 replicates) IL10 concentrations (pg/mL)on the culture supernatants from the TIDCs treated for 24 hours with irrelevant ab (Iso), PD-1 blocking ab (a-PD-1), or PD-L1 blocking ab (a-PD-L1).B, Representative photo of an RT-PCR reaction evaluating both IL10 and b-actin expression in TIDCs that were either treatedwith irrelevant ab (Iso) or a-PD-1 for 24hours in vitro.C, Linegraphof IL10 concentrations (pg/mL) in the culture supernatants from theTIDCs treated for 24hourswith increasing concentrations of anti–PD-1blocking antibody. Each data point is the mean � SEM of triplicate measurements. D, Line graph of IL10 concentrations (pg/mL) in culture supernatants of TIDCscultured in vitro over a time course. Each data point is the mean� SEM of triplicate measurements. E, Bar graph of mean IL10 concentrations (pg/mL;� SEM; n¼ 5)in ascites and blood serum of tumor-bearing mice that were either untreated or treated with PD-1–blocking Ab as described in Materials and Methods. F, Bargraph of mean (� SEM; N ¼ 3 replicates) IL10 concentrations (pg/mL) on the culture supernatants from the IL10-treated BMDCs (PD-1þ) that were treatedfor 24hours as inA.G andH,Representative histograms (G) andbar graph (�SEM,N¼ 3) ofmeanfluorescence intensity (MFI) values of PD-1 (H) onBMDCs thatweretreated with conditioned media (culture supernatants) from BMDCs that were either untreated or treated with isotype Ab or PD-1–blocking Ab as in B. Culturesupernatants were then either pretreated with IL10-neutralizing Ab or untreated before adding on to the fresh cultures of BMDCs. n.s., nonsignificant.

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specific antibodies were observed in ascites of mice from thecombination treatment group compared with the other groups(Fig. 6D). These results demonstrate that the combination ofPD-1 blockade and IL10(R) neutralization modulates the adap-tive immunity by enhancing the infiltration of activated andantigen-specific immune effectors into the TME.

Combination treatment leads to a decrease in infiltration ofMDSCs in the ascites

Immune suppressive cells present in the TME are adept atsuppressing the effector responses against the tumor and hencepromote tumor escape and progression. Myeloid-derived sup-pressor cells (MDSC) are a subset of such suppressive cells that

Figure 4.

Combination of PD-1 blockade andIL10 neutralization reduces tumorburden and enhances survival oftumor-bearing mice. A, Kaplan–Meierplot of ID8 tumor–bearing mice(N ¼ 12-16) that were treatedintraperitoneally with respectiveantibodies starting at day 25 posttumor implantation. B, Mean tumorweights in grams (�SEM, N ¼ 5–6),from different treatment groups, asmeasured at the time of ascitesharvest.

Figure 5.

Combination treatment enhances the infiltration of activated T cells in the ascites of tumor-bearing mice. A, Mean (�SEM, N ¼ 6) number of T cellsper 1 � 105 leukocytes in the ascites of tumor-bearing mice from different treatment groups. B and C, Mean (�SEM, N ¼ 4) percentages of CD8þ T cells (B) andCD4þ T cells (C) among total CD3 cells from the ascites of tumor-bearing mice from treatment groups. D and E, Mean (�SEM, N ¼ 5–7) percentagesof CD69þ cells among CD8þ T cells (D) and CD4þ T cells (E) from the ascites of tumor-bearing mice from treatment groups.

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have been shown to play a major role in immune suppression inthe ovarian cancer microenvironment (31). In a murine model ofbreast cancer, we have shown that a combination regimen ofpeptide vaccine (MHC class I binding immunogenic peptidesfrom rat neu (neu), legumain, and b-catenin targeting tumor cells,cancer stem cells, and tumor-associated macrophages) and PD-1blocking antibody leads to a decrease in the frequency of MDSCsin the tumor (22, 32). In this study, we found that although anti–PD-1 treatment alone did not change the frequency of MDSCs inthe ascites, anti-IL10, and the combination treatment significantlyreduced the frequency of MDSCs in the ascites of tumor-bearingmice (Fig. 7A and B).

DiscussionIn the present work, we have identified the TME-associated

cytokine IL10 as a critical regulator of the PD-1–PD-L1 axis in theTME. First, we found that IL10, a cytokine whose expression inincreased in the TME of several cancers, is capable of increasingPD-1 surface expression in a STAT-3–dependent manner. Second,

we found that blockade of PD-1,with an antagonisticmonoclonalantibody, on DCs led to increased release of IL10 by DC, which isfurther capable of increasing PD-1 expression on DCs, hencecreating a vicious immune escape loop. Lastly, we show thatPD-1 blockade and IL10 signal antagonism as a combinationtherapy, using blocking antibodies, enhances the antitumor T-and B-cell immunity in ovarian cancer-bearing mice, leading tosignificantly improved survival and decrease in tumor burden.

Although the critical role of DCs in initiating and shaping theresponse of immune system against pathogens and malignanciesis widely acknowledged, the contribution of this subset, amongtumor-infiltrating immune cells, in cancer malignancies remainsobscure (33). Despite comprising up to 40% of infiltratingimmune cells in the tumors, studies of the role of DCs in theTME have been conflicting. Recent studies have shown that DCsmay switch phenotype from immune stimulatory to immunesuppressive along the course of disease progression and PD-1expression among TIDCs potentially contributes to this processby paralyzing the cells in a persistent immune suppressive state(4, 16). Various cytokines factors have been shown to upregulate

Figure 6.

Combination treatment enhancesthe antigen-specific antibodyresponse in tumor-bearing mice. A,Representative dot plot. B, Bargraph of the mean frequency (�SEM,N ¼ 4–5 replicates) of CD19þB220þ Bcells among CD45þ cells from theascites of tumor-bearing mice.C and D, Mean levels (ng/mL) of FRa-specific antibodies measured in theserum (�SEM,N¼ 3 replicates; C) andascites (�SEM, N ¼ 7 replicates; D),respectively, ofmice from thedifferenttreatment groups.

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PD-1 expression on T cells, including IFNa, IFNg , andmost of theg chain cytokines (26, 34–37). To the best of our knowledge, thepresent work is the first demonstration that cytokines, particularlyIL10, is able to drive increased expression on DCs derived fromeither BMDCs or TIDCs. IL10 is a cytokine whose levels have beenshown tobe elevated in the TMEandbloodof tumor-bearingmiceas well as ovarian cancer patients (18, 38–40). Despite elevatedlevels of IL10 in sera of both tumor-bearing mice and humans,PD-1 is selectively expressedon the TIDCs andnot their peripheralDCs (4, 21). A potential reason for this observation is that IL10,while elevated in sera, may not be at the levels needed to activatepdcd1 gene expression. Indeed, our recent work shows that whileelevated, IL10 levels reach only about amean of 0.65 ng/mL in the

blood of patients, which may not be enough to mediate PD-1expression and/or systemic immune suppression (38). The IL10-mediated increase in PD-1 expression on DCs was dependent onSTAT-3 activity, as use of STAT-3 inhibitor or siRNA mediatedknockdown of STAT3 both resulted in inability of IL10 to increasePD-1 expression on DCs. These results support the previousfindings implicating STAT-3 in mediating PD-1 expression onCD3/CD28 stimulated T cells upon IL6 treatment (28).

A unique finding from our studies of PD-1 biology on TIDC isthat the blockade of PD-1 results in augmented cytokine releaseincluding high level release of IL10 (4). Thus, the observation thatIL10 drives PD-1 expression and that blockade of PD-1 results inhighly elevated IL10 production and release reveals a potentiallyrapid acting compensatory or adaptive immune regulatory loopthat may foster escape from anti–PD-1 monotherapy. Indeed, wefind that PD-1 blockade-induced IL10 induces further PD-1expression on the DCs favoring continued functional paralysis.Furthermore, IL10 may also result in maintaining the immunesuppressive phenotype of ovarian TIDCs by upregulating surfacePD-L1 expression, which as we previously showed is dominantmeans that TIDCs are able to mediate immune suppression inthe ovarian cancer microenvironment (24). Although PD-1 andPD-L1bind eachother, they eachhaveother ligands theybind (1).In this regard, future studies should examine if PD-L1 blockadein vivo, with or without IL10(R) blockade, results in similartherapeutic efficacy in ovarian cancer as seen with the PD-1blockade. Lastly, another potentially important finding is thatIL10 activated release of soluble PD-L1 (sPD-L1), a form of PD-L1that induces apoptosis of T cells and has been repeatedly shownto be associated with poor outcome in patients with advancedcancers (41–45).

In addition to increased PD-L1 expression, the increased IL10can also act on many if not all immune cells, and blunt theireffector responses (46). Interestingly, Sun and colleagues showedthat blockade of PD-1 signaling led to an increase in IL10Rexpression on tumor antigen-specific CD8þ T cells, hence render-ing them increasing susceptible to IL10-mediated inhibition andsuggesting that compensatory mechanisms can also becomeactivated in T cells following PD-1/PD-L1 axis blockade (47).That IL10 has a major role in evasion of immune-mediatedregression of tumor following checkpoint blockade in the presentmodel is demonstrated by the observation that combinationtreatment, blockade of PD-1 and IL10(R) significantly increasedthe survival of tumor-bearing mice and reduced the tumor bur-den. One potentially key finding from the current study is thatalthough PD-1 was largely ineffective alone as a monotherapy itwas able to nearly quadruple the numbers of T cells and doublethe number of B cells in the TME. Despite that, both subsets ofeffector lymphocytes appeared unable to activate as indicated byCD69 and antibody production for T and B cells, respectively,unless the mice also received anti-IL10(R) therapy. This indicatesthat, in this model, IL10 is a major compensatory suppressionmechanism. These observations suggest that while PD-1 blockadealonemay be ineffective as therapy in some tumors (29) owing tothe induction of secondary suppressive mechanisms such as theone driven by IL10, a combination of PD-1 blockade and dis-ruption of IL10/IL10R signaling enhances the endogenous anti-tumor immunity, resulting in improved survival and reducedtumor burden. Although the paucity of intraperitoneal tumortissuemade it difficult to assess for the functional changes in the Tand B cells induced by the combination therapy, it is notable that

Figure 7.

Combination treatment results in decreased infiltration of MDSCs in theascites of tumor-bearing mice. A, Representative dot plots of CD11bþGr1þ

cells in the ascites from different treatment groups. For analysis, live cellswere pregated on CD45þ cells. B, Mean frequency (�SEM, N ¼ 5–7) ofCD11bþGr1þ cells in the ascites of the different treatment groups.

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in our prior breast cancer studies that both Th1 and Th2 responseswere associated with tumor regression in mice treated with thecombination of vaccine and anti–PD-1 (22). These latter resultssuggest that other phenotypes may contribute to regression ifappropriately coordinated with Th1 cell–mediated immuneresponses.

We have previously shown that PD-1 blockade on TIDCs leadsto an increase in IL6 release (21). In the current study, we showthat IL6 is able to induce PD-1 expression onDCs, albeit the effectwas much less pronounced compared with IL10. Althoughnot studied in ovarian cancer, the combination of IL6 and PD-1/PD-L1 blockade has been evaluated in HCC and PDAC, withresults showing synergistic antitumor effects (48, 49). Futurestudies should determine if IL6 blockade combined with PD-1/PD-L1 blockade results in therapeutic efficacy in ovarian cancercomparable with the effects of IL10 and PD-1 blockade.

The increase in FRa-specific IgG may contribute to antitumorefficacy by directly blocking FRa-dependent tumor cell prolifer-ation and indirectly inducing antibody-dependent cell-mediatedcytotoxicity (ADCC) by binding to Fcg receptors on NK cells asshown in a preclinical study of antifolate receptor alpha antibody(50). Although we showed that IL10 is released by TIDCs at highlevels following PD-1 blockade, there are other IL10 sources, suchasmacrophages, Bregs, and Tregs, that could also contribute to theIL10mediated immune suppression (46). Future studies needs toevaluate if these cells, similarly to TIDCs, respond to PD-1 block-ade by increasing IL10 release. IL10 mediates its immune sup-pressive effects both directly suppressing lymphocyte responsesand indirectly by blocking DC functions (51–54). It is notable,however, that IL10 may under conditions have antitumor activityby enhancing the activity of cytotoxic T cells (55–57).

In conclusion, the approval of anti–PD-1 and anti–PD-L1 forthe treatment of several cancers has generated widespread excite-ment in the field of tumor immunotherapy. But anti–PD-1treatment is still suboptimal in various cancer subtypes and hasnot yet been tested in late-stage clinical trials for ovarian cancer. Ina recent clinical study of 20 patients with platinum-resistantovarian cancer, an overall response rate of 15% was achieved(58). Although these results are encouraging, there is still need forimprovements in these therapies.Our results show that anti–PD-1can induce a compensatory production of IL10 byDCs infiltratingthe ascites of ovarian cancer. Given the presence of IL10 in highlevels in the patients with ovarian carcinoma and its role inimmune suppression, our results implicated it in the regulationof PD-1 expression and consequently PD-1 blockade, resulting in

enhanced IL10 production is significant. These results open uppossibilities to explore if such combination therapy is a viableoption in clinical settings for added benefits in patients receivingPD-1–based therapies. Other mechanisms of resistance to PD-1therapy, including induction of other checkpoint molecules, lackof T-cell infiltration, and low tumor mutational load, have alsobeen identified in various tumors and combinatorial approachesto target these mechanisms are being explored (59).

Disclosure of Potential Conflicts of InterestM.S. Block reports receiving other commercial research support fromMerck.

No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: P. Lamichhane, B. Shreeder, J. Krempski, E.L. Goode,K.L. KnutsonDevelopment of methodology: P. Lamichhane, L. Karyampudi, K.L. KnutsonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P. Lamichhane, B. Shreeder, D. Bahr, J. Daum,K.R. Kalli, K.L. KnutsonAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P. Lamichhane, L. Karyampudi, B. Shreeder, J. Daum,E.L. Goode, K.L. KnutsonWriting, review, and/or revision of the manuscript: P. Lamichhane,L. Karyampudi, B. Shreeder, J. Daum, K.R. Kalli, E.L. Goode, M.S. Block,M.J. Cannon, K.L. KnutsonAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D. Bahr, M.J. Cannon, K.L. KnutsonStudy supervision: K.L. Knutson

AcknowledgmentsThe authors are grateful for the technical support of Laura Lewis-Tuffin and

the Mayo Clinic Florida Flow Cytometry and Cell Analysis Shared Resource.

Grant SupportThis work was supported by the Minnesota Ovarian Cancer Alliance (K.L.

Knutson and L. Karyampudi), the Fred C. and Katherine B. Andersen Founda-tion (K.L. Knutson and K.R. Kalli), theMarsha Rivkin Center for Ovarian CancerResearch (L. Karyampudi, K.L. Knutson, and M.J. Cannon), Mayo ClinicComprehensive Cancer Center grant P30-CA015083 (R. Diasio), and the MayoClinic Ovarian Cancer SPORE (P50-CA136393 to K.L. Knutson, E.L. Goode, M.S. Block, and M.J. Cannon).

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.

ReceivedMarch23, 2017; revised July 24, 2017; accepted September 27, 2017;published OnlineFirst October 9, 2017.

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Published OnlineFirst October 9, 2017; DOI: 10.1158/0008-5472.CAN-17-0740