Analysis of Immune Cells from Human Mammary Ductal ... · using monoclonal antibodies (mAb), or in some cases by syn-thetic compounds. 1Department of Medical Microbiology and Immunology,

Research Article

Analysis of Immune Cells from Human MammaryDuctal Epithelial Organoids Reveals Vd2þ T CellsThat Efficiently Target Breast Carcinoma Cells inthe Presence of BisphosphonateNicholas A. Zumwalde1,2, Jill D. Haag2, Deepak Sharma2, Jennifer A. Mirrielees3,Lee G.Wilke3, Michael N. Gould2, and Jenny E. Gumperz1

Abstract

Developing strategies to enhance cancer prevention is aparamount goal, particularly given recent concerns about sur-gical treatment of preinvasive states such as ductal carcinomain situ. Promoting effective immunosurveillance by leukocytesthat scan for nascent neoplastic transformations represents apotential means to achieve this goal. Because most breastcancers arise within the ductal epithelium, enhancing protec-tive immunosurveillance will likely necessitate targeting one ormore of the distinctive lymphocyte types found in these sitesunder normal conditions. Here, we have characterized theintraepithelial lymphocyte compartment of non-canceroushuman breast tissue and identified a subset of T lymphocytes

that can be pharmacologically targeted to enhance theirresponses to breast cancer cells. Specifically, Vd2þ gd T cellswere consistently present in preparations of mammary ductalepithelial organoids and they proliferated in response to zole-dronic acid, an aminobisphosphonate drug. Vd2þ T cells frombreast ductal organoids produced the antitumor cytokine IFNgand efficiently killed bisphosphonate-pulsed breast carcinomacells. These findings demonstrate the potential for exploitingthe ability of Vd2þ gd T cells to respond to FDA-approvedbisphosphonate drugs as a novel immunotherapeutic approachto inhibit the outgrowth of breast cancers. Cancer Prev Res; 9(4);305–16. �2016 AACR.

IntroductionIdentifying genetic loci associated with reduced risk of breast

cancer may provide novel targets for cancer prevention (1). Suchloci may operate directly within mammary epithelial cells or maybe mediated by the activities of non-mammary cells. We haverecently reported that the rat Mcs5a locus acts via the immunesystem and that the resistant allele of Mcs5a is associated withincreased frequency and functional activity of gd T cells withinspleen andmammary epithelium (2). These findings suggest thatengaging key immune cell types to phenocopy the effects of theresistant Mcs5a allele may represent an effective breast cancerprevention strategy. However, given the substantial differencesbetween humans and muroid rodents in the molecular specifi-

cities of innate immune cells that mediate defense against incip-ient threats, an essential prerequisite to such an effort is identi-fying the immune subsets typically present in human mammaryductal epithelial tissues and determining how these can be med-ically targeted.

Cancerous cells are culled from tissues through the process ofimmunosurveillance, whereby several different types of leuko-cytes continuously scan for neoplastically transformed cells andeliminate them (3). Because breast cancers typically originatefrom the epithelial cells lining the mammary ducts and lobules(4), the immune cells responsible for immunosurveillance oftransformed breast cells are likely to be those that patrol theductal epithelium. Although recent studies have illustrated thepresence of leukocytes in the human breast (5–8) and even inthe epithelium (6, 7), the specific leukocyte subsets within thisspecialized tissue niche have remained poorly characterized.Moreover, a key unanswered question is whether immune cellsare present that can be targeted to promote enhanced immuno-surveillance of precancerous or cancerous cells.

Conserved T lymphocyte populations are particularly attractivefor this type of approach because they recognize non-polymor-phic antigen-presenting molecules and thus are present in allindividuals regardless of human leukocyte antigen (HLA) type,and they can selectively be activated based on features of the T cellreceptor (TCR). Some examples of conserved T lymphocytes aregd T cells, mucosal-associated invariant T (MAIT) cells, andinvariant natural killer T (iNKT) cells. Based on their characteristicTCR chain usages, these types of T cells can be specifically targetedusing monoclonal antibodies (mAb), or in some cases by syn-thetic compounds.

1Department of Medical Microbiology and Immunology, University ofWisconsin School of Medicine and Public Health, Madison,Wisconsin.2McArdle Laboratory for Cancer Research, Department of Oncology,University of Wisconsin School of Medicine and Public Health, Madi-son, Wisconsin. 3Department of Surgery, University of WisconsinSchool of Medicine and Public Health, Madison,Wisconsin.

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

Current address for D. Sharma: Radiation Biology and Health Sciences Division,Bhabha Atomic Research Centre, Trombay Mumbai, India.

Corresponding Authors: Jenny E. Gumperz, 1550 Linden Drive Madison, WI53706. Phone: 608-263-6902; Fax: 608-262-8418; E-mail:[email protected]; and Michael N. Gould, E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-15-0370-T

�2016 American Association for Cancer Research.

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For example, human Vd2þ T cells are selectively activated byFDA-approved aminobisphosphonate (BP) drugs. These com-pounds act on Vd2þ T cells because they block the mevalonatebiosynthetic pathway within target cells, which leads to theaccumulation of a particular metabolic intermediate calledisopentenylpyrophosphate (IPP). IPP associates with the cyto-plasmic tail of a cell-surface protein called butyrophilin 3A1(BTN3A1), causing a recognizable change in molecular featuresof the extracellular domain of BTN3A1 (9, 10). Target cells thatexpress BTN3A1 and that have undergone an intracellularaccumulation of IPP trigger TCR-dependent activation of Vd2þ

T cells (11–13), causing them to proliferate, secrete cytokinessuch as IFNg , and to kill the target cells (14). Thus, BPs maypromote the antitumor functions of human gd T cells in severalways, including (i) by expanding the numbers of Vd2þ T cells;(ii) by promoting their production of the antitumor cytokineIFNg ; and (iii) by promoting their killing of tumor cells.Indeed, administration of BPs to human cancer patients whohad bone metastases (3 females with breast cancer and 6 maleswith prostate cancer) was associated with the expansion of aneffector population of Vd2þ T cells in the blood, and withenhanced IFNg production (15). These findings suggest that BPtreatment may also provide an effective means to enhance theimmunosurveillance functions of human Vd2þ T cells that playa critical role in eliminating nascent neoplastic cells before theycan develop into tumors.

Administration of BP is likely to be particularly effective forcancers located in tissues patrolled by Vd2þ T cells (e.g., highlyvascularized sites, such as bone). However, it is less clear whetherthis strategy represents a viable option for promoting the elim-inationof cancer cells located in epithelial tissues, because the gd Tcells in mucosal tissues mainly belong to other subtypes that donot respond to BPs. Some studies have shown lower prevalence ofpostmenopausal breast cancer in certain populations who havereceived BP treatment (16–20), although other studies found noevidence of decreased breast cancer risk inwomenwhohave taken

BPs (21). Thus, the potential utility of BP administration forpromoting immunological control of incipient breast cancerremains unclear. Here, we have addressed this question by inves-tigating the presence and functionality of targetable T lymphocyteeffector populations from primary human breast epithelialtissues.

Materials and MethodsBreast tissue acquisition and preparation

Non-cancerous breast tissue was obtained from the Coopera-tive Human Tissue Network (CHTN; funded by the NationalCancer Institute) or provided by the University of Wisconsin'sTranslational Science BioCore-BioBank from reduction mammo-plasties or contralateral prophylactic mastectomies (Table 1).Acquisition and analysis of the breast tissue was approvedby the University of Wisconsin Health Services InstitutionalReview Board.

Human breast organoids were isolated as previously pub-lished (22, 23). Briefly, breast tissue was minced and digestedovernight in a 37�C shaker with 1X collagenase/hyaluronidasein Complete EpiCult B HumanMedia (Stem Cell Technologies)supplemented with 5% fetal bovine serum (FBS; Hyclone).After incubation, the digested tissue was spun for 1 minute orless at 80 to 100 � g to form a visible cell pellet enriched forepithelial ductal organoids. This cell pellet was washed and thebreast organoids were collected on a 40-mm filter. In addition, acell pellet containing stromal cells, red blood cells and smallductal epithelial fragments was also collected from the super-natant of the digested tissue. Organoids and stromal cell frac-tions were cryopreserved in 50% FBS/6% dimethyl sulfoxide,and stored in liquid nitrogen until needed. Single-cell suspen-sions from the organoids were prepared for all flow cytometricanalyses and used for in vitro experiments by trypsinizing theorganoids using 2 mL of ethylenediaminetetraacetic acid(EDTA)/trypsin solution for 1 to 2 minutes. EDTA (Thermo

Table 1. Human breast tissue donor information

Sample ID Age, sex, race Elective procedure Pathology report

L625 A1 47 y, female, N/A Prophylactic mastectomy N/AL625 B1 56 y, female, N/A Prophylactic mastectomy N/AL625 C1 36 y, female, N/A Reduction mammoplasty N/AL625 D1 28 y, female, N/A Reduction mammoplasty N/AL625 G1 37 y, female, N/A Prophylactic mastectomy N/AL625 I1 36 y, female, N/A Reduction mammoplasty N/AL625 J1 18 y, female, BL Reduction mammoplasty (macromastia) NormalL625 K1 41 y, female, WH Reduction mammoplasty (macromastia) FPCIL625 L1 23 y, female, BL Reduction mammoplasty (macromastia) NormalL625 M1 36 y, female, WH Reduction mammoplasty (macromastia) Fibrosis; CLI; PASHL625 N1 38 y, female, BL Reduction mammoplasty (macromastia) NormalL625 O1 46 y, female, WH Reduction mammoplasty (macromastia) NormalL625 P1 42 y, female; WH Reduction mammoplasty (macromastia) FibrosisL625 Q1 37 y, female, BL Reduction mammoplasty (macromastia) NormalL625 S1 50 y, female; BL Reduction mammoplasty (macromastia) NormalL625 T1 24 y, female, WH Reduction mammoplasty (macromastia) NormalL625 U1 51 y, female, BL Reduction mammoplasty (macromastia) NormalL625 V1 27 y, female, WH Reduction mammoplasty (macromastia) PASHL625 W1 19 y, female, BL Reduction mammoplasty (macromastia) FibrosisL625 X 1 33 y, female, WH Reduction mammoplasty (macromastia) PASHL625 Y1 21 y, female, BL Reduction mammoplasty (macromastia) NormalL625 Z1 44 y, female, WH Reduction mammoplasty (macromastia) NormalL625 A2 36 y, female; WH Reduction mammoplasty (macromastia) Normal

Abbreviations: CLI, chronic lobular inflammation; FPCI, focal periductal chronic inflammation; PASH, pseudoangiomatous stromal hyperplasia.

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Cancer Prev Res; 9(4) April 2016 Cancer Prevention Research306




Fisher Scientific)/trypsin (Worthington Biochemical Corpora-tion) solutions were made by adding 50 mg EDTA to 25 mLwarm HBSS (Life Technologies) or PBS (Corning) without Ca2þ

/Mg2þ; subsequently 5 mg trypsin was then added to 2 mLof EDTA solution and diluted 1:100 for usage.

Peripheral blood mononuclear cell isolationPeripheral blood mononuclear cells (PBMC) were isolated

from healthy donors according to protocols approved by theUW Health Sciences and Minimal Risk IRBs. Written informedconsent was obtained from all donors. Blood was processedusing Ficoll-paque PLUS (GE Healthcare) and spun for 40minutes without brake or acceleration at 400 RCF. The buffycoat was removed and washed with PBS for 15 minutes at

400 RCF. The supernatant was discarded and pellet resus-pended in PBS and washed for 10 minutes at 300 RCF.

Flow cytometry and intracellular stainingFor surface stains, cells were harvested, washed with PBS,

blocked with 20% human AB serum (Fc block) for 15 minutes,stained with antibodies for 30 minutes at 4�C, washed, resus-pended in PBS, and analyzed on a LSRII (BD Biosciences) withFlowJo analysis software (Version 9.3.1; Tree Star Inc.). Intra-cellular (IFN)-g and IL17A staining was performed according tothe manufacturer's recommendations using the BD Cytofix/Cytoperm kit (BD Biosciences) in the presence of BD GolgiStopprotein transport inhibitor (BD Biosciences). IntracellularFoxP3 staining was performed according to the manufacturer's

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Bisphosphonate Activates Human Breast T Cells

www.aacrjournals.org Cancer Prev Res; 9(4) April 2016 307




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instructions using the True-Nuclear transcription factor bufferset (BioLegend).

Directly conjugated fluorescent antibodies used for flow cyto-metry included: CD3 (clone OKT3; BioLegend), CD45 (HI30;BioLegend), CD4 (RPA-T4; BioLegend), CD8a (RPA-T8; BioLe-gend),CD8b (SIDI8BEE; eBioscience), Vd2 (B6; BioLegend), Va7.2(3C10; BioLegend), NKT cell receptor Va24Ja18 (6B11;BioLegend), Vd1 (REA173; Miltenyi Biotec), CD103 (Ber-ACT8;BioLegend), EpCAM (9C4; BioLegend), CD49f (GoH3;BioLegend), CD24 (ML5; BioLegend), CD29 (TS2/16; BioLegend),CD10 (HI10a; BioLegend), Muc-1 (16A; BioLegend), CD31(WM59; BioLegend), CD27 (O323; BioLegend), CD45RO(UCHL-1; BD Biosciences), LAMP-1 (H4A3; BioLegend), IFNg(4S.B3; BioLegend), IL17A (BL168; BioLegend), FoxP3 (206D;BioLegend),NKG2D(1D11; BioLegend),Butryophilin3A1 (BT3.1;BioLegend), MICA/B (6D4; BioLegend), ULBP1 (170818; R&DSystems), ULBP2/5/6 (165903; R&D Systems), ULBP3 (166510;R&D Systems), ULBP4 (709116; R&D Systems).

Bisphosphonate stimulation and Vd2 T cell expansionPBMCs or single cells prepared from the breast organoids

were exposed to 2.5 mmol/L zoledronate in combination withirradiated feeder PBMCs (exposed to 7 � 103 to 8 � 103 Rad) tostimulate and expand Vd2 gd T cells. Specifically, 2 � 105

isolated PBMCs were cultured with 5 � 105 irradiated feedersin an end volume of 0.2 mL T cell media in 96-well plates.Single-cell preparations from organoids were plated with 5 �105 total irradiated feeders in an end volume of 0.2 mL T cellmedia in 96-well plates. Lymphocyte cultures were maintainedin RPMI 1640 with L-glutamine (Corning) supplemented with15% heat inactivated bovine calf serum (HI-BCS; ThermoFisher Scientific), 3% human AB serum (Atlanta Biologicals),1% penicillin/streptomycin (P/S; Mediatech) and 200 units/mLIL2 (Peprotech) referred to as T cell media. Percentages of Vd2 Tcells were obtained by flow cytometry 2 to 3 weeks afterstimulation and compared with media-only controls; Vd2frequencies above 1% were considered positive for expansion.Alternately, freshly isolated PBMCs were also stimulated with2.5 mmol/L zoledronate in T cell media at 1 � 106 cells/well in24-well plates and expanded for 7 days. Bisphosphonate (BP)expanded cells were used in cytotoxicity assays. BP doseresponse curves were generated using fresh PBMCs with eitherzoledronate or alendronate for 7 days in T cell media. Freshmedia were provided when needed. Zoledronate (NovationLLC and Novartis) and alendronate (Teva PharmaceuticalsUSA) BP were obtained from UW Health Pharmacy Services.

Analysis of Vd2þ T cell responses to tumor cellsMDA-MB-468breast carcinoma cellswere obtained fromATCC

as an authenticated cell line, and maintained in DMEM/F12(Corning) supplemented with 10% HI-BCS (Thermo FisherScientific) and 1% P/S (Mediatech). The MDA-MB-468 cells werepulsed with or without 5 mmol/L zoledronate overnight, then

washed and resuspended at 3 � 106 cells/1.0 mL media. Cellsfrom primary organoids and cell suspensions containing BPexpanded organoid- or blood-derived Vd2 T cells were eachincubated with MDA-MB-468 cells for 4 to 6 hours at 37�C ina total of 0.2mL in 96-well plates. After the incubation, Vd2 T cellswere analyzed for cell-surface LAMP-1 and/or intracellular IFNgexpression by flow cytometry.

To assess gd T cell killing of target cells, MDA-MB-468 cells wereused as targets and day 7 BP expanded PBMC-derived Vd2 T cellswere used as effectors. Briefly, targets were pulsed with or without10 mmol/L zoledronate overnight. At the same time, day 7stimulated Vd2 T cells were cultured with IL2 overnight. Thefollowing day, targets were trypsinized, washed and prepared ata concentration of 1� 105 to 2 � 105 cells/0.1 mL T cell mediawithout IL2. Effectors were prepared without IL2 and co-incubat-ed with targets at different effector-to-target ratios in 96-wellplates at 37�C for 4.5 hours. Additionally, 0.1mLs of 0.1% trypsinwas used for several minutes to recover adherent cells. The cellswere washed with Fc block and stained with CD45 for 30minutesat 4�C to delineate effectors from targets. Samples were washedwith PBS, spun, and resuspended in Annexin V binding buffer(BioLegend). Annexin V (5 mL/tube; BioLegend) and propidiumiodide (10 mL/tube; BioLegend) were added to samples andincubated for 15 minutes in the dark at room temperature.Additional Annexin V binding buffer was added before flowcytometry analysis.

Statistical analysisGraphPad Prism versions 4.0 and 4.0c software (GraphPad

Software) were used to construct data graphs and determinestatistical significance using the Wilcoxon test for paired samplesor the Student unpaired two-tailed t test or the Mann–Whitney.The P value cutoffs and notation were used as follows: �, P < 0.05;��, P < 0.01; ��, P � 0.0001.

ResultsPreparation of human breast tissue yields highly enrichedductal epithelial organoids

Samples of breast tissue from human subjects who had under-gone reduction mammoplasty or prophylactic mastectomywere collected for analysis (see Table 1). The samples wereprepared using a protocol designed to separate tissue fragmentsrepresenting ductal organoids from stromal layers. A representa-tive image of the resulting tissue fragments showing ductal andalveolar structures is shown in Fig. 1A. To confirm that thefragment preparations were enriched for epithelial cells com-pared with the stromal fraction, we utilized multiparameter flowcytometry to assess relative frequencies of cells expressing epi-thelial, endothelial, or hematopoietic markers (Fig. 1B). Cellsthat were double negative for both the hematopoietic linagemarker CD45 and the endothelial marker platelet/endothelialcell adhesion molecule-1 (PECAM-1; CD31; ref. 24) were

Figure 2.Characterization of immune cells from breast-derived organoids yields unique lymphocyte percentages compared with blood. A, gating strategy to delineateCD3þCD45þ T lymphocytes. B, quantification of CD3þCD45þ T lymphocytes. C, expression of the coreceptors CD8aþ and CD4þ by CD3þ T cells. D, quantificationof CD8aþ expression by the CD3þ T cells. E, CD45RO�/þ and CD27�/þ (markers of T cell activation and differentiation) staining of CD8aþ T cells. F,quantification ofCD8aþT effectormemory cells (CD45ROþCD27�). G, expression ofCD103þ (an intraepithelial cell associated integrin) by cells of theCD8aþgate. H,quantification of CD103þ CD8aþ T cells. �� , P < 0.0001 (Student t test).






assessed for expression of the epithelial cell adhesion molecule(EpCAM; refs. 25, 26) to delineate epithelial content (Fig. 1B).Analysis of organoid and stromal fractions from at least 10different breast tissue samples demonstrated that the organoidswere significantly enriched for EpCAMþ cells compared with thestromal cell fractions, 43% and 9% respectively (Fig. 1C).EpCAM-expressing cells within the organoid preparations alsoexpressed other markers associated with mammary gland-derivedepithelial cells, including CD49flow/þ(25, 26), CD10low (25, 26),CD24þ (24), CD29þ (24), and Muc-1int/high (ref. 25; Supple-mentary Fig. S1A–S1E). Organoid preparations contained anaverage of 24% CD45þ cells (leukocytes), and 9% CD31þ cells(endothelial cells; ref. Fig. 1D). These results indicated that ourbreast tissue preparation method successfully enriches for ductalepithelial organoids, and thus the associated leukocytes are likelyto be highly enriched for cells from the ductal epithelial tissue.

Leukocyte populations associated with human breastorganoids differ from those in peripheral blood

We next examined the immune cell subsets within theCD45þ population of the breast organoid preparations. Typ-ically, at least 90% (90.63% � 7.51%) of the CD45þ cells wereCD3þ, indicating that they are T lymphocytes (Fig. 2A and B).In contrast, CD3þ cells made up on average about 60% of theCD45þ cells in the peripheral blood (Fig. 2A and 2B). Of theCD3þ cells in the organoid preparations, about 75% (75.67%� 13.32%) were typically CD8þ, whereas CD3þ cells in theblood typically contained less than 34% CD8þ cells (Fig. 2Cand D). Essentially all of the organoid-derived CD8aþ T cellsco-expressed CD8b, indicating that they are likely distinctfrom the CD8aaþ intra-epithelial lymphocytes that have beenidentified within intestinal epithelium (ref. 27; SupplementaryFig. S2A). Further analysis of the organoid CD8þ T cell pop-ulation demonstrated that it consists almost exclusively ofCD45ROþCD27� cells (Fig. 2E and F), a phenotype thatis characteristic of effector memory T cells (28). In contrast,CD8þ T cells in the blood are composed of almost equalproportions of na€�ve T cells (CD27þCD45RO�; ref. 28), effectorT cells (CD27�CD45RO�; ref. 29), central memory T cells(CD27þCD45ROþ; ref. 28), and effector memory T cells(CD27�CD45ROþ; ref. 28; Fig. 2E and F). Moreover, typically80% (�12.94%) of the organoid preparation CD8þ T cellsexpressed the integrin aE (CD103), which is a marker of intrae-pithelial lymphocytes (30),whereas less than3%of theperipheralbloodCD8þ T cells expressed thismarker (Fig. 2G andH).Most ofthe CD8� organoid T cells expressed intermediate levels of CD4(Fig. 2C) and were essentially all CD45ROþCD27� (Supplemen-tary Fig. S2B). On average about 4% of the organoid CD4þ cellsexpressed FoxP3 (Supplementary Fig. S2C and S2D), which ischaracteristic of a regulatory T cell phenotype. Notably, in contrastto the CD8þ T cells, only a small fraction of organoid-derivedCD4þ T cells expressedCD103 (Supplementary Fig. S2E). Togeth-er, these data demonstrate that the immune cell populationsassociated with our organoid preparations are clearly distinctfrom those of the blood, and are highly enriched for T cells withcharacteristics of IELs.

Conserved T lymphocyte subsets are present in organoids andexpand in response to BP

We next wanted to determine whether we could detectlymphocyte subsets with conserved T cell receptors within the

breast organoids that might be targetable in a chemopreventionstrategy against breast cancer. We screened for two differentsubpopulations of gd T cells (Vd1 and Vd2), MAIT cells, andiNKT cells. Both subsets of gd T cells were clearly detectable inalmost all of the samples analyzed (Fig. 3A and B), althoughthey made up comparatively small percentages of the CD3þ

cells (Vd1 mean ¼ 1.27%; Vd2 mean ¼ 0.42%). MAIT cells,which were identified using the Va7.2 T cell receptor, weredetectable as >3.1% of the CD3þ cells in organoids (Fig. 3Aand B). iNKT cells were detected in some of the samples, but didnot appear to be markedly enriched (data not shown). Thus,although Vd2 cells are predominantly found within the blood,these results indicated that these potentially targetableT lymphocytes are typically present in the breast ductalepithelium.

We next investigatedwhether breast epithelial organoid Vd2þ Tcells are able to respond functionally to an FDA-approved BP.Preliminary studies confirmed that zoledronate more potentlystimulated blood-derived Vd2þ T cells compared with alendro-nate (Supplementary Fig. S3), consistent with prior studies byother groups (15, 31). Therefore, we cultured the total cells fromorganoid preparations with zoledronate or with medium alonefor 2 to 3 weeks, and performed flow cytometric analysis todetermine the relative frequencies within the culture of Vd2þ Tcells or MAIT cells as a control. Exposure to BP resulted inmarkedincreases in the frequency of Vd2þ cells, but not of MAIT cells,within the cultures (Fig. 3C). Increases in total Vd2þ cell numberwere also observed, suggesting that the Vd2þ T cells had prolif-erated (data not shown). Overall, exposure to BP induced clearlydetectable Vd2þ T cell expansion in approximately 45% of theorganoid samples we tested (Fig. 3D). These data demonstratethat organoid-derived Vd2þ T cells can respond to an FDA-approved BP drug.

BP treatment facilitates IFNg production by organoid-derivedVd2þ T cells in response to triple-negative breast carcinomacells

Whereas gd T cells typically have a T helper 1 (TH1) cytokineproduction phenotype that is characterized by the productionof high levels of IFNg , it has recently become clear that lym-phoid cells in and around epithelial tissues are also responsiblefor the production of IL17, a cytokine that promotes epithelialintegrity but that may also play a pathogenic role in tumori-genesis (32). Therefore, we tested cytokine production byprimary T cells in organoid preparations by stimulating themwith PMA and ionomycin, then performing intracellular cyto-kine staining for IFNg and IL17. Detectable populations ofcells expressing IL17 were observed in the non-Vd2 CD8þ andCD8� T cell subsets; however, the Vd2þ T cell populationappeared heavily biased toward production of IFNg with littleor no evidence of IL17-producing cells (SupplementaryFig. S4). Based on these results, we focused our further analyseson IFNg production.

We determined whether the organoid-derived Vd2þ T cellsproduce cytokines in response to a human breast carcinoma cellline, MDA-MB-468, which is triple negative for estrogen recep-tor, progesterone receptor, and HER2/neu. Organoid cells werecultured with BP for 3 to 4 weeks, then exposed to MDA-MB-468 cells that were either pulsed with BP or mock-treated.Intracellular cytokine staining was performed to detect thefrequency of gd T cells expressing IFNg . We observed robust

Zumwalde et al.





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Figure 3.Breast organoids contain specifically targetable lymphocytes that respond toan FDA-approved BP drug. A, flow cytometry analysis of an organoidpreparation from tissue sample L1 (see Table 1) for gd T cells and MAIT cells(Va7.2 T cell receptor). B, quantification of T cell subsets from organoidpreparations from the indicated tissue samples (see Table 1 for tissue donorinformation). Symbols under the dashed line were below the limit ofdetection. C, example of flow cytometry results showing Vd2þ T cellexpansion in the presence of 2.5 mmol/L BP compared with culture mediumalone. Va7.2þMAIT cells were used as a control for nonresponsiveness to BP.D, quantification of Vd2 T cell frequency from organoids compared to PBMCsas a positive control after 2 to 3weeks of culture in the presence of BP. Five of11 samples (45.5%) displayed Vd2þ T cell expansion from the purifiedorganoid fraction. Each symbol represents an independent expansionattempt using the indicated tissue samples. Dashed line indicates thethreshold used to delineate expansion.

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Figure 4.Breast organoid–derived Vd2 gd T cells produce IFNg in response to atriple-negative breast carcinoma cell line pulsed with BP. A, Vd2þ T cellswere expanded in vitro from organoid preparations by exposure to BP.The T cells were coincubated with MDA-MB-468 breast carcinoma cellsthat were pulsed (right) or not pulsed (left) with BP, and intracellularIFNg production was assessed by flow cytometry. Results shown arerepresentative of two independent experiments. B, primary organoid-derived cells were coincubated with MDA-MB-468 cells that were pulsed(right) or not pulsed (left) with BP, and intracellular IFNg production byVd2þ and Vd2� T cells was assessed by flow cytometric analysis.Numbers shown in the gates are the percentage of IFNg–expressingcells from the Vd2þ or Vd2� T cell populations. C, plot showingaggregated results for IFNg production by primary Vd2þ T cells from theindicated breast tissue samples. Normalized mean fluorescence intensity(MFI) for IFNg was determined by dividing the IFNg MFI of the Vd2þ cellsby the IFNg MFI of the corresponding Vd2� T cells in the same sample.Each symbol represents an independent experiment to assess IFNgproduction by T cells from the indicated breast tissue samples. Thedashed line represents a normalization ratio of 1. n.s., not significant; � , P¼ 0.0121 (Mann–Whitney test; plus BP to ex vivo); � , P ¼ 0.0262 (Mann–Whitney test; plus BP to no BP).






IFNg responses to BP-pulsed target cells, but little or no detect-able IFNg staining in response to mock-treated target cells(Fig. 4A). These results demonstrated that organoid-derivedVd2þ T cells that expand in response to BP treatment alsoproduce cytokines in response to BP-pulsed breast cancer cells,but did not clarify whether a significant frequency of theprimary gd T cells within breast organoids can mount similarresponses to transformed cells.

Therefore, we tested the ability of primary Vd2þ T cells withinorganoid cell preparations to produce cytokines in response totumor cells directly ex vivo. Indeed, when we exposed organoid-derived cells to BP-pulsed or mock-treated MDA-MB-468 breastcarcinoma cells, we found that a detectable fraction of the Vd2þ

subset showed intracellular IFNg staining (Fig. 4B). Analysis ofmultiple different primary tissue samples revealed that the nor-malized IFNg mean fluorescence intensity of the Vd2þ cells wasconsistently higher in response to BP-pulsedMDA-MB-468 breastcarcinoma cells than to mock-treated carcinoma cells (Fig. 4C).These results demonstrate that primary gd T cells from breastductal epithelia are able to respond functionally to breast carci-noma cells that have been exposed to BP.

Organoid-derived Vd2þ T cells demonstrate cytotoxicity toBP-treated triple-negative breast carcinomas

To further investigate, we tested the cytotoxic responses of Vd2þ

T cells to MDA-MB-468 breast carcinoma cells. We first analyzedsurface expression of the LAMP-1 protein (CD107a), whichbecomes expressed at the cell surface as a result of vesicle fusionwith the plasmamembrane during a killing response. Vd2þ T cellswithin BP-expanded breast tissue cultures showed robust cellsurface LAMP-1 expression after exposure to BP-pulsed MDA-MB-468 target cells, and a small percentage of the Vd2þ cellstypically also showed cell-surface LAMP-1 expression in responseto mock-treated target cells (Fig. 5A). To confirm that Vd2þ T cellsurface LAMP-1 expression was correlated with target cell killing,we assessed the viability of the MDA-MB-468 carcinoma cells byAnnexin V staining. BP-pulsed MDA-MB-468 carcinoma cellswere efficiently killed upon exposure to in vitro--expanded Vd2þ

T cell cultures (greater than 75% target cell death within 4hours; Fig. 5B). Notably, there was also significant killing ofmock-treated MDA-MB-468 breast carcinoma cells, although thisrequired much higher effector-to-target cell ratios (Fig. 5B). Anal-ysis of T cells within primary organoid cell preparations revealedthat a fraction of the Vd2þ cells typically showed cell-surfaceLAMP-1 expression after exposure toMDA-MB-468 cells (Fig. 5C).Exposure to BP-pulsed target cells elicited detectable cell-surfaceLAMP-1 expression on Vd2þ T cells nearly in 100%of the samplestested, while exposure to mock-treated MDA-MB-468 cellsresulted in cell-surface LAMP-1 expression by Vd2 cells in about

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Figure 5.Cytotoxic functions by breast-derived Vd2 gd T cells in response to atriple-negative carcinoma cell line. A, Vd2þ T cells were expanded in vitrofrom breast tissue preparations by exposure to BP. The T cells werecoincubated with MDA-MB-468 breast carcinoma cells that were pulsed(right) or not pulsed (left) with BP, and cell surface expression of CD107a(LAMP-1, a marker of recent cytotoxic activity) was assessed by flowcytometry after 4 hours. B, MDA-MB-468 cells were pulsed (black squares)or not pulsed (gray squares) with BP, and coincubated with in vitro–expanded Vd2þ T cells at the indicated effector:target ratios. The plotshows cytotoxicity of the target cells as assessed by cell-surfaceupregulation of Annexin V. Similar results were obtained in fiveindependent experiments. C, primary organoid cells were coincubatedwith MDA-MB-468 cells that were pulsed (right) or not pulsed (left) withBP, and CD107a expression by T cells was assessed by flow cytometry.

Numbers in the gates indicate the percentage of CD107a-expressing cellsfrom the Vd2þ or Vd2� T cell populations. The figure shows representativeresults from one out of seven independent experiments. D, plot showingaggregated results for cell-surface CD107a expression by primary T cellsfrom the indicated breast tissue samples after exposure to MDA-MB-468cells that were pulsed or not pulsed with BP, or by primary organoid-derived T cells that were not exposed to tumor cells ("ex vivo"). Eachsymbol represents an independent experiment to assess CD107a cell-surface expression by T cells from the indicated breast tissue samples.n.s., not significant; � , P ¼ 0.0273; �� , P ¼ 0.0078 (Wilcoxon test).

Zumwalde et al.





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Figure 6.Expression of tumor-associated ligands and receptors. A, flowcytometric analysis of MDA-MB-468 cells using antibodies specific forBTN3A1 (Vd2þ T cell receptor ligand), or for the following NKG2Dligands: MICA and MICB, ULBP-1, ULBP-2 (antibody also cross-reactswith ULBP-5 and -6), ULBP-3, or ULBP-4. Filled histograms showstaining with the specific mAb; dashed histograms show staining by anisotype control mAb. B, expression of NKG2D (an activating receptorthat recognizes MICA/B) by Vd2þ T cells, CD8a� T cells, and CD8aþ

T cells from organoid preparations. Median fluorescence intensity (MFI)values for isotype control mAb staining are shown in italics, and specificmAb MFI values are shown in bold.






50% of the samples (Fig. 5D). These results demonstrate that BPtreatment of breast carcinoma cells promotes highly efficientcytotoxic responses by Vd2þ T cells, and indicate that Vd2þ Tcells also mediate less efficient cytotoxicity that is independent ofBP exposure of the breast carcinoma cells.

The functional responses by breast Vd2þ T cellsmay result fromTCR-mediated recognition of BTN3A1 that has associated withIPP (33). Additionally, these responses could be mediated by thebinding of NKG2D receptors on the gd T cells to a family ofligands, calledMICA/B and ULBP1-6, that are upregulated duringcellular stress and that are often expressed by tumor cells (34). Wetherefore investigated the expression of these key tumor controlmolecules. Flow cytometric analysis of the MDA-MB-468 cellsrevealed weakly positive staining for BTN3A1 (Fig. 6A). Stainingfor expression of NKG2D ligands showed clearly positive stainingforMICA/B and at least onemember of theULBP family (Fig. 6A).We also analyzed the organoid-derived lymphocytes for expres-sion of the NKG2D receptor, and found that nearly all Vd2þ andCD8þ T cells, and some of the CD8� T cells were NKG2D positive(Fig 6B). Thus,most primary breast epithelial organoid associatedT cells express receptors for recognition of stressed or neoplasticcells.

DiscussionIt has recently become clear that the role of gd T cells in cancer

can include both antitumorigenic and protumorigenic functions.Anti-neoplastic functions of gd T cells include their critical role intumor immunosurveillance and as early responders to nascenttransformations (35–37). Additionally, gd T cells can mediaterejection (killing) of established tumors (38, 39). Paradoxically,however, studies from murine models have indicated that gd Tcells can also promote the outgrowth of carcinomas as a result ofthe expression of factors that promote epithelial cell growth (40).Moreover, a recent analysis of human breast cancer patientsshowed that the presence of regulatory gd T cells within the tumorwas correlated with poor survival and high risks of relapse (41).These contrasting roles of gd T cells underscore that it is critical tounderstand the characteristics of the immune cells in ductalepithelial tissues in order to design effective immunopreventativestrategies.

Prior analyses of immune populations associated withhuman breast have utilized unfractionated breast tissue thatwas not enriched specifically for the epithelial compartment.For example, Ruffell and colleagues analyzed tissue samplesfrom prophylactic mastectomies and found that 60% to 70%of CD45þ cells were CD3þ T lymphocytes of several differentvarieties (5). The results presented here contrast with earlierstudies of breast tissue and demonstrate that the human breastepithelial organoid represents a specialized immunologicalniche that is comprised mainly of T cells (>90% of theCD45þ cells). In contrast to the composition of T cells fromblood, the breast epithelial T cell population was markedlyskewed toward CD8þ cells, which is consistent with a recentstudy demonstrating that CD8 T cells are directly integratedwithin the breast epithelium (7). Moreover, our observationthat both the CD4þ and CD8þ T cells from the breast orga-noids had a CD27� effector memory phenotype contrastssharply with the dominant phenotypes of T cells from blood.Thus, our analysis clearly establishes that a distinct make-up ofleukocyte subsets is associated with the breast epithelial orga-

noids, and that blood contamination of the samples is not amajor factor.

Given this, it was particularly noteworthy that we detectedVd2þ T cells in 87% of organoids. This gd T cell subset is thoughtto localizemainly to the blood,while other types of gd T cells (e.g.,Vd1þ T cells) are thought to be characteristic of peripheral tissues(42). Because the organoid-associated Vd2þ T cells were notuniformly positive for the epithelial-residency marker CD103(data not shown), some of the Vd2þ T cells we detected mayhave been in the process of transiently trafficking through thetissue rather than permanently residing there. Nevertheless, thepresence of Vd2þ cells as part of the steady-state surveillancepopulation of breast epithelial organoids has important implica-tions for immunotherapeutic enhancement of cancer prevention,because this population is highly targetable.

Vd2þ T cells recognize the accumulation of specific moleculesassociated with the biosynthesis of isoprenoid lipids, and thustheydetect changes to cellularmetabolism that are associatedwithhyper-proliferation and neoplastic transformation (43). As aresult of this, Vd2þ T cells become activated by administrationof FDA-approved BP drugs because these drugs block the farnesylpyrophosphate synthase enzyme and cause accumulation of IPPwithin cells. Interestingly, it has recently been shown that humanbreast cancer cell lines vary in their endogenous accumulation ofIPP after exposure to BP, and that the amount of IPP produced inresponse to BP treatment correlatedwith the ability of Vd2þ T cellsto infiltrate and control the growth of transplanted tumor cells invivo (34). We found that breast-derived Vd2þ T cells showedefficient cytotoxicity toward the MDA-MB-486 triple-negativebreast carcinoma cell line after BP exposure, despite the compar-atively low cell-surface expression of BTN3A1 on the tumor cells.Thus, these tumor cells may be particularly rich in their accumu-lation of IPP after BP exposure. Alternatively, they may elicitstrong cytotoxic responses by gd T cells as a result of their highcell-surface expression of NKG2D ligands such as MICA/B. Theseresults suggest that administering BPs or related compounds tobreast epithelial tissues may be an efficient way to enhance thekilling of nascent neoplastic cells by Vd2þ T cells within the breasttissue. However, because we also found that the Vd2þ T cellsshowed a less potent, but still significant cytotoxic response to thetumor cells that were not treated with BP (an effect that might bedue to endogenous accumulation of IPPwithin the tumor cells, orsimply to their upregulation of MICA/B molecules), it may besufficient to use BP administration as a means to induce theproliferation of Vd2þ T cells and thus to increase their frequencyin vivo, without actually targeting breast epithelial cells for BPuptake.

In addition to Vd2þ cells, we also detected Vd1þ T cells in thebreast epithelial organoid fraction. Vd1 cells have been shown tobe highly antitumorigenic to different tumors such as multiplemyeloma (44), acute myeloid leukemia (45), and acute lympho-blastic leukemia (45). As a result of recent advances in under-standing molecular interactions involved in activating Vd1þ

T cells, such as CD1d-mediated presentation of the cellular lipidsulfatide (46), it may soon be feasible to specifically target thissubset. We also detected a subset of Va7.2 TCR expressing lym-phocytes that have been broadly characterized as MAIT cells (47).MAIT cells recognize vitaminmetabolites that are presentedby thenon-classical antigen-presenting molecule MR-1 (47). Becausemany different microbes produce the chemical compounds rec-ognized by MAIT cells, including Staphylococcus aureus and

Zumwalde et al.





epidermidis, which are the major causative agents for humanmastitis, it is likely that there is an important interplay betweenthe resident microbiota and the immune cells in the ductal net-works of human breast.

Together, our findings demonstrate that the lymphocyte com-partment associated with human breast ductal epithelial orga-noids contains several conserved T cell populations that could betargeted, or in some cases possibly inhibited, to promote theimmune-mediated clearance of nascent neoplastic cells. Such anapproach may be particularly important for prophylacticallytreating women thought to be at high risk for breast cancer, andmay also provide a novel non-invasive means to treat ductalcarcinoma in situ.

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

Authors' ContributionsConception and design: N.A. Zumwalde, J.D. Haag, D. Sharma, M.N. Gould,J.E. GumperzDevelopment of methodology: N.A. Zumwalde, J.D. Haag, D. Sharma,L.G. Wilke

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):N.A. Zumwalde, J.D. Haag, J.A. Mirrielees, L.G. WilkeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): N.A. Zumwalde, J.D. Haag, D. Sharma, M.N. Gould,J.E. GumperzWriting, review, and/or revision of themanuscript:N.A. Zumwalde, J.D.Haag,L.G. Wilke, M.N. Gould, J.E. GumperzAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): N.A. Zumwalde, J.D. Haag, J.A. MirrieleesStudy supervision: M.N. Gould, J.E. Gumperz

Grant SupportMajor funding for this project was from a Collaborative Health Sciences

Program grant from the Wisconsin Partnership Project to M.N. Gould,J.E. Gumperz, and L.G. Wilke. N.A. Zumwalde was supported by NIH T32CA157322. Additional funding was provided by NIH U01 ES019466 toM.N. Gould.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedOctober 21, 2015; revised January 7, 2016; accepted January 8, 2016;published OnlineFirst January 25, 2016.

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2016;9:305-316. Published OnlineFirst January 25, 2016.Cancer Prev Res Nicholas A. Zumwalde, Jill D. Haag, Deepak Sharma, et al. Carcinoma Cells in the Presence of Bisphosphonate

T Cells That Efficiently Target Breast+2δOrganoids Reveals VAnalysis of Immune Cells from Human Mammary Ductal Epithelial

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