t-cell expression of il10 is essential for tumor immune ... · for immune surveillance in the small...

Research Article

T-cell Expression of IL10 Is Essential for TumorImmune Surveillance in the Small IntestineKristen L. Dennis1, Abdulrahman Saadalla2, Nichole R. Blatner1, Shuya Wang1,Vysak Venkateswaran3, Fotini Gounari3, Hilde Cheroutre4, Casey T.Weaver5,Axel Roers6, Nejat K. Egilmez7, and Khashayarsha Khazaie2

Abstract

IL10 is attributed with immune-suppressive and anti-inflam-matory properties, which could promote or suppress cancer inthe gastrointestinal tract. Loss of IL10 exacerbates colonicinflammation, leading to colitis and cancer. Consistent withthis, transfer of IL10-competent regulatory T cells (Treg) intomice with colitis or hereditary polyposis protects against dis-ease, while IL10-deficient mice are predisposed to polyposiswith increased colon polyp load. Little is known about theprotective or pathogenic function of IL10 in cancers of the smallintestine. We found CD4þ T cells and CD4þ Foxp3þ Tregs to bethe major sources of IL10 in the small intestine and responsiblefor the increase in IL10 during polyposis in the APCD468 mousemodel of hereditary polyposis. Targeted ablation of IL10 in T

cells caused severe IL10 deficiency and delayed polyp growth.However, these polyps progressively lost cytotoxic activity andeventually progressed to cancer. Several observations suggestedthat the effect was due to the loss of IFNg-dependent immunesurveillance. IL10-incompetent CD4þ T cells failed to secreteIFNg when stimulated with polyp antigens and were inefficientin T-helper-1 (TH1) commitment. By contrast, the TH17 com-mitment was unaffected. These findings were validated usingmice whose T cells overexpress IL10. In these mice, we observedhigh intra-polyp cytotoxic activity and attenuation of polypo-sis. Thus, expression of IL10 by T cells is protective and requiredfor immune surveillance in the small intestine. Cancer ImmunolRes; 3(7); 806–14. �2015 AACR.

IntroductionIL10 is a key immunosuppressive cytokine that is upregulated

in cancer, and it has been attributed with a role in obstructingtumor lysis (1, 2) and tumor rejection (3, 4). However, reportsalso suggest a protective role for IL10 in cancer (5–7). Theambiguity suggests a complex role for IL10 that may need to beaddressed in defined models of cancer, in a spatial and temporalmanner. We have looked at the function of IL10 during thedevelopment of adenomatous polyps in the small intestine ofAPCD468 mice, a well-characterized mouse model of hereditarypolyposis (8–11). Our findings support a protective role for IL10,specifically in preventing the adenoma to carcinoma transition.

The immune-suppressive functions of IL10 are associated withits effects on antigen-presenting cells as well as T cells. IL10downregulates expression of costimulatorymolecules by dendrit-ic cells (DC) and induces T-cell anergy upon antigen encounter(1, 12). However, the inhibitory effects of IL10 are not alwaysconsistent; for example, IL10 hinders the proliferation of mouseCD4 T cells but not necessarily their expression of cytokines,including IFNg (13). IL10 has differential effects on CD8þ T cells,and unexpectedly it activates and expands tumor-resident T cells(14). The effects of IL10 on T cells may depend on their state ofactivation, which may explain both the enhancing and inhibitoryeffects observed after IL10 treatment in different in vivo experi-mentalmodels (15). The timing of exposure to IL10 appears to becritical, inhibitory at antigen priming, and stimulatory at recall(16). Thus, transgenic expression of IL10 has disparate effects onT-cell response, inhibiting OVA peptide vaccination possibly atthe T-cell priming level, while enhancing rejection of transplantedtumors in mice, potentially by influencing T-cell memory (17).Injectionof soluble IL10 exacerbates graft-versus-host disease (18,19). This observation is in linewithmore recentfindings of criticalneeds for IL10 in cytotoxic T-cell differentiation (20) and IFNg-dependent tumor lysis (14, 21, 22). The anti-inflammatory prop-erties of IL10, particularly in the colon (23), suggest that thecytokine can suppress some immune responses while enhancingothers. Effective immune intervention and therapy requires abetter understanding of the functions of IL10 in specific tissueand disease settings (23). Here, we have examined the role of IL10in small bowel cancer.

To assess the expression of IL10 in the small intestine duringpolyposis, we introduced into APCD468 mice (8) a sensitivereporter transgene that expresses Thy1.1 under the control of the

1Robert H. Lurie Comprehensive Cancer Center, Northwestern Univer-sity Feinberg School of Medicine, Chicago, Illinois. 2Departments ofImmunology and Surgery, Mayo College of Medicine, Mayo Clinic,Rochester, Minnesota. 3Committee on Immunology, Department ofMedicine, The University of Chicago, Chicago, Illinois. 4Division ofDevelopmental Immunology, La Jolla Institute for Allergy and Immu-nology, La Jolla, California. 5Department of Pathology, University ofAlabama at Birmingham, Birmingham, Alabama. 6Institute for Immu-nology, Technical University Dresden, Dresden, Germany. 7Depart-mentofMicrobiologyand Immunology, School ofMedicine,Universityof Louisville, Louisville, Kentucky.

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

CorrespondingAuthor:Khashayarsha Khazaie, MayoClinic, GuggenheimBuild-ing, Room3-42B, 200First Street Southwest, Rochester, MN55905. Phone: 507-284-0545; Fax: 507-284-0981; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-14-0169

�2015 American Association for Cancer Research.

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IL10 gene promoter (IL10Thy1.1; refs. 24, 25). Using this model,we show that T cells are the major cellular source of IL10. Byselectively ablating IL10 in T cells of bone marrow chimericAPCD468 mice (CD4CreIL10fl/flAPCD468), we confirm that IL10 inthe small intestine is almost entirely derived from T cells. Bycomparing the CD4CreIL10fl/flAPCD468 mice with APCD468 micethat overexpress IL10 under the control of the IL2 promoter intheir T cells (APCD468IL2pIL10), we show that expression of IL10by T cells is indispensable for INFg-dependent T-cell cytotoxicityand protective polyp-specific immune surveillance. Our findingsprovide new support for the antitumor properties of IL10.

Materials and MethodsAnimals

B6 (C57BL/6J), CD4Cre, IL10�/�, and Rag1�/� mice werepurchased from The Jackson Laboratory. We generated theAPCD468 mice on a B6 background (26). IL10fl/fl mice weredeveloped by Roers and colleagues (27). IL2pIL10 mice weredeveloped by Hagenbaugh and colleagues (28). IL10Thy1.1

Foxp3GFP reporter mice were developed by Maynard and collea-gues (25). CD4-Cre (29) and IL10-deficient mice have beendescribed (30). All animal work was approved and conductedaccording to the guidelines of the Animal Care and Use Com-mittee (ACUC) of Northwestern University.

Gene expression arrayMicroarray analyses were performed using CD4þ T cells iso-

lated from the small intestine of healthy Foxp3GFP or Foxp3GFP

APCD468 mice. Cells were subjected to negative selection(untouched T-cell isolation kit; Invitrogen), labeled for CD4, anddouble sorted on a Dako MoFlo cell sorter for expression of CD4and exclusion of Foxp3 anddead cells (DAPI). Sorted populations(post-sort purity >98%) were used to prepare RNA for microarrayanalyses according to immgen.org. Array data were normalizedfor background. A GenePattern Expression Dataset file was gen-erated from all array data. These were further compared forexpression enrichment of a predetermined IL10 molecular path-way (Biocarta) using the Gene Set Enrichment program (BroadInstitute). A heatmap of the IL10 pathway genes was generatedusing the ExpressCluster v1.3module of the GenePattern package(Broad Institute).

Histology and immunostainingCleaned intestines were fixed for 2 hours at room temperature

with freshly prepared 2% paraformaldehyde (PFA). The fixedtissues were then washed for 5 minutes with PBS, and thenincubated overnight at 4�C in 20% sucrose (in PBS). The nextday, tissues were washedwith PBS, then rolled inOCT, and frozensections of 4 to 5 mm were prepared for analysis. Foxp3 wasvisualized using GFP reporter activity. IL10 was visualized usingrat anti-mouse Thy1.1-biotin and streptavidin–Alexa Fluor 594.CD4 was visualized using mouse anti-mouse CD4 and anti-mouse-Cy5. Rabbit anti-mouse granzyme B or rabbit IgG controland rat anti-mouse perforin (Abcam) were incubated at 1:100overnight at þ4�C, and then visualized using secondary antibo-dies anti-rabbit Alexa Fluor-594 and anti-rat Alexa Fluor-488(Invitrogen). Rabbit anti-mouse Foxp3 (Abcam) was incubatedat 1:100 overnight at 4�C, then visualized using secondary anti-rabbit Alexa Fluor-594 (Invitrogen). Fluorescent microscopyimageswere taken using a TissueGnosticsmicroscope, and images

were analyzed using ImageJ. Other anti-mouse antibodies usedwere Ki67 (SP6), Granzyme B, IgG, perforin (CB5.4), purchasedfrom Abcam; Foxp3 (FJK-16s) was from eBioscience. Secondaryantibodies used were AlexaFluor-488 and AlexaFluor-594 fromInvitrogen. Apoptosis staining was performed using a TUNELApoptosis Detection Kit (Millipore). Light microscopy photo-graphs were taken using the Leica DCC camera, and images wereanalyzed using the NIH software, ImageJ. Fluorescentmicroscopyimages were taken using TissueGnostics microscope, and imageswere analyzed using ImageJ.

Gut mononuclear cell preparationThe small intestine was harvested, cleaned, and then minced.

The minced tissue was then incubated in 25 mL complete RPMI-1640 with 10 units of collagenase type IV (Worthington Bio-chemical Corporation) for 25 minutes at 37�C with gentle agi-tation. Cells were enriched on a Percoll gradient (44%/67%), andgut mononuclear cells (MNC) at the interface were collected andwashed prior to analysis.

Bone marrow reconstitutionMice (4- to 6-week-old) were lethally irradiated (1000 rad; split

dose). Lin-depleted bone marrow cells were injected into theretro-orbital (RO) sinus. Details are provided in SupplementaryMaterials.

Flow-cytometric staining and analysisCells in suspensionwere transferred to a 96-well round-bottom

plate and stained with extracellular antibodies and Live/DeadViolet dye (Invitrogen) for 20 minutes. Cells were then washedand fixed with 1% PFA (10 minutes), permeabilized (0.3%saponin), and stained with intracellular antibodies (30minutes).Additional details are provided in Supplementary Materials. Datawere acquired on a FACSCanto II instrument and analyzed usingFlowJo software (TreeStar).

Serum and tissue extractsMice were bled from the RO sinus. Blood was left for 15 to 30

minutes at room temperature to clot. Samples were centrifugedfor 15 minutes, and supernatants were collected. Polyps weremicrodissected and separated from the surrounding healthytissue. Tissue was minced and extruded in PBS on ice for 1minute using a 16-gauge needle and syringe. Samples werecentrifuged for 15 minutes, and the extracts were collected andfiltered. Protein concentration was determined using the Brad-ford assay.

ELISA and multiplex ELISAELISAs for IL10 was performed according to themanufacturer's

instructions (eBioscience). Multiplex ELISA was performedaccording to the manufacturer's instructions using a customizedanalyte detection set from Millipore. Data were acquired on aLuminex 100 instrument and analyzed with xPONENT software(Luminex Corporation).

IFNg ELISPOTSpleen-derived DCs were cultured for 1 week with X-Vivo

medium containing mouse rec-GM-CSF (10 ng/mL; Miltenyi) at37�C, 5% CO2. CD11cþ DCs (purified by Miltenyi MACS col-umn) were pulsed with polyp extract (20 mg/100 mL) overnightbefore being cocultured with CD3þ MACS column–purifiedT cells for 40 hours. Anti–IFNg-coated ELISPOT plates (Millipore)

IL10 Is Needed for Tumor Immune Surveillance

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were visualized according to the manufacturer's instructions(Mabtech). Data were acquired on a CTL-Immunospot instru-ment and analyzed using CTL ImmunoSpot software (CellularTechnology Limited).

TH1 polarizationTH1 polarization was performed as described previously (31).

Total cells were harvested from the MLN, and CD4þ respondercells were isolated bymagnetic bead columns (Miltenyi). Spleensfrom syngeneic mice were harvested, red blood cells were lysed,and irradiated with 3,000 rad. Spleen cells and responder cellswere cultured for 7 days with 5 ng/L mouse rec-IL2 (eBioscience),10 mg/mL monoclonal anti-IL4 (clone 11B11; BD Pharmingen),10 ng/mL rec-IL12 (BD Pharmingen), 3 mg/mL anti-CD3(eBioscience), and 3 mg/mL anti-CD28 (eBioscience).

Statistical analysisBox and whisker histograms depict median flanked by the

upper and lower 25% quartiles with whiskers showingmaximumand minimum data points. Statistical analyses were performedwith the use of the Prism 4 software.

Histograms depict mean � SEMStatistical significance was determined using a two-tailed

paired t test (�, P < 0.01; ��, P < 0.001; ��, P < 0.0001; ns, notstatistically significant).

ResultsConventional CD4þ T cells and Tregs are the major source ofIL10 in the small intestine

IL10 was elevated during polyposis, reaching levels that were10- to 20-fold higher in polyps and in healthy intestine tissue ofAPCD468 mice than in age-matched wild-type (WT) B6 mice (Fig.1A). The cellular source of IL10 was visualized using a sensitiveIL10Thy1.1 reporter (25). To distinguish conventional T cells fromTregs, we crossed Foxp3-GFP reporter mice (32) with IL10Thy1.1

mice, and then generated bone marrow chimeric APCD468 micethat expressed Thy1.1 stably under the control of the IL10 pro-moter. Lethally irradiated Thy1.2 B6 and Thy1.2 APCD468 micewere reconstituted with bone marrow from IL10Thy1.1Foxp3GFP

dual-reporter mice. The efficiency of bonemarrow chimerismwas76% or better (Supplementary Fig. S1). Multicolor immunoflu-orescence staining of jelly-roll preparations of the small intestineallowed us to visualize CD4þFoxp3þ Tregs and CD4þFoxp3�

conventional T cells (Supplementary Fig. S2A). Expression of IL10was visualized by staining for Thy1.1 (Supplementary Fig. S2Band S2C). No staining for Thy1.1 was detected in the intestines ofcontrol Thy1.2 mice (Supplementary Fig. S2D). Isotype controlantibodies were used to validate these findings (SupplementaryFig. S3). Strikingly, conventional CD4þ T cells and Tregsaccounted for nearly all IL10-expressing cells infiltrating the smallintestines of healthy (B6) as well as APCD468 mice (margin andpoly; Fig. 1B). In 4-month-old polyp-ridden APCD468 mice, thefrequency of IL10-expressing cells was higher in the margin thanthat inside the polyps (Fig. 1C). This was largely accounted for bythe accumulation of conventional CD4T cells in the polypmargin(Fig. 1D) and the increase in their IL10-competent fraction (insidethe polyps, 22%; in the margin, 27%) relative to that in thehealthy B6 intestine (16%; Fig. 1D). By contrast, Tregs preferen-tially accumulated inside the polyps and produced less IL10

(inside the polyps, 30%; in the polyp margins, 44%) than in thehealthy B6 intestine (58%; Fig. 1D).We analyzed gene expressionin highly purifiedCD4þ T cells using Affymetrix expression arrays,and observed that during polyposis robust activation of genesdownstream of the IL10-receptor (IL10R) were mainly in con-ventional T cells in the margin (P ¼ 0.0018; Fig. 2A). By contrast,Foxp3þ Tregs, primarily those in the polyps, showed only a trendtoward IL10R signaling (P ¼ 0.044; Fig. 2B). These observationsagree with earlier observations of Tregs losing their ability toexpress IL10 during polyposis in mice (10) and also in coloncancer patients (33). Our findings are consistent with T cells andTregs responding to their own IL10 in an autocrine fashion duringpolyposis.

T-cell ablation of IL10 delays polyposis but facilitates cancerprogression

To determine the contribution of T cells to the content andfunction of IL10, we specifically ablated IL10 in the T cells of

Figure 1.CD4þ T cells and Tregs are the source of IL10 in the intestine. A, single ELISAfor IL10 in small intestinal tissue extracts. Microdissected polyps (open bars)were compared with margin (dashed bar) in APCD468 mice, and to healthyintestine of C57/BL6 mice (B6; black bar). Group sizes were as follows: B6(n¼ 5), APCD468 (n¼ 3), IL10�/��APCD468 (n¼ 3), andCD4CreIL10fl/flAPCD468

(n ¼ 5). B, intestine-infiltrating IL10þ cells in IL10Thy1.1Foxp3GFP B6 mice andpolyps and margins or IL10Thy1.1Foxp3GFPAPCD468 mice. Pie charts depict thefrequencies of IL10-expressing CD4þFoxp3þ and CD4þFoxp3� cells relativeto all cells that express IL10. C, frequencies of IL10þ cells were expressed as apercentage of total cells (DAPI). D, frequencies of the indicated cells inhealthy B6 intestine or polyps and margins of the small intestine of CD4Cre

IL10fl/flAPCD468 mice were expressed as a percentage of total DAPI-stainedinfiltrating cells (empty bars); the IL10þ fraction of each cell type isindicated (shaded bars). Mice were lethally irradiated and reconstitutedwith bone marrow from IL10Thy1.1Foxp3GFP reporter mice. IL10þ cells wererevealed by staining excised intestines for Thy1.1. Thy1.2 CD4CreIL10fl/fl B6(n¼ 3) and CD4CreIL10fl/flAPCD468 (n¼ 4). At least 5 representative regionswere counted per mouse. Histogram shows mean � SEM of triplicatesamples. Statistical significance determined with the two-tailed unpairedStudent t test: �� , P < 0.0001; �� , P < 0.001; � , P < 0.01.

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Cancer Immunol Res; 3(7) July 2015 Cancer Immunology Research808




CD4CreIL10fl/flAPCD468 mice. In parallel, for comparison we gen-erated IL10�/�APCD468 mice that were completely deficient forIL10. Ablation in T cells reduced IL10 concentration in theintestine to nearly undetectable levels (Fig. 1A). Together, thereporter and ablation experiments demonstrated that IL10 con-tent of the small intestine was almost entirely derived from the T-cell compartment.

Next, we determined the outcomes of IL10 deficiency in thesmall intestine. APCD468 mice and CD4CreIL10fl/flAPCD468 mice at2 months of age had comparable numbers of aberrant crypts andmicroscopic polyps, detected using a dissection microscope (Fig.3A and B). By 4months of age, APCD468mice developed extensivevisible polyposis in the small intestine, but IL10�/�APCD468 andCD4CreIL10fl/flAPCD468 mice had very few, if any, visible polyps(Fig. 3A). However, by 7 months of age, CD4CreIL10fl/flAPCD468

and IL10�/�APCD468 mice became cachexic. The intestines con-tained large and invasive lesions (Fig. 3B and C). Thus, theinability of T cells to produce IL10 changed both the kinetics andnature of polyposis in the small intestine, favoring cancer pro-gression as the mice aged.

Loss of intra-polyp cytotoxicity inmicewith IL10-ablated T cellsAPCD468 mice exhibit ongoing intra-polyp cytotoxic activity

and polyp-specific TH1 responses (33). To better understandthe impact of ablation of IL10 in T cells on anti-polyp immu-nity, we measured intra-polyp cytotoxic activity by staininghistologic sections for granzyme B and perforin (34, 35). By 2

months of age, the aberrant-crypts and micro-adenomas ofCD4CreIL10fl/flAPCD468 mice had nearly twice as many gran-zyme Bþ and perforinþ infiltrating cells as in the lesions ofAPCD468 mice (Fig 4A and B; Supplementary Fig. S3), but thecytotoxic activity declined sharply by 4 months and wascompletely extinguished by 7 months of age. By comparison,intra-polyp cytotoxicity increased with age in the IL10-compe-tent APCD468 mice (Fig. 4A and B; Supplementary Fig. S3).There were no significant differences or change in cytotoxicactivities within the healthy margin tissues of the IL10-com-petent versus IL10-defective mice (Fig. 4A and B). Furthermore,at all ages examined, CD8 T cells were preferentially excludedfrom the polyps of CD4CreIL10fl/flAPCD468 mice and were notcytotoxic (Fig. 4C). We detected consistent drops in polyp-associated apoptotic events as CD4CreIL10fl/flAPCD468 miceaged (Fig. 5A and B). Thus, we conclude that IL10 is essentialfor anti-polyp cytotoxic activity and tumor lysis, but cannot besure that the actual effectors are CD8þ T cells.

Mice with IL10-ablated T cells have systemic TH1 defectsThe above findings resonate with earlier reports that IL10

is required for antitumor IFNg-dependent cytotoxic response(21, 22). The gradual loss of cytotoxicity implied defects in T-cell help. Therefore, we used ELISPOT assays to compare polyp-specific TH1 responses in 4-month-old APCD468 mice withresponses in age-matched CD4CreIL10fl/flAPCD468 mice. T cellsfrom the spleens were cocultured with DCs that had been

Figure 2.Activation of IL10R in conventionalCD4þ T cells and Tregs of polypsversus healthy intestine. A, Affymetrixexpression arrays of CD4þ Foxp3�

conventional T cells from polyps (P)and healthy surroundingmargin tissue(M) of 4-month-old APCD468 mice andage-matched healthy B6 mice. Arraydata were subjected to IL10 gene setenrichment analysis (GSEA) for theIL10 molecular pathway (Biocarta);(NES ¼ 1.9806725, P ¼ 0.0018, FDR q¼ 0.0018; left). Heatmap of IL10pathway genes (ExpressCluster v1.3module of the GenePattern package;Broad Institute; right). B, the sameanalysis was performed for CD4þ

CD25þ Tregs (NES ¼ 1.5675694,P ¼ 0.044, FDR q ¼ 0.044; left).Heatmap of IL10 pathway genes(right). Cells were FACS sorted twicefrom B6 small intestine and fromAPCD468 microdissected polyps orsurrounding healthy tissue. RNAisolated from three independentpreparations of cells was submitted toamicroarray analysis. Three replicatesof each sample were grouped in orderto calculate the class means.






pulsed with polyp extracts, and IFNg expression by the T cellswas measured. Strong IFNg responses were detected in T cellsisolated from APCD468 mice, but significantly less (P < 0.001,Student t test) response was detected in T cells isolated fromCD4CreIL10fl/flAPCD468 or IL10�/�APCD468 mice (Fig. 6A). Todetermine if this was a T-cell inherent defect, we tested TH1polarization of IL10-proficient and IL10-deficient T cells. CD4 Tcells isolated from CD4CreIL10fl/fl T cells demonstrated poorTH1 commitment compared with T cells from healthy B6 mice(Fig. 6B and C). These findings are consistent with an earlier

report that expression of IL10 by T cells is required for TH1commitment (22). We tested the ability of CD4 T cells tocommit to the TH17 lineage by stimulating them with anti-CD3 in vivo as described previously (36). These cells readilycommitted to the TH17 lineage (data not shown), indicatingthat the drop in TH17 cytokines in 4-month-old mice may havebeen more likely a secondary result of attenuation of polyposisat this age.

Figure 4.IL10 deficiency aborts perforin and granzyme B activities. A, intra-polypcytotoxic activity. Immunofluorescent images of 4-mm paraffin sections ofintestine specimens from APCD468 and CD4CreIL10fl/flAPCD468 mice at2, 4, and 7, months of age; shown are perforin (green) and granzyme B (red)coactivity (yellow) within polyps (�400) and margins (�200). Redarrows, granzyme Bþ cells; green arrows, perforinþ cells; yellow arrows, cellsstaining for both granzyme B and perforin 200�. B, cumulative data.Mean � SEM for polyps were as follows: APCD468 (0.91 � 0.29, 2 mo; 1.68 �0.16, 4 mo) and CD4CreIL10fl/flAPCD468 (2.20� 0.46, 2 mo; 0.25� 0.14, 4 mo;0.073 � 0.044, 7 mo). Mean � SEM for margins were as follows: APCD468

(0.02 � 0.0073, 2 mo; 0.016 � 0.0054, 4 mo) and CD4CreIL10fl/flAPCD468

(0.040� 0.021, 2 mo; 0.063� 0.023, 4 mo; 0.067� 0.030, 7 mo). At least 4fields at 200� magnification were analyzed per slide; APCD468 mice (2 mo,n¼ 2; 4mo, n¼ 5) and CD4CreIL10fl/flAPCD468mice (2mo, n¼ 3; 4mo, n¼ 3; 7mo, n ¼ 3). C, colocalization of cytotoxicity and CD8 cells in polyps andmargins. Top, specimen from the small intestine of a representative 2-month-old CD4CreIL10fl/flAPCD468 mouse stained for granzyme B (green)and CD8 (red); arrows, polyps. CD8 cells were abundant in the margin butabsent from the polyp, and there was no colocalization with granzyme B.Bottom, small intestine specimen of a representative 7-month-old CD4Cre

IL10fl/flAPCD468 mouse stained the same; note the scarcity of granzyme Bþ

cells in general and abundance of CD8 cells in the margin only. Sections werecounterstainedwith DAPI. Two-tailed unpaired Student t test; �� , P <0.0001;�� , P < 0.001; � , P < 0.01.

Figure 3.T cell–specific ablation of IL10 attenuates polyposis but promotes cancer. A,polyp counts in the small intestine of IL10-competent versus IL10-defectiveAPCD468 mice. APCD468 mice were of 2 (n ¼ 10) or 4 (n¼ 29) months of age;thesemiceweremoribundby4months. CD4CreIL10fl/flAPCD468micewere of 2(n¼ 8), 4 (n¼ 23), or 7 (n¼ 7) months of age. For comparison, we measuredpolyposis in IL10�/�APCD468 mice of 4 (n ¼ 11) months of age. P values weredetermined with the two-tailed unpaired Student t test (�� , P < 0.0001). B,representative hematoxylin and eosin–stained small intestine rolls of APCD468

or CD4CreIL10fl/flAPCD468 mice. Age of mice is indicated. Whole rolls at 5�magnification. Arrows, adenomatous polyps. APCD468 (n ¼ 5) and CD4Cre

IL10fl/flAPCD468 (n¼ 6). C, representative histologies of adenomatous polypsof APCD468 or CD4CreIL10fl/flAPCD468 mice. Age of mice is indicated. Note thetop-down distribution of epithelial cells with abnormal nuclei in polyps ofAPCD468mice and extension of the aberrant cells to the submucosal border inCD4CreIL10fl/flAPCD468 mice (insets). In the panel representing the 7-monthmouse, arrows point to areas of invasion.

Dennis et al.





Overexpression of IL10 by T cells increases intra-polypcytotoxicity and attenuates polyposis

Tregs lose expression of IL10 during polyposis, and this may beimportant because the adoptive transfer of fresh IL10-competentTregs from healthy mice to polyp-ridden mice causes polypregression (10). However, the consequences of increased IL10expression by endogenous T cells on polyposis have not beenassessed. To test whether the increased expression of IL10 by Tcells during polyposis is protective or harmful, we crossedIL2pIL10 with APCD468 mice. IL2pIL10APCD468 mice had atten-uated polyposis (Fig. 7A) and increased intra-polyp cytotoxicity(Fig. 7B; Supplementary Fig. S4), as compared with age-matchedAPCD468mice. T cells isolated from the intestines of both IL2pIL10and IL2pIL10APCD468 mice produced high levels of IL10 com-pared with T cells from control B6 or APCD468 mice (Fig. 7C

and D). Furthermore, the adoptive transfer of purified CD4þ Tcells from IL2pIL10 donors to lymphopenic Rag1�/�APCD468

recipients suppressed polyposis (Fig. 7E). These observationsvalidated our findings that expression of IL10 by T cells isprotective and critically contributes to antitumor cytotoxicresponses, and confirming earlier findings by others that IL10boosts immune surveillance (22).

DiscussionProminent cellular sources of IL10 in mice are thought to be

neutrophils (37) and macrophages (38, 39), as well as theintraepithelial lymphocytes of the small intestine and lamina-propria lymphocytes of the colon (40),which include T-repressive(Tr1) cells (41, 42), TH1 cells (43), and Tregs (25). Using asensitive IL10 reporter transgene, we found that conventional Tcells and Tregs are by far the major cellular sources of IL10 in theintestines ofWTB6mice aswell as APCD468mice. Ablation of IL10in T cells reduced the levels of IL10, making the mice practicallyIL10 deficient.

Surprisingly, and in contrast with the colon where T-cell IL10deficiency exacerbates polyposis (11), ablating IL10 expressionin T cells initially attenuated polyposis in the small intestine.Because IL10 is anti-inflammatory, and polyp growth in boththe small and large intestines is inflammation driven (9), thisobservation can be interpreted as a demonstration of thedifference in the nature of inflammation in the two sites. Thedifference could be in dependence on microbes. Polyposis isinhibited in the colon of germfree mice or mice treated withantibiotics (ref. 44; and unpublished observations), but not inthe small intestine (45). The reduced rate in polyp growth was ashort-term benefit, and mice went on to develop large invasivetumors later. Tumor progression correlated with defects in T-cell function and intratumor cytotoxic activity. IL10-ablated Tcells were less efficient in committing to the TH1 lineage thanWT T cells, as assessed by in vitro lineage commitment. The micehad poor systemic TH1 responses to polyps, and intra-polypcytotoxic activity was extinguished with time. TH1 cells are

Figure 5.Small intestinal granzyme B/perforin and TUNEL staining of the smallintestine polyps. A, representative 400� images of intra-polypTUNEL staining of 4-mm sections from the intestines of 2-, 4-, and7-month-old APCD468 and CD4CreIL10fl/flAPCD468 mice. B, quantification ofintra-polyp TUNEL staining in APCD468 (2 mo, n ¼ 3; 4 mo, n ¼ 3) andCD4CreIL10fl/flAPCD468 (2 mo, n¼ 3; 4 mo, n¼ 3; 7 mo, n ¼ 5) mice. At least4 fields at 200� magnification were analyzed per slide. Two-tailedunpaired Student t test; �� , P < 0.0001; ��, P < 0.001; � , P < 0.01.

Figure 6.T-cell IL10 is required for polyp-specific TH1 response and TH1 commitment. A, IFNg ELISPOT assays using mouse spleen–derived CD3þ cells from B6 or APCD468

mice; T cells were cocultured overnight with splenic DCs that either had been pulsed with extract of the healthy B6 intestine, not pulsed, or pulsed withextract of microdissected polyps from APCD468 mice; n ¼ 3 for each source. Two-tailed unpaired Student t test; �� , P < 0.0001; � , P < 0.01. B, representative plotsgated on live, CD4þ lymphocytes expressing IFNg after 7 days of polarization using anti-CD3/CD28, recombinant IL12, and blocking anti-IL4. C, percentage of IFNgþ

cells in the live, CD4þ lymphocyte gate. Each sample (n¼ 3 per group)was run in duplicate. Mean� SEMwere as follows: B6 T cells (51.67�0.67) andAPCD468 T cells(40.67 � 0.74). Two-tailed unpaired Student t test; �� , P < 0.0001.






necessary for the long-term activity of CD8þ cytotoxic T cells(46), and this has been in part attributed to their expression ofIFNg (47–50). The loss of T-cell help is consistent with thegradual loss of intra-polyp cytotoxic activity and tumor apo-

ptosis. Our findings are in line with those of several otherinvestigators in mouse models that link IL10 to T-cell memoryand IFNg-dependent T-cell cytotoxicity in mouse models ofcancer, as reviewed recently (51).

The in vivo effects of upregulation or exogenous administrationof cytokines are not always predictable from the outcome ofdeficiency of the same cytokine. For example, IL23 blockade ordeletion reduces tumor incidence, yet exogenous administrationof IL23 also causes tumor regression (52, 53). Using IL10-defec-tive or IL10-overexpressing T cells, we demonstrated consistencyin the antitumor effects of IL10. The indispensable role of IL10 incancer immune surveillance is also highlighted by the pathologyof patients who are born with IL10 deficiency. A third of thepatients with IL10R deficiency have defective CD8 T-cell tumorimmune surveillance and develop B-cell lymphomas in the firstdecade of their life (54). Together, thesefindings demonstrate thatIL10 is not only required for tumor-specific immune surveillancebut that its overexpression or targeted deliverymay be therapeuticin the cancer setting.

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

Authors' ContributionsConception and design: K.L. Dennis, H. Cheroutre, N.K. Egilmez, K. KhazaieDevelopment of methodology: K.L. Dennis, C.T. Weaver, A. Roers, K. KhazaieAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.L. Dennis, A. Saadalla, N.R. Blatner, S. Wang,V. Venkateswaran, C.T. WeaverAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.L. Dennis, A. Saadalla, N.R. Blatner, S. Wang,V. Venkateswaran, F. Gounari, H. Cheroutre, K. KhazaieWriting, review, and/or revision of the manuscript: K.L. Dennis, N.R. Blatner,H. Cheroutre, N.K. Egilmez, K. KhazaieAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K.L. Dennis, S. Wang, V. Venkateswaran,H. CheroutreStudy supervision: N.R. Blatner, K. Khazaie

AcknowledgmentsThe authors thank Dr. Steven Rosen for being a continuous source of

inspiration and encouragement. Dr. Yang-Xin Fu and Dr. Bana Jabri aregratefully acknowledged for providing valuable advice andmaterial. They thankDr. Alan Pemberton from the University of Edinburgh for the generous dona-tion of the anti-mousemMCP6. The authors also thank Nicoleta Carapanceanufor excellent technical support.

Grant SupportK. Khazaie is the recipient of NIH grants 1R01CA160436-01 and

1R01AI108682-01. F.Gounari is supported by 1R01AI108682-01.N.K. Egilmezis supported by A1092133.

Received September 14, 2014; revised February 28, 2015; acceptedMarch 23,2015; published OnlineFirst April 8, 2015.

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2015;3:806-814. Published OnlineFirst April 8, 2015.Cancer Immunol Res Kristen L. Dennis, Abdulrahman Saadalla, Nichole R. Blatner, et al. Surveillance in the Small IntestineT-cell Expression of IL10 Is Essential for Tumor Immune

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