restoration of natural killer cell antimetastatic activity by il12 … · cells (1 105 4t1) were...

Microenvironment and Immunology

Restoration of Natural Killer Cell AntimetastaticActivity by IL12 and Checkpoint BlockadeIsabel Ohs, Laura Ducimeti�ere, Joana Marinho, Paulina Kulig, Burkhard Becher,and Sonia Tugues

Abstract

Immune checkpoint therapies target tumor antigen-specific Tcells, but less is known about their effects on natural killer (NK)cells, which help control metastasis. In studying the develop-ment of lung metastases, we found that NK cells lose theircytotoxic capacity and acquire a molecular signature defined bythe expression of coinhibitory receptors. In an effort to over-come this suppressive mechanism, we evaluated NK cellresponses to the immunostimulatory cytokine IL12. Exposureto IL12 rescued the cytotoxicity of NK cells but also led to the

emergence of an immature NK cell population that expressedhigh levels of the coinhibitory molecules PD-1, Lag-3, andTIGIT, thereby limiting NK cell–mediated control of pulmo-nary metastases. Notably, checkpoint blockade therapy syner-gized with IL12 to fully enable tumor control by NK cells,demonstrating that checkpoint blockers are not only applicableto enhance T cell–mediated immunotherapy, but also to restorethe tumor-suppressive capacity of NK cells. Cancer Res; 77(24);7059–71. �2017 AACR.

IntroductionMetastasis—the spread of malignant cells from the tumor of

origin to distant sites—represents the main cause of cancer-asso-ciated death (1). Metastasis formation is a tightly regulatedmultistep process involving the crosstalk between tumor cellsand components of the surrounding local microenvironment,which together influence disseminating tumor cells. Especially,leukocytes have been demonstrated to either restrict or potentiatemetastatic growth (2, 3).

As pivotal players of the innate immune defense, NK cells haveshown the potential to recognize and reject tumor cells in varioustumor models, being particularly effective in controlling meta-static dissemination (4, 5). To recognize malignant cells, NK cellsare equippedwith a diverse repertoire of activating and inhibitoryreceptors that tightly regulate their activity (6). Thus, whereashealthy cells avoid NK cell–mediated killing through the engage-ment of inhibitory receptors byMHC class Imolecules,malignantcells may either lose this signal or upregulate activating receptorsas a result of cellular stress (7), leading to tumor cell eliminationby cytotoxic granules and death receptors (8).

Evidence for NK cell–mediated tumor surveillance has alsobeen reported in cancer patients, with correlative studies asso-

ciating high levels of tumor-infiltrating NK cells with favorableprognosis (9–12). However, due to the low NK cell numbersfound within the tumor microenvironment (TME; refs. 13, 14)and the tumor-induced NK cell suppression (15–19), the extentto which they contribute to immune surveillance against pri-mary tumors is unclear. Attempts to sustain antitumor NK cellresponses have focused on their ex vivo expansion and activa-tion using cytokines such as IL2, IL15, or IL12 (20). Althoughpilot studies showed a therapeutic potential of cytokine-activated NK cells, their antitumor activity is restricted by theTME (21), which comprises several suppressive immune celltypes and tumor-secreted factors (22).

Checkpoint blockers, which target coinhibitory receptors ortheir ligands, are among the most promising approaches aimedto antagonize TME-mediated immune suppression and have bynow yielded significant clinical benefit against several types ofcancer (23). Even though checkpoint blockade has been mainlyexploited to liberate tumor antigen-specific T cells, evidence fora role of immune checkpoints on other tumor-infiltratingleukocytes, such as NK cells, is only now emerging (24). Thiscould be of particular importance in distal metastatic sites suchas the lung, an organ where NK cells are abundant (25).However, the application of checkpoint blockade therapy tospecifically target metastases requires a better understandingof the mechanisms regulating NK cell antitumor responsesoperating at late-stage disease.

In preclinical mouse models of metastasis, we show that lungNK cells are rendered dysfunctional during the course of cancerprogression. Concomitant with their impaired ability to elim-inate metastasizing tumor cells, lung NK cells displayedreduced levels of activating receptors, whereas the expressionof coinhibitory receptors, classically described on T cells, wasincreased. We used IL12 to therapeutically boost lung NK cellcytotoxicity, but the antimetastatic activity was limited througha further dramatic increase of checkpoint receptors on the NKcell surface. Coadministration of checkpoint blockers against

Inflammation research, Institute of Experimental Immunology, University ofZurich, Switzerland.

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

S. Tugues and B. Becher share last authorship of this article.

Corresponding Authors: Sonia Tugues, Inflammation Research, Institute ofExperimental Immunology, University of Zurich, 8057 Zurich, Switzerland.Phone: 41-44-635-37-09; E-mail: [email protected]; and BurkhardBecher, [email protected]

doi: 10.1158/0008-5472.CAN-17-1032

�2017 American Association for Cancer Research.

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PD-1 or Lag-3 increased the efficacy of IL12 immunotherapy inan NK cell–dependent manner. Taken together, these datademonstrate that blocking immune checkpoints on NK cellscan be exploited to complement NK cell–based antimetastatictherapies.

Materials and MethodsAnimals

Female (6–10 weeks old) BALB/c and C57BL/6 mice wereobtained from Janvier labs. MMTV-PyMT (FVB/N-Tg(MMTV-PyVT)634Mul/J) mice were obtained from The JacksonLaboratory. C57BL/6 Ncr1cre/wt mice (NKp46iCre) were provid-ed by Eric Vivier and Rosa26iDTR mice by Ari Waisman (Mainz,Germany). Nkp46iCre mice were crossed to Rosa26iDTR miceto obtain NKp46iCre/wtRosa26iDTR/wt (Nkp46iCreR26RiDTR) orNKp46iCre/wtRosa26wt/wt controls (Nkp46iCre). Mice homozy-gous for the Tcrdtm1Mom targeted mutation (Tcrd�/� mice) werefrom The Jackson Laboratory (Stock #002120; ref. 26). Allanimals were kept in house according to institutional guide-lines under specific pathogen-free conditions. All animalexperiments were approved by the Swiss cantonal veterinaryoffice (licenses 147/2012 and 142/2015).

Murine tumor cell lines4T1 cells were provided by M. Detmar (Institute of Pharma-

ceutical Sciences, ETH,Zurich, Switzerland), YAC-1 cells byM. vanden Broek (Institute of Experimental Immunology, UniversityZurich, Zurich, Switzerland), and 4T1-mCherry cells by NicolaAceto (Department of Biomedicine, University of Basel, Basel,Switzerland). LLCs and B16F10 melanoma cells were purchasedfrom ATCC. All cancer cells were used between passages 2 and 6.Cell lines have been tested by Mycoscope PCR Detection Kit(Genlantis) and authenticated according to ATCC STR database.

Expression and purification of IL12FcIL12Fc expressed in 293T cells was purified from supernatant

using a protein A column (1 mL, HiTrap, GE Healthcare). Afterelution with 0.1 mol/L citric acid, pH 3.0 using a purifier(€AktaPrime) and dialysis for 40 hours in PBS, pH 7.4, theconcentration and purity of IL12Fc were measured using themouse IL12 (p40) ELISA kit (BD OptEIATM, 555165) and silverstaining (Pierce Silver Stain Kit, Thermo Scientific), respectively.

Models of tumor metastasisCells (1 � 105 4T1) were injected in the second mammary fat

pad of 6- to 10-week-old female BALB/c mice. Lung metastaseswere analyzed after approximately 23 days. To quantify NK cellligands on 4T1 cells, 4T1-mCherry cells were injected. For exper-imental metastasis, Balb/c mice were injected intravenously with1 � 105 4T1 cells into the tail vein and C57BL/6 WT and Tcrd�/�

mice with 4 � 105 LLCs or 3 � 105 B16F10 melanoma cells. Forthe resection experiments, 1 � 105 4T1 cells were injected in thefourth mammary fat pad of 10-week-old female Balb/c mice. Toresect primary tumors, mice were intraperitoneally (i.p.) injectedwith fluniximin (Biokema; 5 mg/kg body weight) before beinganesthetized with 3% to 5% isoflurane (Minrad) in an inductionchamber. Anesthesia was maintained at 3% isoflurane deliveredthrough a nose adaptor. Tumors weighing about 0.5 to 0.8 g wereremoved by blunt dissection using sterilized instruments.

Administration of IL12 and checkpoint blockersA total of 200 ng of IL12Fc or the IgG fragment as control (Ctrl)

diluted in 25 mL PBS were intranasally (i.n.) administered permouse. MMTV-PyMT mice were treated three times per weekstarting at week 6. Balb/c mice were treated one day beforeprimary tumor resection and subsequently 3 times per weekstarting 1 day after resection. 4T1 tumor–bearing mice weretreated i.n. with 50 ng of IL12Fc in combination with i.p. admin-istration of 200 mg of anti–Lag-3 antibodies (C9B7W, BioXCell),anti–PD-1 antibodies (RMP1-14, BioXCell) or correspondingisotype IgG (2A3, BioXCell) at day 12 after tumor cell injection.Mice were treated three times per week and studies were termi-nated at day 25. In the experimental metastasis model, mice weretreated with 150 mg of anti–Lag-3 antibodies (C9B7W, BioXCell),anti–PD-1 antibodies (RMP1-14, BioXCell), or correspondingisotype IgG (2A3, BioXCell) and 200 ng of IL12Fc.

Antibody depletionBalb/c mice were treated i.p. with 50 mL of anti-asialo GM1

antibodies (Wako Pure Chemical Industries) or rabbit IgG(Sigma) at days 3, 7, 10, and 16 after tumor cell injection.MMTV-PyMT mice were treated i.p. with 50 mL of anti-asialoGM1 antibodies (Wako Pure Chemical Industries) or rabbit IgG(Sigma) twice a week starting at week 9 of age. Lung metastasesin MMTV-PyMT mice were quantified at 14 weeks of age viaIndia ink. In the LLC experimental metastasis model, 50 mL ofanti-asialo GM1 antibodies (Wako Pure Chemical Industries)were administered i.p. at day �1 and day 7 after tumor cellinjection. Anti-NK1.1 antibody (200 mg/mouse, clone PK136,BioXCell) or Diphtheria Toxin from Corynebacterium diphthe-ria (Calbiochem, 250 ng/mouse for initial depletion and125 ng/mouse for the following injections) diluted in PBSwere injected i.p. at days �1, 2, 5, 8, and 11 after tumorinoculation. Anti-CD4 (100 mg/mouse, GK1.5, BioXCell) wasinjected i.p. at day �1, 6, 12, and 17 after tumor inoculation.In Tcrd�/� mice inoculated with B16F10 melanoma cells, lungcolonies were counted 21 days after tumor inoculation. Deple-tion efficiency was monitored during the studies by bleedingthe mice and subsequent flow-cytometric analysis.

Quantification of lung metastasesPulmonary metastases were quantified by intratracheal injec-

tion of India ink (15% India ink in PBS). India ink–injected lungswere fixed in Feket's solution (300 mL 70% ethanol, 30 mL 37%formaldehyde, and 5mL glacial acetic acid) overnight.White lungmetastases were counted under a dissection microscope. Meta-static index was calculated as the number of lung metastasesdivided by the primary tumor weight.

Flow cytometryFlow-cytometric analysis of lungs from 4T1 tumor–bearing

mice was performed at day 10 or day 20 after tumor cell injection.Lungs were harvested, digested with Collagenase IV (0.4 mg/mL)for 45 minutes at 37�C and erythrocytes were lysed with ACK(ammonium–chloride–potassium) lysis buffer. Tibiae and fem-ora were flushed with PBS and erythrocytes were lysed subse-quently. Cellswere incubated for 20minutes in Fc-blocking buffer(2.4G2) before being stained with the following antibodies: anti-CD45 (30-F11, BioLegend), anti-CD49b (Dx5, BioLegend), anti-NKp46 (29A1.4, eBioscience), anti-CD3 (17A2, eBioscience),anti–Gr-1 (6-8C5, BioLegend), anti-CD11b (M1/70, BioLegend),

Ohs et al.

Cancer Res; 77(24) December 15, 2017 Cancer Research7060




anti-CD27 (LG.3A10, BioLegend), anti-NKG2D (CX5, eBio-science), anti–DNAM-1 (10E5, BioLegend), anti-KLRG1 (2F1,eBioscience), anti-Ly49G2 (eBio4D11, eBioscience), anti-TIGIT(1G9, BioLegend), anti–Lag-3 (eBioC9B7W, eBioscience), anti–PD-1 (29F.1A12, BioLegend), anti-Ly6G (1A8, BioLegend),anti-SiglecF (E50-2440, BD), anti-F4/80 (CI:A3-1, AbD Serotec),anti-Ly6C (HK1.4, BioLegend), anti-CD11c (N418, BioLegend),anti-MHC II (10F.9G2, BioLegend), anti-H60 (205326, R&D),anti–Rae-1 (186107, R&D), anti-CD155 (690912, R&D), anti–PD-L1 (10F.9G2, BioLegend), anti-MHC I (34-2-12, BioLegend)anti-NK1.1 (PK136, Biolegend), anti-CD94 (18d3, Biolegend),anti-NKG2A/C/E (20d5, BD Bioscience) and anti-Ly49E/F (CM4,eBioscience). To exclude dead cells, we used the Zombie Aquafixable viability kit (BioLegend). Doublets were excluded by FSC-A/FSC-H gating. For intracellular cytokine staining, cells werestimulated for 4 hours at 37�C and 5% CO2 in RPMI 1640medium containing 10% FCS, 50 ng/mL PMA, 500 ng/mL iono-mycin and 1 mL/mL GolgiPlug (BD Bioscience). For detection ofintracellular IFNg or granzyme B, cells were fixed after surfacestaining, permeabilized with Cytofix/Cytoperm (BD Biosciences)and stained with an anti-IFNg mAb (XMG1.2, BD) or an anti-granzyme B mAb (GB11, BD Bioscience). Acquisition was per-formed on an LSRII Fortessa flow cytometer (BD Bioscience), anddata were analyzed using FlowJo Version X (TreeStar). Absolutecell numbers were quantified using AccuCheck Counting beads(Life Technologies).

Next-generation sequencingRNA from NK cells (CD45þCD3�Ly6G�NKp46þCD49bþ)

isolated at days 10 and 20 after 4T1 tumor cell injection wasamplified using the SMART-Seq v4 Ultra Low Input RNA Kit(Clontech) at the New York Genome Center. After synthesis,cDNAwas sheared to a size of approximately 350 bp, and librarieswere generated using the KAPA Hyper DNA Library Prep accord-ing to the manufacturer's instructions. Final libraries were quan-tified using the KAPA Library Quantification Kit (KAPA Biosys-tems), Qubit Fluorometer (Life Technologies) and Agilent 2100BioAnalyzer, and were sequenced on an Illumina HiSeq2500sequencer (v4 chemistry) using 2 � 125 bp cycles aiming for30 million reads per sample. Differential gene expression for NKcells isolated from day 10 versus day 20 was calculated usingDESeq2, and differentially expressed genes with a P value of lessthan 0.05 were used for hierarchical clustering.

Using the SMART-seq2 Amplification Kit (Clontech), RNApurified from NK cells (CD45þCD3�Ly6G�CD122þCD49bþ)isolated from Ctrl- or IL12-treated lungs was converted intocomplementary DNA libraries, amplified and sequenced for200 to 250 million reads using 50 bp paired-end read at theQuantitative Genomics Facility in Basel. FastQC was used toquality check the reads. Low-quality ends were clipped (3 basesfrom the start and 10 bases from the end). Trimmed reads werealigned to the reference genome and transcriptome (FASTA andGTF files were downloaded from the UCSC mm10 repository)using STAR version 2.3.0e_r291 with default settings. Distribu-tion of the reads across genomic isoform expression was quan-tified using the R package GenomicRanges from Bioconductor(Version 3.0). Differentially expressed genes were identified usingthe Rpackage edgeR fromBioconductor (Version 3.0). By setting aminimummean of expression of 100 reads, very lowly expressedgenes were filtered out. Sequencing information is available at theEuropean Bioinformatics Institute (EBI; ENA: PRJEB15668).

Gene ontology analysisDatabase for Annotation, Visualization, and IntegratedDiscov-

ery (DAVID; http://david.abcc.ncifcrf.gov) was used to identifyenriched biological functions and assess gene lists for enrichmentof genes annotated with specific Gene Ontology Project (GO)biological process terms (http://www.geneontology.org). Asinput for DAVID ontology analysis, a list of 1,722 genes thatwere differentially expressed in NK cells day 10 compared to day20 was created (P < 0.05). Heat maps corresponding to thedifferent functional categories within this list were generatedusing the R package and an adjusted P value [false discovery rate(FDR)] < 0.1.

Killing assayLung NK cells (live, CD45þCD3�NKp46þCD49bþCD11bþ

CD27�) were sorted with a BD FACSAria III sorter. YAC-1 cellswere stained with PKH26 Red Fluorescent Cell Linker Mini Kit(Sigma, MINI26-1KT). NK cells were incubated with YAC-1target cells at effector:target ratios of 1:1, 3:1, and 6:1 for 5 hoursat 37�C in 5%CO2. After removal ofmedium, Topro (0.8mmol/L)was added to the cells and cells were acquired on an LSRIIFortessa flow cytometer (BD). Data were analyzed using FlowJoVersion X (TreeStar). The percentage of specific lysis was calcu-lated as follows: [(Experimental lysis � spontaneous lysis)/(maximum lysis � spontaneous lysis)] � 100%. Alternatively,sorted NK cells were added to 4T1-mCherry cells at effector:target ratios of 1:1, 5:1, and 10:1 and incubated for 10 hours at37�C in 5% CO2. After removal of medium, mCherry fluores-cence was detected with a microplate reader (Tecan InfiniteM200 PRO, M€annedorf, Switzerland). The percentage of lysiswas calculated as followed: [100 � (Sample fluorescence �100/4T1 alone fluorescence]. Cell supernatants were collectedand subjected to IFNg analysis by ELISA.

Quantitative real-time PCRLung NK cells (CD45þCD3�Ly6G�CD49bþNKp46þ) from

mice being treated with either Ctrl or IL12 together with eitherCtrl IgG, anti–PD-1 or anti–Lag-3 mAb after i.v. injection of 4T1tumor cells were purified with an AriaIII Sorter. RNA was isolatedfrom NK cells using the Qiagen Micro Kit according to the man-ufacturer's protocol. Random primers (Invitrogen) were used forsynthesis of complementary DNA and the following primers forquantitative real-time PCR using a CFX384 Cycler (Bio-Rad Lab-oratories): Prf1 50-TCTTGGTGGGACTTCAGCTT-30 and 50-GAG-CAGGGACAGGTCGTG-30, Ifng 50-GCATTC ATGAGTATTGCCA-AG-30 and 50-GGTGGACCACTCGGATGA-30, PolII 50-CTGGTCC-TTCGAATCCGCATC-30 and 50-GCTCGATACCCTGCAGGGTCA-30, Pdcd1 50-CGTCCCTCAGTCAAGAGGAG-30 and 50-GTCCCTA-GAAGTGCCCAACA-30, Cd226 50-TCGCTCAGAGGCCATTACAG-30 and 50-CCCTGGGCTCTTTAAGTGGAA-30, Csf2 50-TGGAAG-CATGTAGAGGCCATCA and 50-GCGCCCTTGAGTTTGGTGAA-AT-30, Cxcr6 50-TACGATGGGCACTACGAGGGAG-30 and 50-GCA-AAGAAACCAACAGGGAGACCAC-30, Cx3cr1 50-GGTCTGGTG-GGAAATCTGTTGG-30 and 50-GAAGAAGGCAGTCGTGAGCTTG-30, Ccr5 50-ACTGCTGCCTAAACCCTGTCA-30 and 50-GTTTTC-GGAAGAACACTGAGAGATAA-30, Cxcr4 50-GAAGTGGGGTCTG-GAGACTAT-30 and 50-TTGCCGACTATGCCAGTCAAG-30, Il4ra 50-ACACTACAGGCTGATGTTCTTCG-30 and 50-TGGACCGGCCTAT-TCATTTCC-30, Gzmb 50-TGGCCTCCAGGACAAGGCAG-30 and 50-GCCTCAGGCTGCTATCCTT-30, Il10 50-GGTTGCCAAGCCTTAT-CGGA-30 and 50-ACCTGCTCCACTGCCTTGCT-30. RT2 qPCR

Harnessing NK Cell–Mediated Immunity against Metastasis

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Ohs et al.





Primer Assay (Qiagen) were used for Tigit (PPM68038A) and Lag3(PPM03649A). Subsequent analyses were performed with Excelcalculating the dCt values.

Immunofluorescent stainingUnfixed lungs were frozen in OCT Tissue Tek. Sections (8 mm

thick)werefixedwith ice-coldmethanol before being stainedwithrat anti-mouse NKp46 (eBioscience, 1:200), rabbit anti-mouseCytokeratin 8 (Abcam, 1:200) as well as the secondary antibodiesgoat anti-rat IgG Alexa 546 (Invitrogen, 1:300) and goat anti-rabbit IgG Alexa Fluor 488 (Life Technologies, 1:300), respective-ly. Images were acquired using a CLSM SP5 Leica microscope andImageJ was used for further data analysis.

ELISAIFNg levels were measured using a mouse IFNg ELISA kit

according to the user manual (BD OptEIATM, 555138).

Statistical analysisP values were calculated using GraphPad statistical soft-

ware (GraphPad Software Inc.). P values smaller than 0.05 wereconsidered significant. �, P < 0.05; ��, P < 0.01; ��, P < 0.001.If no asterisks are indicated, no statistically significant differencewas found.

ResultsLung NK cells become dysfunctional at advanced stages ofbreast cancer

Although much effort has been invested in elucidating therole of adaptive immunity in tumor immunosurveillance (27),the contribution of the innate immune system to this process isstill poorly understood. Recently, we reported an importantrole of NK cells in control of lung metastases in the 4T1 mousemodel of metastatic breast cancer (5). Consistent with theresults obtained in Rag2�/�Il2rg�/� mice, anti-asialo GM1-mediated depletion of NK cells increased the metastatic burdenin 4T1 tumor–bearing mice and in mammary tumor virus–polyoma middle T (MMTV-PyMT) transgenic mice of sponta-neous mammary carcinoma (Fig. 1A and B). The critical role ofNK cells in metastatic control was confirmed using anti-NK1.1antibody or Nkp46iCreR26RiDTR mice, which resulted in highermetastatic load in mice bearing Lewis lung carcinoma (LLC;Supplementary Fig. S1A–S1C). In contrast, the depletion of

CD4 T cells (Supplementary Fig. S1D) and the lack of T, B, andNKT cells in Rag1�/� mice (5) or gd T cells in Tcrd�/� mice(Supplementary Fig. S1E) did not alter lung metastatic burden.

Because metastases still arose in the presence of NK cells incontrol mice, we asked the question as to how these tumor cellsescaped immunosurveillance. One potential mechanism is theexclusion of effector cells from the vicinity of cancer cells. How-ever, whereas NK cells were sparse in primary tumors (data notshown), they were found to infiltrate lung metastases, decreasingonly slightly during tumor progression (Fig. 1C and D). Cancersescape immune-mediated rejection by rendering NK cells dys-functional (15, 18). Indeed,NK cells isolated from lungs at day 20after tumor cell injection (4T1 d20), resembling a progressedtumor stage, were impaired in their lytic activity against YAC-1and 4T1 cells when compared with lung NK cells from naïvemiceor isolated at an earlier stage (4T1 d10; Fig. 1E and F). In addition,the production of IFNg by these cells (4T1 d20) in coculture withtumor cells was severely compromised (Fig. 1G and H), althoughtheir capacity to produce IFN-g after stimulation with PMA andionomycin was not affected (Supplementary Fig. S1F). Concom-itant with their impaired cytotoxicity, NK cells isolated from lungsat an advanced tumor stage acquired a less differentiated pheno-type (CD11bhighCD27high; Fig. 1I).

Together, these findings highlight the crucial contribution ofNK cells to metastatic control and further indicate that, despitetheir presence in the metastatic microenvironment, they fail tokill tumor cells over time, resulting in metastatic outgrowth.

Dysfunctional NK cells express distinctive patterns of activatingand inhibitory receptors

To reveal the molecular mechanisms by which NK cellsbecome impaired during advanced stages of tumor progression,we performed deep transcriptome profiling comparing themwith early-stage functional NK cells. Hierarchical clustering andprincipal component analysis displayed a close relationshipamong the samples within each time point as well as a distinctgene profile of dysfunctional compared to functional NK cells(Fig. 2A and B). Gene ontology analysis revealed a differentialexpression of genes involved in cell adhesion, secretion ofchemokines and cytokines, cytotoxic mediators, and, mostimportantly, regulation of cell activation (Supplementary Fig.S2A; Fig. 2C). Within the latter category, the checkpoint inhi-bitors TIGIT (Tigit) and Lag-3 (Lag3), negative regulators of Tcell function (28), were among the most differentially

Figure 1.NK cells show an impaired capacity to control metastases with disease progression. A, Tumor weight and metastatic index of a-asialo GM1 or rabbitIgG-treated 4T1 tumor–bearing Balb/c mice at day 21 after tumor cell injection. Data shown are pooled from two independent experiments with 3 mice pergroup. B, Number of lung metastases in 13-week-old MMTV-PyMT mice treated with a-asialo GM1 or rabbit IgG. C, Representative fluorescence images oflungs from naïve or 4T1 tumor–bearing Balb/c mice, stained with anti-NKp46 (red), anti-Cytokeratin8 (green), and DAPI (blue). Scale bar, 75 mm. NK cells innonmetastatic and metastatic areas were quantified in 7 fields per lung from 5 4T1 tumor–bearing Balb/c mice. D, Gating strategy and quantification ofabsolute NK cell (CD45þCD3�NKp46þCD49bþ) numbers in lungs of naïve and 4T1 tumor–bearing Balb/c mice at days 10 and 20 after 4T1 injection.Data are pooled from three independent experiments with at least 4 mice per group. E, Specific lysis of YAC-1 cells by lung NK cells isolated fromnaïve or 4T1 tumor–bearing Balb/c mice at day 10 or 20 after tumor cell injection. Data are representative of three independent experiments. F, Lysis of4T1-mCherry cells by lung NK cells isolated from naïve or 4T1 tumor–bearing Balb/c mice at day 20 after tumor cell injection. Data are representativeof two independent experiments. G, IFNg production by lung NK cells isolated from naïve or 4T1 tumor–bearing Balb/c mice at day 20 after tumor cellinjection upon coculture with 4T1-mCherry cells. Data are representative of two independent experiments. H, IFNg production by lung NK cells isolated fromnaïve or 4T1 tumor–bearing Balb/c mice at day 10 or at day 20 after tumor cell injection upon coculture with YAC-1 cells. I, Representative plots andquantification of CD11bhighCD27high and CD11bhighCD27low lung NK cells from naïve and 4T1 tumor–bearing Balb/c mice at day 10 or day 20 after tumorcell injection. Data are representative of three independent experiments with at least 4 mice per group. Error bars,� SEM. For A, B, and E, an unpaired Studentt test and for D and F–I, one-way ANOVA with post hoc Bonferroni test were used. � , P < 0.05; ��, P < 0.01; �� , P < 0.001.






expressed genes in dysfunctional lung NK cells (Fig. 2C–E;Supplementary Table S1). In addition, lung NK cells atadvanced stages of breast cancer upregulated inhibitory Ly49receptors (Klra5 (Ly49E), Klra17, Klra7 (Ly49G2); Fig. 2C andD; Supplementary Fig. S2B; Supplementary Table S1), whereasthe levels of the activating receptors DNAM-1 and NKG2D wereclearly decreased (Fig. 2C and F; Supplementary Table S1).Other canonical NK cell receptors such as NKp46, CD94,NKG2A/C/E, and Ly49A remained unaltered (SupplementaryFig. S2C and S2D). Increased expression of inhibitory receptorsand the concomitant loss of activating receptors were alsodetected on circulating and bone marrow NK cells of 4T1tumor–bearing mice at advanced stages of disease (Supplemen-tary Fig. S2E–S2L), indicating a systemic impairment of thesecells during disease progression.

These results show that the diminished lytic activity that NKcells acquire during tumor development is associated with adifferential expression of activating and inhibitory receptors.Therefore, restoration of NK cell function may improve thesuppression of metastatic spread.

IL12 activates NK cells but also induces the expression ofcoinhibitory receptors

Cytokine therapy has been used to harness the cytotoxicpotential of NK cells against cancer (21). Especially, IL12 caninitiate NK cell–dependent antimetastatic responses in the 4T1model when delivered directly to the lung via i.n. administra-tion (5). Also in the transgenic mouse model of breast cancermetastasis (MMTV-PyMT) and in a postsurgical 4T1 breastcancer metastasis model, IL12 led to a potent reduction ofpulmonary metastases (Fig. 3A and B). IL12 increased theexpression of the activating receptor NKG2D, induced NK celldifferentiation towards CD27lowKLRG1þ NK cells, boostedIFN-g production and prevented the decline in the lytic activityof NK cells observed in Ctrl-treated mice (Supplementary Fig.S3A–S3D). To gain further insights into the effects of IL12 onNK cells, we performed genome-wide transcriptome analysis oflung NK cells from Ctrl- or IL12-treated mice. Analysis ofdifferentially expressed genes revealed increased levels of cyto-toxic mediators (Prf1, Gzmk), activating receptors (Tnfsf4,Cd160, Cd226), proinflammatory cytokines (Csf2, Ifng), adhe-sion molecules (Itgax, Icam1, Itgam), and chemokine receptors(Cxcr6, Cx3cr1, Ccr5) on lung NK cells upon IL12 treatment(Fig. 3C; Supplementary Table S2). The differential expressionof these transcripts was confirmed in lung NK cells from 4T1tumor–bearing mice by qPCR (Supplementary Fig. S3E).

Strikingly however, the beneficial effects of IL12 on NK cellactivation were hindered by the concomitant induction of theimmune inhibitory receptors PD-1 (Pdcd1), Lag-3 (Lag3), andTIGIT (Tigit) on lung NK cells (Fig. 3C; Supplementary Table S2),which was also observed in tumor-bearing mice (SupplementaryFig. S3E; Fig. 3D and E). Comparative analyses of the PD1þ versusthe PD1� population showed that PD-1þ lung NK cells resemblean immature population of NK cells, indicated by an increase ofCD27highKLRG1� cells, and a high expression of CD94 andNKG2A/C/E (Fig. 3F–H). Of note, PD-1þ NK cells were alsoexpressing the highest amounts of Lag-3 (Fig. 3I) but low levelsof granzyme B (Fig. 3J). Overall, IL12 partially restores thecytotoxic capacity of NK cells, but also leads to the emergenceof a less mature NK cell population with elevated expression of

coinhibitory receptors, whichmay limit the therapeutic efficacy ofthis cytokine.

The metastatic TME shows differential expression patterns ofNK cell ligands

Tumor cells and their surrounding environment have beenreported to express stress-induced ligands that modulate NK cellactivity (18, 29). We used fluorescent mCherry-tagged 4T1 tumorcells to characterize NK cell–ligand expression in tumor cells andthe TME during cancer progression (Fig. 4A and B). The NKG2DligandsH60 andRae-1 as well as CD155 (ligand to bothDNAM-1and TIGIT) were mainly expressed by 4T1 cells, with higherexpression of H60 and Rae-1 on metastasized compared withprimary tumor cells (Fig. 4C and D). Albeit at lower amounts,CD155was also expressed on several populations ofmyeloid cells(monocytes, macrophages, and granulocytes) from primarytumors or naïve and metastatic lungs (Fig. 4D), indicating thatcancer-associated myeloid cells could modulate NK cell immuneresponses. Even though several tumor cell types have been shownto express PD-L1 (30, 31), 4T1 tumor cells are essentially PD-L1negative. PD-L1 was, however, prominently expressed by macro-phages both in primary tumors andmetastatic lungs (Fig. 4E). Theinhibitory capacity of the metastatic TME was further supportedby elevatedMHC I andMHC II (themain ligand for Lag-3, ref. 32)on metastasized tumor cells compared with the primary tumor(Fig. 4F and G). Interestingly, i.n. administration of IL12 not onlyreduced Rae-1 and H60 on tumor cells, it also increased theexpression of PD-L1 and MHC II (Fig. 4C, E and G). Together,these "side" effects of IL12 likely limit the capacity of NK cells toeliminate cancer cells.

Overall, the differential expression pattern of NK cell ligandswithin the metastatic microenvironment suggests that mainlytumor cells but also myeloid cells are potentially equipped toimpede NK cell function. Hence, we propose that blocking theinteraction ofNK cell receptors with their inhibitory ligands couldimprove the efficacy of IL12 immunotherapy.

IL12 treatment synergizeswith checkpoint blockade inNK cell–mediated metastasis control

Recently, immune checkpoints including PD-1, Lag-3, andTIGIT were revealed to exert a negative effect on NK cell activityin vitro (33–35). Given that IL12 treatment enhances the expres-sion of these coinhibitory molecules on NK cells, we assessedwhether their neutralization could increase the antimetastaticeffects of IL12. Notably, the combined treatment of IL12 witheither anti–Lag-3 (a-Lag-3) or anti–PD-1 (a-PD-1) antibodiessignificantly reduced lung metastases when compared withmonotherapy, while primary tumor weight was unaltered regard-less of the therapeutic intervention used (Fig. 5A).

It is feasible that the combination therapy could influence theprimary tumoror thedisseminationof cancer cells. Todismiss thispossibility, we injected 4T1 cells directly i.v., which leads to theirpredominant accumulation in the lung. Also, in the absence of theprimary tumor, coadministration of IL12 and a-Lag-3 or a-PD-1antibodies was far superior to either monotherapy (Fig. 5B).Importantly, whereas checkpoint blockade traditionally targetsT cells (36), we could here demonstrate the dependence on NKcells, because NK cell depletion alone reversed the antimetastaticeffect of both combinatorial therapies (Fig. 5C). Moreover, thecombinatorial treatment was also shown to efficiently reduce thenumber of pulmonary metastases in Lewis lung carcinoma

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Figure 2.

Phenotypic changes of lung NK cells during tumor progression. A, Heat map displaying cluster analysis of differentially expressed genes (with an adjustedP value of less than 0.05) of lung NK cells isolated at days 10 and 20 after 4T1 tumor cell injection. Presented data are based on the analysis of atleast three independent biological replicates per time point. B, Principal component analysis (PCA) of lung NK cells isolated at days 10 and 20 after 4T1 tumorcell injection. Presented data are based on the analysis of at least three independent biological replicates per time point. C, Heat map of selected genesdifferentially expressed in lung NK cells isolated at day 10 or 20 after 4T1 tumor cell injection (adjusted P value of less than 0.1). Presented data are based onthe analysis of three independent biological replicates per time point. Expression levels of TIGIT, Ly49G2 (D), NKG2D, DNAM-1 (E), and Lag-3 (F) on lung NKcells isolated from naïve and 4T1 tumor–bearing Balb/c mice at day 10 or 20 after tumor cell injection (mean fluorescence intensity, MFI). Data arerepresentative of three independent experiments with at least 4 mice per group. Error bars, �SEM. One-way ANOVA with post hoc Bonferroni test was used.�� , P < 0.001.






Figure 3.

Impact of IL12 on lung NK cell activation. A, IL12 treatment schedule of MMTV-PyMT mice. Quantification of pulmonary metastases at 13 weeks of age. Data arepooled from two independent experiments with at least 4 mice per group. B, IL12 treatment schedule of Balb/c mice with resected 4T1 primary tumorsat day 28 after tumor cell injection. Quantification of pulmonary metastases at day 35. Data are representative of two independent experiments withat least 4 mice per group. C, Heat maps of selected differentially expressed genes in lung NK cells (CD45þCD3�Ly6G�CD122þCD49bþ) isolated fromnontumor-bearing Balb/c mice treated three times i.n. with 200 ng of IL12 or Ctrl. Three biological replicates were used per group. D, Expression levels of PD-1and E, Mean fluorescence intensity (MFI) of TIGIT and Lag-3 on lung NK cells isolated from 4T1 tumor–bearing mice treated three times per week i.n. with 200ng of IL12 or Ctrl. Cells were analyzed at day 23 after tumor cell injection. Data represent two independent experiments with 5 mice per group. Expressionlevels of CD27 and KLRG1 (F), CD94 (G), and NKG2A/C/E (H), MFI of Lag-3 (I), and MFI of granzyme B on PD-1þ compared with PD-1� lung NK cells (J) isolatedfrom 4T1 tumor–bearing mice treated three times per week i.n. with 200 ng of IL12 or Ctrl. Cells were analyzed at day 23 after tumor cell injection. Datarepresent two independent experiments with 5 mice per group. Error bars, �SEM. An unpaired Student t test with Welch correction was used. �� , P < 0.01; �� ,P < 0.001.

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Figure 4.

Expression profile of NK cell ligands on tumor cells and tumor-associated myeloid cells. Gating strategy of 4T1-mCherry tumor cells and several myeloidsubsets in primary tumors (A) and metastatic lungs (B) at day 30 after 4T1-mCherry injection. Immune cells were pregated on live and CD45þ cells.C, Mean fluorescence intensity (MFI) of H60 and Rae-1 on the indicated myeloid cells and 4T1 tumor cells (black, cells isolated from primary tumor; dark gray,cells isolated from metastatic lung; light gray, cells isolated from metastatic lung treated with IL12; white, cells isolated from naïve lung). D and E, MFIof CD155 (D) and PD-L1 (E) on myeloid cells and 4T1 tumor cells (black, cells isolated from primary tumor; dark gray, cells isolated from metastatic lung; lightgray, cells isolated from metastatic lung treated with IL12; white, cells isolated from naïve lung). F, MFI of MHC I on tumor cells isolated from primarytumors (black) and metastatic lungs (dark gray). G, MFI of MHC II on 4T1 tumor cells isolated from primary tumors (black), metastatic lungs (dark gray),and metastatic lungs treated with IL12 (light gray) of Balb/c mice. Error bars, � SEM. Data are representative of two independent experiments with at least4 mice per group. An unpaired Student t test with Welch correction was used. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.






Figure 5.

Combined treatment of IL12 andcheckpoint blockers reducespulmonary metastases. A, Combinedtreatment schedule of 4T1 tumor–bearing mice. Primary tumor weightand metastatic index of 4T1 tumor–bearing mice at day 25 after tumor cellinjection. Representative pictures areshown. Data are pooled from twoindependent experiments with at least4 mice per group. B, Combinedtreatment schedule of mice with 4T1experimental metastasis. Number ofmetastases and size of metastaticnodes were quantified at day 14 afteri.v. injection of 4T1 tumor cells bystaining with India ink. Representativepictures are shown. Data are pooledfrom two independent experimentswith at least 5 mice per group.C, Combined treatment schedule ofmice with 4T1 experimental metastasisupon NK cell depletion with a-asialoGM1 antibody. Number of metastasesand size of metastatic nodes werequantified at day 14 after tumor cellinjection by staining with India ink.Representative pictures are shown.D, Combined treatment schedule ofmice with LLC experimentalmetastasis. Number ofmetastaseswasquantified at day 15 after i.v. injectionof LLC tumor cells by staining withIndia ink. Representative pictures areshown. Error bars,� SEM. An unpairedStudent t test with Welch correctionwas used. � , P < 0.05; �� , P < 0.01; �� ,P < 0.001.

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(LLC; Fig. 5D), a preclinical model of lung cancer in whichcheckpoint inhibitors were also induced on lung NK cells uponIL12 administration (Supplementary Fig. S4A–S4C). Mechanis-tically, the coadministration of IL12 and a-Lag-3 or a-PD-1slightly altered total numbers of proliferating NK cells (Fig. 6Aand B), but most notably resulted in increased frequencies ofhighly differentiated (CD11bhighCD27lowKLRG1þ) NK cells(Fig. 6C and D), producing higher amounts of granzyme B,perforin and also IFNg (Fig. 6E–G), a central mediator of IL12-mediated antimetastatic immune responses in the 4T1model (5).

Hence, the specific blockade of immune checkpoints on cytokine-activated NK cells reactivates their antitumor properties, leadingto a potent control of metastasis.

DiscussionThe potential capacity of NK cells in targeting disseminating

tumor cells is undisputable (4), and therapeutic approachesdirected to increase their antitumor activity are of great clinicalvalue. A successful utilization of these cells to control metastatic

Figure 6.

Combined treatment of IL12 andcheckpoint blockers increasesdifferentiation and granzymeexpression on lung NK cells. A,Quantification of absolute lung NK cellnumbers in mice being treated witheither Ctrl or IL12 together with eitherCtrl IgG, a-PD-1, or a-Lag-3mAbafter i.v.injection of 4T1 tumor cells. Data arerepresentative of two independentexperiments with at least 3 mice pergroup. B, Proliferation of lung NK cellsisolated from mice being treated witheither Ctrl or IL12 together with eitherCtrl IgG, a-PD-1, or a-Lag-3mAbafter i.v.injection of 4T1 tumor cells. Data arerepresentative of two independentexperiments with at least 3 mice pergroup.C andD,KLRG1 (C) and CD11b (D)expression on lung NK cells isolatedfrom mice being treated with either Ctrlor IL12 together with either Ctrl IgG, a-PD-1, or a-Lag-3 mAb after i.v. injectionof 4T1 tumor cells. E,Mean fluorescenceintensity (MFI) of granzyme B on lungNK cells isolated from mice beingtreated with either Ctrl or IL12 togetherwith either Ctrl IgG, a-PD-1 or a-Lag-3mAb after i.v. injection of 4T1 tumorcells. Data are representative oftwo independent experiments with atleast 3 mice per group. Lung NK cellswere isolated from mice being treatedwith either Ctrl or IL12 together witheither Ctrl IgG, a-PD-1, or a-Lag-3 mAbafter i.v. injection of 4T1 tumor cells andmRNA expression levels of Prf1 and Ifng(F) were quantified by qRT-PCR. G, 5 �106 lung single cells isolated from micebeing treated with either Ctrl or IL12together with either Ctrl IgG, a-PD-1, ora-Lag-3 mAb after i.v. injection of 4T1tumor cells were cultured for 24 hours incomplete RPMI before IFNg levels insupernatant were determined by ELISA.Error bars, � SEM. One-way ANOVAwith post hoc Bonferroni test was used.� , P < 0.05; �� , P < 0.01; �� , P < 0.001.






disease can, however, be achieved only by a detailed understand-ing of their functions at advanced stages of cancer, when facing thecomplexity of tumor–stromal interactions in distant sites. Here,we report an impaired capacity of lung NK cells to eliminatemetastatic tumor cells in preclinical models of metastasis. Evi-dence of defective cytotoxicity and altered expression of NK cellreceptors was previously observed in tumor-associated NK cells(15, 18, 37, 38). In patients with non–small cell lung carcinoma,for example, the TME in the lung led to a local downregulation ofan array of activating receptors on NK cells (39). We here dem-onstrate that dysfunctional lung NK cells not only downregulateactivating receptors, but acquire high expression of coinhibitoryreceptors such as TIGIT and Lag-3, but not PD-1 (SupplementaryFig. S5). Given that a tightly regulated balance between activatingand inhibitory receptors determines the outcome of NK–targetcell interactions (6), the vast array of receptors with negativeregulatory function overexpressed on lungNK cells during diseaseprogression may explain their inability to clear disseminatedtumor cells.

An additional layer of regulation of NK cell receptors arisesfrom their own ligands, which are often induced in tumor cellsupon cellular stress (40). In metastatic lungs, we detected theNKG2D ligands Rae-1 and H60 exclusively on tumor cells.Whether this is translated into enhanced NK cell functions is notclear, because sustained expression of NKG2D ligands or theirshedding from the cell surface has been shown to cause NK celldesensitization (41–43). Tumor-derived CD155 and MHC II aswell as PD-L1 were also found at high amounts within themetastatic microenvironment. This is of special interest, becauseTIGIT can bind CD155 with greater affinity than DNAM-1 (28)and potentially counteract signaling through this activating recep-tor. The engagement of the Lag-3/MHC II and PD-L1/PD-1 path-ways on lung NK cells may also constitute mechanisms ofimmune evasion at this particular metastatic site.

External control of NK cell activity is orchestrated throughproinflammatory cytokines such as IL2, IL18, IL15, and IL12(21, 44). We selected IL12 to reactivate dysfunctional NK cells.Despite the inefficiency of this cytokine to reverse NK cell anergyin MHC Iþ lymphomas (45), we could show that NK cell func-tions can be regulated by IL12 in the 4T1model. Remarkably, thestimulating effects of IL12 on lung NK cells coincided with theinduction of coinhibitory receptors, such as TIGIT, PD-1, and Lag-3.Within theNK cell population, the expression of these receptorswas found to be higher in a unique subset of immature IL12-induced NK cells, closely resembling the previously describedemergency NK cells (eNK cells; ref. 5). Hence, we suggest that thehigh expression of checkpoint blockers on cytokine-activated NKcells arrests these cells at a stage that prevents them from excessiveactivation, hindering the release of their full cytotoxicity. There isnow mounting evidence from in vitro studies that coinhibitoryreceptors canbe induced inNK cells. For instance, the engagementof PD-1 on NK cells from multiple myeloma patients reducedtheir cytotoxic potential (34). Also, CD96 and TIGIT, two inhib-itory receptors that counteract the activity of DNAM-1 (6), wereshown to be expressed on resting and activated NK cells, respec-tively (46). Even though CD96, rather than TIGIT, was shown to

play an important role in the control of lung metastasis (24), wecould not independently verify this, due to the lack of commer-cially available reagents.

However, whereas individual blockade of PD-1 or Lag-3 failedto exhibit any clinical benefit, the combination of checkpointblockade with IL12 exerted a potent antimetastatic effect. Theprofound synergy between anti–PD-1 or anti–Lag-3 with IL12 inpreclinical models of breast cancer was entirely dependent on NKcells. To translate this to human cancers, where checkpointblockade activates tumor antigen dependent T-cell responses(23), the combinatorial treatment proposed here may concom-itantly boost NK cell immunity against metastasis.

Collectively, our data demonstrate that a suitable immunomo-dulation of NK cells in metastatic lungs can be exploited forsuccessful antimetastatic therapy. So far, the adoptive transfer ofex vivo expanded NK cells has not led to objective clinicalresponses in patients with solid tumors (47). We thus proposethat a break release on these cells via blocking specific checkpointreceptors can be beneficial not only to boost cytokine-activatedNK cells, but also to reactivate the endogenous NK cell compart-ment at the metastatic sites.

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

Authors' ContributionsConception and design: I. Ohs, P. Kulig, B. Becher, S. TuguesDevelopment of methodology: I. Ohs, J. Marinho, S. TuguesAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): I. Ohs, L. Ducimeti�ere, P. Kulig, S. TuguesAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): I. Ohs, L. Ducimeti�ere, J. Marinho, P. Kulig, S. TuguesWriting, review, and/or revision of the manuscript: I. Ohs, L. Ducimeti�ere,B. Becher, S. TuguesAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): I. Ohs, J. Marinho, S. TuguesStudy supervision: B. Becher, S. Tugues

AcknowledgmentsWe thank Sabrina Nemetz, Mirjam Lutz, Nicole Burkhalter, and the Flow

Cytometry Facility, University of Zurich for assistance. We also thank IvaLelios for advice on the analysis of Next Generation Sequencing data.

Grant SupportThis work was supported by grants from the Swiss National Science

Foundation (310030_146130 and 316030_150768 to B. Becher andCRSII3_136203 to B Becher), the University Research Priority Project"Translational Cancer Research" (B. Becher and S. Tugues), the Forschung-skredit of the University of Zurich (grant no. FK-14-024 to I. Ohs), theEuropean Union FP7 project TargetBraIn 279017, NeuroKine316722, andATECT602239 (to B. Becher).

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 April 6, 2017; revised August 15, 2017; accepted October 11, 2017;published OnlineFirst October 17, 2017.

References1. Massagu�e J, Obenauf AC. Metastatic colonization by circulating tumour

cells. Nature 2016;529:298–306.2. DeNardo DG, Johansson M, Coussens LM. Immune cells as mediators of

solid tumor metastasis. Cancer Metastasis Rev 2008;27:11–8.


Ohs et al.




3. Kitamura T, Qian B-Z, Pollard JW. Immune cell promotion of metastasis.Nat Rev Immunol 2015;15:73–86.

4. Krasnova Y, Putz EM, Smyth MJ, Souza-Fonseca-Guimaraes F. Bench tobedside: NK cells and control of metastasis. Clin Immunol 2015;177:50–59.

5. Ohs I, vandenBroekM,NussbaumK,M€unzC,Arnold SJ,Quezada SA, et al.Interleukin-12 bypasses common gamma-chain signalling in emergencynatural killer cell lymphopoiesis. Nat Commun 2016;7:13708.

6. Martinet L, Smyth MJ. Balancing natural killer cell activation throughpaired receptors. Nat Rev Immunol 2015;15:243–54.

7. Jaeger BN, Vivier E. Natural killer cell tolerance: control by self or self-control? Cold Spring Harb Perspect Biol 2012;4:a007229–9.

8. Smyth MJ, Cretney E, Kelly JM, Westwood JA, Street SEA, Yagita H, et al.Activation of NK cell cytotoxicity. Mol Immunol 2005;42:501–10.

9. Coca S, Perez-Piqueras J, Martinez D, Colmenarejo A, Saez MA, Vallejo C,et al. The prognostic significance of intratumoral natural killer cells inpatients with colorectal carcinoma. Cancer 1997;79:2320–8.

10. Ishigami S, Natsugoe S, Tokuda K, Nakajo A, Che X, Iwashige H, et al.Prognostic value of intratumoral natural killer cells in gastric carcinoma.Cancer 2000;88:577–83.

11. Villegas FR, Coca S, Villarrubia VG, Jim�enez R, Chill�onMJ, Jare~no J, et al.Prognostic significance of tumor infiltrating natural killer cells subsetCD57 in patients with squamous cell lung cancer. Lung Cancer 2002;35:23–8.

12. Pasero C, Gravis G, Granjeaud S, Guerin M, Thomassin-Piana J, Rocchi P,et al. Highly effective NK cells are associated with good prognosis inpatients with metastatic prostate cancer. Oncotarget 2015;6:14360–73.

13. Halama N, BraunM, Kahlert C, Spille A, Quack C, Rahbari N, et al. Naturalkiller cells are scarce in colorectal carcinoma tissue despite high levels ofchemokines and cytokines. Clin Cancer Res 2011;17:678–89.

14. Esendagli G, Bruderek K, Goldmann T, Busche A, Branscheid D, Vollmer E,et al. Malignant and non-malignant lung tissue areas are differentiallypopulated by natural killer cells and regulatory T cells in non-small celllung cancer. Lung Cancer 2008;59:32–40.

15. Carrega P, Morandi B, Costa R, Frumento G, Forte G, Altavilla G, et al.Natural killer cells infiltrating human nonsmall-cell lung cancer areenriched in CD56 bright CD16(-) cells and display an impaired capabilityto kill tumor cells. Cancer 2008;112:863–75.

16. GarnerWL,Minton JP, James AG,HoffmannCC.Humanbreast cancer andimpaired NK cell function. J Surg Oncol 1983;24:64–6.

17. DewanMZ, TakadaM, TerunumaH,Deng X, Ahmed S, YamamotoN, et al.Natural killer activity of peripheral-blood mononuclear cells in breastcancer patients. Biomed Pharmacother 2009;63:703–6.

18. Mamessier E, Sylvain A, Thibult M-L, Houvenaeghel G, Jacquemier J,Castellano R, et al. Human breast cancer cells enhance self tolerance bypromoting evasion from NK cell antitumor immunity. J Clin Invest2011;121:3609–22.

19. French LE, Tschopp J. Defective death receptor signaling as a cause oftumor immune escape. Semin Cancer Biol 2002;12:51–5.

20. Guillerey C, Huntington ND, Smyth MJ. Targeting natural killer cells incancer immunotherapy. Nat Immunol 2016;17:1025–36.

21. Childs RW, Carlsten M. Therapeutic approaches to enhance natural killercell cytotoxicity against cancer: the force awakens. Nat Rev Drug Discov2015;14:487–98.

22. Balkwill FR, Capasso M, Hagemann T. The tumor microenvironment at aglance. J Cell Sci 2012;125:5591–6.

23. Pardoll DM. The blockade of immune checkpoints in cancer immuno-therapy. Nat Rev Cancer 2012;12:252–64.

24. Blake SJ, Stannard K, Liu J, Allen S, Yong MCR, Mittal D, et al.Suppression of metastases using a new lymphocyte checkpoint targetfor cancer immunotherapy. Cancer Discov 2016; DOI: 10.1158/2159-8290.CD-15-0944.

25. Bj€orkstr€om NK, Ljunggren H-G, Micha€elsson J. Emerging insights intonatural killer cells in human peripheral tissues. Nat Rev Immunol 2016;16:310–20.

26. Itohara S, Mombaerts P, Lafaille J, Iacomini J, Nelson A, Clarke AR, et al. Tcell receptor delta gene mutant mice: independent generation of alphabeta T cells and programmed rearrangements of gamma delta TCR genes.Cell 1993;72:337–48.

27. Galon J, Angell HK, Bedognetti D, Marincola FM. The continuum ofcancer immunosurveillance: prognostic, predictive, and mechanisticsignatures. Immunity 2013;39:11–26.

28. Yu X, Harden K, Gonzalez LC, Francesco M, Chiang E, Irving B, et al. Thesurface protein TIGIT suppresses T cell activation by promoting thegeneration of mature immunoregulatory dendritic cells. Nat Immunol2009;10:48–57.

29. Hasmim M, Messai Y, Ziani L, Thiery J, Bouhris J-H, Noman MZ, et al.Critical Role of Tumor Microenvironment in Shaping NK Cell Functions:Implication of Hypoxic Stress. Front Immunol 2015;6:482.

30. Driessens G, Kline J, Gajewski TF. Costimulatory and coinhibitory recep-tors in anti-tumor immunity. Immunol Rev 2009;229:126–44.

31. Topalian SL, Drake CG, Pardoll DM. Targeting the PD-1/B7-H1(PD-L1)pathway to activate anti-tumor immunity. Curr Opin Immunol 2012;24:207–12.

32. Hemon P, Jean-Louis F, Ramgolam K, Brignone C, Viguier M, Bachelez H,et al. MHC class II engagement by its ligand LAG-3 (CD223) contributesto melanoma resistance to apoptosis. J Immunol 2011;186:5173–83.

33. Stanietsky N, Rovis TL, Glasner A, Seidel E, Tsukerman P, Yamin R, et al.Mouse TIGIT inhibits NK-cell cytotoxicity upon interaction with PVR.Eur J Immunol 2013;43:2138–50.

34. Benson DM, Bakan CE, Mishra A, Hofmeister CC, Efebera Y, Becknell B,et al. The PD-1/PD-L1 axis modulates the natural killer cell versusmultiple myeloma effect: a therapeutic target for CT-011, a novel mono-clonal anti-PD-1 antibody. Blood 2010;116:2286–94.

35. Miyazaki T, Dierich A, Benoist C, Mathis D. Independent modes of naturalkilling distinguished in mice lacking Lag3. Science 1996;272:405–8.

36. Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age.Nature 2011;480:480–9.

37. CarlstenM, Norell H, Bryceson YT, Poschke I, Schedvins K, LjunggrenH-G,et al. Primary human tumor cells expressing CD155 impair tumor targetingby down-regulating DNAM-1 on NK cells. J Immunol 2009;183:4921–30.

38. Fauriat C, Just-Landi S, Mallet F, Arnoulet C, Sainty D, Olive D, et al.Deficient expression of NCR in NK cells from acute myeloid leukemia:evolution during leukemia treatment and impact of leukemia cells inNCRdull phenotype induction. Blood 2007;109:323–30.

39. Platonova S, Cherfils-Vicini J, Damotte D, Crozet L, Vieillard V, Validire P,et al. Profound coordinated alterations of intratumoral NK cell phenotypeand function in lung carcinoma. Cancer Res 2011;71:5412–22.

40. ChanCJ, SmythMJ,Martinet L.Molecularmechanisms of natural killer cellactivation in response to cellular stress. Cell Death Differ 2014;21:5–14.

41. Groh V, Wu J, Yee C, Spies T. Tumour-derived soluble MIC ligands impairexpression of NKG2D and T-cell activation. Nature 2002;419:734–8.

42. Reiners KS, Topolar D, Henke A, Simhadri VR, Kessler J, Sauer M, et al.Soluble ligands for NK cell receptors promote evasion of chronic lympho-cytic leukemia cells from NK cell anti-tumor activity. Blood 2013;121:3658–65.

43. Salih HR, Rammensee H-G, Steinle A. Cutting edge: down-regulation ofMICA on human tumors by proteolytic shedding. J Immunol 2002;169:4098–102.

44. Tugues S, Burkhard SH, Ohs I, VrohlingsM, Nussbaum K, Berg Vom J, et al.New insights into IL-12-mediated tumor suppression. Cell Death Differ2015;22:237–46.

45. Ardolino M, Azimi CS, Iannello A, Trevino TN, Horan L, Zhang L, et al.Cytokine therapy reverses NK cell anergy in MHC-deficient tumors. J ClinInvest 2014;124:4781–94.

46. Chan CJ, Martinet L, Gilfillan S, Souza-Fonseca-Guimaraes F, Chow MT,Town L, et al. The receptors CD96 and CD226 oppose each other in theregulation of natural killer cell functions. Nat Immunol 2014;15:431–8.

47. Geller MA,Miller JS. Use of allogeneic NK cells for cancer immunotherapy.Immunotherapy 2011;3:1445–59.






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