reprogramming tumor-inﬁltrating dendritic cells for …€¦ · reprogramming tumor-inﬁltrating...

Research Article

Reprogramming Tumor-Infiltrating Dendritic Cells forCD103þCD8þ Mucosal T-cell Differentiation and BreastCancer Rejection

Te-Chia Wu1,4, Kangling Xu1,4, Romain Banchereau1, Florentina Marches1, Chun I. Yu1, Jan Martinek1,Esperanza Anguiano1, Alexander Pedroza-Gonzalez1, G. Jackson Snipes2, Joyce O'Shaughnessy3,Stephen Nishimura5, Yong-Jun Liu1, Virginia Pascual1, Jacques Banchereau1, Sangkon Oh1, andKarolina Palucka1,6

AbstractOur studies showed that tumor-infiltrating dendritic cells (DC) in breast cancer drive inflammatory Th2 (iTh2)

cells and protumor inflammation. Here, we show that intratumoral delivery of the b-glucan curdlan, a ligand ofdectin-1, blocks the generation of iTh2 cells and prevents breast cancer progression in vivo. Curdlan reprogramstumor-infiltrating DCs via the ligation of dectin-1, enabling the DCs to become resistant to cancer-derived thymicstromal lymphopoietin (TSLP), to produce IL-12p70, and to favor the generation of Th1 cells. DCs activated viadectin-1, but not those activated with TLR-7/8 ligand or poly I:C, induce CD8þ T cells to express CD103 (aEintegrin), a ligand for cancer cells, E-cadherin.Generationof thesemucosal CD8þT cells is regulated byDC-derivedintegrin avb8 and TGF-b activation in a dectin-1–dependent fashion. These CD103þCD8þ mucosal T cellsaccumulate in the tumors, thereby increasing cancer necrosis and inhibiting cancer progression in vivo in ahumanized mouse model of breast cancer. Importantly, CD103þCD8þ mucosal T cells elicited by reprogrammedDCs can reject established cancer. Thus, reprogramming tumor-infiltrating DCs represents a new strategy forcancer rejection. Cancer Immunol Res; 2(5); 487–500. �2014 AACR.

IntroductionIn recent years, we have witnessed an improved understand-

ing of the critical roles that the tumormicroenvironment playsin cancer growth, evasion from host immunity, and resistanceto therapeutic agents (1). A better definition of the molecularand cellular components of the tumor microenvironment willenhance the clinical efficacy of current immunotherapyapproaches and enable tailoring of specific therapeutic strat-egies. Breast and pancreatic cancers are characterized byinfiltration of inflammatory Th2 (iTh2) cells, which coexpressinterleukin (IL)-4/IL-13 and TNF-a but not IL-10 (2, 3). Clin-ically, the Th2 signature in breast cancer (4, 5) and theexpression of the Th2 master regulator GATA-3 in pancreaticcancer (6) are associated with poor outcomes.

Experimentally, iTh2 cells accelerate tumor development inhumanizedmousemodels of breast cancer through the activityof IL-13 (2). In genetically engineered mouse models of mam-mary cancer, iTh2 cells accelerate the development of pulmo-nary metastasis via IL-4 (7). IL-4 and IL-13 exert protumoractivity through several pathways, including (i) the triggeringof TGF-b secretion (8), (ii) the upregulation of antiapoptoticpathways in cancer cells (9), and (iii) the generation of type II–polarized macrophages that foster tumor growth directly viathe secretion of growth factors, and indirectly via the inhibitoryeffects on CD8þ T-cell function (10). Indeed, CD8þ T cells areessential for tumor rejection through the generation of cyto-toxic effectors. The presence of CD8þT cells in primary tumorsis associated with the long-term survival of patients withcolorectal and breast cancer (10, 11). Thus, iTh2 cells have abroad and profound impact on the tumor microenvironmentand cancer progression.

The generation of iTh2 cells in breast cancer depends on thepresence of mature tumor-infiltrating OX40Lþ dendritic cells(DC; ref. 3). In experimental models of breast cancer, this DCphenotype is driven by cancer-derived thymic stromal lympho-poietin (TSLP; refs. 3, 12). Previous studies have demonstratedthat dectin-1, an innate immune receptor with activatingmotifs[immunoreceptor tyrosine-based activation motif (ITAM)], canreprogram DCs from inducing Th2 responses into Th1responses (13, 14). We therefore investigated whether curdlan,a natural ligandof dectin-1 (15), could reprogram the functionofbreast tumor-infiltrating DCs to enable cancer rejection.

Authors' Affiliations: 1RalphSteinmanCenter forCancer Vaccines, BaylorInstitute for Immunology Research; 2Baylor University Medical Center,Sammons Cancer Center; 3Texas Oncology, US Oncology, Dallas; 4Insti-tute ofBiomedical Studies, BaylorUniversity,Waco, Texas; 5Department ofPathology, UCSF, San Francisco, California; and 6Department of Onco-logical Sciences, Mount Sinai School of Medicine, New York, New York

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

Corresponding Author:Karolina Palucka, Baylor Institute for ImmunologyResearch, 3434 Live Oak, Dallas, TX 75204. Phone: 214-820-7450;Fax: 214-820-4813; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-13-0217

�2014 American Association for Cancer Research.

CancerImmunology

Research

www.aacrjournals.org 487

on June 5, 2017. © 2014 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst March 4, 2014; DOI: 10.1158/2326-6066.CIR-13-0217

http://cancerimmunolres.aacrjournals.org/

Materials and MethodsCells, tissues, and reagents

Breast cancer cell lines, Hs587T andMCF-7, were purchasedfrom the American Type Culture Collection; MDA-MB-231 waspurchased from Xenogen and cultured in nonselecting media.All lines are banked as low-passage stock from which workingbanks are periodically renewed. All lines were verified by genemicroarrays twice in the past 7 years. Morphology, in vitrogrowth rate, and in vivo growth rate were the same as theoriginal lines. The Mycoplasma test was performed regularly,and the cell lines wereMycoplasma-free for each in vitro and invivo experiment.

Cell lines were cultured in RPMI [plus glutamine, 2 mmol/L;penicillin, 50 U/mL; streptomycin, 50 mg/mL; minimum essen-tial medium (MEM) nonessential amino acids, 0.1 mmol/L;HEPES buffer, 10 mmol/L; and sodium pyruvate, 0.1 mmol/L]and 10% fetal calf serum in T150 flasks at a seed density of 2�106 cells/25 mL. At 90% confluence, fresh medium was added,and cells were cultured for an additional 48 hours. Supernatantwas centrifuged and stored at �80�C.

Peripheral blood mononuclear cells (PBMC) were obtainedby leukapheresis from healthy donors (Institutional ReviewBoard approved). Primary tissues from patients were obtainedfrom the BUMC Tissue Bank and are exempt. Animal experi-ments were carried out with permission from the InstitutionalAnimal Care and Use Committee.

b-Glucan, curdlan (Wako Pure Chemical Industries), was inPBS at a working concentration of 100 mg/mL. The workingconcentrations of theneutralization antibodieswere as follows:20 mg/mL for anti–dectin-1 (clone 259931; R&D Systems), 10mg/mL anti–IL-12 (clone 20 C2; Thermo Scientific), 100 mg/mLanti–TGF-b (clone 1D11; R&D Systems), 50 mg/mL anti-CD103(clone Ber-ACT8; BioLegend), and 100 mg/mL anti-b8 (clone37E1). Curdlan was labeled with aminofluorescein (5-DTAF;Molecular Probes–Invitrogen).

Dendritic cellsDCs were enriched from PBMCs obtained after Ficoll-Paque

Plus density gradient centrifugation (Stemcell Technologies) bynegative selection with monoclonal antibodies (mAb) to CD3,CD9, CD14, CD16, CD19, CD34, CD56, CD66b, and glycophorin A(Human pan-DC Pre-Enrichment Kit; Stemcell Technologies).Cells were labeled with anti-human lineage cocktail-FITC (CD3,CD14, CD16, CD19, CD20, and CD56), CD123-PE (9F 5), CD11c-APC (S-HCL-3; BD Biosciences), and HLA-DR-APC-eflour780(LN3; Sigma-Aldrich); lin�CD123�HLA-DRþCD11cþ DCs weresorted with FACSAria (BD Bioscience). DCs were seeded at 100� 103 cells per well in 200 mL of RPMI with 10% human ABserum, and culturedwithmedium alone or in the presence of 20ng/mL of rhTSLP (R&D Systems), or tumor-derived products.After 48 hours, DCs were harvested, washed, and analyzed orused in experiments.

CD11c green

Dectin-1 red

DAPI blue

Cytokeratin red

Dectin-1 green

DAPI blue

CD83 red

Dectin-1 green

DAPI blue

CD20 green

Dectin-1 red

DAPI blue

Figure 1. Dectin-1–expressing cells infiltrate primary breast tumors. Immunofluorescence staining on frozen tissue sections from patients' primary cancers.Left to right, CD11c (green)/dectin-1 (red); cytokeratin (red)/dectin-1 (green); CD83 (red)/dectin-1(green); CD20 (red)/dectin-1 (green). Top to bottom, singlefluorescence for each indicated antibody and overlay. Blue, nuclear staining with DAPI. Representative of 27 tumors analyzed. Bar, 90 mm.

Wu et al.

Cancer Immunol Res; 2(5) May 2014 Cancer Immunology Research488




Figure 2. Curdlan blocks iTh2 in humanbreast cancer. A, Hs578Tbreast tumor-bearingNOD/scid/b2 nullmicewere reconstitutedwithmonocyte-derivedDCsand autologous T cells isolated from the same donor, with or without treatment with curdlan (100 mg/mL) or anti-TSLPR antibody (200 mg/mouse). Emptycircles, PBS; red, DCþT; blue, DCþTþcurdlan; and black, DCþTþanti-TSLPR. B, mean value from five independent experiments, 23 mice per group.C, cytokines in the activated tumor supernatant measured by Luminex. Single points indicate individual mouse. D, sorted blood DCs were pretreated with100mg/mLcurdlan, incubatedwith supernatant ofMDA-MB231breast cancer cell line (BCsup) for 48hours, andcoculturedwith allogeneic naïveCD4þTcells.Cells were restimulated for intracellular cytokine staining at day 7. E, summary of different experiments. Single points represent the percentage from individualexperiment with blood DCs from 13 different healthy donors. ��, P < 0.005; ��, P < 0.0001. n.s., not statistically significant.

Reprogramming Tumor DCs for Cancer Rejection

www.aacrjournals.org Cancer Immunol Res; 2(5) May 2014 489




A

B C

D

E F

Wu et al.





ImmunofluorescenceOptimum cutting temperature (OCT)–embedded (Sakura

Finetek USA), snap-frozen tissues were cut at 6 mm and air-dried on Superfrost slides (Cardinal Health). Frozen sectionswere fixed with cold acetone for 10 minutes. Dectin-1 wasstained with mAbs prepared in-house (clone12.2D8.2D4) fol-lowed by Alexa Flour 488 or 568 goat anti-mouse immuno-globulinG 1 (IgG1; Invitrogen). Cytokeratin 19was labeledwithclone A53-BA2 (Abcam) followed by Alexa Fluor 568 goat anti-mouse IgG2a (Invitrogen). CD83 was stained with clone HB15a(Immunotech) followed by Alexa Fluor 568 goat anti-mouseIgG2b (Invitrogen). CD20 was stained with clone L26 (Dako)followed by Alexa Fluor 488 goat anti-mouse IgG2a (Invitro-gen). Directly labeled antibodies used were fluorescein iso-thiocyanate (FITC) anti–HLA-DR (clone L243; BD Biosciences)and FITC anti-CD11c (clone KB 90; Dako). Finally, sectionswere counterstained for 2 minutes with the nuclear stain 40,6-diamidino-2-phenylindole (DAPI; 3mmol/L in PBS; Invitrogen–Molecular Probes).

Flow cytometrymAbs to human OX40L-PE (clone Ik-1), HLA-DR (clone

L243), lineage cocktail-FITC (CD3, CD14, CD16, CD19, CD20,and CD56), CD11c-APC (clone S-HCL-3), CD3-PerCP (cloneSK7), CD4-PE-Cy7 (clone SK3), CD8-APC-Cy7 (clone SK1),CD80-PE (L307.4), CD86-FITC [clone 2331(FUN-1)], CD70-PE(Ki-24), CD83-FITC (HB15e), IL-13-PE (JES10-5A2), TNF-a-PECy7 (mAb11), IFN-g–Alexa Flour 700 (B27), pSTAT4-FITC(38/p-stat4), pSTAT6-PE (J91-99358.11), pSTAT3-AF647 (4/pStat3), and pSTAT5-AF647 (16) were obtained from BD Bios-ciences. mAb toMHC class I-PE (W6/32) was fromDako. IL-10-Pacific blue (JES3-9D7) and Perforin-PE (dG9) were obtainedfrom eBioscience. mAbs to IL-17A-PerCP Cy5.5 (BL168), CD103-Alexa Flour 647 (Ber-act8), Granzyme A-Pacific blue (GB9), andGranzyme B-Alexa Flour 700 (GB11) were obtained from Bio-Legend; anti-integrinb8 (14E5)was conjugatedwithAlexa Fluor488 in-house.For surface staining, cells were incubated with antibodies

for 30 minutes at 4�C in the dark, washed and fixed with 1%paraformaldehyde (PFA), events of stained cells wereacquired with FACSCanto or LSR-II (BD Biosciences), andanalyzed with the FlowJo software (TreeStar). For intracel-lular cytokines, cells were stained using the BD Cytofix/Cytoperm Fixation/Permeabilization Kit according to themanufacturer's instructions. For pSTATs staining, cells werefixed with 2% to 4% formaldehyde for 10 minutes at 37�C and

permeabilized with ice-cold methanol for 30 minutes at 4�C.Cells were washed and stained with mAbs to pSTAT3,pSTAT4, pSTAT5, and pSTAT6 for 30 minutes at roomtemperature.

CytokinesT cells from DC-T cocultures were resuspended at 106 cells/

mL in medium and activated for 5 hours with phorbol 12-myristate 13—acetate (PMA) and ionomycin (Iono). Brefeldin A(GolgiPlug; BD Biosciences) and monensin (GolgiStop; BDBiosciences) were added for the last 2.5 hours. The BD Cyto-fix/Cytoperm Fixation/Permeabilization Kit was used accord-ing to the manufacturer's instructions. Labeled samples wereacquired with FACSCanto or LSR-II (BD Biosciences). Whole-tissue fragments of tumors from humanized mice (4 mm �4 mm � 4 mm, 0.015–0.030 g, approximately) were placed inculture medium with 50 ng/mL of PMA (Sigma-Aldrich) and1 mg/mL of ionomycin (Sigma-Aldrich) for 18 hours. Cytokineproduction was analyzed in the culture supernatant byLuminex.

DC-T cell coculturesTotal T cells were enriched from apheresis using magnetic

depletion of other leukocytes (EasySep Human T Cell Enrich-ment Kit; Stemcell Technologies). Blood DCs cultured withmedium, TSLP, or tumor-derived factors were cocultured withna€�ve allogeneic T cells in a ratio of 1:5. For curdlan treatment,DCs were preincubated with curdlan for 3 minutes at roomtemperature.

Humanized miceNOD.Cg-Prkdc(scid)b2m(tm1Unc)/J, abbreviated NOD/

scid/b2 null mice were irradiated the day before tumorimplantation. Tumors were injected with 1 � 106 mono-cyte-derived DCs, and with autologous T cells: 10 � 106

CD4þ T cells admixed with 10� 106 CD8þ T cells. Monocyte-derived DCs were generated by culturing the adherentfraction of PBMCs with 100 ng/mL of granulocyte macro-phage colony-stimulating factor (GM-CSF; Genzyme) and 10ng/mL of IL-4 (R&D Systems). CD4þ and CD8þ T cells fromthe same donor as DCs were positively selected from thawedPBMCs according to the manufacturer's instructions (Mil-tenyi Biotec) to >90% purity. Tumor volume was monitoredevery 2 to 3 days: [(short diameter) 2 � long diameter]/2.Tumors were injected with 100 mg/mL of curdlan at days 3, 6,and 9 after implantation.

Figure 3. Curdlan reprograms the function of DC in breast cancer via dectin-1. A, sorted blood DCs were exposed to BCsup for 48 hours, and OX40Lþ orOX40L� DCs were sorted by fluorescence-activated cell sorting (FACS) analysis. OX40Lþ DCs were recultured with or without curdlan for 24 hours andthen cocultured with naïve allogeneic total T cells. After 7-day culture, cells were collected and restimulated for ICS. B, sorted blood DCs were exposed toBCsup for 48 hours. The curdlan (100 mg/mL) treatment is for 3 minutes at room temperature before adding BCsup. Each point indicates the percentage ofOX40LþDCs from10 independent experiments analyzedby flowcytometry. C, sortedbloodDCswere exposed to recombinant humanTSLP (20ng/mL) for 48hours with or without 3 minutes pretreatment with curdlan (100 mg/mL) and analyzed by flow cytometry. D, DCs were pretreated with anti–dectin-1-neutralizing antibody, followed by curdlan and BCsup. OX40L expression on DCs by flow cytometry. Representative of four independent experiments.E, summary of the four experiments. Each line indicates an independent experiment. F, Hs578T-bearing NOD/scid/b2 null mice were reconstituted withmonocyte-derived DCs and autologous T cells isolated from the same donor. b-Glucan (curdlan; 100 mg/mL) or anti–dectin 1 mAb plus curdlan werecoinjected with DCs and T cells. Tumor size (ordinate) was monitored at indicated days (abscissa). ��, P < 0.005; ��, P < 0.0001. White circle, PBS; blacksquare, DCþT; blue triangle, DCþTþcurdlan; red triangle, DCþTþanti-dectinþcurdlan; and white square, curdlan. Ab, antibody.






100

80

60

40

20

0

100

80

60

40

20

0

100

80

60

40

20

0

100

80

60

40

20

0

100

80

60

40

20

0

100

80

60

40

20

0

100

80

60

40

20

0

100

80

60

40

20

0

100

80

60

40

20

0

% o

f M

ax

% o

f M

ax

% o

f M

ax

% o

f M

ax

% o

f M

ax

% o

f M

ax

% o

f M

ax

% o

f M

ax

% o

f M

ax

CD83 CD80 CD86 CD70 MHC class I

pSTAT3 pSTAT4 pSTAT5 pSTAT6

−−− −

−−

−−−−

−− −

−−− −

A

B

C D

E

Figure4. CurdlanmodulatesDCmaturationinbreastcancer.A,sortedbloodDCswereharvestedafter48hoursof incubationunderthe indicatedconditions,andtheexpressionofCD83, CD80, CD86, CD70, andMHCclass I were analyzed.Gray, isotype control; blue, BCsup-DC; and green, BCsup/curdlan-DC.Representativehistogramsof three independent experiments.B, sortedbloodDCswereharvestedafter 1 hourof incubationunder the indicatedconditions, and theexpressionofpSTAT3, pSTAT4, pSTAT5, and pSTAT6were analyzed by intracellular staining and flowcytometry. Gray, isotype control; red, BCsup-DC; blue, BCsup/curdlan-DC; and black, BCsup/aDectin/curdlan-DC. C, the supernatant from DCs culture was collected for IL-12p70 examination. (Continued on the following page.)

Wu et al.





Stamper–Woodruff binding assayCD8þ T cells (20,000) sorted from DC-T cocultures and

labeled with carboxyfluorescein succinimidyl ester (CFSE)were put on acetone-fixed breast tumor sections and incubat-ed at 37�C. After 1 hour, the slides were washed to remove theunbound cells, fixed with 4% PFA for 10 minutes, treated withbackground buster for 30 minutes at room temperature,stained with cytokeratin, and finally counterstained for 2minutes with the nuclear stain DAPI.

T-cell retention in vivoNOD/scid/b2 null mice were subcutaneously injected with

10� 106 MDA-MB231 cells. CD8þ T cells (500,000) sorted fromDC-T cocultures and labeled with CFSE were injected into thetumors. After 3 days, the tumors were harvested and frozenwith OCT or digested with collagenase (2.5 mg/mL; RocheDiagnostics), and processed to single-cell suspension. Somegroups of mice were left for tumor growth monitoring.

MicroarraysTotal RNA was purified using the mirVana miRNA Isola-

tion Kit (Invitrogen). RNA integrity was assessed using theBioanalyzer 2000 (Agilent). Target labeling was carried outusing the TargetAmp Nano-g Biotin-aRNA Labeling Kit forthe Illumina System (Epicentre). Labeled RNA was hybrid-ized onto HumanHT-12 v4 Expression BeadChips (Illumina).Illumina GenomeStudio version 1.9.0 software was usedto subtract background and scale samples to the globalaverage signal intensity. Ingenuity pathway analysis (IPA)was applied to reveal transcriptional networks as describedpreviously (17).

ResultsCurdlan inhibits the generation of iTh2 cells and breasttumor developmentImmunofluorescence analysis of tissues from 27 primary

breast cancers (Supplementary Table S1) revealed the presenceof b-glucan receptor dectin-1 in all samples with CD11cþ

CD20�HLA-DRþCD83þ mature DCs; dectin-1–positive cellswere found in the peritumoral areas (Fig. 1).To establish whether the ligation of dectin-1 by curdlan in

the tumor microenvironment might affect breast cancer pro-gression in vivo, we used a humanized mouse model of humanbreast cancer that we have described earlier (2, 3). Intratu-moral administration of 10 mg of curdlan prevented breastcancer progression (Hs578T breast cancer cell line) and was aseffective as the neutralizing anti-TSLP receptor antibody(Fig. 2A). The antitumor effect of curdlan has been observed

in five independent experiments with a total of 23 mice thathad been grafted with monocyte-derived DCs and autologousT cells obtained from several donors (Fig. 2B). Breast tumorprogression in this model is dependent on IL-13; as tumors donot grow in the absence of IL-13 or in the PBS control (2, 3), weanalyzed IL-13 production by breast cancers that were har-vested from humanized mice and activated with PMA/Iono.When compared with controls, curdlan-treated tumors pro-duced significantly less IL-13 (DCþT: 1,038 � 115 pg/mL;DCþTþcurdlan: 361 � 62 pg/mL; n ¼ 23; P < 0.0001) butsimilar levels of IFN-g (DCþT: 6,880 � 1,796 pg/mL;DCþTþcurdlan: 10,669 � 2,081 pg/mL; n ¼ 23; P ¼ 0.17) andIL-10 (DCþT: 41� 7.7 pg/mL; DCþTþcurdlan: 38� 8 pg/mL;n¼ 23; P¼ 0.83; Fig. 2C).We have shown earlier that bloodDCsas well as monocyte-derived DCs exposed to breast cancer cellsupernatants (BCsups), such as MDA-MB231, Hs578T, andMCF-7 (Supplementary Table S2), which express and secreteTSLP, can induce the differentiation of na€�ve T cells into iTh2cells (2, 3). To determine whether curdlan prevents the breastcancer–induced polarization of DCs, purified blood Linneg

CD123lowHLA-DRþCD11cþ DCs were exposed for 48 hours toBCsups with and without curdlan, and subsequently cocul-tured in vitro with na€�ve allogeneic CD4þ T cells for 7 days.Thereafter, T cells were activated for 5 hours with PMA/Ionoand analyzed using intracellular cytokine staining (ICS) andflow cytometry (Fig. 2D). As expected, CD4þ T cells exposedto DCs that had been pretreated with BCsups alone producedboth IL-13 and TNF-a (22%� 3% of CD4þ T cells). In contrast,T cells exposed to DCs treated with both BCsups andcurdlan produced less IL-13 (6% � 0.3% of CD4þ T cells;n ¼ 13; P < 0.0001; Fig. 2E). In both cases, CD4þ T cellsproduced IFN-g (þBCsup-DC: 26% � 0.5%; and þBCsup/curdlan-DC: 33% � 1.5% of CD4þ T cells, respectively; n ¼13; P ¼ 0.0002; Fig. 2E). Thus, curdlan inhibits the progressionof human breast cancer by preventing the generation ofprotumor iTh2 cells.

Ligation of dectin-1 with curdlan results inreprogramming of breast cancer DC maturation

To determine whether curdlan can reprogram the functionof tumor-conditioned DCs, we sorted OX40Lþ and OX40L�

DCs that arise in response of blood DCs to BCsups. The sortedDCs were then exposed to curdlan for 24 hours, washed andcocultured with na€�ve allogeneic T cells. As expected, OX40Lþ

DCs induced T cells to express IL-13, whereas OX40L�DCs didnot. Treatment of OX40LþDCswith curdlan altered their T-cellpolarization capacity as no IL-13 was induced (Fig. 3A). Addingcurdlan to DCs also prevented the induction of OX40L by

(Continued.) Singlepoints indicate samples from independent experiments.D, sortedbloodDCswerepretreatedwith curdlan andexposed toBCsup.The anti–IL-12-neutralizingantibodywasusedtopretreattheDCs,whichwereharvestedandcoculturedwithallogeneicnaïveTcells.Thepercentageof IFN-gþTcellsandTNF-aþIL-13þTcellsdefinedatday7byICSfromthree independentexperiments.E,transcriptionalprofilesofBCsupDCsfrom3donorsculturedfor6hours invitro inthepresence of lipopolysaccharide (LPS)-induced TLR4-activation inhibitor Polymyxin B (PMB); PMB þ curdlan or BCsup alone. Of note, 314 transcriptsoverexpressed1.5-foldincurdlanþPMBtreatmentwithBCsupalone(Welch t test0.05)wereidentified.Atotalof873transcriptsunderexpressed1.5-foldincurdlan+PMBtreatmentcomparedwithBCsupalone (Welch t test0.05).Sampleswerenormalized toeachdonor's untreated referencesample. IPAof the314 transcriptsidentifiedintheright.Thecolorscale representsthe foldchangeof themoleculesselectedintheaverageofPMBþcurdlan–treatedDCsascomparedwithuntreatedreference samples. The major overexpressed regulators are represented in the center of the network. Edges represent literature-based connections betweenmolecules (full, direct connection; dashed, indirect connection). IPA of the 873 transcripts identified in the left. �, P < 0.05. n.s., not statistically significant.






A B

C D

E

F

Wu et al.





BCsups (BCsups DCs: 25%� 2%, n¼ 10; BCsups DCþcurdlan:6%� 1.1%, n¼ 10; P < 0.0001; Fig. 3B). The inhibition of OX40Lexpression by curdlan was also observed when DCs weretreated with human recombinant TSLP (Fig. 3C). Conversely,the addition of anti–dectin-1 antibodies, which block thebinding of curdlan to DCs (13), before curdlan treatment,allowedOX40L expression byDCs exposed toBCsups in severalindependent experiments (Fig. 3D and E), demonstrating thatcurdlan does indeed engage dectin-1. In vivo the administra-tion of anti–dectin-1 antibodies to developing breast cancertumors prevented the protective effect of curdlan (Fig. 3F).These results confirm that curdlan acts through the dectin-1expressed by tumor-infiltrating DCs in breast cancer.We then observed that DCs treatedwith BCsups and curdlan

showed high levels of CD83, CD80, CD86, CD70, andMHC classI, indicating that curdlan is able to induceDCmaturation in thepresence of breast cancer–derived factors (Fig. 4A; ref. 18).Thus, curdlan blocks specifically OX40L expression withoutinterfering with the other components of the DC-maturationprogram. OX40L transcription in DCs depends upon the phos-phorylation of STAT5 and STAT6 (19). As we showed earlier,STAT6 in both the leukocyte infiltrate and the cancer cells isactivated in the breast cancer microenvironment (13). Expo-sure of BCsup-DC to curdlan led to enhanced phosphorylationof STAT4 and decreased phosphorylation of STAT6 (Fig. 4B),thereby resulting in an increase in the pSTAT4:pSTAT6 ratio.This switch in the activation pattern of STATs was associatedwith increased secretion of IL-12p70 by curdlan-treated DCs(Fig. 4C). Adding IL-12–neutralizing antibodies to cocultures ofna€�ve allogeneic T cells with curdlan-treated BCsup-DCrestored the generation of iTh2 cells (Fig. 4D). Thus, curdlanenables STAT4 activation in BCsup-DC, which is associatedwith increased IL-12 production and subsequent Th1 response.Transcriptome analysis revealed the overexpression of 314

transcripts and the underexpression of 873 transcripts bycurdlan-treated BCsup-DC (Fig. 4E). IPA of the overexpressedtranscripts revealed networks centered on NF-kB, IL-6, andTNF (Fig. 4E). Theunderexpressed transcripts formednetworkscentered on several transcription factors (Fig. 4E). Curdlan-exposedDCs showed abundant transcription ofDC-maturationmarkers, such asCD86 andTNFSF9 (4-1BBL); cytokines, such asGM-CSF, TNF, IL-6, IL-12, IL-15, and IL-23; integrins, includingITGB8 that is involved in the activation of TGF-b (20);and severalmolecules thatmight facilitatemigration, includingmatrix metalloproteinase 7 (MMP7; Supplementary Table S3).MMP7 might facilitate DC migration to the draining lymphnodes, a feature that seems blocked in breast cancer–infiltrat-ing DCs (21). Conversely, curdlan-exposed DCs underexpressedCD14, CD68, and CSF1R, all of which are associated with animmature DC phenotype. Consistent with DC maturation,

CCR6, which contributes to immature DC retention at thetumor site by binding toMIP3-a (21), was also underexpressed.Thus, curdlan prevents the polarization of DCs induced bysoluble tumor factors and TSLP.

Dectin-1 signal blocks Tc2 differentiation and enablesgeneration of effector CD8þ T cells

As CD8þ T cells are essential effectors of antitumor immu-nity, na€�ve allogeneic CD8þ T cells were cocultured withBCsup-DC, exposed or not exposed to curdlan. ICS at day 7revealed that upon PMA/Iono restimulation, CD8þ T cellscultured with BCsup-DC produce IL-13 (þBCsup-DC: 23% �1.3%; n¼ 9), IFN-g , and TNF (Fig. 5A and B), indicating a partialtype II polarization. However, CD8þ T cells cultured withcurdlan-treated BCsup-DC displayed a type I phenotype withfew IL-13–producing CD8þ T cells (þBCsup-DC: 23% � 1.3%;þBCsup/curdlan-DC: 2% � 1%; n ¼ 9; P < 0.0001), andpredominantly IFN-g–producing CD8þ T cells (þBCsup-DC:53% � 1%; þBCsup/curdlan-DC: 68% � 1.6%; n ¼ 9; P <0.001; Fig. 5A and B).

CD8þ T cells cultured with BCsup-DC expressed high levelsof perforin but low levels of granzymes A and B (Fig. 5C and D).Similar to monocyte-derived DCs (13, 22), curdlan-exposedBCsup-DC allowed the generation of CD8þ T cells expressinghigh levels of granzymes A and B (Fig. 5C and D). To test theireffector function, CD8þ T cells were labeled with CFSE andculturedwith BCsup-DCwith orwithout curdlan treatment for6 days. Then, proliferating CFSE-negative CD8þ T cells weresorted and injected into breast cancer tumors established inimmunodeficient mice. At day 3 after injection, CD8þ T cellsgenerated with curdlan-treated BCsup-DC persisted in thebreast cancer microenvironment better than CD8þ T cellsgenerated by BCsup-DC (Fig. 5E). CD8þ T-cell persistencewithin the tumor was associated with tumor necrosis (Fig.5F). Curdlan exposure of BCsup-DC resulted in the enhancedtranscription of IL-15, IL-15-RA, and 4-1BBL (SupplementaryTable S3), molecules that are known to play important roles inthe generation of high-avidity CD8þ effector T cells, facilitatingcancer rejection (23–25).

Dectin-1 signal enables breast cancer DCs to promotegeneration of mucosal CD8þ T cells via TGF-b

The accumulation and persistence of CD8þ T cells in cancernests is critical for cancer rejection. CD103 integrin allows theretention of effector and memory T cells (26) via binding to E-cadherin expressed on epithelial cells (27–30). DCs exposed tocurdlan showed an increased ability to induce CD103 on CD8þ

T cells (Fig. 6A and B). To assess whether these CD103þCD8þTcells adhered to breast cancer, we used a modified Stamper–Woodruff tissue binding assay (21). Proliferating CFSE-

Figure 5. Curdlan enables generation of CTL able to induce tumor necrosis. A, curdlan/BCsup-DC were cocultured with allogeneic naïve T cells. ICS at day 7;gated AquanegCD3þCD8þ T cells. B, summary of nine independent experiments. C, Granzyme (Gzm) A, Granzyme B, and perforin (PFN) in CD8þ

T cells elicited by DCs pretreated under the indicated conditions. D, summary of three independent experiments. E, CD8þ T cells were sorted fromin vitro culture and injected into MDA-MB231 breast tumors in NOD/scid/b2 null mice. Single-cell suspensions from tumors harvested at day 3 were stainedwith anti-human-CD45 mAb and analyzed by flow cytometry. The percentage of CD45þ cells from four independent experiments. SSC, side scatteredlight. F, hematoxylin and eosin (H&E) stainingof tumor sections. The necrosis level is rated andcalculated in each region. Summary of points� areas/total areain each section ¼ necrosis index. Necrosis index from five sections. �, P < 0.05; ��, P < 0.005; ��, P < 0.0001.






0 102 103 104 105

0

102

103

104

105

0 102 103 104 105

0

102

103

104

105

0 102 103 104 105

0

102

103

104

105

0 102 103 104 105

0

102

103

104

105

A B

C D

E F

G H

Figure 6. Curdlan enables generation of CD103þCD8þ T cells with enhanced binding to cancer cells and rejects established breast cancer. A, curdlan/BCsup-DCs were cocultured with allogeneic naïve T cells. CD103 expression on CD8þ T cells at day 6. B, summary of seven independent experiments.C, CD8þ T cells were sorted from DC-T cocultures, labeled with CFSE, and used to overlay on frozen breast tumor sections. The sections were fixedand stained with cytokeratin (red). The CD8þ T cells (green) were counted in each 0.15 mm2 cytokeratinþ area. (Continued on the following page.)


Wu et al.




negative allogeneic CD8þ T cells were sorted from cocultureswith DCs, relabeled with CFSE (green), and overlaid on frozenbreast cancer tissue sections to allow adherence. After 60minutes, tissue sections were washed and counterstained withanti-cytokeratin mAb (red) to visualize cancer cells. Numbersof bound T cells per 0.15mm2 cytokeratinþ areas were countedon a series of consecutive tissue sections. CD8þT cells exposedto curdlan-treated DCs adhered significantly more to frozenbreast cancer tissue sections (Fig. 6C; þBCsup-DC: 7 � 1;þBCsup/curdlan-DC: 26 � 2; n ¼ 20; P < 0.0001) and blockingCD103 with an mAb decreased their numbers (Fig. 6C;þBCsup/curdlan-DCþisotype antibody: 22 � 3; þBCsup/cur-dlan-DCþaCD103: 2 � 0.5; n ¼ 20; P < 0.0001). The binding ofCD8þ T cells to breast cancer tissue sections was alsodecreased when BCsup-DCs were pretreated with anti–dec-tin-1 antibody before curdlan treatment (Fig. 6D; þBCsup/curdlan-DC: 32� 4;þBCsup/aDectin/curdlan-DC: 15� 3; n¼10; P ¼ 0.003). Thus, curdlan exposure enables DCs to inducethe differentiation of CD103þCD8þ T cells in a dectin-1–dependent manner.Intratumoral injection of curdlan increased the frequency of

CD103þCD8þ T cells in breast cancer tumors in vivo (Fig. 6E;DCþT: 9%� 0.3% of CD8þ T cells, n¼ 3; DCþTþcurdlan: 31�1.2 of CD8þT cells, n¼ 4; P < 0.0001). When sorted, these CD8þ

T cells triggered tumor necrosis upon transfer into tumorsestablished in immunodeficient mice (Fig. 6F). To establishwhether the activated CD8þ T cells can inhibit the devel-opment of highly proliferative tumors, we used MDA-MB231breast cancer cells that can grow in an immune microen-vironment–independent manner. A single injection of CD8þ

T cells elicited by BCsup-DC treated with curdlan completelyinhibited breast cancer progression in a manner dependentupon the expression of CD103 (Fig. 6G). Indeed, breastcancer tumors only grew in mice that received controlCD8þ T cells or in the presence of CD103-blocking antibody(Fig. 6G). Finally, CD8þ T cells elicited by curdlan-treatedDCs were able to reject established breast cancers in vivoupon repeated adoptive transfer of as few as 0.5 � 106 T cells(Fig. 6H).To determine the mechanism by which DCs enabled the

induction of CD103 expression inCD8þT cells, we analyzed therole of TGF-b1 as it induces CD103 expression on T cells (31,32). Using TGF-b1–neutralizing antibodies and pharmacologicblockade of TGF-b1 by the TGF-bRI kinase inhibitor II (33), theability of curdlan-treated DCs to induce the differentiation of

CD103þCD8þ T cells was substantially reduced (Fig. 7A).Transcriptional profiling revealed that curdlan exposureenables the overexpression of ITGB8 in DCs (SupplementaryTable S3). The product of this gene is a cell-surface receptor forthe latent domain (LAP) of TGF-b (34). The binding to theintegrin avb8 with subsequent metalloproteolytic cleavage ofLAP represents a major mechanism of TGF-b activation invivo (35). Consistent with RNA expression, curdlan-treatedBCsup-DC showed increased cell-surface expression of avb8(Fig. 7B). Adding antibodies that neutralize avb8 to CD8þ T-cell cocultures with curdlan-treated BCsup-DC resulted inthe complete inhibition of CD103 expression by CD8þ T cellstriggered as the result of DC exposure to curdlan (Fig. 7C).Thus, curdlan-treated DCs activate TGF-b1 through avb8 toinduce CD103þCD8þ T cells that reject breast cancer cells.The impact of curdlan on DCs is unique as DCs activatedwith the TLR8 ligand or poly I:C do not express avb8 (Fig.7D).

Thus, reprogramming tumor-infiltrating DCs via dectin-1ligation enables the simultaneous blockade of Th2 inflamma-tion and induction of mucosal CD8þ T cells that are able toreject established cancers in vivo. This opens a novel avenue forimmunotherapy of breast and pancreatic cancer, where linksbetween type II inflammation and poor prognosis have beendemonstrated.

DiscussionOur previous studies have established the roles of tumor

cells, DCs, and iTh2 cells in the progression of breast cancer (2,3). Here, we show an immunotherapy strategy for breast cancerbased on the reprogramming of tumor-infiltrating DCs in situby targeting pattern-recognition receptor dectin-1. Indeed, thedirect engagement of dectin-1 via intratumoral delivery of itsligand (b-glucan) initiated the reprogramming of DC matura-tion, resulting in the broad modulation of tumor-infiltratingCD4þ and CD8þ T-cell function leading to breast cancerrejection. The key principle is a simultaneous blockade ofprotumor iTh2 response, a switch to Th1 immunity, and anamplification of a potent antitumor CD8þ T-cell immunity.The direct binding ofb-glucan to tumor-infiltrating DCs allowsthe reprogramming of their function, including the blockade ofiTh2 cells secreting IL-4 and IL-13 in favor of the generation ofIFN-g–secreting CD4þ T cells, thus corroborating results fromearlier studies (36, 37). b-Glucan–exposed DCs induced thegeneration of CD8þ T cells expressing CD103, a ligand for E-

(Continued.) Twenty fields were counted from two individual breast tumor sections. The T cells were preincubated with anti-CD103 or isotype controlantibodies and then overlaid on breast tumor sections and processed as above. D, CD8þ T cells were cocultured with DCs that were pretreated with anti–dectin-1-neutralizing antibody, followed by curdlan and BCsup. CD8þ T cells were sorted fromDC-T coculture and labeled with CFSE for Stamper–Woodruffassay as described above. Ten fields were counted from two individual breast tumor sections. E, Hs578T-bearing NOD/scid/b2 null mice were reconstitutedwithmonocyte-derived DCs and autologous T cells with or without curdlan (100 mg/mL). Gating of single-cell suspension for humanCD45þ cells andCD103þ

CD8þ T cells. Single dots represent the percentage of CD103þCD8þ T cells from each mouse. F, CD8þ T cells sorted from the tumor-cell suspensions wereinjected intoMDA-MB231 tumors inNOD/scid/b2nullmice. Frozen sections from the tumors atday3, hematoxylin andeosin (H&E) staining. Thenecrosis levelis rated and calculated as in Fig. 5. G, the NOD/scid/b2 null mice were injected subcutaneously with 10 � 106 MDA-MB231 cells. A total of 500,000 sortedCD8þ T cells were injected into tumors. Black circles, PBS, n¼ 6; red squares, CD8þ T cells from BCsup-DC-T coculture, n¼ 6; blue triangles, CD8þ T cellsfromBCsup/curdlan-DC-T coculture, n¼7; green triangles, CD8þT cells fromBCsup/curdlan-DC-T coculturewith pretreatmentwith anti-CD103mAb,n¼8.H, NOD/scid/b2 null mice were injected subcutaneously with 10� 106MDA-MB231 cells. Sorted CD8þ T cells (500,000) were injected into tumors starting atday 40 after tumor implantation (insert plot, tumor size around 150mm3) every other day for a total of three injections. Black squares, PBS, n¼ 6; red triangles,CD8þ T cells from BCsup-DC-T coculture, n ¼ 6; blue triangles, CD8þ T cells from BCsup/curdlan-DC-T coculture, n ¼ 7. ��, P < 0.005; ��, P < 0.0001.






cadherin, with superior capacity to accumulate in and to rejectbreast cancer in vivo.

Suppression of type II responses is linked with enhanced IL-12 production by DCs. Interestingly, the blockade of IL-12 inDC-T-cell cocultures restored iTh2 differentiation, eventhough we have shown earlier that iTh2 differentiation isdependent on OX40L (3). A possible explanation is that whenIL-12 is blocked and the CD40L signal is provided by T cells, theOX40L can be expressed and drive T-cell polarization (38).Whereas the abundance of IL-12 upon curdlan exposure wasexpected, genomic profiling revealed several intriguing tran-scripts, includingNotch 2 and IL1F9 (IL-36g). In themouse, DC-specific deletion of the Notch2 receptor caused a reduction ofDC populations in the spleen, and was associated with the lossof CD11bþCD103þ DCs in the intestinal lamina propria and acorresponding decrease of IL-17–producingCD4þTcells in theintestine (39). The IL-36 receptor pathway has been suggestedin the regulation of IFN-g secretion bymurine CD4þT cells (40,41). Furthermore, IL-36g has been shown as downstream of thedectin-1/Syk signaling pathway upon exposure to Aspergillusfumigatus (42). Thus, curdlan exposure in the presence oftumor-derived factors leads to phenotype switch, and enables

DC commitment to induce IFN-g–secreting CD4þ T cells.Although assessment of the global IFN-g secretion at the tumorlevel does not reveal a difference between curdlan-treated and–untreated tumors, we observe a clear difference at the level ofCD4þ T cells. These results suggest that other cells mightcontribute to IFN-g secretion in untreated tumors.

The impact on mucosal CD8þ T-cell differentiation wasspecific to curdlan-dectin-1 signaling and could not be inducedby exposure of DCs to TLR8 ligand CL075 or to poly I:C (43).Dectin-1–dependent mucosal marker of CD8þ T cells is aCD103 integrin aE, which forms a heterodimer with theintegrin b7 allowing peripheral CD8þ T cells to be retainedin the epithelial compartments (44, 45). CD103 specificallybinds E-cadherin that is expressed on murine and humanepithelial cancer cells (27, 28). The expression of CD103 onCD8þ T cells seems to depend mostly upon TGF-b1 (31, 32).Studies on GVHD in mice lacking TGF-b receptor signalingdemonstrated that the effector CD8þ T cells infiltrating theintestinal epithelium did not express CD103 and were lesspathogenic (46).We have shown previously that humanCD1cþ

DCs use activated membrane-bound TGF-b1 to induce CD103expression on proliferating CD8þ T cells, both in the allogeneic

A B

C D

Figure 7. CD103þCD8þ T cellsinduction is TGF-b–dependentand mediated by avb8. A, DCspretreated with anti–TGF-b1 andTGF-b1 receptor kinase inhibitorfor 30 minutes were coculturedwith T cells. Single pointsrepresent independentexperiments. B, avb8 staining.Gray, isotype control; red, BCsup-DC; and blue, curdlan/BCsup-DC.C, sorted blood DCs werepretreated with curdlan, activatedby BCsup for 48 hours, pretreatedwith anti-b8 mAb, and coculturedwith allogeneic naïve T cells.Single point indicates thepercentage of CD103þCD8þ Tcells from three individual DCdonors at day 6. D, avb8 staining.Gray, isotype control; red, BCsup-DC; and blue, poly I:C or CL075/BCsup-DC. Ab, antibody.�, P < 0.05; ��, P < 0.0001.


Wu et al.




and autologous influenza-specific T-cell responses (47). Here-in, we find that in the breast cancer environment, CD1cþ DCsare largely inhibited by TSLP in their capacity to generateCD103-expressing CD8þ T cells. However, dectin-1 engage-ment enables DCs to express integrin b8, thereby facilitatingTGF-b1 activation. Accordingly, whereas in the influenzamodel CD103 expression was contact-dependent (47), herethe activity could be transferred by exposure to DCsupernatant.In the context of cancer, CD103 expression by CTLmediates

adherence to E-cadherin, resulting in cancer rejection (29).Indeed, mucosal homing and retention of CD8þ T cells isimportant for mucosal cancer vaccines (16). For example, onlyintranasal vaccination elicited mucosal-specific CD8þ T cellsexpressing the mucosal integrin CD49a (16). These resultsconfirm the critical role of the route of immunization for thetrafficking of effector T cells (48, 49) and the critical role oftissue DCs in imprinting the trafficking patterns of elicited Tcells (50). Here, we provide anothermechanismbywhich CD8þ

T cells can be equipped with molecules allowing mucosalretention.In summary, our studies have identified a number of

targets generated by tumor-infiltrating DCs and T cells, theligation of which results in tumor destruction in vivo by thehuman immune system in humanized mice. These includeOX40L, IL-13, and now dectin-1; these agents act in a uniquepathway that we have characterized. Whereas we need tocharacterize the impact of dectin-1 engagement on othercells present in the tumor microenvironment, in diseaseswhere the iTh2 signature is associated with poor outcomes,as is the case in breast (4, 5) and pancreatic (6) cancers, the

prevention of cancer-promoting effects combined with theexpansion of potent CD8þ T-cell immunity might representa novel option for these patients.

Disclosure of Potential Conflicts of InterestS. Nishimura has received a commercial research grant from MedImmune,

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

Authors' ContributionsConception and design: T.-C. Wu, K. Xu, C.I. Yu, A. Pedroza-Gonzalez,J. Banchereau, S. Oh, K. PaluckaDevelopment ofmethodology:T.-C.Wu, K. Xu, F. Marches, C.I. Yu, J. Martinek,A. Pedroza-Gonzalez, S. Nishimura, Y.-J. Liu, K. PaluckaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T.-C. Wu, K. Xu, F. Marches, J. Martinek, E. Anguiano,G.J. Snipes, J. O'Shaughnessy, K. PaluckaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T.-C. Wu, K. Xu, R. Banchereau, J. Martinek,E. Anguiano, J. O'Shaughnessy, V. Pascual, K. PaluckaWriting, review, and/or revision of the manuscript: T.-C. Wu, K. Xu,R. Banchereau, F. Marches, E. Anguiano, A. Pedroza-Gonzalez, J. O'Shaughnessy,V. Pascual, J. Banchereau, K. PaluckaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K. Xu, E. AnguianoStudy supervision: K. Palucka

AcknowledgmentsThe authors thank the patients and the volunteers; Luz S. Muniz, Joseph Fay,

and the Cores at BIIR, including Clinical, Apheresis, Flow Cytometry, andImagingCore and theAnimal Facility; and Jennifer L. Smith for the help provided.K. Palucka acknowledges the support from the BIIR, Baylor University MedicalCenter Foundation, Cancer Prevention Research Institute of Texas, and NIH/NCI.

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

Received December 5, 2013; revised January 29, 2014; accepted February 20,2014; published OnlineFirst March 4, 2014.

References1. DeNardo DG, Andreu P, Coussens LM. Interactions between lympho-

cytes and myeloid cells regulate pro- versus anti-tumor immunity.Cancer Metastasis Rev 2010;29:309–16.

2. Aspord C, Pedroza-Gonzalez A, Gallegos M, Tindle S, Burton EC, SuD, et al. Breast cancer instructs dendritic cells to prime interleukin 13-secreting CD4þ T cells that facilitate tumor development. J Exp Med2007;204:1037–47.

3. Pedroza-GonzalezA,XuK,WuTC,AspordC,TindleS,MarchesF, et al.Thymic stromal lymphopoietin fosters human breast tumor growthby promoting type 2 inflammation. J Exp Med 2011;208:479–90.

4. Teschendorff AE, Gomez S, Arenas A, El-Ashry D, Schmidt M, Gehr-mann M, et al. Improved prognostic classification of breast cancerdefined by antagonistic activation patterns of immune response path-way modules. BMC Cancer 2010;10:604.

5. Kristensen VN, Vaske CJ, Ursini-Siegel J, Van Loo P, Nordgard SH,Sachidanandam R, et al. Integrated molecular profiles of invasivebreast tumors and ductal carcinoma in situ (DCIS) reveal differentialvascular and interleukin signaling. Proc Natl Acad Sci U S A 2012;109:2802–7.

6. De Monte L, Reni M, Tassi E, Clavenna D, Papa I, Recalde H, et al.Intratumor T helper type 2 cell infiltrate correlates with cancer-asso-ciated fibroblast thymic stromal lymphopoietin production andreduced survival in pancreatic cancer. J Exp Med 2011;208:469–78.

7. DeNardo DG, Barreto JB, Andreu P, Vasquez L, Tawfik D, Kolhatkar N,et al. CD4(þ) T cells regulate pulmonary metastasis of mammarycarcinomas by enhancing protumor properties of macrophages. Can-cer Cell 2009;16:91–102.

8. Terabe M, Berzofsky JA. Immunoregulatory T cells in tumor immunity.Curr Opin Immunol 2004;16:157–62.

9. Zhang WJ, Li BH, Yang XZ, Li PD, Yuan Q, Liu XH, et al. IL-4-inducedStat6 activities affect apoptosis and gene expression in breast cancercells. Cytokine 2008;42:39–47.

10. DenardoDG,BrennanDJ, Rexhepaj E,Ruffell B, ShiaoSL,MaddenSF,et al. Leukocyte complexity predicts breast cancer survival and func-tionally regulates response to chemotherapy. Cancer Discov 2011;1:54–67.

11. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A,Mlecnik B, Lagorce-Pages C, et al. Type, density, and location of immune cells withinhuman colorectal tumors predict clinical outcome. Science 2006;313:1960–4.

12. Olkhanud PB, Rochman Y, Bodogai M, Malchinkhuu E, Wejksza K, XuM, et al. Thymic stromal lymphopoietin is a key mediator of breastcancer progression. J Immunol 2011;186:5656–62.

13. Gringhuis SI, den Dunnen J, Litjens M, van der Vlist M, Wevers B,Bruijns SC, et al. Dectin-1 directs T helper cell differentiation bycontrolling noncanonical NF-kappaB activation through Raf-1 andSyk. Nat Immunol 2009;10:203–13.

14. Baran J, Allendorf DJ, Hong F, Ross GD. Oral beta-glucan adjuvanttherapy converts nonprotective Th2 response to protective Th1 cell-mediated immune response in mammary tumor-bearing mice. FoliaHistochem Cytobiol 2007;45:107–14.

15. Goodridge HS, Reyes CN, Becker CA, Katsumoto TR, Ma J, Wolf AJ,et al. Activationof the innate immune receptorDectin-1 upon formationof a 'phagocytic synapse'. Nature 2011;472:471–5.






16. Sandoval F, Terme M, Nizard M, Badoual C, Bureau MF, FreyburgerL, et al. Mucosal imprinting of vaccine-induced CD8þ T cells iscrucial to inhibit the growth of mucosal tumors. Sci Transl Med2013;5:172ra20.

17. Obermoser G, Presnell S, Domico K, Xu H, Wang Y, Anguiano E, et al.Systems scale interactive exploration reveals quantitative and quali-tative differences in response to influenza and pneumococcal vac-cines. Immunity 2013;38:831–44.

18. Min L, Isa SA, Fam WN, Sze SK, Beretta O, Mortellaro A, et al.Synergism between curdlan and GM-CSF confers a strong inflamma-tory signature to dendritic cells. J Immunol 2012;188:1789–98.

19. ArimaK,WatanabeN, Hanabuchi S, ChangM, SunSC, Liu YJ. Distinctsignal codes generate dendritic cell functional plasticity. Sci Signal2010;3:ra4.

20. Markovics JA, Araya J, Cambier S, Jablons D, Hill A, Wolters PJ, et al.Transcription of the transforming growth factor beta activating integrinbeta8 subunit is regulated by SP3, AP-1, and the p38 pathway. J BiolChem 2010;285:24695–706.

21. Bell D, Chomarat P, Broyles D, Netto G, HarbGM, Lebecque S, et al. Inbreast carcinoma tissue, immature dendritic cells reside within thetumor, whereasmature dendritic cells are located in peritumoral areas.J Exp Med 1999;190:1417–26.

22. Leibundgut-Landmann S, Osorio F, Brown GD, Reis e Sousa C.Stimulation of dendritic cells via the dectin-1/Syk pathway allowspriming of cytotoxic T-cell responses. Blood 2008;112:4971–80.

23. Dubsky P, Saito H, Leogier M, Dantin C, Connolly JE, Banchereau J,et al. IL-15–induced human DC efficiently prime melanoma-specificnaive CD8(þ) T cells to differentiate into CTL. Eur J Immunol2007;37:1678–90.

24. Romano E, Cotari JW, Barreira da Silva R, Betts BC, Chung DJ,Avogadri F, et al. Human Langerhans cells use an IL-15R-alpha/IL-15/pSTAT5-dependent mechanism to break T-cell toleranceagainst the self-differentiation tumor antigen WT1. Blood 2012;119:5182–90.

25. Wolfl M, Kuball J, Ho WY, Nguyen H, Manley TJ, Bleakley M, et al.Activation-induced expression of CD137 permits detection, isolation,and expansion of the full repertoire of CD8þ T cells responding toantigen without requiring knowledge of epitope specificities. Blood2007;110:201–10.

26. Cepek KL, Shaw SK, Parker CM, Russell GJ, Morrow JS, Rimm DL,et al. Adhesion between epithelial cells and T lymphocytes mediatedby E-cadherin and the alpha E beta 7 integrin. Nature 1994;372:190–3.

27. Agace WW, Higgins JM, Sadasivan B, Brenner MB, Parker CM. T-lymphocyte–epithelial-cell interactions: integrin alpha(E)(CD103)beta(7), LEEP-CAM and chemokines. Curr Opin Cell Biol 2000;12:563–8.

28. Schon MP, Arya A, Murphy EA, Adams CM, Strauch UG, AgaceWW, et al. Mucosal T lymphocyte numbers are selectively reducedin integrin alpha E (CD103)-deficient mice. J Immunol 1999;162:6641–9.

29. Le Floc'hA, Jalil A, Vergnon I, LeMauxChansac B, Lazar V, BismuthG,et al. Alpha E beta 7 integrin interaction with E-cadherin promotesantitumor CTL activity by triggering lytic granule polarization andexocytosis. J Exp Med 2007;204:559–70.

30. Le Floc'h A, Jalil A, Franciszkiewicz K, Validire P, Vergnon I, Mami-ChouaibF.Minimal engagement ofCD103oncytotoxic T lymphocyteswith an E-cadherin-Fcmolecule triggers lytic granule polarization via aphospholipase Cgamma-dependent pathway. Cancer Res 2011;71:328–38.

31. Parker CM, Cepek KL, Russell GJ, Shaw SK, Posnett DN, SchwartingR, et al. A family of beta 7 integrins on human mucosal lymphocytes.Proc Natl Acad Sci U S A 1992;89:1924–8.

32. Rihs S, Walker C, Virchow JC Jr, Boer C, Kroegel C, Giri SN, et al.Differential expression of alpha E beta 7 integrins on bronchoalveo-lar lavage T lymphocyte subsets: regulation by alpha 4 beta 1-

integrin crosslinking and TGF-beta. Am J Respir Cell Mol Biol1996;15:600–10.

33. Ito T, Hanabuchi S, Wang YH, Park WR, Arima K, Bover L, et al. Twofunctional subsets of FOXP3þ regulatory T cells in human thymus andperiphery. Immunity 2008;28:870–80.

34. Kitamura H, Cambier S, Somanath S, Barker T, Minagawa S, Marko-vics J, et al. Mouse and human lung fibroblasts regulate dendritic celltrafficking, airway inflammation, and fibrosis through integrin alphav-beta8-mediated activation of TGF-beta. J Clin Invest 2011;121:2863–75.

35. Li MO, Flavell RA. TGF-beta: a master of all T cell trades. Cell2008;134:392–404.

36. Dragicevic A, Dzopalic T, Vasilijic S, Vucevic D, Tomic S, Bozic B, et al.Signaling through Toll-like receptor 3 and Dectin-1 potentiates thecapability of human monocyte-derived dendritic cells to promote T-helper 1 and T-helper 17 immune responses. Cytotherapy 2012;14:598–607.

37. Lin JY, Chen JS, Chen PC, Chung MH, Liu CY, Miaw SC, et al.Concurrent exposure to a dectin-1 agonist suppresses the Th2response to epicutaneously introduced antigen in mice. J Biomed Sci2013;20:1.

38. Ohshima Y, Tanaka Y, Tozawa H, Takahashi Y, Maliszewski C, Dele-spesse G. Expression and function of OX40 ligand on human dendriticcells. J Immunol 1997;159:3838–48.

39. Lewis KL, Caton ML, Bogunovic M, Greter M, Grajkowska LT, Ng D,et al. Notch2 receptor signaling controls functional differentiation ofdendritic cells in the spleen and intestine. Immunity 2011;35:780–91.

40. Vigne S, Palmer G, Lamacchia C, Martin P, Talabot-Ayer D, RodriguezE, et al. IL-36R ligands are potent regulators of dendritic and T cells.Blood 2011;118:5813–23.

41. Vigne S, Palmer G, Martin P, Lamacchia C, Strebel D, Rodriguez E,et al. IL-36 signaling amplifies Th1 responses by enhancing prolifer-ation and Th1 polarization of naive CD4þ T cells. Blood 2012;120:3478–87.

42. Gresnigt MS, Rosler B, Jacobs CW, Becker KL, Joosten LA, van derMeer JW, et al. The IL-36 receptor pathway regulates Aspergillusfumigatus-induced Th1 and Th17 responses. Eur J Immunol 2012;43:416–26.

43. Agrawal S, Gupta S, Agrawal A. Human dendritic cells activated viadectin-1 are efficient at priming Th17, cytotoxic CD8 T and B cellresponses. PLoS ONE 2010;5:e13418.

44. Shaw SK, Brenner MB. The beta 7 integrins in mucosal homing andretention. Semin Immunol 1995;7:335–42.

45. Sheridan BS, Lefrancois L. Regional and mucosal memory T cells. NatImmunol 2011;12:485–91.

46. El-Asady R, Yuan R, Liu K, Wang D, Gress RE, Lucas PJ, et al. TGF-{beta}-dependent CD103 expression by CD8(þ) T cells promotesselective destruction of the host intestinal epithelium during graft-versus-host disease. J Exp Med 2005;201:1647–57.

47. Yu CI, Becker C, Wang Y, Marches F, Helft J, Leboeuf M, et al. HumanCD1c(þ) dendritic cells drive the differentiation of CD103(þ) CD8(þ)mucosal effector T cells via the cytokine TGF-beta. Immunity 2013;38:818–30.

48. Mullins DW, Sheasley SL, ReamRM,Bullock TN, Fu YX, Engelhard VH.Route of immunization with peptide-pulsed dendritic cells controls thedistribution of memory and effector T cells in lymphoid tissues anddetermines the pattern of regional tumor control. J ExpMed 2003;198:1023–34.

49. Sheasley-O'Neill SL, Brinkman CC, Ferguson AR, Dispenza MC,Engelhard VH. Dendritic cell immunization route determines integrinexpressionand lymphoid andnonlymphoid tissuedistribution ofCD8Tcells. J Immunol 2007;178:1512–22.

50. Mora JR, BonoMR, Manjunath N,Weninger W, Cavanagh LL, Rosem-blatt M, et al. Selective imprinting of gut-homing T cells by Peyer'spatch dendritic cells. Nature 2003;424:88–93.


Wu et al.




2014;2:487-500. Published OnlineFirst March 4, 2014.Cancer Immunol Res Te-Chia Wu, Kangling Xu, Romain Banchereau, et al. Mucosal T-cell Differentiation and Breast Cancer Rejection

+CD8+Reprogramming Tumor-Infiltrating Dendritic Cells for CD103

Updated version

10.1158/2326-6066.CIR-13-0217doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerimmunolres.aacrjournals.org/content/suppl/2014/03/04/2326-6066.CIR-13-0217.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerimmunolres.aacrjournals.org/content/2/5/487.full#ref-list-1

This article cites 50 articles, 25 of which you can access for free at:

Citing articles

http://cancerimmunolres.aacrjournals.org/content/2/5/487.full#related-urls

This article has been cited by 1 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department

Permissions

[email protected]

To request permission to re-use all or part of this article, contact the AACR Publications Department at



http://cancerimmunolres.aacrjournals.org/lookup/doi/10.1158/2326-6066.CIR-13-0217

http://cancerimmunolres.aacrjournals.org/content/suppl/2014/03/04/2326-6066.CIR-13-0217.DC1

http://cancerimmunolres.aacrjournals.org/content/2/5/487.full#ref-list-1

http://cancerimmunolres.aacrjournals.org/content/2/5/487.full#related-urls

http://cancerimmunolres.aacrjournals.org/cgi/alerts

mailto:[email protected]

mailto:[email protected]


reprogramming tumor-inﬁltrating dendritic cells for …€¦ · reprogramming tumor-inﬁltrating...

Documents