bcell derivedil35drivesstat3-dependentcd8þt …...cancer immunology research | research article...

CANCER IMMUNOLOGY RESEARCH | RESEARCH ARTICLE

Bcell–Derived IL35Drives STAT3-DependentCD8þT-cellExclusion in Pancreatic Cancer A C

Bhalchandra Mirlekar1, Daniel Michaud2, Samuel J. Lee1, Nancy P. Kren1, Cameron Harris1, Kevin Greene3,Emily C. Goldman4, Gaorav P. Gupta1,4, Ryan C. Fields5, William G. Hawkins5, David G. DeNardo6,Naim U. Rashid1,7, Jen Jen Yeh1,8, Autumn J. McRee1,9, Benjamin G. Vincent1,9,10, Dario A.A. Vignali11, andYuliya Pylayeva-Gupta1,12

ABSTRACT◥

Pancreatic ductal adenocarcinoma (PDA) is an aggressivemalignancy characterized by a paucity of tumor-proximal CD8þ

T cells and resistance to immunotherapeutic interventions. Can-cer-associated mechanisms that elicit CD8þ T-cell exclusion andresistance to immunotherapy are not well-known. Here, using aKras- and p53-driven model of PDA, we describe a mechanism ofaction for the protumorigenic cytokine IL35 through STAT3activation in CD8þ T cells. Distinct from its action on CD4þ

T cells, IL35 signaling in gp130þCD8þ T cells activated thetranscription factor STAT3, which antagonized intratumoralinfiltration and effector function of CD8þ T cells via suppressionof CXCR3, CCR5, and IFNg expression. Inhibition of STAT3signaling in tumor-educated CD8þ T cells improved PDA growthcontrol upon adoptive transfer to tumor-bearing mice. We

showed that activation of STAT3 in CD8þ T cells was drivenby B cell– but not regulatory T cell–specific production of IL35.We also demonstrated that B cell–specific deletion of IL35facilitated CD8þ T-cell activation independently of effector orregulatory CD4þ T cells and was sufficient to phenocopy ther-apeutic anti-IL35 blockade in overcoming resistance to anti–PD-1 immunotherapy. Finally, we identified a circulating IL35þ

B-cell subset in patients with PDA and demonstrated thatthe presence of IL35þ cells predicted increased occurrence ofphosphorylated (p)Stat3þCXCR3�CD8þ T cells in tumors andinversely correlated with a cytotoxic T-cell signature in patients.Together, these data identified B cell–mediated IL35/gp130/STAT3 signaling as an important direct link to CD8þ T-cellexclusion and immunotherapy resistance in PDA.

IntroductionInfiltration of cytotoxic T cells into the tumor parenchyma corre-

lates with better outcomes in a variety of tumor types, especially in the

context of immunotherapy (1). Thus, better understanding of themechanisms that modulate T-cell trafficking and function in tumors isnecessary to overcome inefficient immune responses. This is partic-ularly relevant in pancreatic ductal adenocarcinoma (PDA), an aggres-sive and deadly disease often characterized by lack of infiltration and/or dampened functionality ofCD8þTcells (2, 3). In the setting of PDA,immunotherapy has been unsuccessful (4, 5). The mechanisms thatrestrict tumor-directed CD8þT-cell function in PDA are thought to belinked to immunosuppression (6–8). Significant research efforts havedescribed a variety of tumor cell-intrinsic or extrinsicmechanisms thatmay act to restrict CD8þ T-cell activity in the PDA tumor microen-vironment (TME), among which are myeloid cell recruitment andpolarization, expansion of regulatory and gd T cells, as well asmodulation of T-cell infiltration by tumor cells themselves (9–17).These studies suggest that reversing immunosuppression in pancreaticcancer could improve endogenous T-cell activity. Although it isbecoming clear that contribution from both tumor cell-intrinsic andextrinsic mechanisms may dictate the type of immunosuppressionpresent in the TME, the mechanisms that these various immunosup-pressive arms utilize to directly control T-cell function in PDA remainpoorly defined.

We previously demonstrated that the cytokine IL35 promotespancreatic tumorigenesis (18, 19). IL35 is a member of the IL12family of cytokines, forms via heterodimerization of p35 and Ebi3,and is thought to signal in na€�ve CD4þ T cells through the IL35receptor (IL35R, consisting of IL12Rb2 and gp130 subunits). Thisactivates STAT1 and STAT4 signaling pathways (20). Elevated IL35can be detected in lymphoma cells and lung cancer, and predicts pooroutcome in cases of leukemia, colorectal, and pancreatic cancer (20).Regulatory T cells (Treg), na€�ve CD4þ T cells (iTr35), dendritic cells(DC), and B cells are known to produce IL35 (11, 19, 21–25), whoseexpression has been linked to its ability to modulate immune

1Lineberger Comprehensive Cancer Center, The University of North Carolina atChapel Hill School of Medicine, Chapel Hill, North Carolina. 2Department of CellBiology, The University of North Carolina at Chapel Hill School of Medicine,Chapel Hill, North Carolina. 3Department of Pathology and LaboratoryMedicine,The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill,North Carolina. 4Department of Radiation Oncology, The University of NorthCarolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina. 5Depart-ment of Surgery, Barnes-Jewish Hospital and the Alvin J. Siteman Comprehen-sive Cancer Center, Washington University School of Medicine, St. Louis, Mis-souri. 6Department ofMedicine, Barnes-JewishHospital and theAlvin J. SitemanComprehensive Cancer Center, Washington University School of Medicine, St.Louis, Missouri. 7Department of Biostatistics, The University of North Carolina atChapel Hill School of Medicine, Chapel Hill, North Carolina. 8Department ofSurgery, The University of North Carolina at Chapel Hill School of Medicine,Chapel Hill, North Carolina. 9Department of Medicine, The University of NorthCarolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina. 10Depart-ment of Microbiology and Immunology, The University of North Carolina atChapel Hill School of Medicine, Chapel Hill, North Carolina. 11Department ofImmunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsyl-vania. 12Department of Genetics, The University of North Carolina at Chapel HillSchool of 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]

Cancer Immunol Res 2020;8:292–308

doi: 10.1158/2326-6066.CIR-19-0349

�2020 American Association for Cancer Research.

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responses via diverse mechanisms (20). IL35 expression in Tregspromotes CD8þ T-cell exhaustion in melanoma and colorectalcancer (21) and has also been shown to facilitate tumor growth viaaccumulation of myeloid-derived suppressor cells (MDSC) andinduction of angiogenesis (26). IL35 expression in B cells alsodampens T-cell activity independently of Tregs in experimentalautoimmune encephalomyelitis by suppressing macrophages andinflammatory CD4þ T cells (24). Because of these effects, it seemslikely IL35 may impact functionality of diverse immune cell popula-tions (20). We previously showed that IL35 exerts immunosuppres-sive effects in PDA and is primarily expressed by two immune cellpopulations, regulatory T and B cells (19). However, the mechanismsunderlying IL35 function in PDA remain unclear, and are addressedin the current study.

Materials and MethodsMice

All mouse protocols were reviewed and approved by the Institu-tional Animal Care and Use Committee of the University of NorthCarolina at ChapelHill (ChapelHill, NC). Animalsweremaintained ina specific pathogen-free facility. Six- to 7-week-old wild-type CD45.2þ

(WT) C57Bl/6J mice were purchased from The Charles River Labo-ratories (#027). The KrasLSL-G12D/þ;Trp53LSL-R172H/þ and p48Cre/þ

(KPC) mice have been described previously (27). Six- to 7-week-oldB6 CD45.1þ (B6.SJLPtprca.Ptprcb/BoyJ) mice were purchased from TheJacksonLaboratory (#002014).Ebi3Tom.L/L and Foxp3Cre-YFP;Ebi3Tom.L/L

mice were obtained from D. Vignali (University of Pittsburgh,Pittsburgh, PA; refs. 21, 28–31). CD19CreEbi3L/L mice weregenerated by crossing CD19Cre mice (32) to Ebi3Tom.L/L mice inour colony for two generations to obtain homozygosity at Ebi3locus. Resulting mice lacked expression of IL35 in either Bcells (BEbi3�/�) or Tregs (TregEbi3�/�). Littermates were used ascontrols.

BWT and Bp35�/� mice were obtained by a mixed bone marrowchimera method using lethally irradiated (1,000 cGy radiationdelivered from cesium source) C57BL/6J mice as recipients (Sup-plementary Table S1; ref. 24). Bp35�/� or control BWT mice wereobtained by reconstituting recipient mice with a mixture of bonemarrow cells from B cell–deficient mMT mice (The Jackson Lab-oratory, #002288) or WT C57BL/6J mice (80%), respectively, andp35�/� mice (20%; The Jackson Laboratory, #002692). A total of10 � 106 bone marrow cells were injected intravenously into theWT recipients irradiated at 1,000 cGy. The chimeras were used after8 weeks and specific deletion of p35 in B cells was confirmed byPCR. Reconstitution was confirmed using flow cytometry for majorimmune subtypes.

Validation of cell type–specific knockoutsTo validate that the deletion of p35 or Ebi3 genes was specific to the

B-cell lineage, splenic CD19þB cells, CD11bþmyeloid cells, andCD4þ

T cells were isolated by FACS fromBp35�/�, BWT, BEbi3þ/�, andBEbi3�/�

mice (please see “Lymphocyte isolation” section). To verify specificdeletion of Ebi3 gene from Tregs, Foxp3þ (YFPþ) Tregs, Foxp3�

(YFP�) conventional T cells (Tcon), and CD19þ B cells werepurified from TregEbi3þ/� and TregEbi3�/� mice by FACS. All sortedpopulations and remaining non-T, -B, or -myeloid cells were lysedand genomic DNA was extracted using the DNeasy Kit (Qiagen).PCR was used to check the presence of WT p35 or Ebi3 allele in theimmune cells (primer sequences are listed in SupplementaryTable S2).

Cell linesThe murine PDA cell line KPC 4662 was derived from a primary

pancreatic tumor of C57Bl/6J KPC mice by Dr. Vonderheide'slaboratory (33). GFP-labeled KPC cells were generated as describedpreviously (18). Cells were maintained at 37�C and 5% CO2 incomplete DMEM (#11995-065, Gibco, 10% FCS and 1� penicillin–streptomycin #15140-122, Gibco) and were confirmed to beMycoplasma and endotoxin free. The cells were used at <16 passages.Cells were confirmed to contain Kras, Cre, and p53 mutant alleles/transgene by genotyping.

Tumor growth and antibody-blocking experimentsFor intrapancreatic injection of cancer cells, mice were anesthetized

using a ketamine (100 mg/kg)/xylazine (10 mg/kg; Med-Vet Interna-tional) cocktail. The depth of anesthesia was confirmed by verifying anabsence of response to toe pinch. An incision in the leftflankwasmade,and 75,000 KPC cells in ice-cold PBS mixed at 1:1 dilution withMatrigel (#354234, Corning) in a volume of 50 mL were injected usinga 28-gauge needle into a tail of the pancreas. The wound was closed intwo layers, 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;Med-Vet International) once subcutaneously after orthotopic surgery.For therapeutic experiments, mice received antibody treatment usinganti-IL35 (V1.4C4.22) at 200 mg/week for 3 weeks, anti-IL27 (MM27-7B1) at 200 mg/week for 3 weeks, and/or anti–PD-1 (RMP1-14, Bio XCell) at 200 mg/injection on days 7, 9, and 11, or their respective IgGisotype controls once an orthotopic tumor reached 4 to 5 mm (day 7;Supplementary Table S3). Tumor growth was monitored by ultra-sound, as described below. Three doses of antibody were given in total,on days 7, 9, and 11 after injection of KPC cells.

Adoptive transfer of CD8þ T cellsSpleens were harvested from orthotopically injected CD45.2þ mice

after 3 weeks after KPC 4662 cell injections. CD8þ T cells were sortedfrom the spleens after red blood cell lysis using a BD FACS-ARIA IIIsorter, and purity of CD8þT cells was >98%. Sorted CD8þT cells weretreated with plate-bound anti-CD3 (1 mg/mL), soluble anti-CD28(2 mg/mL) as control or plate-bound anti-CD3 (1 mg/mL), solubleanti-CD28 (2 mg/mL), and STA-21 (20 mmol/L) to inactivate pSTAT3in CD8þ T cells for 48 hours. After 48 hours, 10 � 106 STA-21– orcontrol-treated CD8þ T cells were adoptively transferred via tail veininjection into CD45.1þ mice. One day after adoptive transfer, 75,000KPC 4662 cells were orthotopically transplanted into the pancreas ofCD45.1þmice. The recipient mice were sacrificed 21 days post-tumorcell injections, tumor size and weight were measured, and spleens andtumors were collected for further processing and analysis.

Depletion of CD8þ T cells, CD4þ T cells, and Tregs in vivoFor CD4þ and CD8þ T-cell depletion studies, 200 mg of anti-CD4

(Bio X Cell, BP0003-1, clone GK1.4) and 200 mg of anti-CD8 (Bio XCell, BP0004-1, clone 53-6.7) or an IgG isotype control rat IgG2b, kand rat IgG2a, k (Bio X Cell), respectively, were administered intra-peritoneally daily starting 3 days prior to tumor cell injection and twicea week after tumor cell injection. Tregs were depleted using 500 mg ofanti-CD25 clone PC 61.5.3 (Bio X Cell, BE0012), injected intraperi-toneally on day 3 and day 1 before pancreatic tumor implantation andrepeated every 5 days for the duration of the experiment. Control micereceived rat IgG1l (Bio X Cell) isotype control. Mice were sacrificed21 days after tumor implantation, tumor size, and weight weremeasured, and spleen and tumor samples were collected for further

STAT3-Mediated T-cell Exclusion Promotes PDA

AACRJournals.org Cancer Immunol Res; 8(3) March 2020 293




processing. Depletion of cells was confirmed by flow cytometry at theend of the experiment.

Ultrasound imagingMonitoring of orthotopic pancreatic tumors was preformed

weekly on the Vevo 2100 Imaging System (VisualSonics Inc.) usingthe MS550D transducer. Mice were anaesthetized using isoflurane(2%) throughout the procedure. Hair was removed from left flank ofeach mouse by electric razor and hair removal cream (Nair, Church& Dwight). Images were taken at 11 mm image depth, andmeasurements were calculated using the Vevo LAB software(VisualSonics Inc.).

Lymphocyte isolationSingle-cell suspensions were prepared from tumors and spleens

isolated from orthotopic and/or adoptive transfer models. Spleenswere mechanically disrupted using the plunger end of a 5 mL syringeand resuspended in 1% FBS/PBS. Spleen samples were processedfollowing RBC lysis (eBioscience; 00-4333-57). For isolation oftumor-infiltrating lymphocytes, tumor tissue was minced into 1 to2 mm pieces and digested with collagenase IV (1.25 mg/mL;#LS004188, Worthington), 0.1% soybean trypsin inhibitor (#T9128,Sigma), hyaluronidase (1 mg/mL; #LS002592, Worthington), andDNase I (100 mg/mL; #LS002007, Worthington) in complete DMEMfor 30minutes at 37�C. Cell suspensions were passed through a 70-mmcell strainer (Falcon) and resuspended in RPMI media (Gibco).Lymphocytes were isolated from processed tumor tissues by OptiPrep(Sigma) density gradient centrifugation. MACS isolation of totalCD45þ leukocytes (MACS Miltenyi Biotec #130-052-301) was per-formed on the leukocyte-enriched fraction according to MiltenyiBiotec's protocol, and the purity was >90%. Cells were stained withfluorophore-labeled antibodies (Supplementary Table S3) for 30 min-utes on ice in FACS buffer (PBSwith 3%FCS and 0.05% sodium azide).After staining, cells were washed twice with FACS buffer and resus-pended in sorting buffer (PBS with 1% FCS and 0.05% sodium azide).Cell sorting using a BD FACS ARIA III sorter was performed toisolate CD19þCD21hiCD5þCD1dþ regulatory B cells (Breg),CD19þCD21loCD5�CD1d� conventional B cells (Bcon), CD4þYFPþ

Tregs, and CD8þT cells. Cells were collected in complete RPMImediacontaining 10% FCS with 1� penicillin–streptomycin (#15140-122,Gibco) antibiotics. More than 97% purity was achieved.

B-cell and T-cell in vitro culturesFor in vitro CD8þ T-cell culture, splenic CD8þ T cells specific for

the OVA257-264 (InvivoGen) antigen were sorted (>98% purity) fromWT mice immunized with OVA257-264 for 1 week (10 mg/mouse). Tcells were cultured with plate bound anti-CD3 (1 mg/mL, Bio X Cell)and soluble anti-CD28 (2 mg/mL, Bio X Cell), with the addition ofrecombinant IL35 (rIL35, 50 ng/mL; Chimerigen Laboratories; CHI-MF-11135) and OVA257-264 (2 mg/mL) for 48 hours. For blockingSTAT3 or STAT4 activity, cells were cultured with 50 mmol/L fludar-abine (Selleckchem), 20 mmol/L STA-21, or 100 mmol/L lisofylline(Santa Cruz Biotechnology) for 48 hours (see Supplementary Table S3for a list of antibodies and reagents).

Mouse splenic Bregs (CD19þCD21hiCD5þCD1dþ) were sorted byflowcytometry fromspleensof tumor-bearingWT,p35�/�, andEbi3�/�

mice (>97% purity), as described above. A total of 100,000 Bregs orBcon cells and 100,000 CD8þ T cells (1:1 ratio) were cocultured inthe 96-well Transwell plates, with B cells occupying the top chamberand CD8þ T cells the bottom chamber (Corning; 3381) for 48 hours.B cells were activated by anti-CD40 (1 mg/mL, eBioscience) and LPS

(2 mg/mL, Sigma) for 48 hours, and CD8þ T cells were activated byplate bound anti-CD3 (1 mg/mL) and soluble anti-CD28 (2 mg/mL)with OVA257-264 (2 mg/mL). Cytokine secretion of CD8þ T cells wasevaluated by flow cytometry, as described below.

Intracellular cytokine and transcription factor stainingFor ex vivo stimulation, sorted cells from tumors or spleens of

orthotopic and/or adoptive transfer models (except for B cells, whichwere cultured in LPS and anti-CD40 prior to this step) were incubatedwith PMA (50 ng/mL; Sigma, #P8139) and ionomycin (200 ng/mL;Sigma, #I0634) in the presence of Golgistop Brefeldin A (1X,BioLegend) in complete RPMI medium for 5 hours at 37�C. Cellswere washed and blocked with anti-CD16/CD32 (Fc Block, BDBiosciences, 0.1 mg/100,000 cells) for 5 minutes on ice. Viability wasassessed using the Live/Dead 7AAD (BioLegend; 420404) stain solu-tion or Live/Dead Aqua cell stain kit (Life Technologies). Cells werethen washed and stained with labeled antibodies against surfacemarkers on ice for 30 minutes in FACS buffer (PBS with 3% FCS and0.05% sodium azide). After surface staining, cells were washed, fixed,and permeabilized using cytofix/cytoperm buffer (BD, 554714) for 15minutes at 4�C in the dark. Intracellular staining was performed usingfluorophore-conjugated cytokine antibodies for 1 hour at 4�C in thedark. After intracellular staining, cells werewashed and resuspended inFACS buffer for acquisition by flow cytometry. Intracellular stainingfor Foxp3 was performed using a Foxp3 staining kit (eBioscience,catalog no. 00-5523). For staining of phosphoproteins, cells were fixedwith fixation buffer (BioLegend; 420801) at room temperature for 10minutes and permeabilized with true-phos perm buffer (BioLegend;425401) at �20�C overnight. Cells were then washed twice andresuspended in cell staining buffer (BioLegend; 420201). Fluoro-phore-conjugated phosphoprotein cocktail antibodies or isotype con-trols were added and incubated for 60 minutes at 4�C. After incuba-tion, cells were washed, resuspended in FACS buffer, and samples wereacquired on LSR II and LSRII Fortessa (BD Biosciences) and analyzedwith FlowJo version 10.2 (TreeStar, Inc.). The human peripheral bloodmononuclear cell (PBMC) samples were processed as described above,but the blocking step was done with human BD Fc Block (BDBiosciences, 564219, 0.1 mg/100,000 cells) for 5 minutes on ice. Allantibodies and reagents are listed in Supplementary Table S3.

ELISAPeripheral blood samples were collected from mice using the retro-

orbital method, and serum samples were prepared by centrifugingsamples at 1,000 rpm for 5 minutes at 4�C. All serum samples werestored at �80�C. Fifty microliters of serum sample was used to detectcirculating IL35. The concentration of serum IL35 was measured byusing the LegendMaxmouse IL35 heterodimer ELISAKit (BioLegend,IL35 precoated ELISA Kit, p35, EBI-3), and IL35 concentration wasdetermined based on the recombinant IL35 standard that came withthe kit. Absorbance were read at 450 nm within 15 minutes usingPerkinElmer Enspire multimode reader. All samples were assayed intriplicates.

Human samplesThe study was carried out in accordance with The University of

North Carolina at Chapel Hill School of Medicine and WashingtonUniversity School of Medicine guidelines and was approved byinstitutional review board ethics committees. Informed consent wasobtained from the patients and healthy donors before blood donation.The study conducted in accordance to ethical standards such as theDeclaration of Helsinki.

Mirlekar et al.

Cancer Immunol Res; 8(3) March 2020 CANCER IMMUNOLOGY RESEARCH294




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Figure 1.

IL35 mediates activation of STAT3, and suppression of IFNg and chemotactic receptors CXCR3 and CCR5 in gp130þCD8þ T cells. A, Representative flow cytometryhistograms (left) and quantification (right) of phospho(p)-STAT1, pSTAT3, and pSTAT4 in CD3þCD8þ T cells activated with anti-CD3 (1 mg/mL) and anti-CD28(2 mg/mL) in the presence of OVA (2 mg/mL) with and without recombinant (r)IL35 (50 ng/mL). Proportion of CD8þ T cells is indicated. B, Representative flowcytometry plots and histograms (left) and quantification (right) of IFNg production and expression of CXCR3 and CCR5 in CD3þCD8þ T cells activated as in A. C,Representativeflowcytometry histograms (left) andquantification (right) of pSTAT1, pSTAT3, andpSTAT inCD3þCD8þTcells sorted for IL12Rb2þor gp130þ subsets(single positive) activated as indicated in A. Proportion of CD8þ T cells is shown. D, Representative flow cytometry plots and histograms (left) and quantification(right) of IFNg production and expression of CXCR3andCCR5 in CD3þCD8þT cells sorted for IL12Rb2þor gp130þ subsets (single positive) activated as indicated inA.Proportion of CD8þ T cells is shown. Error bars indicate SEM; P values were calculated using Student t test (unpaired, two-tailed); NS, not significant. � , P < 0.05;�� , P < 0.01; �� , P < 0.001. Experiments were conducted using 6- to 8-week-old C57B6 mice with 6 mice per group in triplicate. Data represent three independentexperiments.






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Mirlekar et al.





PBMC isolation and B-cell enrichmentBlood samples were collected from 30 treatment-na€�ve patients with

pancreatic cancer and 30 healthy controls. PBMCs were obtained byseparating the whole blood samples via density gradient centrifugation(Lymphoprep, Axis-shield). Briefly, blood samples were collected inyellow top ACD tubes (BD Vacutainer, 364606;�20 mL each). Bloodwas diluted by addition of an equal volume of 0.9%NaCl. About 20mLof diluted blood was layered on top of 10 mL Lymphoprep andcentrifuged at 800� g for 20 minutes at 18�C. The mononuclear cellswere removed from the sample/medium interface using a Pasteurpipette. Cells were collected by centrifugation at 250� g for 5 minutesandwere washed three times with 1�RPMI1640. The isolated PBMCswere then stained with anti-human CD19 (HIB19; BioLegend),CD24 (ML5; BioLegend), and CD38 (HB-7; BioLegend) in FACSbuffer for 20 minutes on ice. CD19þCD24hiCD38þ Bregs andCD19þCD24loCD38� Bcon cells were sorted using a BD FACSARIAIII, and cells were collected in complete RPMI media. Morethan 97% cell purity was achieved.

PDA patient samplesFor the purposes of analyzing CD8þ T-cell and IL35þ immune cell

infiltration, we examined 11 samples containing PDA lesions. Samplesconsisted of 5-mm sections that were cut from formalin FFPE blocksprovided by theTissue Pathology Laboratory (TPL) of theUNCSchoolof Medicine and were obtained after informed written consent. Thisstudy was conducted in accordance with the Declaration of Helsinki.All samples were anonymized prior to being transferred to theinvestigator's laboratory and therefore met exempt human subjectresearch criteria. Samples were selected on the basis of patient diag-nosis with PDA.

qPCR analysis for gene expressionRNA was prepared from sorted CD19þCD24hiCD38þ Bregs and

CD19þCD24loCD38� Bcon cells from human PBMCs using theRNeasy Micro Kit (Qiagen). CDNA was generated using High-Capacity cDNA-RT Kit (Invitrogen). qPCR analysis (with 100 ngDNA template) was performed using the SSO advanced universalSYBR green super-mix reagent (Bio-Rad) and Applied Bio-Systemplatform. Results were normalized to the expression of b-actin, andeach sample was run in triplicate. Gene expression was determined bythe DDCt method (2–DDCt). Primer sequences are listed in Supplemen-tary Table S2.

IHC and immunofluorescenceMouse splenic and tumor tissues for all models and treatments

analyzed were fixed in 10% buffered formalin (Thermo Fisher Scien-tific) for 48 hours and embedded in paraffin. Six-micrometer sectionswere deparaffinized and rehydrated. A solution of 1% hydrogenperoxide (stock of 30% hydrogen peroxide, Sigma) in methanol at

room temperature for 10 minutes was used to quench endogenousperoxidase activity. Antigen retrieval was done in a 10 mmol/L sodiumcitrate plus 0.05% Tween-20 solution (pH 6.1) for 15 minutes in amicrowave oven. Blocking was performed for 1 hour at room temper-ature in a solution of 10%goat serum (Vector Laboratories), 10mmol/LTris–HCl, 0.1 mol/L magnesium chloride, 1% BSA, and 0.5% Tween-20. Sections were incubated with primary rat anti-CD8a (clone 53-6.7,BD Pharmingen) 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 (final concentrationof 3.75mg/mL) and incubated for 1 hour at room temperature. TertiaryABC solution was prepared according to the manufacturer'sinstructions (Vectastain ABC Kit, Vector Laboratories) and incubatedfor 45 minutes at room temperature. Sections were developed using a3,30-diaminobenzidine tetrahydrochloride kit (DAB Peroxidase Sub-strate Kit, Vector Laboratories). Slides were then counterstained withHarris hematoxylin (Sigma), dehydrated, and mounted with DPXmounting media (Sigma). Images were acquired using Nikon EclipseNi-U microscope, with NIS-Elements software (Nikon).

For immunofluorescence, mouse pancreata were fixed, rehydrated,andsubjected toantigenretrieval asdescribedabove.Blockingwasdonefor 1hour (10mmol/LTris-HCL, 0.1mol/LMgCl2, 0.5%Tween-20, 1%BSA, 10% chicken serum; Vector Laboratories), primary antibodieswere applied overnight at 4�C. Anti-mouse antibodies includedphospho-Histone 3 (Millipore, 06-570) and CK8 (DSHB, Troma-I).Secondaryantibodieswereusedat1:200 for1hourat roomtemperaturefollowed by incubationwith 1 mg/mLDAPI for 10minutes. Slides werewashed andmounted with ProLongGold anti-fademountingmedium(Invitrogen), and images were acquired with an Olympus BX61.

For the purposes of analyzing status of pSTAT3 and CXCR3expression on CD8þ T cells, 11 human PDA lesion samples wereanalyzed. Samples consisted of 5-mm sections that were cut from FFPEblocks provided by the TPL of the UNC School of Medicine. Allsamples were anonymized prior to being processed. Triple IF (3-plexIF) stains were carried out in the Leica Bond-Rx fully automatedstaining platform (Leica Biosystems Inc.). Slideswere dewaxed inBondDewax solution (AR9222) and hydrated in Bond Wash solution(AR9590; all from Leica Biosystems). Epitope retrieval for all targetswas done for 20 minutes in Bond-epitope retrieval solution 1 pH 6.0(AR9661) or solution 2 pH9.0 (AR9640; both from Leica Biosystems).The epitope retrieval was followed with 10 minutes of endogenousperoxidase blocking using Bond peroxide blocking solution (DS9800;Leica Biosystems). Positive and negative controls (no primary anti-body) and single-stain controls were done for 3-plex IF where oneprimary antibody was omitted to make sure that cross-reactivitybetween the antibodies did not occur. The following antibodies wereused: CD8 (4B11, Leica). CD8þ T cells were counted per 10� field-of-view (FOV), counting 3-6 FOV per tumor sample. Stained slides werecounterstained with Hoechst 33258 (# H3569) and mounted with

Figure 2.STAT3 activation in CD8þ T cells is suppressive in vitro and in vivo.A,Representative flow cytometry plots and histogramsof IFNg production and expression of CXCR3,CCR5, phospho(p)-STAT1, pSTAT3, and pSTAT4 in CD3þCD8þ T cells stimulated with anti-CD3 (1 mg/mL) and anti-CD28 (2 mg/mL) in the presence of OVA (2 mg/mL),rIL35 (50 ng/mL), and/or STA-21 (20 mmol/L) inhibitor. Proportion of CD8þ T cells is indicated. B, Quantification of IFNg production and expression of CXCR3, CCR5,pSTAT1, pSTAT3, and pSTAT4 in CD3þCD8þ T cells from A. C, Experimental schema used to test the role of Stat3 activation in CD8þ T cells. D,Quantification of tumorweight fromCD45.1þmice 3weeks after orthotopic adoptive transfer of tumor-educated CD45.2þCD8þ T cells pretreatedwith anti-CD3/CD28with andwithout STA-21CD45.2þCD8þ T cells and injectionwithKPC cells (n¼ 6mice/group).E,Numbers (no.) of adoptively transferredCD45.2þ (left) andendogenous CD45.1þ (right) tumor-infiltratingCD3þCD8þ T cells 3weeks after orthotopic adoptive transfer to the CD45.1þ recipients indicated inD. F,Representative flowcytometry histograms (left) andquantification (right) of pSTAT1, pSTAT3, andpSTAT4 in intratumoral CD45.2þCD8þ T cells 3weeks after orthotopic adoptive transfer to CD45.1þ recipients indicated inD. G, Representative flow cytometry plots and histograms (left) and quantification (right) of IFNg production and expression of CXCR3 and CCR5 in intratumoralCD45.2þCD8þ T cells 3 weeks after orthotopic adoptive transfer to CD45.1þ recipients indicated in D. 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 three independent experiments.






ProLong Diamond Antifade Mountant (#P36961; Life Technologies).We previously estimated the average value of Ebi3þ cells/FOV acrossall tumor samples as 20 Ebi3þ cell/FOV. FOVs with number of Ebi3þ

cells <20 were classified as Ebi3low and FOVs with number of Ebi3þ

cells �20 were classified as Ebi3hi, as reported by Mirlekar andcolleagues (19).

Analysis of Ebi3, p35, and cytokeratin immunofluorescence inhuman PDA

Slides containing fluorescently labeled tissue sections were scannedin the Aperio ScanScope FL (Leica Biosystems) using the 20� objectiveand images were archived in TPL's eSlide Manger database (LeicaBiosystems). Images were manually annotated to demarcate tumor (allimages) and lymphoid regions (present on three images) using AperioImageScope (v12.4.2.700). To reduce operator bias, annotations inlymphoid regions were made using the Hoechst nuclear counterstainlayer (EBI3 and IL12a image layers were not visible). Annotated imageswere then analyzed using Tissue Studio software (Tissue Studio version2.7withTissue Studio Library version 4.4.2;Definiens Inc.). For analysisof tumors, regions positive for cytokeratin were digitally separated fromthe surrounding stroma (Tissue StudioComposer software) followedbycellular analysis for coexpression of EBI3, IL12a, and cytokeratin. Inlymphoidareas, cellular analysis for coexpressionofEBI3 and IL12awasperformed on all cells within the annotation regions. Analysis dataincluded the percentage of co-expressing cells and the average cellularfluorescent intensities for each antibody marker.

Generation and analysis of RNA sequencing data from humanB cells

An RNA sequencing (RNA-seq) library preparation for humanPBMC B-cell populations derived from three healthy volunteers andthree treatment-na€�ve patients with PDAwas generated. PDA sampleswere preselected on the basis of positivity for p35 and Ebi3 expression,as assessed by qPCR.

Samples were sorted for humanBregs and Bconmarkers (12 samplestotal), as described above, and template RNA was derived from freshlysorted single-cell suspensions using the Illumina TruSeq strandedlibrary preparation protocol. Sequencing was done on the IlluminaHiSeq4000 platform using 150 bp paired-end chemistry and targeting 9� 107 reads per sample. TCGA expression matrices were accessed athttp://firebrowse.org. FASTQ files were aligned to the human referencegenome using STAR v2.4.2. The BAMoutput files were then quantifiedusing Salmon v0.8.2. FastQC v0.11.7 and MultiQC v1.5 were used togenerate quality assurance reports (Supplementary File S1). Statisticalanalyses were executed in R v3.3.3. Differential gene expression analysiswas conducted on the resulting expressionmatrices using theDESeq2Rpackage. Genes that were found to be differentially upregulated intumor-associated Breg subtypes compared with Bcon subtypes, with aBenjamin–Hochberg corrected P value of less than 0.1, were identified.A tumor-infiltrating Breg signature was calculated by taking thegeometric mean of the expression values of the identified genes. TheBreg signature was then calculated for TCGApancreatic cancer tumors.Gene signatures for CD8þ T cells and cytotoxicity were calculated bytaking the geometric mean of the CD8 T-cell gene signature genes (asper ref. 34) and the genes PRF1 and GZMA as per Rooney andcolleagues (35), respectively. A scatterplot was generated from thetumor-infiltrating Breg signature and the ratio of the CD8þ T-cell tocytotoxicity signature using the ggplot2 R package. These gene signa-tures were calculated across tumor types in TCGA. Correlation coeffi-cients were generated for each tumor type, and a volcano plot depictingthe results was created using the ggplot2 R package. Gene set enrich-

ment analysis (GSEA v3.0) was used comparing different subtypes forgene sets, C2 andC7, from theMolecular SignaturesDatabase (MSigDBv6.2). Raw sequencing patient data can be found at NIH Gene Expres-sion Omnibus (GEO) repository under accession #GSE144504. Rawsequencing data for healthy controls cannot be deposited due toInstitutional IRB Certification guidelines. Raw data for controls areavailable upon request from the corresponding author.

Statistical analysisAt least 9 to 21 mice were used in each group, with a minimum of 6

mice in each group per experiment, and the experiments were repeateda minimum 3 times to validate reproducibility. Before analysis, datawere examined for quality. Groupmeans were compared with Studentt tests. Significance in variations between two groups was determinedby unpaired Student t test (two-tailed). Statistical analysis was per-formed usingGraphPad Prism software. Data are presented asmean�SEM. P < 0.05 was considered statistically significant.

ResultsIL35 suppresses chemotactic receptor expression and effectorfunction in CD8þ T cells

IL35-mediated tumor growth is associated with changes inT-effector and Treg subsets (19). To understand the cellular basis ofIL35 immunosuppression, we depleted CD4þ T cells or CD25þ Tregsin p35�/� mice orthotopically injected with primary syngeneicKrasG12D;Trp53R172H;p48Cre/þ (KPC) mouse pancreatic cancer cells(Supplementary Fig. S1A–S1D; ref. 19). Neither one of these depletionstrategies rescued tumor growth, whereas, as previously shown, deple-tion of CD8þ T cells fully restored tumor growth, prompting us to askwhether IL35 may directly signal to CD8þ T cells (SupplementaryFig. S1A–S1D; ref. 19). IL35 has been shown to signal through theSTAT1/STAT4 heterodimer in CD4þ T cells. However, the mecha-nism of IL35 signaling in CD8þ T cells is not known (20). We foundthat recombinant IL35 elicited an increase in phosphorylation ofSTAT1, STAT3, and STAT4 in CD8þ T cells, raising the possibilitythat these pathways could regulate IL35-dependent CD8þ T-cellfunction (Fig. 1A). Because one of the features of IL35 loss is increasedinfiltration and activation of CD8þ T cells in the tumor parenchy-ma (19), we asked whether IL35 could directly affect the expression ofchemokine receptors and/or IFNg production by cytotoxic T lym-phocytes by examining the expression of CXCR3 and CCR5, whichhave been implicated as important facilitators of intratumoral CD8þ

T-cell infiltration (17, 36). We found that incubation of activatedOVA-specific CD8þ T cells with recombinant IL35 resulted in sig-nificantly attenuated expression of IFNg and both CXCR3 and CCR5,consistent with the observation that p35 expression correlates withdecreased CXCR3 in autoimmunity (Fig. 1B; ref. 37).

In CD4þ T cells, IL35 mediates function via receptor chainsIL12Rb2 and gp130, which signal to activate STAT1 and STAT4 butnot STAT3 (38). To understand how IL35R contributes to STAT3activation in CD8þ T cells, we sorted T cells based on singularexpression of either IL12Rb2 or gp130 (Supplementary Fig. S2A). Wefound that when treated with IL35, IL12Rb2þCD8þ T (subset-1) cellsactivated STAT4, and the gp130þ subset (subset-2) activated bothSTAT1 and STAT3 (Fig. 1C; Supplementary Fig. S2A). Consistentwith this observation, treatment with rIL35 activated all three STATsin IL12Rb2þgp130þCD8þ T cells (Supplementary Fig. S2B and S2C).Modulation in IFNg , CXCR3, and CCR5 correlated with expression ofthe gp130þ subunit, although IL12Rb2þCD8þ T cells somewhatdownregulated IFNg and CXCR3 but not CCR5 (Fig. 1D). Thus,

Mirlekar et al.




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B cell–derived IL35 promotes tumor growth andmediates suppression of IFNg and chemotactic receptors CXCR3 and CCR5 in CD8þ T cells.A, Experimental schemaused to generate tumor-bearing mixed bone marrow chimeras containing B cell–specific deletion of p35 (Bp35�/�) and corresponding control BWT mice. B,Quantification of tumor weight from BWT and Bp35�/� mice 3 weeks post-orthotopic injection with KPC4662 cells (n ¼ 6 mice/group). C, Representative flowcytometry histograms (left) and quantification (right) of intracellular p35 expression in intratumoral and intrasplenic CD19þCD21hiCD5þCD1dhi Bregs frommice in B.Proportion of p35þBregs is indicated.D,Representativeflowcytometry histograms (left) andquantification (right) of intratumoral CD45þCD3þCD4þCD25� effectorT cells in BWT and Bp35�/� mice from B. E, Representative flow cytometry plots (left) and quantification (right) of intracellular IFNg (isotype; rat IgG1, k) and TNFa(isotype; rat IgG1, k) expression by CD3þCD4þ intratumoral T cells from mice in B. Proportion of CD4þ T cells is indicated. F, Representative flow cytometryhistograms (left) and quantification (right) of intratumoral CD3þCD4þFoxp3þ Tregs from mice in B. Proportion of CD4þ T cells is indicated. G, Representative flowcytometry histograms (left) and quantification (right) of intracellular p35 (isotype; rat IgG2a, ) and IL10 (isotype; rat IgG2b, k) expression by intratumoral CD3þCD4þ

T cells frommice inB.H,Quantification of frequency of tumor-infiltrating CD45þCD3þCD8þ T cells fromBWT andBp35�/�mice fromB determined by flow cytometry.I,Representative flow cytometry plots (left) and quantification (right) of IFNg (isotype; rat IgG1, k) in intratumoral CD45þCD3þCD8þ T cells frommice inB. J,Ratio ofmean CD3þCD4þCD25� effector T cells (Teff) to Tregs (left) and ratio of mean CD3þCD8þ cytotoxic T cells to Tregs (right) were calculated on the basis of thepercent-positive lymphocyte population determined by flow cytometry. Error bars, SEM; P valueswere calculated using Student t test (unpaired, two-tailed); NS, notsignificant. �� , P < 0.01; �� , P < 0.001. Data represent three independent experiments.






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IL35 signaling in gp130þCD8þ T cells impeded expression of CXCR3,CCR5, and IFNg .

STAT3 inhibition in tumor-educated CD8þ T cells reducespancreatic tumor growth

To understand which STAT signaling pathway was responsible forregulating IFNg and/or chemokine receptors on CD8þT cells, we usedinhibitors of pSTAT1, pSTAT3, and pSTAT4 (fludarabine, STA-21, orlisofylline, respectively; refs. 39–41) in vitro on antigen-directed CD8þ

T cells in the presence or absence of rIL35 (Fig. 2A; SupplementaryFig. S3A–S3D). Phospho-STAT3 was not affected in antigen-stimulated CD8þ T cells, and treatment with STA-21 did not affectIFNg production or CXCR3/CCR5 expression (Fig. 2A). However,reduction of IFNg , as well as CXCR3 and CCR5 caused by treatmentwithOVAþ rIL35was rescued by addition of STA-21 (Fig. 2A andB).STAT1 and STAT4 activation was needed for sustaining IFNg expres-sion following antigenic stimulation of CD8þ T cells, likely reflectingsignaling by antigen-presenting cells via IL12 (SupplementaryFig. S3A–S3D; ref. 41). However, neither STAT1 nor STAT4 inhibitionin activated CD8þT cells treated with recombinant IL35 was sufficientto rescue IFNg production and chemokine receptor expression (Sup-plementary Fig. S3A–S3D). Overall, these data suggest that IL35-mediated activation of STAT3 in CD8þ T cells impeded multiple axesrequired for their functionality.

We reasoned that if IL35-driven activation of STAT3 in CD8þ Tcells was an important factor driving CD8þ T cell exclusion andinactivity in PDA, then inhibition of STAT3 activation in tumor-educated immunosuppressed CD8þ T cells could confer antitumorresponses. To test this, we isolated splenic CD8þ T cells fromWT CD45.2þ mice harboring KrasG12D;Trp53R172H;p48Cre/þ (KPC)orthotopic tumors, and treated T cells with either control or STA-21ex vivo (Fig. 2C). Control or inhibitor-treated T cells were thenadoptively transferred to WT CD45.1þ mice, followed by orthotopicinjection ofKPC tumor cells.We observed significantly reduced tumorgrowth in mice that received STA-21–pretreated CD8þ T cells(Fig. 2D). This was accompanied by increased infiltration ofCD45.2þCXCR3þCCR5þCD8þ T cells and significant increases inIFNg production but no differences in infiltration by endogenousCD45.1þCD8þTcells (Fig. 2E–G). Lethally irradiatedmice adoptivelyinfused with STA-21–treated CD8þ T cells were also able to bettercontrol tumor growth (Supplementary Fig. S4). These experimentssuggested that activation of STAT3 in CD8þ T cells may regulate theirinfiltration and antitumor activity in vivo.

IL35 production by Tregs is dispensable for pancreatic tumorgrowth

Both Bregs and Tregs can produce IL35 in a variety of diseasesettings, including PDA (19, 20). IL35 production by Foxp3þ Tregs isimportant for tumor growth in models of melanoma and colorectalcancer (21). To determine whether Treg-derived IL35 confers

immunosuppression in PDA, we used mice with Treg-specificFoxp3Cre-YFP-driven deficiency in Ebi3 (TregEbi3-/�; SupplementaryFig. S5A and S5B; refs. 21, 28–31). We found that Treg-specificdeficiency in IL35 did not alter orthotropic tumor growth, as KPCcells injected into either Treg

Ebi3þ/� or TregEbi3�/� littermates grew to a

comparable tumor size, even though Ebi3 expression in CD4þ T cellswas reduced to background levels (Supplementary Fig. S5C and S5D).Intratumoral and splenic frequency of total Foxp3þ Tregs, as well ascytokine production by B cells, was not altered in mice with Treg-specific deletion of Ebi3, suggesting that endogenous production ofIL35 by Tregs in PDA does not control Treg or IL35þ B-cell expansionat the tumor site (Supplementary Fig. S5E–S5H). The frequency andactivity of CD4þ and CD8þ T effector cells and frequencies of myeloidsubsets were similar between Treg

Ebi3þ/� or TregEbi3�/� animals

(Supplementary Fig. S5I–S5R). These observations highlight intrinsicdifferences in how different tumors types co-op IL35 and suggest thatin the presence of IL35þ B cells, reduction of IL35 expression by Tregcells is not sufficient to alter PDA growth.

IL35þ B cells establish immunosuppression in PDATo determine whether B cell–derived IL35 confers immunosup-

pression in PDA, we first generatedmice with B-cell specific deficiencyin the p35 (Bp35�/�) subunit of IL35 using a mixed bone marrowchimera approach (Fig. 3A; Supplementary Fig. S6A). Orthotopicinjections of KPC cells resulted in attenuated PDA growth comparedwith controls (Fig. 3B). Analysis of IL35 expression in B cells con-firmed near complete reduction in p35 protein in both splenic andintratumoral Bregs (Fig. 3C; Supplementary Fig. S6B and S6C).Previously, we observed that B cell–specific loss of IL35 resulted indecreased proliferation of cells transformed by KrasG12D alone (18). Incontrast,KPC cells did not require B cell–derived IL35 for proliferationin vivo (Supplementary Fig. S6D). These findings, together with ourobservations suggesting that IL35 affected antitumor immuneresponses, prompted investigation into the immune functionalityfollowing B cell–specific deletion of IL35 (19).

Intratumoral immune profiling revealed elevated frequency ofIFNgþ and TNFaþCD4þ effector T cells, as well as decreased inintratumoral but not splenic FoxP3þ Tregs, in Bp35�/� and BEbi3�/�

mice compared with BWT mice, suggesting that B cell–derived IL35may directly or indirectly regulate Treg frequency locally but notsystemically (Fig. 3D–F; Supplementary Fig. S6E). Because it has beenproposed that exogenous IL35 may promote expansion of suppressiveTregs by inducing its own expression in CD4þ T cells (20), weexamined the ability of Tregs to produce IL35 in the context of Bcell–specific deletion of this cytokine. Although the overall frequencyof intratumoral Tregs was reduced, the ability of Tregs to produce IL35remained intact (Fig. 3G; Supplementary Fig. S6E). Overall, thissuggested that although B cell–directed IL35 signaling may regulatenumbers of CD4þ T-cell subsets in PDA, IL35 expression by Tregscould be maintained in a cell autonomous fashion. The changes in the

Figure 4.Production of IL35 by B cells impedes anti–PD-1 therapy. A, Experimental schema used to generate tumor-bearing mice that contain B cell–specific deletion of Ebi3(BEbi3�/�) and corresponding control BEbi3þ/� mice. B, Quantification of tumor weight from anti–PD-1–treated (200 mg) and IgG-treated (200 mg) BEbi3þ/� andBEbi3�/�mice 3weeks after orthotopic injectionwith KPC cells (n¼ 6mice/group). C,Quantification of tumor growth by ultrasound from BEbi3þ/� and BEbi3�/�micefrom B. D, Quantification of frequency of tumor-infiltrating CD45þCD3þCD8þ T cells from BEbi3þ/� and BEbi3�/� mice from B determined by flow cytometry. E,Representative flow cytometry plots of intratumoral CD45þCD3þCD8þ T cells stained for IFNg (isotype; rat IgG1, k) frommice in B. F,Quantification of intratumoralCD8þIFNgþT cells fromallmice inB.G,Representative flowcytometry histograms (left) and quantification (right) of expressionof CXCR3, CCR5, phospho(p)-STAT1,pSTAT3, and pSTAT4 in intratumoral CD3þCD8þT cells isolated fromBEbi3þ/� andBEbi3�/�mice fromB.H,Representative flowcytometry plots andhistograms (left)and quantification (right) of IFNg production and expression of CXCR3, CCR5, pSTAT1, pSTAT3, and pSTAT4 in CD3þCD8þ T cells 48 hours after coculturewith Bregsin 1:1 ratio derived from the indicated mice. Experiment were done in triplicate with 6 mice per group. 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. Data represent three independent experiments.






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Mirlekar et al.





T-cell landscape were accompanied by increased infiltration and IFNgexpression by effector CD8þT cells, and overall, there was a significantincrease in the ratio of CD4þ and CD8þ effector T cells to Tregs(Fig. 3H–J; Supplementary Fig. S6F and S6H). Concordant with ourfindings using global genetic loss of IL35, the frequency of myeloid cellsubsets was not affected by B-cell loss of IL35 (Supplementary Fig. S6I–S6L). These results suggested that cancer-associated IL35 expression inB cells was sufficient to dampen productive T cell–mediated immuneresponses in PDA.

Depletion of IL35 fromB cells sensitizes PDA to immunotherapyAnti–PD-1 checkpoint blockade does not impede murine PDA

growth, and the response rate in human PDA has been poor (42). Wehave reported that complete genetic deficiency in IL35 confersincreased PDA sensitivity to anti–PD-1 treatment, presumablythrough increased numbers of infiltrating CD8þ T cells. However,the cell type–specific contribution to the establishment of IL-35–driven immunosuppression and resistance to anti–PD-1 remainsunclear (19). To develop a tractable model of B cell–specific loss ofIL35, we generated a model where B cells lacked expression of Ebi3(BEbi3�/�) subunit (Fig. 4A; Supplementary Fig. S7A) by crossing amouse with a conditional deletion of floxed Ebi3 allele with a mousecoding for B cell–specific Cre recombinase (CD19Cre; refs. 21, 32). Tounderstand whether B cell–specific deficiency in IL35 was sufficient toconfer sensitivity to anti–PD-1, we treated BEbi3þ/� and BEbi3�/�micewith anti–PD-1 or isotype control (Fig. 4A). Orthotopic injections ofKPC cells phenocopied our results with Bp35�/� mice and resulted indecreased PDA growth (Fig. 4B). Combination of anti–PD-1 with Bcell–specific loss of IL35 conferred additional significant reduction intumor growth compared with BEbi3þ/�mice treated with either isotypeor anti–PD-1 or BEbi3�/� mice treated with isotype control (Fig. 4Band C). Reduced Ebi3 protein was seen in splenic and intratumoralBregs (Supplementary Fig. S7B and S7C).We also found thatKPC cellswere not dependent on B cell–derived Ebi3 for proliferation in vivo(Supplementary Fig. S7D). Loss of B cell–derived Ebi3 increased CD4þ

effector T cells and decreased intratumoral Treg frequency (Supple-mentary Fig. S7E–S7G) and increased infiltration and IFNg expressionby effector CD8þ T cells (Fig. 4D–F). Anti–PD-1–treated BEbi3�/�

mice exhibited slightly upward trending changes in effector CD4þ

T cells, comparable with those in IgG-treated BEbi3�/�mice. The ratioof CD4þ and CD8þ effector T cells to Tregs was significantly higher inBEbi3�/�mice treated with anti–PD-1 compared with the BEbi3�/� IgGcohort, and may contribute to better tumor control, but thefrequency of myeloid cell subsets was not affected by B-cell lossof Ebi3 (Supplementary Figs. S7I and S7J and S8A–S8E). Overall,these data suggested that elimination of IL35 expression from B cellsalone may benefit anti–PD-1 efficacy by potentiating effector T-cellresponses.

To determine which T-cell subset was responsible for reducedtumor growth when IL35 was deleted from B cells, we depletedeither CD4þ T cells, CD25þ Treg, or CD8þ T cells (SupplementaryFig. S9A-I). Tumor growth was rescued only under conditions ofCD8þ T-cell depletion, suggesting that B cells may utilize IL35 todirectly control CD8þ T-cell functionality (Supplementary Fig. S9A–S9I).We then analyzed the role of B cell–derived IL35 in suppression ofCD8þT-cell function in vivo. In addition to altered expression of IFNg(Fig. 3I, Fig. 4E and F), B cell–specific deletion of IL35 correlated withincreased expression of both CXCR3 and CCR5 and a concordantdecrease of pSTAT1, pSTAT3, and pSTAT4 in CD8þ T cells (Fig. 4G;Supplementary Fig. S10A and S10B). These results suggested that Bcell–derived IL35 in PDA acted to restrain infiltration and effectorfunction of cytotoxic CD8þ T cells.

To understand whether IL35 production by B cells was sufficient tosuppress CD8þ T-cell function, we established coculture assays wherecytokine production in splenic B cells derived from tumor-bearinganimals was stimulated by addition of LPS and anti-CD40. Activated Bcells were seeded in Transwells and cocultured with activated OVA-directed CD8þT cells. Activated B cells inhibited IFNg production andchemokine receptor expression by CD8þ T cells in an IL35-dependentmanner (Fig. 4H). This functional suppression correlated withincreased phosphorylation of STAT1, STAT3, and STAT4 in CD8þ

T cells (Fig. 4H).

Pharmacologic blockade of IL35 reduces pancreatic tumorgrowth

Our published data indicate that global genetic deficiency in IL35increases intratumoral cytotoxic CD8þ T cells and suggests thattargeting IL35 genetically may be an effective strategy to convert PDAfrom an immunologically “cold” to a “hot” tumor (19). Treatmentsthat increase tumor infiltration by CD8þ T cells represent a promisingstrategy for use in combination with checkpoint blockade. To deter-mine how pharmacologic blockade of IL35 affected PDA tumorgrowth and responsiveness to anti–PD-1, WT mice were orthotopi-cally injected with KPC cells and treated with monotherapies orcombination therapy (Fig. 5A). Blockade of IL35 resulted in significantreduction of serum IL35 and a concordant reduction of tumor growth(Fig. 5B; Supplementary Fig. S11A). Treatment with combinationanti–PD-1 and IL35 blockade further improved tumor growth control(Fig. 5B). Because anti-IL35 targets the Ebi3 subunit, which is also asubunit of IL27, we tested whether depletion of IL27 affected PDAgrowth. Anti-IL27 (against the p28 subunit) did not elicit reducedtumor growth, suggesting that the action of anti-IL35 was on target(Supplementary Fig. S11B). Measurements of tumor growth duringtreatment with anti-IL35 revealed that both single-agent and combi-nation with anti–PD-1 had an effect on tumor growth approximately14 days post-treatment initiation (Fig. 5C). Survival of orthotopically

Figure 5.IL35 blockade relieves immunosuppression of CD8þ T cells and synergizes with anti–PD-1. A, Schematic of the antibody treatment regimen. Anti-IL35 (200 mg firstdose, followed by 100 mg/week) or control IgG antibody was administered in therapeutic schedule (1 week after tumor cell injection on days 7, 11, and 15).Administration of anti–PD-1 (200 mg) was initiated on day 7 after tumors reached approximately 4 to 5 mm in diameter. Two more doses of anti–PD-1 wereadministered on days 9 and 11. Micewere sacrificed 3weeks after tumor cell injection or assessed for survival.B,Quantification of tumorweight fromWTmice treatedwith therapeutic anti-IL35, anti–PD-1, or combination (as inA) 3weeks after orthotopic injectionwithKPC cells (n¼6mice/group).C,Quantification of tumor growthby ultrasound from WT mice in B. D, Survival plot of orthotopically injected WT mice from B treated with the indicated therapeutic antibodies. E, Quantification oftumor growth by ultrasound from spontaneous KPCmice treated with therapeutic anti-IL35, anti–PD-1, or combination as in A. Treatment was initiated when tumormeasuring approximately 5 mmwas visualized by ultrasound (n¼ 6 mice/group). Data represent three independent experiments. F,Quantification of frequency oforthotopic tumor-infiltrating CD45þCD3þCD8þ T cells frommice in B.G, Representative flow cytometry plots of intracellular IFNg in intratumoral CD45þCD3þCD8þ

T cells from the mice in B. Proportion of CD8þ T cells is indicated. H,Quantification of intratumoral CD8þIFNgþ T cells from the mice in B. I,Quantification of CXCR3,CCR5, pSTAT3, and pSTAT4 expression in intratumoral CD3þCD8þ T cells from mice in B. 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. Data represent three independent experiments.






Figure 6.

Identification of IL35-producing B cells in patients with PDA. A, Quantification of CD19þCD24hiCD38hi B cells in peripheral blood of healthy volunteers (n¼ 30) andtreatment-na€�ve PDA patients (n ¼ 30). Proportion of CD19þ cells is indicated. B, Fold change in Il10, p35, and Ebi3 from sorted CD19þCD24hiCD38hi Bregs orCD19þCD24loCD38lo Bcon cells from healthy volunteers or patients with PDA (n ¼ 5 samples/B-cell group) determined by qPCR. Fold change determined bycomparing with healthy Bcon cells. (Continued on the following page.)

Mirlekar et al.





injected mice treated with combination anti-IL35 and anti–PD-1 wasalso significantly improved (Fig. 5D). Treatment of animals bearingspontaneous KrasG12D;Trp53R172H;p48Cre/þ (KPC)-driven pancreaticcancer with either anti-IL35 alone or combinationwith anti–PD-1 alsosignificantly reduced tumor growth (Fig. 5E). Reduced tumor growthin the orthotopic setting was accompanied by increased infiltrationand activation of effector T cells, as well as a reduction in Tregfrequency, corroborating our observations in models of completeor B cell–specific IL35 deficiency (Fig. 5F–H; SupplementaryFig. S11C–S11E). Mechanistically, administration of anti-IL35 signif-icantly reduced the frequency of IL35-producing CD4þ T cells andBregs, suggesting that IL35 may regulate its own expression insuppressive cell types (Supplementary Fig. S11F and S11G). We alsofound that the frequency of PD-1þCD8þ T cells increased intreated animals, suggesting that anti-IL35 blockade in PDA resultedin increased effector function of CD8þ T cells (SupplementaryFig. S11H). We confirmed that expression of CXCR3 and CCR5 wasincreased on CD8þ T cells derived from anti-IL35–treated animals,concordant with decreases in pSTAT3 and pSTAT4 activation,although no additional changes were observed upon addition ofanti–PD-1 (Fig. 5I; Supplementary Fig. S11I).

Immature B cells produce IL35 and IL10 in human PDATo understand which human B-cell subset may correspond to

murine IL35þIL10þ B cells, we analyzed peripheral blood of healthydonors and treatment-na€�ve patients with PDA for presence ofpreviously reported human Breg markers (Supplementary Fig. S12A;refs. 43–47). CD19þB cells fromperipheral bloodwere sorted and lysedto assess their ability to express cytokines. QPCR revealed that theCD19þCD24hiCD27þ B10 subset produced IL10, whereas theCD19þCD24hiCD38hi immature B-cell subset was most similar to themurine Bregs and produced both IL10 and IL35 (SupplementaryFig. S12B). Analysis of themajor immune cell subtypes fromperipheralblood of patients with PDA by flow cytometry was concordant withdata frommousemodels (19) and indicated that IL35 (p35þEbi3þ) wasprimarily expressed by B cells and CD4þ T cells, with a largercontribution from B cells (Supplementary Fig. S12C and S12D).Neither murine plasma cells nor human plasmablasts produced IL10nor IL35, indicating that the cytokine-producing B cells in PDAdid notdevelop from plasma cells, as has been suggested in prostate cancer(Supplementary Fig. S13A–S13D; refs. 24, 48).

Overall, the frequency of CD19þCD24hiCD38hi immature B cells inthe peripheral blood of patients with PDAwas significantly higher thanin healthy controls (Fig. 6A). We found that CD19þCD24hiCD38hi

peripheral B cells from patients with PDA expressed significantlyhigher IL35 and IL10 compared with healthy controls and Bcon cells(Fig. 6B). This observation is consistent with the notion that IL35 isinduced in the context of inflammation (20). There was heterogeneityamong patients with regards to frequency of peripheral blood imma-

ture IL35þ B cells, suggesting heterogeneity of Breg responses in PDApatient population (Fig. 6A–C). Thus, at least some patients with PDAexhibit B cell–driven immunosuppressive mechanisms, as evidencedby increased Breg activity.

IL35þ immune cells correlatewith pSTAT3þCXCR3�CD8þ T cellsin human PDA

We previously documented the presence of intratumoral IL35þ Bcells and T cells in human PDA (19). Consistent with our hypothesisthat IL35 may suppress CD8þ T-cell infiltration, we reported thatIL35þ immune cells negatively correlatedwithCD8þT-cell infiltrationinto human tumor cell nests (19). To interrogate the relationshipamong IL35 expression, STAT3 activation, and CD8þ T-cell infiltra-tion, we used multiplexed immunofluorescence to analyze primaryhuman PDA tissues for CD8þ T cell–specific expression of pSTAT3andCXCR3 in IL35 high (Ebi3hi) or IL35 low (Ebi3lo) regions (Fig. 6D;ref. 19).We found that the majority of IL35þ cells resided at theinterface between immune aggregates (IA) and tumor parenchyma,suggesting that IAs might serve as sites of active immune suppressionin PDA (Fig. 6E). Consistent with the idea that IL35 may induceSTAT3 activation inCD8þT cells, we found that the presence of IL35þ

immune cells positively correlated with pSTAT3 activation in CD8þ

T cells, and there was very little STAT3 activation in the regions thatcontained few or no IL35þ cells (Fig. 6F). Overall, the percentage ofpSTAT3þCD8þ cells was not high, but specifically correlated withpresence of IL35þ immune cells. On the contrary, the presence ofCXCR3þCD8þ T cells, overall, negatively correlated with IL35 expres-sion (Fig. 6G). Analysis of IL35þ regions revealed a significant negativeassociation between pSTAT3 activation and CXCR3 expression inCD8þ T cells (Fig. 6H). We also examined coexpression of Ebi3 andp35 proteins on archived human PDA samples and found that,consistentwith reports, Ebi3 andp35were coexpressed in cytokeratinþ

cells at frequencies ranging from 0.37% to 89.26% of all cytokeratinþ

cells, with a median value of 18.27% (Supplementary Fig. S14A andS14B; ref. 49). Although the consequences of epithelial-specific IL35expression in PDA on immune function are not yet clear, our initialanalysis did not find a correlation between the frequency of IL35þ

cancer cells and CD8þ T-cell infiltration into the tumor parenchyma(Supplementary Fig. S14C). These results suggested that IL35þ

immune cells in the human PDA TME may restrict CD8þ T-cellinfiltration into the tumor parenchyma by suppressing expression ofCXCR3 via STAT3 activation.

To understand how human IL35þ B cells correlated with overallcytotoxic responses in human PDA, we performed bulk RNA-seq onsorted peripheral Bregs and Bcon cells from patients with PDA orhealthy volunteers. A tumor-associated Breg signature was calculatedas described in the Materials and Methods. This gene signaturecorrelated with the ratio of a CD8 signature and a previously publishedcytotoxicity score (50). Specifically, the cancer-derived Breg signature

(Continued.) C, Fold change in expression of Il10, p35, and Ebi3 from sorted CD19þCD24hiCD38hi Bregs in healthy donors or patients with PDA determined by qPCR.D, Representative immunofluorescence staining for CD8, CXCR3, and pSTAT3 in samples of human PDA. Arrow, pSTAT3þCD8þ T cells; arrowhead, pSTAT3�CD8þ Tcells; and asterisks, pSTAT3þCD8� cells. Scale bars, 25 mm. E, Proportion of Ebi3-high and -low tumor regions as a function of immune aggregates (IA). Data werederived fromcounting 3 to 6field-of-view (FOV)/tumor sample (n¼ 11 tumor samples). F,Proportion of pSTAT3þCD8þT cells as function of low versus high numbersof Ebi3þ immune cells. Eachdatapoint is the percentageof pSTAT3þCD8þT cells out of all CD8þT cells/20�FOV.Datawere derived fromcounting 3 to 6FOV/tumorsample (n¼ 11 tumor samples).G,Proportion of CXCR3þCD8þ T cells as function of low versus high numbers of Ebi3þ immune cells. Each data point is the percentageof CXCR3þCD8þ T cells out of all CD8þ T cells/20x FOV. Data were derived from counting 3 to 6 FOV/tumor sample (n ¼ 11 tumor samples). H, Proportion ofCXCR3þCD8þT cells as function of pSTAT3þ versus pSTAT3� in tumor regions high for Ebi3þ immune cells. Each data point is the percentage of CXCR3þCD8þT cellsout of all CD8þ T cells/20� FOV. Data were derived from counting 3 to 6 FOV/tumor sample (n¼ 11 tumor samples). I, Correlation of the cancer Breg signature withthe cytotoxic CD8þ T-cell index in PAAD from TCGA. J, Correlation of the cancer Breg signature with the cytotoxic CD8þ T-cell index across TCGA subtypes. Errorbars, SEM; P values were calculated using Student t test (unpaired, two-tailed). � , P < 0.05; �� , P < 0.01; �� , P < 0.001. Data represent three independent experiments.






inversely correlated with the ratio of the T-cell cytotoxicity score to theCD8 score in The Cancer Genome Atlas pancreatic adenocarcinoma(TCGAPAAD) pancreatic cohort (Fig. 6I).We also found that severaladditional cancer types in TCGA presented with an upregulated Bregsignature that negatively correlated with antitumor T-cell activity,suggesting that the role of IL35þ B cells in regulating T-cell responsesin cancer was broader than previously appreciated (Fig. 6J).

DiscussionT cell–based immunotherapy is at the frontlines of clinical care for

several types of cancer. Unfortunately, PDA thus far has failed torespond to checkpoint blockade therapy (42). Effective responses toimmunotherapeutics has been shown to correlate with preexistingintratumoral effector T-cell infiltration, which is lacking in a largeproportion of PDA cases (1). Establishment of diverse immunosup-pressive networks and physical barriers, such as dense desmoplasia,has been implicated in restricting endogenous antitumor responsesand immunotherapy efficacy in PDA (6). Considerable effort has beenput into delineating the contribution of some major immune infil-trating cells, such as myeloid cells and Tregs. Recruitment andprotumor polarization of macrophages and MDSCs have been shownto constrain CD8þ T-cell responses by engaging GM-CSF and CSFR1axes (11, 51). Tolerogenic CD206þMHCIIlow tumor-associatedmacrophages can also promote Treg expansion (52). Tregs, whichare well-known drivers of suppression of tumor-directed CD8þ T-cellresponses, are shown to also act on DCs and prevent productiveantigen presentation (14). Investigations into rare subsets of immunecells present in PDA TME, such as gd T cells, have yielded importantinsights into contributions of these cells to biology of PDAdevelopment (16).

A number of cell types have been shown to produce immunosup-pressive IL35 in cancer, including Treg and B cells, as well as DCs (20).A study has suggested that a subset of patients with PDA haveupregulated IL35 in tumor cells, although it is not clear what effectthis has on immune function (49). Murine KPC cells used in our studydo not produce IL35 and, therefore, recapitulate tumor biology whereIL35 is sourced from the stromal microenvironment. Here, we showedthat B cell–specific production of IL35 suppressed endogenous T-cellresponses in PDA.Genetic or pharmacologic targeting of IL35 resultedin decreased tumor growth associated with increased effector T-cellinfiltration and IFNg production. We uncovered that sensitivity toanti–PD-1 was evident in the context of targeting IL35.We found thatB cell–derived IL35 directly suppressed infiltration and activation ofCD8þ T cells by downregulating chemotactic receptors CXCR3 andCCR5 and effector cytokine IFNg , and this was dependent on IL35-induced phosphorylation of STAT3 in CD8þT cells. Ex vivo inhibitionof pSTAT3 activated CD8þT cells frommice bearing PDA tumors andconferred antitumor activity upon adoptive transfer of inhibitor-treated T cells. These data point to a previously unappreciated roleof B cells in directly suppressing infiltration and activity of tumor-directed CD8þ T cells in PDA.

Although B cells can account for up to 25% of immune infiltratein some solid tumors and up to 15% in human PDA (53, 54), theprecise mechanisms of B-cell contribution to tumor growth are notwell established. In concert with the idea that B lymphocytes playversatile roles in solid cancer biology, we and others have demon-strated that B cells promote PDA growth in coordination withhypoxia via immunoglobulin deposition or by production ofIL35 (18, 54, 55). These findings have led to initiation of severalclinical trials in patients with PDA using combination chemother-

apy and ibrutinib (an inhibitor of Btk found in both activated B cellsand macrophages), marking an important foray into testing thecontribution of B-cell and macrophage activation in human PDA(NCT02436668). However, given the diverse repertoire of B-cellfunction in cancer, it is important to better understand the con-sequences of disrupting the functionality of individual B-cell subsetsin PDA.

Our data suggested that IL35þ B cells directly regulate infiltrationand function of CD8þ T cells in PDA in a STAT3-dependent manner.CXCR3 and CCR5 are chemokine receptors expressed on effectorT cells, can be upregulated by ligands CXCL9, CXCL10, and CXCL11,and have been shown to be important for T-cell infiltration into thetumor bed (36). Studies in melanoma and lung cancer models havefound that genetic lack of STAT3 in T cells results in increasedmigration and effector function (56, 57). These observations, togetherwith our data and studies showing that STAT3 regulates proliferation,survival, cytotoxic gene expression, and memory function in CD8þ

T cells that respond to viral infections, suggest that STAT3 could bea possible therapeutic target for enhancing antitumor CD8þ T cell–mediated responses (58, 59). Further work needs to be done toestablish if IL35 blockade and/or inhibition of STAT3 in effectorT cells generates persistent memory responses and may providefurther insight into strategies for combination immunotherapy.STAT inhibitor studies performed by us at this point were doneex vivo and are limited to proof-of-concept because all threeinhibitors used (fludarabine, STA-21, and lisofylline) may haveoff-target effects (60–62).

Concordant with previous studies suggesting that B cells may influ-ence Treg numbers in cancer, we found that B-cell production of IL35was important for localized expansion of Foxp3þ Tregs (63). Specifictriggers for IL35 production by Tregs in cancer have not been well-defined, although prior reports suggest that recombinant IL35 caninduce IL35 in Tregs and na€�ve CD4þCD25�Foxp3� T cells (20). Ourcurrent data showed that IL35 in B cells does not affect production ofIL35 by Tregs, so we can rule out B cells as being an initiating source ofIL35 for T-cell lineages. It is possible that Treg-specific production ofIL35 is sufficient to keep its expression going in a cell-autonomousmanner. It is also currently not known which cancer-associated inflam-matory mediators induce IL35 production in B cells, and additionalresearch dissecting the mechanisms of IL35 induction in immunosup-pressive cells may provide novel targets for therapy.

With regards to immune cell–specific contribution of IL35 totumor growth, our findings contrast data in mouse models ofmelanoma and colon cancer, which demonstrate that Treg-specific expression of IL35 is important in tumor growth con-trol (21). It is possible that the difference in overall levels of IL35produced by B cells and Tregs in distinct tumor models mayaccount for this effect. We observed that in PDA, approximately20% of Bregs and roughly 8% of CD4þ T cells produced IL35. This isin contrast with murine melanoma, where up to 40% of tumor-infiltrating Tregs are reported to produce IL35 (21).

Our study supports the idea that B cell–derived IL35 promotestumorigenesis via inhibition of CD8þ T-cell infiltration and effectorcytokine production, and we observed an increase in CD8þ T-cellPD-1 expression following anti-IL35 treatment, in concordancewith an idea that newly activated T cells upregulate PD-1 expressionovertime (27). It is also possible that physical location of immu-nosuppressive cells, with respect to tumor-reactive CD8þ T cells,may influence the nature of immunosuppression. Although ourpreliminary analysis did not indicate alterations in myeloid popula-tions in either B cell– or Treg-specific deletion of IL35, it is still

Mirlekar et al.





possible that IL35 may act on other immune cells apart from B cellsand T cells.

Validation that IL35þ B cells have a role in inducing immunosup-pression in human PDA was performed. We analyzed human B cellsfrom patients with PDA using markers CD24 and CD38, previouslyused to identify IL10-producing B cells in patients with autoimmunity,and established that the CD24þCD38þ subset produced IL35 and waspresent at higher frequencies in patients with PDA, suggesting disease-instigated expansion of this cell subtype (64). We also demonstratedthat the presence of IL35þ B cells inversely correlated with antitumorcytotoxic T-cell activity and expansion of Bregs across distinct cancertypes, suggesting that Bregs are major drivers of immunosuppressionin a variety of cancers.

Disclosure of Potential Conflicts of InterestG.P. Gupta is a consultant/advisory board member for and has ownership interest

(including patents) in Naveris Inc. D.A.A. Vignali is a scientific advisory boardmember for, reports receiving a commercial research grant from, and has ownershipinterest (including patents) in Tizona Therapeutics. No potential conflicts of interestwere disclosed by the other authors.

Authors’ ContributionsConception and design: B. Mirlekar, D.G. DeNardo, Y. Pylayeva-GuptaDevelopment of methodology: B. Mirlekar, D. Michaud, Y. Pylayeva-GuptaAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): B. Mirlekar, D. Michaud, E.C. Goldman, G.P. Gupta, R.C. Fields,J.J. Yeh, A.J. McRee, Y. Pylayeva-GuptaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): B. Mirlekar, D. Michaud, S.J. Lee, N.P. Kren,N.U. Rashid, B.G. Vincent, Y. Pylayeva-Gupta

Writing, review, and/or revision of the manuscript: B. Mirlekar, S.J. Lee, K. Greene,E.C. Goldman, R.C. Fields, W.G. Hawkins, J.J. Yeh, A.J. McRee, B.G. Vincent,D.A.A. Vignali, Y. Pylayeva-GuptaAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): B. Mirlekar, N.P. Kren, C. Harris, E.C. Goldman,R.C. Fields, W.G. Hawkins, B.G. Vincent, Y. Pylayeva-GuptaStudy supervision: E.C. Goldman, Y. Pylayeva-GuptaOther (pathology): K. GreeneOther (provision of key reagents and technical advice): D.A.A. Vignali

AcknowledgmentsWe thank B. Savoldo and G. Dotti for discussions and help with manuscript

preparation. This work was supported in part by R37 CA230786 (Y. Pylayeva-Gupta),University Cancer Research Fund at The University of North Carolina (UNC) atChapel Hill (Y. Pylayeva-Gupta); AACR–PanCAN Pathway to Leadership Grant 13-70-25-PYLA (Y. Pylayeva-Gupta), V Foundation for Cancer Research grant V2016-016 (Y. Pylayeva-Gupta), Concern Foundation Conquer Cancer Now Award(Y. Pylayeva-Gupta), the Washington University in St. Louis SPORE in PancreaticCancer (R.C. Fields and W.G. Hawkins; P50CA196510), the WUSTL SPORE CareerEnhancementAward grant 1P50CA196510-01A1 from theNCI (Y. Pylayeva-Gupta),Cancer Cell Biology Training Program (CCBTP) grant 2T32CA071341-21(D. Michaud), and R01 CA203689 (D.A.A. Vignali). The UNC Flow CytometryCore Facility, the UNC Translational Pathology Laboratory, the Animal Histopa-thology and Laboratory Medicine Core, and the UNC Lineberger Animal StudiesCore are supported in part by P30 CA016086 Cancer Center Core Support Grant tothe UNC Lineberger Comprehensive Cancer Center.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 15, 2019; revised September 13, 2019; accepted December 9, 2019;published first February 5, 2020.

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Mirlekar et al.




2020;8:292-308. Published OnlineFirst February 5, 2020.Cancer Immunol Res Bhalchandra Mirlekar, Daniel Michaud, Samuel J. Lee, et al. Exclusion in Pancreatic Cancer

T-cell+Derived IL35 Drives STAT3-Dependent CD8−B cell

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