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Microenvironment and Immunology

Inherent and Tumor-Driven Immune Tolerance inthe Prostate Microenvironment Impairs NaturalKiller Cell Antitumor ActivityChristine Pasero1,2, Gwena€elle Gravis2, Mathilde Guerin2, Samuel Granjeaud1,Jeanne Thomassin-Piana2, Palma Rocchi1,2, Maria Paciencia-Gros2, Flora Poizat2,M�elanie Bentobji2, Francine Azario-Cheillan2, JochenWalz2, Naji Salem2, Serge Brunelle2,Alessandro Moretta3, and Daniel Olive1,2,4

Abstract

The field of immunotherapy for solid tumors, such as prostatecancer, has been recently focusing on therapies that can countertumor-mediated immunosuppression. Precise quantification andcharacterization of the immune infiltrates in tumors is crucial toimprove treatment efficacy. Natural killer (NK) cells, major com-ponents of the antitumor immune system, have never beenisolated from prostate tumors, despite their suspected role indisease progression. Here, we examined the frequency, pheno-type, and functions of NK cells infiltrating control and tumorprostate tissues. NK cell infiltrates in prostate tissues were mainlyCD56 (NCAM1)-positive and displayed an unexpected imma-ture, but activated, phenotype with low or no cytotoxic potential.Furthermore, we show that TGFb1 (TGFB1) is highly secreted intothe prostate environment and partly mediates the immunosup-pressive effects on NK cells. In addition to this basal level of

immunotolerance toNK cells, the prostate environment becamefurther resistant to NK cell–mediated immunity upon cancercell infiltration. Coculture experiments revealed that prostatecancer cells induced the expression of inhibitory receptor (ILT2/LILRB1) and downregulated the expression of activating recep-tors NKp46 (NCR1), NKG2D (KLRK1), and CD16 (FCGR3) byNK cells, thus preventing their recognition of tumor cells.Notably, blood levels of NKp46 were also decreased in prostatecancer patients and were inversely correlated with levels ofprostate-specific antigen, the main prognostic factor in prostatecancer. Our study shows that a strong immunosuppressiveenvironment impairs NK cell function at multiple levels inprostate cancer and provides a rationale for the design oftherapies that restore NK cell efficiency in the prostate tumormicroenvironment. Cancer Res; 76(8); 2153–65. �2016 AACR.

IntroductionProstate cancer is one of the most frequent causes of cancer

mortality among men (1) and is still incurable once it becomesmetastatic. Androgen-deprivation therapy (ADT) is the mainstayof treatment for advanced prostate cancer, but resistance tocastration inevitably develops. Immunotherapy has been intro-duced as an alternative therapeutic approach with Sipuleucel-Tand GVAX-PCa cell-based cancer vaccines, or Ipilimumab (anti-CTLA-4) mAb (2). Over the past few years, the immune infiltrateand its prognostic significance have attracted a lot of attention (3);however, infiltrating lymphocytes are poorly characterized inprostate. Thus, the interaction between immune system and

cancer cells within the patient's tumor needs to be better under-stood in order to develop clinically effective immunotherapies.

Natural killer (NK) cells are innate cytotoxic lymphocytesthat play a fundamental role in antitumor immunity (4). Micelacking NK cells or defective for NK cell receptors have a higherincidence of spontaneous tumors (5). A 11-year follow-upstudy reported that a low degree of NK cell cytotoxicity wascorrelated with increased cancer risk in men (6). NK cells can bebroadly divided in CD56dim cytotoxic subset, which representthe majority of NK cells in the blood, and in CD56bright

cytokine producer subset (7). The activation of NK cells isregulated by a balance between activating and inhibitory sig-nals transmitted through surface receptors that determine thecytotoxic response (8). Inhibitory receptors include the killerimmunoglobulin-like receptors (KIR), CD94/NKG2A, andILT2/CD85j. The main activating receptors of NK cells are thenatural cytotoxicity receptors (NCRs: NKp46, NKp30, NKp44),NKG2D and DNAM-1 (DNAX accessory molecule-1).

Tumor cells have developed several mechanisms to evade fromNK immunosurveillance, through the modulation of cell surfacemolecules or the release of immunosuppressive soluble factorssuch as PGE2 or TGFb1 in the tumor microenvironment (9, 10).Alterations of NCRs have been observed in blood from acutemyeloid leukemia patients (11), and recent studies show thatintratumoral NK cells display phenotypic and/or functionalalterations compared with peripheral NK cells in breast cancer(12, 13), lung cancer (14–16), colorectal cancer (17), renal cell

1Centre de Recherche en Canc�erologie de Marseille, INSERM U1068/CNRS U7258, Marseille, France. 2Institut Paoli-Calmettes, Marseille,France. 3D.I.M.E.S., University di Genova, Genova, Italy. 4Aix MarseilleUniversit�e, Marseille, France.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Daniel Olive, Institut Paoli Calmettes Inserm U1068,232 bd Sainte Marguerite, Marseille 13009, France. Phone: 334-8697-7271; Fax:334-9122-3610; E-mail: [email protected]; and Christine Pasero,[email protected]

doi: 10.1158/0008-5472.CAN-15-1965

�2016 American Association for Cancer Research.

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carcinoma (18), and gastrointestinal stromal tumors (GIST;refs.19, 20).

In prostate cancer, the ligands for NKp30 and NKp46 werefound to be expressed on primary tumors but not on benignprostate hyperplasia (21). Importantly, increasedNKcell infiltratewithin prostate tumors was found to be associated with a lowerrisk of progression through IHC, providing evidence that NK cellsmay have a protective role against prostate cancer in humans (22).However, prostate-infiltrating NK cells have never been isolatedand their phenotype and functions remain unknown. Indeed, theisolation of tumor-infiltrating immune components is problem-atic due to the small size of prostate samples and the infiltrativegrowth of the tumor within the gland.

We, therefore, set out to analyze and compare for the first timeNK cell phenotype and functions in tumor but also controlprostate tissues, in comparison with NK cells isolated fromperipheral blood of prostate cancer patients (pB-PC). Our resultsreveal two unexpected levels of tissue tolerance to NK cells inprostate cancer; first, NK cells infiltrating the prostate glanddisplay immature phenotype with hyporesponsive functionalstatus; second, the tumor breaks the balance between activatingand inhibitory NK cell receptors. Together, these alterations leadto a profound immunosuppressive environment that should betargeted to restore NK cell cytotoxicity against prostate cancer.

Patients and MethodsPatients

Prostate cancer patients were prospectively recruited in a com-prehensive cancer center (Institut Paoli-Calmettes IPC, Marseille,France) and provided written informed consent. The project wasreviewed by the internal reviewboard and the studywas approvedby a central national ethics committee.

Tumor tissues were obtained from 21 prostate cancer patients(mean age 71.9 years) including n ¼ 16 localized (Loc) orlocoregional (LCR) tumors and n ¼ 5 metastatic (M) tumors.Clinical characteristics of the tumors can be found as Supplemen-tary Table S1. The mean follow-up period was 12.1 months.Control prostate tissues (n ¼ 20, mean age 66.9 years) werecollected from prostate cancer patients, in an area selected outof the tumor site by pathologists, or from bladder cancer patientsat the time of cystoprostatectomy. All sections were strictly select-ed by a single pathologist (J. Thomassin-Piana). Blood samples(pB-PC) were obtained from 18 patients with localized prostatecancer (mean age 61 years) and from 30 patients with metastaticprostate cancer (mean age 65 years). Blood samples from healthymen volunteers (mean age 51 years) were used as controls(Etablissement Francais du Sang (EFS, Marseille, France).

Isolation of lymphocytes from bloodPeripheral blood mononuclear cells (PBMC) were purified

from blood samples by Lymphoprep (lymphocyte separationmedium) density gradient centrifugation. PBMCs were washedand resuspended in RPMI (Gibco, Life Technologies) medium forfurther analysis.

Isolation of lymphocytes from prostate tissuesFresh prostate tissues were extemporaneously treated and

mechanically minced using scalpels in RPMI medium. Cell sus-pensions obtained were filtered successively through 70- and 30-mmcell strainers (Miltenyi Biotec). The supernatant was harvestedand centrifuged at high speed to remove residual cellular ele-

ments. This supernatant, referred as "dissociation supernatant"was aliquoted and frozen at�20�C until further use in functionalexperiments. Cell suspension was washed and used directly forflow cytometry staining.

Cell linesPC3 cell line is derived from a late-stage castrate-resistant

metastatic prostate cancer sample andwas purchased fromATCC.PC3 was cultured in DMEM (Gibco Life Technologies) supple-mented with 10% fetal calf serum (FCS, Lonza). K562 cell line isderived from human erythroleukemia cell line (ATCC) and wascultured in RPMI supplemented with 10% FCS.

Flow cytometryCells were incubated with conjugated antibodies or isotypic

controls (listed in Supplementary Table S2) for 30minutes at 4�C,washed and extemporaneously collected on a BD LSR Fortessacytometer (BD Biosciences), and analyzed with FACSDiva (BDBiosciences) or FlowJo (TreeStar Inc.) softwares. For NK cells, thegating strategy consisted on the elimination of the doublets basedon FSC-A/FSC-H parameters, followed by the removal of deadcells using a cell viability marker. NK cell population was definedas CD3�CD56þ cells within the lymphocyte gate. The phenotypicdata in the article are represented as percentage of cells positive fora given marker (bimodal expression); or the mean ratio fluores-cence intensity (RMFI, unimodal expression), that is, the ratiobetween the mean fluorescence intensity (MFI) of cells stainedwith the selected mAb and that of cells stained with isotypiccontrol alone.

CD107 degranulation and IFNg/TNFa productionPBMCs were activated overnight in IL2 (100 UI/mL; Chiron)

and IL15 (10 ng/mL; Miltenyi Biotec), and incubated with K562target cells at effector: target (E:T) ratio of 10:1 during 4 hours,with monensin (Golgi Stop), FITC-conjugated anti-CD107a(LAMP1), and FITC-conjugated anti-CD107b (LAMP2) mAbs(BD Biosciences). Then PBMCs were stained for 30 minutes at4�Cwith PerCP-Cy5.5–conjugated anti-CD3 andPC7-conjugatedanti-CD56 antibodies and permeabilized with Cytofix/Cytoperm(BD Biosciences). Intracellular antibodies were next added (IFNgand TNFa) before cell analysis.

51Cr experimentsBlocking mAbs directed against NK cell receptors or related

ligands were produced in A. Moretta's laboratory: anti-NKp30(F252), anti-NKp46 (KL247), anti-2B4 (CO54), anti-DNAM(F5),anti-NKG2D (ON72). Purified NK cells from healthy donors orpatients were incubated at various effector: target (E:T) ratios(10:1, 5:1, 2:1) in triplicate, with 51Cr-labeled K562–positivecontrol cell line, in the presence of blocking mAbs or saturatingconcentrations of appropriate isotype controls. Four hours later,supernatants were harvested and assayed for the release of 51Cr.Spontaneous and total lysis were measured by incubating targetcells in the absence of NK cells and in the presence of NP40,respectively. The percentage of specific lysis was measured asfollows: (experimental lysis – spontaneous lysis)/(maximumlysis-spontaneous lysis) � 100.

Detection of soluble factors and cytokinessMICA, ADAM17, TGFb1, and PGE2 secretion were evaluated

by ELISA (R&D Systems). IL1ra, MCP-1, RANTES, and IL8 secre-tion were evaluated in a 9-plex multiple human cytokine assay

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Cancer Res; 76(8) April 15, 2016 Cancer Research2154




(Bio-Plex; Bio-Rad). IL1b, IL4, IL6, IFNg , and TNFa were notdetected in the supernatants. This assay was run according to themanufacturer's recommended procedure; beads were read on theBio-Plex suspension array system and data were analyzed usingBio-Plex manager software with 5PL curve fitting.

IHCImmunohistochemical staining was performed on 3-mm thick

sections of formalin-fixed paraffin-embedded prostate tissuesfixed in 4% formol alcohol. Slides were incubated for 20 minutesin a 98�C water bath (pH 9 for both NKp46 and CD3). Afterneutralization of the endogenous peroxidase (EnVision FLEXPeroxidase blocking reagent; Dako) and saturation, sections werelayered with the anti-NKp46 polyclonal antibody (R&D Systems,1/40e) or the anti-CD3 polyclonal antibody (Dako, FLEX Ready-to-use). The primary antibody was incubated for 6 hours (anti-NKp46 antibody) or 20 minutes (anti-CD3 antibody). Sectionswere revealed with the EnVision FLEX/HRP Kit (Dako), DABsubstrate Kit (Dako), counterstained with the EnVision FLEXhematoxylin substrate buffer (Dako), and mounted using Pertex(Histolab). Controls were performed by incubating slides withsecond antbody alone.

Statistical analysesAll statistical analyses were performed using GraphPad Prism

software (GraphPad). The nonparametric tests (paired Wilcoxontest or unpaired Mann–Whitney test) are indicated in the figurelegends. Only P values inferior to 0.05 were considered as signif-icant. Heatmaps were performed using multi-experiment viewerof the TM4 micro-array software suite (TMeV); version 4.9 wasretrieved from http://www.tm4.org/mev.html.

ResultsNK cells infiltrating the prostate gland display peculiarphenotype and low cytotoxic potential

We first investigated NK cell infiltration on paraffin-embeddedprostate tissues by immunohistochemical staining using the NKcell–specific marker NKp46 (Fig. 1A). The results show thatprostate tissues are poorly infiltrated by NK cells, in comparisonwith T cells that were detected with anti-CD3 antibody (Fig. 1B).Both populations were mainly localized in the stroma surround-ing normal and tumor prostatic glands. We prospectively collect-ed tumor (n ¼ 21) and control prostate tissues (n ¼ 20) toinvestigate infiltrating NK cell phenotype, by comparison withNK cells isolated fromperipheral bloodof prostate cancer patients(pB-PC, n ¼ 10). Lymphocytes were isolated by mechanicaldissociation of fresh tissues, and NK cells were extemporaneouslyanalyzed by flow cytometry. We found low frequency of NK cellswithin control (2.9 � 0.6%, 1.47 � 105 NK cells/g) and tumorprostate tissues (3.6� 0.5% of living lymphocytes, 2.2� 105 NKcells/g), compared with the proportion of NK cells in peripheralblood (12.7 � 2.3%; Fig. 1C). We analyzed NK cell subsets inblood and tissues by flow cytometry, according to CD56 expres-sion (Fig. 1D). We found that the pattern of distribution ofCD56dim and CD56bright subsets was significantly perturbed intumor as well as in control tissues. In peripheral blood fromprostate cancer patients, the CD56dim subset prevailed over theCD56bright subset, as described in peripheral blood from healthydonors. In contrast, the CD56bright subset was significantlyincreased both in control and tumor tissues. Then maturation

and activationmarkers were measured by flow cytometry (Fig. 2Aand B). CD57 has been associated with the terminal differenti-ation stage of CD56dim NK cells (23) and CD57þ cells displayedhigher cytotoxic capacity and decreased capacity to proliferate(24).We found that CD57 expressionwas significantly reduced ininfiltrating NK cells compared with peripheral NK cells. In con-trast, the activation markers CD69 and NKp44 were significantlyincreased in NK cells infiltrating control and tumor tissues com-pared with pB-PC. To note, CD57 decreased expression was notonly due to the lower proportion of CD56dim subset; indeed,CD57 was decreased also within CD56dim subset both in controland tumor tissues. CD69 and NKp44 were mostly increased inCD56bright subset (Fig. 2C).

To further explore prostate-infiltrating NK cells, we measuredex-vivo CD107 degranulation of overnight IL2/IL15 activated NKcells in response toK562 erythroleukemia cell line (Fig. 2DandE).Interestingly, NK cells isolated from both control (bottom) andtumor (top) prostate tissues showed significantly low or nulldegranulation potential (measured as CD107 assay) but highactivated phenotype (CD69 expression) compared with periph-eral NK cells.

Together, these results show that immature but activated NKcells are overrepresented in prostate gland, regardless of whetherthe tissue is invaded by the tumor or not. Moreover, prostate-infiltrating NK cells display hyporesponsive functional status inthe presence of target cells. This highlights a mechanism of tissuetolerance in prostate gland that impairs NK cell phenotype andfunctions.

Tumor-induced alterations on activating and inhibitoryNK cell receptors

To explore what happens in cancer context, we conducted acomparative analysis between NK cells infiltrating tumor andcontrol prostate tissues (Fig. 3). We found that the activatingreceptors NKp46 and NKG2D were significantly decreased inNK cells infiltrating tumor compared with control tissues (Fig.3A). There was also a trend towards a lower expression ofNKp30 and DNAM-1 in tumors. In contrast, the inhibitoryreceptor ILT2 showed higher expression in NK cells infiltratingtumor compared with control tissues (Fig. 3B). Interestingly,CD16, which is involved in antibody-dependent cellular cyto-toxicity (ADCC), is clearly decreased in tumor compared withcontrol tissues (Fig. 3C). Representative flow cytometry profilesare shown in Fig. 3D. We observed, as previously published inbreast, lung, and colorectal cancer (12, 14, 17), that pairedperipheral and intratumoral NK cells from the same patientsdisplayed drastic differences in their phenotype (Supplemen-tary Fig. S1) but our study identifies which ones are due to thetumor and not to tissue localization. We further explored NKcell alterations depending on the stage of prostate cancer:localized (Loc) or locoregional (LCR; i.e., tumor with extra-capsular extension, or seminal vesicle/regional lymph nodeinvasion, but has not spread into other organs; n ¼ 16), ormetastatic prostate cancer for which tumor has already spreadto other organs (n ¼ 4; see Supplementary Table S1). Asdepicted in Fig. 3E, the decreased expression of activatingNKp46 and NKG2D, and the increased expression of inhibitoryILT2 were more pronounced in NK cells infiltrating metastaticthan localized or LCR tumors. These observations prompted usto investigate whether the phenotype of infiltrating NK cellswould correlate with clinical outcome. We considered median

Tissue and Tumor NK Cell Alterations in Prostate Cancer

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Figure 1.Frequency and phenotype of NK cell infiltrate in tumor and control prostate tissues. A and B, immunohistochemical staining of infiltrating immune cells on paraffin-embedded control or tumor prostate tissues. A, NKp46þ NK cells. B, CD3þ T lymphocytes. Brown, positively stained cells. NG, normal prostatic gland; TG,tumor prostatic gland. Original magnifications, �10, �20, �40, followed by computer magnification. C and D, flow cytometry analysis of infiltratingNK cells after tissue dissociation. C, frequency of CD3�CD56þNK cells within prostate tumors (n¼ 21, gray circle) and control prostate tissues (n¼ 20, white circle)gated among living lymphocytes. In parallel, white squares show the frequencyofNK cells in peripheral blood fromprostate cancer (PC) patients (pB-PC, n¼ 10). Thehorizontal bar represents the mean value � SEM. D, frequency of CD56dim and CD56bright NK subsets in tumor and control prostate tissues compared withperipheral blood from patients (pB-PC). The scatter plots (left) report frequency for CD56dim and CD56bright subsets for all patients tested. The horizontal barrepresents the mean value � SEM. Representative examples (right) for flow cytometry gating of CD3�CD56þ NK cell subsets are shown in blood, control, andtumor prostate tissues in two patients with prostate cancer. Two regions were selected to represent CD56dim and CD56bright populations. Statistical analyses weredone using nonparametric Mann–Whitney test. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.

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Figure 2.NKcells infiltrating both control and tumor prostate tissues display altered profile and poor cytotoxic potential. A, expression ofCD57, CD69, andNKp44gated onNKcells isolated fromcontrol (n¼ 20,white circle) and tumor prostate tissues (n¼ 21, gray circle) for all patients tested. In parallel, white squares show the expression ofeach marker on NK cells from peripheral blood (pB-PC, n ¼ 10). The horizontal bar represents the mean value � SEM. Statistical analyses were done usingnonparametric Mann–Whitney test. � , P < 0.05; �� , P < 0.01; �� , P < 0.001. B, representative dot plots for CD57/CD56, CD69/CD56, and NKp44/CD56 profiles oncontrol and tumor prostate tissues are presented in comparison with peripheral blood. The quadrant marker has been set according to the isotype controls. C,expression of CD57, CD69, and NKp44 on CD56dim and CD56bright NK cell subsets from control (gray column) and tumor (black column) prostate tissues comparedwith pB-PC (white column). D and E, functionality of NK cells infiltrating control and tumor prostate tissues compared with NK cells from peripheral blood. CD107degranulation was assessed by flow cytometry on NK cells incubated overnight in medium complemented with IL2 (100 UI/mL) and IL15 (10 ng/mL) beforeincubationwith K562 cell line according to 10:1 E:T ratio for 4 hours. D, dot plot representation of CD107 (degranulation) and CD69 (activation) expression in NK cellsinfiltrating prostate tumor (red) compared with peripheral NK cells from the same patient (top); and NK cells from control prostate tissue (blue) comparedwith peripheral NK cells from healthy donor (bottom). Numbers in dot plots indicate the percentage of CD107 or CD69þ cells within CD56þ NK cells. E, summary ofCD107 andCD69expression obtained onNK cells isolated fromperipheral blood (white circle) and tissues (control, blue circle; tumor, red circle) Bar graphs representmean � SEM. Statistical analyses were done using nonparametric Mann–Whitney test. �, P < 0.05; �� , P < 0.01; �� , P < 0.001.






values for tumor samples (n ¼ 21) as cut-off to determineNKp46high (cutoff ¼ 10.6) and NKG2Dhigh (cutoff ¼ 9.9) expres-sion. We looked at the distribution of patients according to theirPSA value and clinical status at last follow-up (Fig. 3F). Interest-ingly, patients with infiltrating NK cells displaying NKp46high

(dashedcolumns)and/orNKG2Dhigh (white columns)expressionall belonged to groups with low (<10 ng/mL) to intermediary(comprised between 10 and 20 ng/mL) PSA levels. None of

these patients were found in the poor prognosis group corre-sponding to high PSA levels. In addition, most patients withhigh expression of NKp46 and NKG2D on intratumoral NKcells at surgery displayed a high tendency toward no progres-sion 1 year after.

Together, these data show that the tumor specifically targets thebalance between activating and inhibitory receptors involved inNK cell cytotoxicity, with underexpression of activating receptors

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Figure 3.NK cells infiltrating prostate tumor show specific alterations on activating/inhibitory receptors compared with control tissues. A–D, the expression of activating andinhibitory NK cell receptorswas analyzed by flow cytometry on NK cells infiltrating control (n¼ 20, white circle) and tumor (n¼ 21, gray circle) prostate tissues aftermechanical dissociation. Scatter dot plots show the expression of activating receptors NKp46, NKG2D, NKp30, DNAM-1 (A); inhibitory receptor ILT2 (B);and cytotoxicmolecule CD16 (C) involved in ADCC. The horizontal bar represents themean value� SEM. Statistical analyseswere done using nonparametric Mann–Whitney test. � , P < 0.05; �� , P < 0.01; �� , P < 0.001. D, representative histogram profiles for each receptor expressed on NK cells from control (top) andtumor (bottom) prostate tissues. The gating strategy and isotype controls are shown in the figure. E, expression of NKp46, NKG2D, and ILT2 compared betweenlocalized (Loc) or locoregional (LCR) tumors (n ¼ 16, gray box plot) and metastatic (M) prostate tumors (n ¼ 4, dark box plot). Data are represented asbox and whisker (min to max; horizontal lines represent median values) graphs and scatter dot plots. Statistical analyses were done using nonparametric Mann–Whitney test. � , P < 0.05; �� , P < 0.01; �� , P < 0.001. F, distribution of patients with high expression of NKp46 (dashed column) or NKG2D (white column) ontumor-infiltrating NK cells according to the measurement of PSA at the time of sample: PSA < 10 ng/mL, PSA comprised between 10 and 20 ng/mL orPSA > 10 ng/mL and the progression or nonprogression of cancer after a median follow-up of 12.1 months. The Fisher exact test was significant (P < 0.05).

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andoverexpressionof inhibitory receptors, andmore pronouncedalterations along with metastatic progression. High expression ofNKp46 or NKG2D on infiltrating NK cells correlated with goodprognosis, suggesting that NK cells could control tumor escape.

The cytokinemilieu in prostate tissue environment impacts NKcell functions partly through TGFb1

To explore the cytokine environment in prostate gland, wequantified soluble factors released in dissociation supernatantfrom control and tumor tissues (Fig. 4A and B). We found highlevels of TGFb1, known for its immunosuppressive propertieson NK cells, in control supernatants and the secretion wassignificantly increased in the tumor. PGE2 secretion was alsoincreased in tumor compared with control milieu. Other sol-

uble factors such as Rantes and IL8 were found in tumor milieuand may account for inflammatory signals observed in cancercontext. Then, NK cells from healthy donors were exposed todissociation supernatants from control or tumor tissues for 24hours to test the effect of cytokine environment on NK cells(Fig. 4C and D). We found that cytokine milieu from tumorinduced a profound inhibition of CD107 degranulation com-pared with control supernatants. Finally, blocking TGFb1 effi-ciently restored CD107 degranulation in control condition (by63% on average) but only partially when NK cells were exposedto tumor supernatants (by 18%). Our results show that cyto-kine milieu in the prostate gland contains immunosuppressivesoluble factors, including TGFb1, that can affect NK cell func-tions. These observations further strengthen and reproduce the

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Figure 4.Analysis of cytokine milieu in control and tumor prostate tissues. A, soluble factor production in dissociation supernatant from control (white column, n ¼ 8) andtumor (black column, n ¼ 8) prostate tissues. The histograms represent the secretion of IL1ra, MCP-1, RANTES, IL8, evaluated by multiplex assays, and sMICA,ADAM17, PGE2, TGFb1, evaluated by ELISA, asmean� SEM. The levels of cytokines have been corrected for cell number after tissue dissociation and are representedas pg/mL for 1 million of cells. B, scatter dot plot representation of TGFb1 secretion (pg/mL for 1 million of cells) in supernatants from control and tumor prostatetissues by ELISA. Data are shown as mean � SEM. Statistical analyses were done using nonparametric Mann–Whitney test. �, P < 0.05; �� , P < 0.01; �� , P < 0.001.C, impact of the cytokine milieu on NK cells. Peripheral NK cells from three independent healthy donors were exposed for 24 hours to individual dissociationsupernatants (dilution one-fourth) from control (n ¼ 6) or tumor (n ¼ 5) prostate tissues. To evaluate the role of TGFb1, dissociation supernatants werepreincubated in parallel with neutralizing TGFb1 antibody (10 mg/mL) for 30 minutes before exposure to NK cells. NK degranulation was assessed against K562 cellline in a 4-hour assay (E:T ratio ¼ 1:1) and the percentage of NK cells positive for CD107 was analyzed by flow cytometry. Data are shown as the percentageof CD107 inhibition calculated as [% positive control (without supernatant) � % in the condition with supernatant]/% positive control � 100 � SEM.Statistical analyses were done using Mann–Whitney test. � , P < 0.05; �� , P < 0.01; �� , P < 0.001. D, representative flow cytometry analyses of CD107 degranulationafter exposure to supernatant from control or prostate tissue, and recovery of degranulation after TGFb1 blockade. The gate has been set according tomedium condition without K562 target cell line.






low or null degranulation potential of NK cells isolated fromcontrol and tumor tissues (Fig. 2D).

Prostate cancer cells alter the receptors involved in theirrecognition

To explore the mechanism that may account for phenotypicaldifferences between NK cells infiltrating control and tumor tis-sues, we performed transwell coculture experiments with PC3prostate cancer cell line (Fig. 5A). NK cells from healthy donorswere exposed to PC3 cell line through direct or indirect (throughtranswell membrane) contact for 48 hours. We found that PC3cells induced a significant downregulation of NKp46, NKG2D,and DNAM-1 after cell-to-cell contact (Fig. 5B). There was also atrend towards a decreased expression of NKp30 and CD16. Incontrast, ILT2 (inhibitory receptor) andCD69 (activationmarker)

expression on NK cells were significantly higher after contact withPC3 cell line.

Because NK cell cytotoxicity is known to depend on the engage-ment of different triggering receptors, we screened the expressionof ligands forNK cell receptors onPC3 cell line (Fig. 5C). PC3 cellsexpressed high level of ligands for DNAM-1 (Nectin-2 andCD155/PVR). The expression of ligands for NKG2D was hetero-geneous (detection with antibodies against MICA/B and ULBP1/2/3, or with NKG2D-Fc recombinant protein). In contrast,NKp30-ligand and NKp46-ligand expression were relativelyweak, but Fc-fusion proteins have lower affinity than that ofspecific mAbs. The expression of HLA family molecules washeterogeneous. To assess the specific involvement of NK cellreceptors during the recognition of prostate tumor cells, wepreincubated resting NK cells from healthy donors with blocking

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Figure 5.Prostate tumor cells alter the expression of activating/inhibitory NK cell receptors through direct contact. A, peripheral PBMCs from healthy donorswere exposed toPC3 prostate cell line through direct or indirect (Tw-PC3 for "transwell membrane," Greiner Bio One, 0.4 mm) contact, according to a 10:1 E:T ratio. After 48 hours, NKcells were harvested and phenotyped for NK cell receptors by flow cytometry. B, the graphs show the expression of NK cell receptors on NK cells exposedto PC3 prostate tumor cell line directly or through transwell membrane, compared with nonexposed NK cells. Results are representative of three independentexperiments (n ¼ 7 healthy donors in total). Statistical analyses were done using nonparametric Wilcoxon matched-pairs signed rank test. � , P < 0.05; �� , P < 0.01;�� , P < 0.001. C, expression of NK cell ligands on PC3 prostate cell line by flow cytometry for three independent experiments. Bar graphs report the expression ofligands for DNAM-1, NKG2D, NKp30, NKp46, and the expression of HLA molecules. Data are shown as mean � SEM. D, cytotoxicity of resting NK cellsagainst PC3 (right) or K562 cell line (positive control, left) was evaluated in a 4-hour 51Cr assay in the presence of blocking antibodies for NKp30, NKp46, 2B4, DNAM-1, ILT2, NKG2D, or irrelevant isotype control mAb. Target cell lysis wasmeasured by Cr51 release. Results are shown as the percentage of cytotoxic decrease inducedby the blocking antibodies compared with the irrelevant control mAb on three independent experiments. Statistical analyses were done using nonparametricWilcoxon matched-pairs signed rank test. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.

Pasero et al.





antibodies to NK cell receptors, and then used them in a 51Crcytotoxic assay against PC3 or K562 cell lines (Fig. 5D).We foundthat blocking NKp46, DNAM-1, or NKG2D significantlydecreased the cytotoxicity of NK cells against the PC3 cell line.Blocking NKp30 decreased cytotoxicity to a lower extent, corrob-orating NKp30 ligand expression. As a control, NKp30, DNAM-1,and to a lesser extentNKG2Dwere themajor receptors involved inNK cell cytotoxicity against K562 cell line, as described in theliterature. In conclusion, our findings show that prostate cancercells downregulate the activating receptors involved in theirrecognition, by mechanism dependent on cell-to-cell contact.Interestingly, these are precisely the receptors we found dysregu-lated in human prostate tumors. Mechanistically, we found anincreased pool of intracellular NKp46 in NK cells isolated fromtumors compared with peripheral NK cells, suggesting thatNKp46 could be internalized but not degraded in response toits ligand in tumor (Supplementary Fig. S2A). We did not detectintracellular NKG2D, but sMICA (�P < 0.05) was significantlyincreased in sera from prostate cancer patients compared withhealthy donors, and sMICB (��P < 0.01) was significantlyincreased in patients with metastatic disease stage, thus sheddingof MICs could lead to internalization and degradation of NKG2D(Supplementary Fig. S2B).

Peripheral NK cells from metastatic patients reflect theintratumoral alterations

To strengthen these observations, we enlarged our analysis toblood samples frompatientswith localized prostate cancer (Loc, n¼ 18), metastatic prostate cancer (Meta, n ¼ 30), and healthydonors as controls (HD, n ¼ 20; Fig. 6A). The expression ofactivating receptors NKp46 and NKG2D was significantly lowerin patients with metastatic prostate cancer than in patients withlocalized prostate cancer or HD. There was also a trend towards adecreased expression of NKp30 and DNAM-1 in metastatic com-pared with localized prostate cancer patients. In contrast, theexpression of inhibitory receptor ILT2 was significantly upregu-lated in metastatic prostate cancer patients compared with local-ized patients or HD. Hence, NK cells from peripheral blood ofmetastatic prostate cancer patients reflect the same phenotypicalterations, targeted on NKp46, NKG2D, and ILT2 than NK cellsinfiltrating the tumor site. In line with this point, intratumoralNKp46 expression was positively correlated with paired periph-eral NKp46 expression from the same patients (n¼ 7; R¼ 0.56, P¼ 0.07; Fig. 6B). We submitted the median expression of eachmarker for each group to TMEV software representation (Fig. 6C),which clearly emphasized that localized patients showed anintermediary phenotype, whereas the metastatic group displayedan inverted profile compared with HD. Kruskal–Wallis ANOVAanalysis found that NKp46, NKG2D, and ILT2 significantly dis-criminatedHD, localized, andmetastatic prostate cancer patients.Other inhibitory receptors such as NKG2A and CD158b (KIRfamily) were increased in prostate cancer patients compared withHD. The upregulation of NKp44 in peripheral NK cells frommetastatic patients showed that these cells were indeed activated.Interestingly, NKp46 expression in blood from prostate cancerpatients was significantly inversely correlated with PSA measure-ment (P < 0.01) at sample, the major clinical marker of poorprognosis in prostate cancer, and Gleason score (Fig. 6D).

We next assessed the functionality of overnight IL2/IL15 acti-vated peripheral NK cells from prostate cancer patients throughtheir capacity to degranulate (CD107 positivity). We found that

degranulation efficiency was significantly reduced in localizedand metastatic prostate cancer patients compared with HD (Fig.6E), and that CD107 response was positively correlated withNKp46 (R ¼ 0.51, P ¼ 0.0002) and NKp30 (R ¼ 0.50, P ¼0.0002) expression at the surface of NK cells (Fig. 6F). IFNg andTNFa production were also reduced in patients compared withHD (Supplementary Fig. S3A). NK cells that can exert multiplefunctions, in contrast with monofunctional NK cells, are morepotent effector cells (25). Thus, we looked at the multifunctionalpotential of peripheral NK cells using the Boolean gating strategy.We found that NK cells from metastatic prostate cancer patientsweremostlymonofunctional cells, exertingCD107degranulationor IFNg or TNFaproduction, thanNKcells from localizedprostatecancer patients or HD (Supplementary Fig. S3A). Redirected lysisagainst P815 target cell line using anti-CD16, anti-NKp30, or anti-NKp46 antibody was also reduced in prostate cancer patientscomparedwith healthy donors (Supplementary Fig. S3B). Finally,effective killing of K562 cell line was significantly reduced inresting NK cells from metastatic prostate cancer patients com-pared with localized prostate cancer patients or HD (Fig. 6G).Altogether these results show that phenotypic alterations ofperipheral NK cells in metastatic prostate cancer are associatedwith major impaired functions.

DiscussionOur study highlights a peculiar patternwhere CD56bright subset

is increased in the prostate gland, suggesting an immature butactivated phenotype compared with blood. NK cells infiltratingcontrol and tumor prostate tissues are not able to exert potentfunctionality by ex vivo exposure to tumor cells, and this wasreproduced by cytokine milieu, strengthening the tolerogenicenvironment in the prostate gland. This pattern was not observedin previous studies: CD56brightCD16� cells were found to beenriched in lung and breast cancer (16, 26) but NK cells infiltrat-ing healthy lung or breast sharedmore similarities with bloodNKcells (12, 14). A recent report showed that NK cell subset distri-bution is organ specific, with CD56bright predominant in liver,adrenal gland, colorectal tissues, and CD56dim predominant inlung and breast tissues (27). One hypothesis is that prostategland appears to be poorly infiltrated by immune cells at basallevel; thus, immune infiltration would rather be related to intra-prostatic inflammation. Thus, the phenotype observed could bea consequence of NK cell attraction from the periphery andsubsequent activation. Indeed, the enrichment of CD56bright

subset suggests either that this subset is specifically attractedfrom the periphery to organs, or that the tissue environmentcan have an influence, that is block or reverse the maturation ofNK cells. In either scenario, our data suggest that NK cellsinfiltrating prostate gland would be poorly armed in case oftumor development. We found that TGFb1, known to alterlymphocyte functions (28), is secreted in high amounts innormal prostate and increased in cancer. Interestingly, a higherexpression of TGFb1 was observed by qRT-PCR in prostatetumors with higher Gleason scores (29). In the literature, ithas been shown that the uterine decidua is populated byCD16� NK cells (30), and that TGFb1 results in conversion ofCD16þ into CD16� cells in vitro, highlighting the influence ofTGFb1 on the genesis of NK subsets (30, 31). Specific expansionof circulating CD56bright/dimCD16� NK cells has been reportedin metastatic melanoma; and this increase was associated with






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Figure 6.Peripheral blood NK cells are tumor-induced subsetswith altered phenotype and functions. A, NK cells were collected from blood samples of patients with localized(n ¼ 18, gray circle) and metastatic prostate cancer (PC; n ¼ 30, black circle) and were compared with NK cells from healthy donors (n ¼ 20, white circle) by flowcytometry. Scatter dot plots report the expression of NKp46, NKG2D, NKp30, DNAM-1 (as MFI ratio compared with isotypic control) and ILT2 (as the percentage ofpositive NK cells for the marker). The horizontal bar represents the mean value. B, Spearman correlation between the intratumoral expression of NKp46and the expression in peripheral blood from the same patients. C, summary of the median expression of the 15 NK cell receptors tested in blood of patients withprostate cancer compared with HD. Values represent the ratio between the median value for given marker in a group compared with the median value in all groupsincluded. Statistical analyses were done using Kruskal–Wallis (KW) ANOVA. � , P < 0.05; �� , P < 0.01; �� , P < 0.001. D, correlation between the expression of NK-cellreceptors in peripheral blood and the clinical parameters (GS, PSA) for patients with prostate cancer. The vertical bar represents the Spearman correlationcoefficients and the P values are indicated in each case if correlation was significant. E, CD107 degranulation of peripheral NK cells isolated from patients withlocalized (n ¼ 13, gray column) or metastatic prostate cancer (n ¼ 23, black column), compared with NK cells from healthy donors (HD; n ¼ 18, white column).Overnight IL2/IL15-activated NK cells were incubated with K562 target cell line according to 1:1 E/T ratio for 4 hours, and CD107 expression was assessed by flowcytometry. Data are shown as mean � SEM. F, Spearman correlation of CD107 response with the expression of NKp46 and NKp30 in peripheral NK cells. Each dotis coded for healthy donors (white circle), localized (orange square), or metastatic patients with prostate cancer (PC; blue diamond). Spearman correlationcoefficients and P values are indicated. G, the cytotoxicity of resting NK cells from patients with prostate cancer was evaluated in a 4-hour 51Cr assay againstK562 cell line (E:T ratio ¼ 1:1) and compared with healthy donors (HD). Target cell lysis was measured by Cr51 release. Statistical analyses were done usingnonparametric Mann–Whitney test. � , P < 0.05; ��, P < 0.01; �� , P < 0.001.

Pasero et al.





elevated plasma TGFb1 (32). Besides alterations on NK cellsubsets, TGFb1 can induce the downregulation of NKG2D andNKp30 expression on NK cells, leading to a decreased ability ofNK cells to kill target cells (33). Phase I studies with fresoli-mumab (human anti-TGFb mAb) are currently ongoing inmelanoma and renal cell carcinoma (34) and our findingsstrongly suggest that clinical strategies targeting TGFb1 couldbe of major interest in prostate cancer. Interestingly, weextracted mRNA expression from a public database including498 localized prostate tumors, and we found that TGFb1 waspositively correlated with some markers of tumor aggres-siveness in prostate cancer such as Gleason score, residualtumor, and pathologic T (Supplementary Fig. S4). CD57 wasinversely correlated with Gleason score and pathologic T,sustaining that lack of maturity of immune cells, and conse-quently lack of cytotoxic functions are associated with aggres-sive prostate cancer disease.

Besides this first level of tissue tolerance, prostate cancer cellsinduce specific alterations on activating (NKp46, NKG2D) andinhibitory (ILT2) receptors involved in cytotoxicity. Interestingly,our study points out that the receptors involved in cancer recog-nition are specific according to the tumor type: here, we identifyNKp46, NKG2D, and DNAM-1 as the main receptors involved inthe killing of prostate cancer cells. In melanoma, NKp30, NKp44,and NKG2D are the receptors modulated by coculture of NK cellswith melanoma cell lines (10, 35). In non–small cell lung carci-noma, a cluster of five receptors (NKp30, DNAM-1, NKp80,CD16, and ILT-2) was reduced in intratumoral NK cells. In breastcancer, the main receptors involved in NK cell cytotoxicity areDNAM-1 and NKG2D (13). Hence, prostate tumor cells couldescape from NK cell antitumor immunity by altering the pheno-type of NK cells and by decreasing their cytotoxic potential, asobserved in several malignancies (11, 35–38). The molecularmechanisms underlying the receptor downmodulation couldeither be chronic ligand exposure (38), or cytokine-induceddownregulation. Peripheral NK cells reflect these phenotypic andfunctional alterations and may represent tumor-induced subsetsas reported in breast cancer (26). In line with these results,impaired NK cell activity measured as IFNg levels has beenrecently described in blood from prostate cancer patients (39).To note, we found an overexpression of NKp44 that might seemcontrary to what would be expected for an activating receptor butthis upregulationwas also observed in breast and lung cancer (12,14), and seems rather be a hallmark of NK cell activation.Interestingly, it has been recently described thatNKp44 recognizestwo distinct ligands resulting in either activation or inhibition ofNK cell effector responses in response to tumor cells (40). Ourstudy provides evidence for the importance of NKp46 andNKG2D in human prostate cancer. The involvement of NKG2Din prostate cancer has been explored in animal models: NKG2D-deficient transgenic adenocarcinoma of the mouse prostate(TRAMP) mice develop more aggressive prostate tumor thanNKG2DWT TRAMP counterparts (5). In humans, MIC is widelyexpressed in prostate adenocarcinoma; however, significantserum levels of soluble MIC were observed in patients withadvanced prostate cancer, indicating that prostate tumorsmay counteract NKG2D-mediated immunity via MIC shedding(41, 42). Ligands for NKG2D (MICA/B andULBP1) are expressedon circulatingmonocytes from prostate cancer patients and couldbe responsible for NKG2Ddownregulation onNK cells (43). Thisis the first time to our knowledge that the major role of NKp46 in

prostate cancer is pointed out. Therefore, counteracting tumor-induced downregulation of surface NKp46 and NKG2D or sus-taining NKp46 and NKG2D-mediated immune response couldrepresent new therapeutic option for metastatic prostate cancerpatients. We have recently retrospectively studied a cohort ofmetastatic prostate cancer patients, highlighting an associationbetween NK cell markers in blood, including NKp46, and clinicaloutcomes (44).

Importantly, it has been shown that some treatments inprostate cancer can affect the immune infiltrate: ADT increasessignificantly the infiltration of CD3þ and CD8þ T lymphocytes,as well as CD68þmacrophages within the tumor (22). Systemicadministration of Sipuleucel-T prior to radical prostatectomyincreases CD3þ, CD4þ, FoxP3�, CD8þ T cells in tissues com-pared with pretreatment biopsies (45). These observationstogether with our findings suggest that combined treatmentsto increase the recruitment of immune infiltrate, and to restoreNK cell efficiency at the same time could represent promisingapproach in prostate cancer. Furthermore, our data show adecreased expression of CD16 on intratumoral NK cells, sug-gesting that the ability to perform ADCC would be compro-mised as confirmed by redirect lysis results on peripheral NKcells from prostate cancer patients. Thus, preventing CD16downregulation or shedding could improve the efficacy oftherapeutic antibody-mediated effects.

Through an integrative view of the literature, it seems obviousthat prostate cancer builds a strong immunosuppressive micro-environment: the tumor cells may recruit and facilitate the accu-mulation of Treg and TH17 lymphocytes (46, 47), which, in turn,could suppress the antitumor immunity; the CD8þ T cells infil-trating prostate tumors become tolerized (48) and are induced tobecome suppressor cells (49); and here we demonstrate that NKcells are switched toward a noncytotoxic profile. Interestingly,these events are all mediated in part through TGFb1. To ourknowledge, this is the first report demonstrating multiple levelsof tolerance to NK cells in a solid tumor, with tissue- and tumor-induced alterations, that together strongly impair NK cell antitu-mor immunity in prostate cancer. Besides shedding light on thephenotype and functions of prostate-infiltrating NK cells innormal and tumor context, our data pave the way for developingtherapies aimed to restore NK cell efficiency in the prostate tumormicroenvironment.

Disclosure of Potential Conflicts of InterestAlessandro Moretta is a founder and shareholder of Innate-Pharma (Mar-

seille, France). No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: C. Pasero, G. Gravis, P. Rocchi, M. Paciencia-Gros,F. Poizat, A. Moretta, D. OliveDevelopment of methodology: C. Pasero, G. Gravis, M. Paciencia-Gros,F. Poizat, D. OliveAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Pasero, G. Gravis, M. Guerin, J. Thomassin-Piana,M. Paciencia-Gros, F. Poizat,M. Bentobji, J.Walz, N. Salem, S. Brunelle, D.OliveAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Pasero, G. Gravis, S. Granjeaud, P. Rocchi,M. Paciencia-Gros, F. Poizat, M. Bentobji,. Salem, S. Brunelle, A. Moretta,D. OliveWriting, review, and/or revision of the manuscript: C. Pasero, G. Gravis,M. Guerin, M. Paciencia-Gros, F. Poizat, J. Walz, A. Moretta, D. Olive






Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): G. Gravis, P. Rocchi, M. Paciencia-Gros,F. Poizat, J. Walz, D. OliveStudy supervision: G. Gravis, P. Rocchi, F. Poizat, D. Olive

AcknowledgmentsThe authors thank Philippe Livrati and Sylvaine Just-Landi for excel-

lent technical support; the Departments of Surgical Oncology, Pathol-ogy, and the Outpatient Unit at the Institut Paoli-Calmettes and thepatients for their contribution to this work; and platforms from CRCM(Centre de Recherche en Canc�erologie de Marseille, France): the Cyto-metry facility, CRCM's Integrative Bioinformatics (Cibi) platform, andICEP (IPC/CRCM Experimental Pathology) platform, especially EmilieAgavnian-Couquiau.

Grant SupportThis work was supported by grants from FrenchCancer Institute (INCa, PAIR

prostate program #R10111AA) and SIRIC (INCa-DGOS-Inserm 6038 grant).The team of D. Olive was labeled "Equipe FRM DEQ 201 40329534" and D.Olive is a Senior Scholar of InstitutUniversitaire de France. C. Paserowas fundedby FrenchCancer Institute for 2 years. A.Morettawas supported byAssociazioneItaliana Ricerca per la Ricerca sul Cancro (AIRC)-Special Project 5�1000 no.9962 and IG 2014 Id. 15704.

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.

Received July 17, 2015; revised November 22, 2015; accepted December 22,2015; published OnlineFirst April 5, 2016.

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2016;76:2153-2165. Published OnlineFirst April 5, 2016.Cancer Res Christine Pasero, Gwenaëlle Gravis, Mathilde Guerin, et al. Microenvironment Impairs Natural Killer Cell Antitumor ActivityInherent and Tumor-Driven Immune Tolerance in the Prostate

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