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Research Article

IL35 Hinders Endogenous Antitumor T-cellImmunity and Responsiveness to Immunotherapyin Pancreatic CancerBhalchandra Mirlekar1,2, Daniel Michaud2,3, Ryan Searcy1,2, Kevin Greene2,4,and Yuliya Pylayeva-Gupta1,2

Abstract

Although successes in cancer immunotherapy have gener-ated considerable excitement, this form of treatment has beenlargely ineffective in patients with pancreatic ductal adenocar-cinoma (PDA). Mechanisms that contribute to the poor anti-tumor immune response in PDA are not well understood.Here, we demonstrated that cytokine IL35 is amajor immuno-suppressive driver in PDA and potentiates tumor growth viathe suppression of endogenous antitumor T-cell responses.The growth of pancreatic tumors in mice deficient for IL35was significantly reduced. An analysis of tumor-infiltratingimmune cells revealed a role for IL35 in the expansion ofregulatory T cells and the suppression of CD4þ effector T cells.

We also detected a robust increase in both the infiltration andactivation of cytotoxic CD8þ T cells, suggesting that targetingIL35 may be an effective strategy to convert PDA from animmunologically "cold" to "hot" tumor. Although PDA istypically resistant to anti–PD-1 immunotherapy, we demon-strated robust synergistic reduction in tumor growth whenIL35 deficiency was combined with anti–PD-1 treatment.These findings provide new insight into the function of IL35in the pathogenesis of pancreatic cancer and underscore thepotential significance of IL35 as a therapeutic target for use incombination immunotherapy approaches in this deadlymalignancy. Cancer Immunol Res; 6(9); 1014–24. �2018 AACR.

IntroductionPancreatic ductal adenocarcinoma (PDA) carries one of the

worst prognoses of human cancers. It is a disease characterized bylate diagnosis and a poor response to therapy (1–3). The forma-tion of PDA is accompanied by changes in stromal compositionand dampened immune surveillance, which are now recognizedas some of the major drivers in PDA tumor evolution andresistance to therapy (4–8). Components of the PDA tumormicroenvironment (TME) have been described in human patientsamples and mouse models of PDA and include infiltratingleukocytes from both the lymphoid and myeloid lineages(9, 10). Tumor-infiltrating immune cells with known immuno-suppressive capabilities, such as tumor-associated macrophagesand myeloid-derived suppressor cells, as well as regulatoryT cells (Treg) and B cells, play important roles in pancreatictumor growth (11–16). These cells are thought to contribute to

an ineffective initial antitumor response and create barriersfor immunotherapeutic approaches in established tumors,although the mechanisms by which immune cells in the PDATME operate in successful establishment of tumor growth remainto be fully understood.

We previously demonstrated that interleukin-35 (IL35)-producing regulatory B cells (Breg) constitute part of the PDATME (13). Although B cells can support tumor growth in anIL35-dependent manner, the mechanism of IL35 function inpancreatic cancer remains unclear. IL35 belongs to the IL12family of cytokines and is formed by the heterodimerizationof the subunits p35 and Ebi3 (17). IL35 expression is inducedunder conditions of elevated inflammatory signaling, andstudies in lung cancer, leukemia, lymphoma, colorectal, andpancreatic cancers have documented elevated IL35 and pre-dicted poor patient outcome (18–23). To date, the majorstromal sources of IL35 were shown to be T cells (predomi-nantly Tregs) and B cells (13, 24–28). The mechanisms thatIL35 acts upon in cancer have not been well established. In amodel of melanoma, IL35 promoted tumor growth viaenhanced accumulation of myeloid-derived suppressor cellsand increased angiogenesis (29), whereas in a study in mela-noma and colon cancer models, IL35 expression in Tregs waslinked to the exhaustion of CD8þ effector T cells (30). It is likelythat depending on disease setting, IL35 may affect functionalityof diverse immune cell populations (17, 31).

In this study, we demonstrated that IL35 promotes pancre-atic tumor growth by antagonizing endogenous antitumorT-cell immune responses. Using established mouse models ofPDA combined with deficiency for IL35, we demonstratedacute dependency of PDA growth on IL35. IL35 enhanced theTreg response and downregulated endogenous effector T-cell

1Department of Genetics, The University of North Carolina at Chapel Hill Schoolof Medicine, Chapel Hill, North Carolina. 2The Lineberger Comprehensive CancerCenter, TheUniversity ofNorthCarolina atChapelHill School ofMedicine, ChapelHill, North Carolina. 3Department of Cell Biology and Physiology, The Universityof North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina.4Department of Pathology, TheUniversity ofNorth Carolina at Chapel Hill Schoolof Medicine, Chapel Hill, North Carolina.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

CorrespondingAuthor:Yuliya Pylayeva-Gupta, TheUniversity ofNorth Carolinaat Chapel Hill, 450 West Drive, Chapel Hill, NC 27599. Phone: 919-962-8296;Fax: 919-966-8212; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-17-0710

�2018 American Association for Cancer Research.

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responses. We also found that deficiency in IL35 renders PDAsensitive to anti–PD-1 immunotherapy, suggesting that IL35expression in PDA confers resistance not only to endogenousantitumor responses but also to current immunotherapeuticapproaches.

Materials and MethodsAnimal models

All mouse protocols were reviewed and approved by theInstitutional Animal Care and Use Committee of the Universityof North Carolina at Chapel Hill. Animals were maintained in aspecific pathogen-free facility. Six- to 8-week-old wild-type (WT)C57Bl/6J mice were purchased from The Charles River Labora-tories (stock #027). Six- to 8-week-old p40–/– (stock #002693),Il10–/– (stock #002251), p35–/– (stock #002692), Il27ra–/– (stock#018078), and Ebi3–/– (stock #008691) mouse strains werepurchased from The Jackson Laboratory. All of these strains wereon C57Bl/6J background. Both male and female mice were usedfor orthotopic injections of PDA cells. Briefly, for injection ofcancer cells into the pancreas, mice were anesthetized using aketamine (100 mg/kg)/Xylazine (10 mg/kg) cocktail adminis-tered via intraperitoneal injection. The depth of anesthesia wasconfirmed by verifying an absence of response to toe pinch.An incision in the left flank was made, and 75,000 KPC cells inice-cold PBS mixed at 1:1 dilution with Matrigel (#354234,Corning) in a volume of 50 mL were injected using a 28-gaugeneedle into a tail of the pancreas. The wound was closed in twolayers, with running 5-0 Vicryl RAPIDE sutures (Ethicon) for thebody wall, and 5-0 PROLENE sutures (Ethicon) for the skin. Allanimals were given the pain reliever buprenorphine (0.1 mg/kg)subcutaneously once, directly after the conclusion of surgical pro-cedure. Mice were euthanized by carbon dioxide–induced narco-sis at 3 weeks after injection of cancer cells. Tumors were weighedandmeasured using digital caliper (VWR) at the endpoint. Part ofthe tumor tissue was fixed in 10% buffered formalin phosphate(Fisher) and reserved for histology, and the rest was processed forflow cytometry as described below in the "Lymphocyte isolation"section. Spleens were harvested for flow cytometry analysis as alsodescribed in "Lymphocyte isolation" section. The LSL-KrasG12D/þ,LSL-Trp53R172H/þ, and p48Cre/þ strains have been described pre-viously (32–35). These mouse strains were bred at The CharlesRiver Laboratories for Dr. Bar-Sagi, prior to being transferredto the University of North Carolina at Chapel Hill.

Cell linesMurine PDA cell lines 4662 or 2173were derived from primary

pancreatic tumors of LSL-KrasG12D/þ; LSL-Trp53R172H/þ; p48Cre/þ

(KPC) mice, as reported in Bayne and colleagues (12). KPCcells were a kind gift from Dr. Vonderheide (University ofPennsylvania Perelman School of Medicine, Philadelphia, PA)and were derived from C57Bl6/J background strain (12). GFP-labeled 4662 and 2173 KPC lines were generated as previouslydescribed (11). Cell lines used for implantation studies weretested and confirmed to be mycoplasma free (MycoAlert PlusMycoplasma detection kit, Lonza) and were used at <10 passages.All cell lines were maintained at 37�C and 5% CO2 in completeDMEM, which contained Dulbecco's Modified Eagle medium(Gibco) supplemented with 10% FBS (Sigma), 1% L-glutamine,and gentamicin (50 mg/mL; Thermo Scientific).

ImmunohistochemistryMouse splenic and tumor tissues were fixed in 10% buffered

formalin (Fisher Scientific) for 48 hours and embedded inparaffin at UNC's Animal Histopathology and LaboratoryMedicine Core. Six-micrometer sections were deparaffinized andrehydrated. A solution of 1% hydrogen peroxide (stock of 30%hydrogen peroxide, Sigma) in methanol at room temperature for10 minutes was used to quench endogenous peroxidase activity.Antigen retrieval was done in a 10 mmol/L sodium citrate plus0.05%Tween-20 solution (pH6.1) for 15minutes in amicrowaveoven. Blockingwas performed for 1hour at room temperature in asolution of 10% goat serum, 10 mmol/L Tris–HCl, 0.1 mol/Lmagnesium chloride, 1% BSA, and 0.5% Tween-20. Sectionswere incubated with primary rat anti-CD8a (clone 53-6.7, BDPharmingen) diluted in 2% BSA/PBS (final concentration of2.5 mg/mL) overnight at 4�C. Secondary biotinylated goat anti-rat (Vector Laboratories) was diluted in 2% BSA/PBS (finalconcentration of 3.75 mg/mL) and incubated for 1 hour at roomtemperature. Tertiary ABC solution was prepared according tothe manufacturer's instructions (Vectastain ABC kit, Vector Lab-oratories) and incubated for 45 minutes at room temperature.Sections were developed using a 3,30-diaminobenzidine tetrahy-drochloride kit (DAB peroxidase substrate kit, Vector Laborato-ries). Slides were then counterstained with Harris hematoxylin(Sigma), dehydrated, and mounted with DPX mounting media(Sigma). Slides were scanned using the Aperio ScanScope XT atUNC's Translational Pathology Lab, and slides were examinedand images were generated using ImageScope Viewer (LeicaBiosystems).

Immunofluorescence (IF) on human samplesFor the purposes of analyzing CD8þ T-cell and IL35þ immune

cell infiltration, we examined 11 samples containing PDA lesions.Samples consisted of 5-mm sections that were cut from FFPEblocks provided by the Tissue Pathology Laboratory of the UNCSchool of Medicine and were obtained after informed writtenconsent. This study was conducted in accordance with the Dec-laration of Helsinki. All samples were anonymized prior to beingtransferred to the investigator's laboratory and, therefore, metexempt human subject research criteria. Samples were selected onthe basis of patient diagnosis with PDAC. Triple IF (3-plex IF)stains were carried out in the Leica Bond-Rx fully automatedstaining platform (Leica Biosystems Inc.). All steps were carriedout at room temperature, except for the epitope retrieval, whichwas done at 100�C. Slides were dewaxed in Bond Dewax solution(#AR9222) and hydrated in Bond Wash solution (#AR9590).Epitope retrieval for all targets was done for 20 minutes in Bond-epitope retrieval solution 1, pH6.0 (#AR9661) or solution 2,pH9.0 (#AR9640). The epitope retrieval was followed with 10minutes of endogenous peroxidase blocking using Bond peroxideblocking solution (#DS9800). Each set of primary and secondaryantibody staining was completed sequentially, followed by incu-bation with TSA reagent for 15 minutes. Stained slides were thencounterstainedwithHoechst 33258 (#H3569) andmountedwithProLong Diamond Antifade Mountant (#P36961, Life Technol-ogies). Positive and negative controls (no primary antibody) andsingle stain controls weremade for the 3-plex IF, with one primaryantibody omitted to make sure that cross-reactivity between theantibodies did not occur. The following primary antibodies wereused: CD20 (1:100, clone MJ1, Leica), CD8 (1:100, clone 4B11,Leica), CD4 (1:100, clone 4B12, Leica), Ebi3 (1:100, clone

IL35 Regulates Antitumor Immune Response in PDA

www.aacrjournals.org Cancer Immunol Res; 6(9) September 2018 1015




HPA046635, Sigma), CK (1:500, clone Zo622, Agilent/DAKO),and IL12p35 (1:100, clone HPA001886, Sigma). For secondary:EnVisionþ System HRP, Labeled Polymer, Anti-mouse #K400(1:50, Agilent/DAKO), EnVisionþ SystemHRP, Labeled Polymer,Anti-mouse #K4001 (1:50, Agilent/DAKO), ImmPRESS HRPAnti-Rabbit Polymer #MP-7401-15 (1:50, Vector Labs). CD8þ

T cells in the tumor cell nests (i.e., directly adjacent to or incontact with tumor cells) and Ebi3þ immune cells were countedper 10� field of view (FOV), counting 3 to 6 FOVs per tumorsample (n¼ 11 tumor samples). Average value of Ebi3þ cells/FOVacross all tumor samples was estimated as 20 Ebi3þ cell/FOV.FOVs with the number of Ebi3þ cells < 20 were classified as Ebi3þ

low and FOVs with the number of Ebi3þ cells� 20were classifiedas Ebi3þ high.

Lymphocyte isolationSingle-cell suspensions were prepared from tumors, spleens,

and pancreatic lymph nodes. Spleens were mechanically dis-rupted using a plunger end of a 10 mL syringe and resuspendedin 1% FBS/PBS. For isolation of tumor-infiltrating lymphocytes,tumor tissue was minced into 1 to 2mmpieces and digested withcollagenase IV (1.25 mg/mL; #LS004188, Worthington), 0.1%trypsin inhibitor from soybean (# T9128, Sigma), hyaluronidase(1mg/mL; # LS 002592,Worthington), andDNase I (100 mg/mL;# LS002007, Worthington) in complete DMEM for 30minutes at37�C. Cell suspensions were passed through a 70-mm strainer andresuspended in RPMI media (Gibco). Lymphocytes were isolatedfrom processed tumor tissues by OptiPrep (Sigma) density gra-dient centrifugation.MACS isolation of total lymphocytes (MACSMiltenyi #130-052-301) was performed on the lymphocyte-enriched fraction according to Miltenyi's protocol, and the purityof MACS-enriched B cells and T cells was >85%. Cell sorting usinga BD FACS ARIA II sorter was performed to isolate Bregs, con-ventional B cells (Bcon), and CD4þ and CD8þ T cells, and >95%purity of sorted cells was achieved.

Flow cytometryFor ex vivo stimulation, 100,000 cells were incubated with PMA

(50 ng/mL; Sigma, #P8139) and ionomycin (200 ng/mL; Sigma,#I0634) in thepresence of 1XGolgistopBrefeldinA (Biolegend) incomplete RPMImedium for 5 hours at 37�Cbefore staining. Cellswere washed and blocked with anti-CD16/CD32 (Fc Block, BDBiosciences, 0.1 mg per 100,000 cells) for 5 minutes on ice andthen stained with labeled antibodies against surface markers orcytokines on ice for 30minutes in FACS buffer (PBS with 3% FCSand 0.05% sodium azide). For intracellular staining, cells werefixed, permeabilized, and stained with labeled intracellular anti-bodies using theBD cytofix/cytopermkit (BDBiosciences, cat.No.554714). Intracellular staining for Foxp3 was performed using aFoxp3 staining kit (eBioscience, cat. No. 00-5523). The followingmonoclonal antibodies directed against mouse antigens wereused for flow cytometry: Biolegend—CD45 (clone 30-F11),CD11b (clone M1/70), F4/80 (clone BM8), Ly6G (clone 1A8),Ly6C (clone HK1.4), CD206 (clone C068C2), MHCII (cloneM5/114.15.2), CD3 (clone 17A2), CD4 (clone GK1.5), IFNg(clone XMG1.2), TNFa (clone MP6-XT22), Foxp3 (clone FJK-16S), CD8 (clone 53-6.7), IL12p40 (clone C15.6), IL27p28(clone MM27-7B1), CD19 (clone 6D5), CD45RB (clone C363-16A), CD21 (clone 7E9); eBioscience—IL35p35 (clone4D10p35); R&D Systems—IL35EBi3 (clone 355022); BD Bios-ciences—CD1d (clone 1B1). Viability of cells was determined

by staining with either 7-aminoactinomycin (Biolegend) orLive/Dead Aqua cell stain kit (Life technologies). All sampleswere acquired on LSR II and LSRII Fortessa (BD Bioscience) atUNC Flow Cytometry Core Facility and analyzed by FlowJoversion 10.2 (TreeStar, Inc.).

Anti–PD-1 treatment and CD8þ T-cell depletionMice were orthotopically injected into the pancreas with KPC

cells. On day 7 after injection, mice were intraperitoneally admin-istered either 200 mg anti–PD-1 (Bio X Cell, clone RMP1-14) orIgG control (BioXCell, clone 2A3)diluted in 100mLof sterile PBS.Three doses of antibody were given in total, on days 7, 9, and 11after injection of KPC cells. For CD8þ T-cell depletion studies,200 mg of anti-CD8 (Bio X Cell, clone 53-6.7) or an IgG isotypecontrol (Bio X Cell, clone 2A3) diluted in 100 mL of sterile PBSwere administered intraperitoneally daily starting 3 days priorto tumor cell injection and twice a week after tumor cell injec-tion. Efficiency of CD8þ T-cell depletion was assessed by flowcytometry.

Statistical analysisAt least 9 to 21mice were used in each group, with aminimum

of 3mice in each group per experiment, and the experiments wererepeated a minimum 3 times to validate reproducibility. Groupmeans were compared with Student t tests. Significance in varia-tions between twogroupswas determinedby anunpaired Studentt test (two-tailed). Statistical analyses were performed usingGraphPad Prism software (version 7.0d), and data are presentedas mean � SEM. P < 0.05 was considered statistically significant.

ResultsIL35 is expressed by multiple immune cell subsets andpromotes PDA growth

To understand the functional significance of IL35 in pancreatictumor growth, we first investigated the source of IL35 expressionin the tumor microenvironment. To do this, we used well-characterized mouse models of PDA: a spontaneous model ofpancreatic tumorigenesis KrasG12D/þ;p48Cre/þ (KC), as well asKrasG12D;Trp53R172H;p48Cre/þ –expressing primary mouse pancre-atic cancer cells (KPC), which give rise to pancreatic tumors uponorthotopic injection into the pancreata of syngeneic mice (12, 13,32, 33, 36). We assessed the frequency of IL35þ immune cellsusing flow cytometry–based detection of cells that were doublepositive for both p35 and Ebi3 subunits (Fig. 1A). At 3 weeks afterorthotopic injection of KPC cells into the pancreata of WT mice,the abundance of IL35þ cells in tumors was similar to that of4-month-old KC mice (Fig. 1A–C). Both models displayedincreased intrapancreatic and splenic accumulation of IL35þ cellscompared with normal WT pancreas and spleen, which is inagreement with previously reported increases in IL35þ in cancer(18–23). We detected two dominant immune cell populationsexpressing IL35: CD19þ B cells and CD4þ T cells (Fig. 1A–C).Consistent with our previous report, the expression of IL35 in Bcells was restricted primarily to Bregs (identified here asCD19þCD1dhiCD21hiCD5þ cells; Fig. 1D; ref. 13). Among CD4þ

T cells, the expression of IL35 was primarily restricted toCD4þFoxp3þ andCD4þFoxp3– T cells (Fig. 1E and F). Altogether,these observations suggest that in the context of pancreaticcancer, CD4þ T cells and Bregs are the primary sources of thecytokine IL35.

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Cancer Immunol Res; 6(9) September 2018 Cancer Immunology Research1016




Because several immune cell types could produce IL35, weelected to use mouse models that completely lack IL35 in all hostcells in order to examine the role of host-derived IL35 in tumorgrowth. To that end,weused p35–/– andEbi3–/– knockout animals,both of which lack IL35. GFP-labeled KPC 4662 cells wereimplanted into the pancreata of p35–/–, Ebi3–/–, or control syn-geneic WT animals. The analysis of pancreata at 3 weeks afterinjection revealed a significant reduction in tumor size in p35–/–

and Ebi3–/– mice compared with WT mice (Fig. 2A and B). Theseresults were recapitulated using the additional syngeneic pancre-atic cell line KPC 2173 (Fig. 2C). In addition to IL35, immuno-suppressive cytokine IL10 is also produced by Bregs and Tregs inthe PDA TME (13, 37). We previously showed that in contrastto IL35, the production of IL10 by B cells had no effect onpancreatic tumorigenesis, suggesting that the IL35 signaling axis

is preferentially used in PDA (31). Here, KPC cells implanted intoIl10–/– mice grew to a size comparable with that of KPC cellsimplanted into WT mice, suggesting that in the context of IL35upregulation, absence of IL10 is not sufficient to alter pancreatictumor growth (Fig. 2A–C).

IL35 modifies effector CD4þ T-cell and regulatory T-cellresponses in PDA

To understand the underlying mechanism for reduced tumorgrowth in the absence of IL35, we examined whether IL35 con-tributes to the activation or recruitment of effector CD4þ T cellsand Tregs. Deficiency in IL35 resulted in significantly increasedtumor-infiltrating effector CD4þ T cells (Fig. 2D and E). We alsoobserved significant increases in the frequency of intratumoralIFNgþ and TNFaþCD4þ effector T cells in p35–/– and Ebi3–/–mice

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Figure 1.

IL35 is upregulated in CD4þ T cells andB cells in PDA. A, Representative flowcytometry plots of CD4þ and CD19þ

cells isolated from the spleens ofWTorKC mice at 4 months of age. Cellswere processed for intracellularstaining with anti-p35 and anti-Ebi3.Percentage of p35þEbi3þCD4þ T cellsand p35þEbi3þCD19þ B cells isindicated. B, Percentage of IL35þ

splenicTcells,Bcells,ormyeloidcells inWT (n¼ 12) or splenic and intratumoralIL35þTcells,Bcells,ormyeloidcellsKCmice (n ¼ 12). C, Percentage of IL35þ

splenicTcells,Bcells,ormyeloidcells inWT or splenic and intratumoral IL35þ

splenicTcells,Bcells,ormyeloidcells inmice orthotopically injected with KPC4662 cells and collected 3 weeksafter tumor cell injection. D,Representative flow cytometry plots ofintratumoral B conventional (Bcon;CD19þCD21þCD5–CD1d–) and Bregs(CD19þCD21hiCD5þCD1dhi) from KCmice at 4 months of age or miceorthotopically injected with KPC cellsand collected 3 weeks after tumor cellinjection. Cells were analyzed bystaining with anti-p35 antibody.Percentage of p35þ Bcon or Bregs isindicated. E, Representative flowcytometry plots of intratumoralCD4þFoxp3– T cells and CD4þFoxp3þ

Tregs fromKCmice at 4months of age.Cells were analyzed by staining withanti-p35 antibody. Percentage of p35þ

CD4þFoxp3– or Tregs is indicated.F, Representative flow cytometry plotsof intratumoral CD4þFoxp3– T cellsand Tregs from mice orthotopicallyinjected with KPC cells and collected3 weeks after tumor cell injection. Cellswere analyzed by staining with anti-p35 antibody. Percentage of p35þ

CD4þFoxp3–orTregs is indicated. errorbars, SEM. p-values were calculatedusing Student t test (unpaired, two-tailed); NS, not significant; � , P < 0.05;�� , P < 0.001. Data represent 3independent experiments.






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Figure 2.

IL35 regulation of tumor growth is accompanied by suppression of CD4þ effector T-cell activity and expansion of Tregs. A, Representative images ofpancreatic tumors from WT, Il10–/–, p35–/–, and Ebi3–/– mice orthotopically injected with KPC 4662 cells and collected at 3 weeks after injection of tumor cells.B,Quantification of tumorweights fromWT, Il10–/–, p35–/–, and Ebi3–/– (N¼ 12; n¼ 3/group)mice.C,Quantification of tumorweights fromWT (n¼ 9), Il10–/– (n¼ 9),and p35–/– (n ¼ 9) mice orthotopically injected with KPC 2173 cells and collected 3 weeks after tumor cell injection. D, Representative flow cytometry plotsof gating strategy for identifying CD4þ T cells and frequency of intratumoral CD4þCD25– effector T cells isolated from WT, Il10–/–, p35–/–, and Ebi3–/– miceorthotopically injected with KPC 4662 cells and collected 3 weeks after injection of tumor cells. Percentage of CD45þCD4þ cells is indicated. E, Quantificationof the frequency of CD45þCD4þ T cells from WT, Il10–/–, p35–/–, and Ebi3–/– (n ¼ 3/group) mice. F, Representative flow-cytometric plots of CD4þIFNgþ andCD4þTNFaþ T cells isolated from WT, Il10–/–, p35–/–, and Ebi3–/– mice orthotopically injected with KPC 4662 cells and collected 3 weeks after injection oftumor cells. G, Quantification of the frequency of CD4þIFNgþ T cells (left graph, % of CD4þ T cells) and CD4þTNFaþ T cells (right graph, % of CD4þ T cells)from WT, Il10–/–, p35–/–, and Ebi3–/– (n ¼ 3/group) mice. H, Representative flow-cytometric plots of intratumoral CD4þFoxp3þ Tregs isolated from WT, Il10–/–,p35–/–, and Ebi3–/– mice. I, Quantification of the frequency of CD4þFoxp3þ (% of CD4þ T cells) from WT, Il10–/–, p35–/–, and Ebi3–/– (n ¼ 3/group) mice.J, Mean CD4þ effector T cell to Treg ratio was calculated based on the percentage of positive lymphocyte population determined by flow cytometry.Error bars, SEM. P values were calculated using Student t test (unpaired, two-tailed); NS, not significant; � , P < 0.05; �� , P < 0.01; �� , P < 0.001. Datarepresent 3–4 independent experiments.

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comparedwith control animals (Fig. 2F andG).We then analyzedthe frequencies of intratumoral CD4þFoxp3þ Tregs. As shownin Fig. 2H and I, we observed a significant reduction in thefrequency of tumor-infiltrating Tregs in animals deficient for IL35.The ratio of CD4þ effector T cells to Tregs was also significantlyincreased in the setting of IL35 deficiency (Fig. 2J). We did notobserve significant differences in abundance or polarization ofmyeloid cells in the absence of IL35 (Supplementary Fig. S1A–S1F). Altogether, these data suggest that IL35 expression in PDAdampens the activity of effector CD4þ T cells and promotes therecruitment and/or proliferation of Tregs, overall contributing tothe suppression of antitumor immune responses.

Because p35–/– and Ebi3–/– also lack cytokines IL12 and IL27,we assessed both of their expression in the PDA TMEand functional relevance to pancreatic tumor growth. Flowcytometric analysis of immune cells in KC mice using anti-bodies against p40 and p28 subunits showed no appreciableexpression of IL12 and IL27, respectively, in B and T lympho-cytes (Supplementary Fig. S2A–S2D). Expression of p40 andp28 subunits in CD11bþ cells was reduced further in thecancer-associated microenvironment, which is consistent withthe notion that proinflammatory functions of macrophages aresuppressed in cancer (Supplementary Fig. S2A–S2D). Weobserved minor differences in tumor burden when KPC cellswere injected into p40–/– animals, consistent with the notionthat IL12 is known to exhibit mostly proinflammatory effectorfunction (Supplementary Fig. S3A and S3B). IL27, however,has been previously linked to immunosuppression (17). Toascertain that the reduced tumor growth observed in IL35-nullanimals is not attributed to a loss of IL27 signaling, weimplanted KPC cells into Il27ra–/– mice. As shown in Fig. 3Aand B, we did not observe differences in tumor growth betweencontrol WT and Il27ra–/– animals, suggesting that IL27 signalingis not essential for potentiating immune responses in PDA.Consistent with our observation that the loss of IL12 or IL27signaling was not consequential for tumor growth, we did notobserve significant differences in effector T-cell infiltration orthe activation or recruitment of Tregs to tumors in p40–/– orIl27ra–/– animals (Fig. 3C–G; Supplementary Fig. S3C–S3F).These observations suggest that the tumor growth effectsobserved in p35–/– and Ebi3–/–mice were due to IL35 deficiency.

IL35 deficiency increases CD8þ effector T-cell activity andefficacy of immunotherapy

Because we observed decreased Treg infiltration and concom-itant improved function of CD4þ effector T-cell responses in theabsence of IL35, we asked whether antitumor CD8þ T-cellresponses might be affected as well. To this end, we quantifiedthe extent of intratumoral infiltration and the activation of CD8þ

T cells. A reduction in tumor growth in p35–/– and Ebi3–/– micecorrelatedwith a significant increase in infiltratingCD8þ cytotoxicT cells (Fig. 4A and B). The ratio of CD8þ T cells to Tregs wassignificantly increased in the absence of IL35 (Fig. 4C). Tumor-infiltrating CD8þ T cells in IL35-deficientmice showed significantincreases in IFNg production compared with WT or Il10–/– mice,indicating an increase in the activation status of these cells (Fig. 4Dand E). To interrogate the relationship of IL35þ T cells, B cells, andCD8þ T-cell infiltration in human disease, we examined primarytumor samples of patients with PDA.Using immunofluorescence,we detected both B cell– and T cell–specific expression of IL35 intumors (Fig. 4F; Supplementary Fig. S4A and S4B). Consistent

with the idea that IL35 may suppress CD8þ T-cell infiltration andactivity, we found that presence of IL35þ immune cells negativelycorrelated with CD8þ T-cell infiltration into tumor cell nests(Fig. 4G; Supplementary Fig. S4C). These results suggest that IL35in the PDA TME acts to restrain the infiltration and activation oftumor-directed CD8þ T cells.

Studies suggest that immunosuppression andpoorCD8þT-cellinfiltration may be the primary cause of resistance to immuno-therapy in pancreatic cancer (36, 38). Preexisting intratumoralinfiltration of CD8þ T cells has been found to be the bestcorrelative marker for successful response to immunotherapy inmelanoma (39, 40). We reasoned that because we observed arobust intratumoral presence of CD8þ T cells in the setting of IL35deficiency, we could potentially achieve better control of tumorgrowth with checkpoint blockade. To this end, we treatedWT andp35–/– animals bearing established PDA tumors with anti–PD-1or isotype control antibodies (Fig. 5A). Consistent with previousreports, the treatment of WT mice with anti–PD-1 alone did notdecrease tumor growth (Fig. 5B). However, compared with IL35deficiency alone, the combination of anti–PD-1 with IL35 defi-ciency resulted in significant inhibition of tumor growth (Fig. 5B).Wenextwanted to understandwhich additional changes in T cell–mediated immune responses accompanied improved control oftumor growth in the setting of IL35 deficiency and anti–PD-1combination. We observed no significant differences in eithertumor-infiltrating effector CD4þ T-cell cytokine production orFoxp3þ Treg responses between p35–/– animals treated withcontrol or anti–PD-1 (Fig. 5C–F). We next assessed the impactof anti–PD-1 treatment on intratumoral CD8þ T-cell populationsin the context of IL35 deficiency. Compared with both WT andp35–/– tumors, an additional significant increase in the percentageof infiltrating CD8þ T cells was observed (Fig. 5G). IntratumoralCD8þT cells fromanti–PD-1 treated p35–/–mice alsodisplayed anactivated phenotype, indicated by a significantly increased per-centage of CD8þIFNgþ cells, although this percent was compa-rable with p35–/– alone (Fig. 5H and I). To understand whetherCD8þ T cells were responsible for the phenotypes observed, weanalyzed additional cohorts of animals where CD8þ T cells weredepleted from WT and p35–/– mice bearing PDAC tumors andtreated with either anti–PD-1 or control (Fig. 5A; SupplementaryFig. S5A). Because only 2% of live cells in PDA tumors from WTmice were CD8þ T cells (Fig. 5G), it was not surprising that CD8þ

T-cell depletion had little effect on WT PDA tumor size (Fig. 5B).However, the depletion of CD8þ T cells in p35–/– animalsrescued tumor growth to a similar extent seen in WT animals,demonstrating that cytotoxic T cells are responsible for theobserved decreases in tumor burden (Fig. 5B). These resultswere recapitulated using an independent KPC 2173 cell line(Supplementary Fig. S5B).

DiscussionEffective response to immunotherapeutics, such as inhibition

of checkpointmechanisms on T cells, has been shown to correlatewith preexisting intratumoral effector T-cell infiltration (39, 40).Both human and murine PDAs fail to respond to checkpointblockade therapy, as the majority of PDA tumors contain lownumbers of infiltrating CD8þ T cells (36, 41–48). In the raresubset of PDA cases that contain T cells, infiltration does notcorrelate with increases in mutation load or neoepitope presence(38), suggesting that evasion of endogenous antitumor responses






in PDA might be governed by the establishment of immunosup-pressive networks.Our study provides evidence that IL35 serves asa crucial immunosuppressive node that attenuates the endoge-nous antitumor immune response to PDA. Expression of IL35 byimmune cells inmurine PDAwas elevated, predominantly withinthe CD4þ T-cell and B-cell subsets. IL35 deficiency severelycompromised tumor growth and correlated with increased intra-tumoral infiltration of effector CD4þ and CD8þ T cells, as well asdecreased CD4þFoxp3þ regulatory T cells. Intratumoral CD8þ

T cells and CD4þ effector T cells expressed elevated IFNg , suggest-ing that IL35 suppresses endogenous antitumor T-cell activity,and treatment with anti–PD-1 was effective in curbing PDAgrowth only in the context of IL35 deficiency. Together, our datasuggest that the IL35 pathway is an attractive therapeutic target to

augment effector T-cell infiltration and potentiate an endogenousantitumor response in order to improve the efficacy of immuno-therapeutic agents in PDA.

Themechanisms that underlie the immune suppressive activityof IL35 in PDA remain poorly understood. Several immune celltypes have been reported to produce IL35 in the context ofautoimmunity and cancer. These include CD4þFoxp3þ Tregs,CD4þFoxp3– T cells, dendritic cells, and B cells (13, 24–28,31, 49). We demonstrated that both PDA-associated B cells andCD4þ T cells were capable of producing IL35.Mechanistically, it ispossible that immunosuppression in the PDA TME is establishedby concerted output of IL35 from both T and B cells or by aselective and dominant ability of either B cells or T cells toestablish an IL35-driven immunosuppressive network. IL35 itself

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IL27 signaling does not confer immunosuppression in PDA. A, Representative images of pancreatic tumors from WT, Il27ra–/–, and p35–/– mice orthotopicallyinjected with KPC 4662 cells and collected at 3 weeks after injection of tumor cells. B, Quantification of tumor weights fromWT, Il27ra–/–, and p35–/– (n¼ 3/group)mice. C, Representative flow-cytometric plots of intratumoral CD4þIFNgþ, CD4þTNFaþ, and CD4þFoxp3þ T cells isolated from WT, Il27ra–/–, and p35–/– mice.D, Quantification of the frequency of intratumoral CD4þIFNgþ T cells (left graph, % of CD4þ T cells), CD4þTNFaþ T cells (middle graph, % of CD4þ T cells),or CD4þFoxp3þ regulatory T cells (right graph, % of CD4þ T cells) from WT, Il27ra–/–, and p35–/– (n ¼ 3/group) mice. E, Quantification of the frequencyof intratumoral CD45þCD8þ T cells from WT, Il27ra–/–, and p35–/– (n ¼ 3/group) mice determined by flow cytometry. F, Representative gating strategy forsorting CD8þ T cells and flow cytometry plots of intratumoral CD8þIFNgþ T cells isolated from WT, Il27ra–/–, and p35–/– mice. Percentage of CD8þ T cellsis indicated. G, Quantification of the frequency of intratumoral CD8þIFNgþ T cells (% of CD8þ T cells) from WT, Il27ra–/–, and p35–/– (n ¼ 3/group) mice.Error bars, SEM. P values were calculated using Student t test (unpaired, two-tailed); NS, not significant; �� , P < 0.01; �� , P < 0.001. Data represent 3 independentexperiments.

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has been shown to support proliferation of Tregs, trigger de novoIL35 upregulation in Tregs and B cells, as well as promoteconversion of effector CD4þ T cells into inhibitory IL35þFoxp3–

T cells, indicating that a single source of IL35might be sufficient toinitiate establishment of immunosuppression. In support of this

idea, a study in melanoma implicated IL35þ Tregs in promotingtumor growth (30). This, together with our findings that IL35þ Bcells supported growth of pancreatic neoplasia (13), suggests thatdistinct IL35þ immune subtypes may confer a protumorigenicadvantage and highlight tumor-specific differences in how

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IL35 suppresses cytotoxic CD8þ T-cell infiltration and activity in PDA. A, Immunohistochemical detection of CD8þ T cells by DAB in tumor tissues from WT,Il10–/–, p35–/–, and Ebi3–/– mice orthotopically injected with KPC 4662 cells and collected at 3 weeks after injection of tumor cells. Scale bars, 100 mm. B,Quantification of the frequency of tumor-infiltrating CD45þCD8þ T cells from WT, Il10–/–, p35–/–, and Ebi3–/– (n ¼ 3/group) mice determined by flow cytometry.C, Mean CD8þ cytotoxic T cell to Treg ratio was calculated based on the percentage of positive lymphocyte population determined by flow cytometry. D,Representative flow cytometry plots of intratumoral CD8þIFNgþ T cells isolated from WT, Il10–/–, p35–/–, and Ebi3–/– mice. Percentage of CD8þ T cells is indicated.E, Quantification of frequency of CD8þIFNgþ T cells (% of CD8þ T cells) from WT, Il10–/–, p35–/–, and Ebi3–/– (n ¼ 12) mice. F, Representative immunofluorescencestaining. Top, Ebi3 (red) and CD20 (green); bottom, Ebi3 (red) and CD4 (green) in samples of human PDA. Scale bars, 25 mm. G, Number of CD8þ T cells intumor cell nests as a function of low versus high numbers of Ebi3þ immune cells. Each point is the number of tumor cell adjacent CD8þ T cells per 10� FOV.Data were derived from counting 3 to 6 FOV per tumor sample (n ¼ 11 tumor samples). Error bars, SEM. P values were calculated using Student t test(unpaired, two-tailed); NS, not significant; �� , P < 0.01; �� , P < 0.001. Data represent 3–4 independent experiments.






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Deficiency in IL35 potentiates efficacy of anti–PD-1 checkpoint blockade. A, Schematic of the antibody treatment regimen. Anti-CD8 or control IgG (redarrows) was administered for 3 days prior to tumor cell injection and then twice weekly on days 3, 5, 9, 11, 15, and 17. Administration of anti–PD-1 (greenarrows) was initiated on day 7 after tumors reached approximately 4 mm in diameter (blue circle). Twomore doses of anti–PD-1 were administered on days 9 and 11.Mice were sacrificed 3 weeks after tumor cell injection. B, Quantification of tumor weights from WT and p35–/– (n ¼ 3/group) mice orthotopically injectedwith KPC 4662 cells and treated with antibodies as described in A. C, Representative flow-cytometric plots of intratumoral CD4þIFNgþ, CD4þTNFaþ, andCD4þFoxp3þ T cells isolated fromWTand p35–/–mice. Percentage of CD4þ T cells is indicated.D,Quantification of the frequency of intratumoral CD4þTNFaþ T cells(% of CD4þ T cells) from WT and p35–/– (n ¼ 3/group) mice. E, Quantification of the frequency of intratumoral CD4þIFNgþ T cells (% of CD4þ T cells) fromWT and p35–/– (n¼ 3/group)mice. F,Quantification of the frequency of intratumoral CD4þFoxp3þ regulatory T cells (% of CD4þ T cells) fromWT and p35–/– (n¼ 9)mice. H, Quantification of the frequency of tumor-infiltrating CD45þCD8þ T cells by flow cytometry from WT and p35–/– (n ¼ 3/group) mice. I, Representativeflow-cytometric plots of CD8þIFNgþ T cells isolated from WT and p35–/– mice. Percentage of CD8þ T cells is indicated. J, Quantification of the frequency ofCD8þIFNgþT cells (%of CD8T cells) fromWTandp35–/– (n¼ 3/group)mice. Error bars, SEM.P valueswere calculated using the Student t test (unpaired, two-tailed);NS, not significant; �, P < 0.05; �� , P < 0.01; �� , P < 0.001. Data represent 3 independent experiments.

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IL35 promotes cancer. Understanding the role of B cell– andT cell–specific contribution to the establishment of IL35-drivenimmunosuppression will be an important and necessary steptoward creating precisely targeted therapies.

Our findings suggest that the mechanism by which hostproduction of IL35 promotes pancreatic tumorigenesis was bylimiting endogenous antitumor T-cell responses. It is not yetclear how IL35 controls infiltration and function of T-cellsubsets in the setting of PDA, and it is possible that thesuppressive effects exerted on CD8þ T cells is mediated directlyor via modulation of CD4þ T effector cells or regulatory T cells.Any direct effects of IL35 on proliferative, migratory, orfunctional properties of target T cells likely depend on theexpression of IL35 receptor chains, such as IL12Rb2/gp130 orWSX1/gp130 heterodimers or IL12Rb2/IL12Rb2 and gp130/gp130 homodimers (17). Of these, IL12Rb2 and WSX1 can beinduced by inflammatory cues. Additional experiments areneeded to characterize expression of IL35 receptor chains onT cells in the PDA TME and to understand how their expressionregulates function and migration of target T cells in vivo. A studyin a model of experimental autoimmune uveitis revealed thatexpression of p35 correlated with a decrease in chemokinereceptor CXCR3, previously implicated in intratumoral traffick-ing of effector T cells (50, 51). These observations suggest thatIL35 could directly regulate exclusion of T cells from the tumorparenchyma, and, at least partially, explain how IL35 sup-presses endogenous antitumor CD8þ T-cell responses.

Mechanistic contribution of IL35 to immunotherapy resistanceis not clear. A study by Turnis and colleagues suggests that IL35may direct expression of the checkpoint molecules PD-1, LAG3,and TIM3onCD8þ T cells, thus facilitating T-cell exhaustion (30).It is possible that downregulation of PD-1 expression on CD8þ

T cells in PDA is transient and can be upregulated by additionalmechanismsonce the T cells infiltrate the tumor parenchyma. Thispossibility is supported by data suggesting progressive exhaustionof antitumor-targeted T cells and may explain why CD8þ T cellscould still respond to anti–PD-1 therapy in the context of IL35deficiency (52). Future studies aimed at understanding the cross-

talk between IL35 expression and T-cell exhaustion may providefurther insight into the strategies that would harness the mosteffective combinations targeting immunosuppressive mechan-isms in PDA.

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

Authors' ContributionsConception and design: B. Mirlekar, Y. Pylayeva-GuptaDevelopment of methodology: B. Mirlekar, Y. Pylayeva-GuptaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B. Mirlekar, D. Michaud, R. Searcy, K. GreeneAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): B. Mirlekar, D. Michaud, Y. Pylayeva-GuptaWriting, review, and/or revision of the manuscript: B. Mirlekar, K. Greene,Y. Pylayeva-GuptaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): B. Mirlekar, R. SearcyStudy supervision: Y. Pylayeva-Gupta

AcknowledgmentsWe thank N. Kren for discussions, P. Patel and Animal Studies Core for

help with mouse colony maintenance, and Sebastien Coquery and EvanTrudeau for help with FACS. The UNC Flow Cytometry Core Facility, theUNC Translational Pathology Laboratory, the Animal Histopathology andLaboratory Medicine Core, and the UNC Lineberger Animal Studies Coreare supported in part by P30 CA016086 Cancer Center Core Support Grantto the UNC Lineberger Comprehensive Cancer Center.

This work was supported by AACR–PanCAN Pathway to Leadership Grant13-70-25-PYLA (Y. Pylayeva-Gupta), UCRF (Y. Pylayeva-Gupta), V Foundationfor Cancer Research grant V2016-016 (Y. Pylayeva-Gupta), and the WUSTLSPORE Career Enhancement Award grant 1P50CA196510-01A1 from the NCI(Y. Pylayeva-Gupta).

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 December 6, 2017; revised April 24, 2018; accepted June 25, 2018;published first July 6, 2018.

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Mirlekar et al.




2018;6:1014-1024. Published OnlineFirst July 6, 2018.Cancer Immunol Res Bhalchandra Mirlekar, Daniel Michaud, Ryan Searcy, et al. Responsiveness to Immunotherapy in Pancreatic CancerIL35 Hinders Endogenous Antitumor T-cell Immunity and

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