A Chimeric Receptor with NKG2D Speciﬁcity Enhances Natural Killer Cell Activation ... · Microenvironment and Immunology A Chimeric Receptor with NKG2D Speciﬁcity Enhances Natural

Microenvironment and Immunology

A Chimeric Receptor with NKG2D Specificity EnhancesNatural Killer Cell Activation and Killing of Tumor Cells

Yu-Hsiang Chang1, John Connolly3, Noriko Shimasaki1, Kousaku Mimura2, Koji Kono2, and Dario Campana1

AbstractNatural killer (NK) cells rely on surface receptors to distinguish healthy cells from cancer cells. We designed a

receptor termed NKG2D-DAP10-CD3z that is composed of the NK cell activating molecule NKG2D plus 2 keysignaling molecules, DAP10 and CD3z, and evaluated its capacity to promote cancer cell killing. Retroviraltransduction of NKG2D-DAP10-CD3zmarkedly increased NKG2D surface expression in NK cells, which becameconsistently more cytotoxic than mock-transduced cells against leukemia and solid tumor cell lines. In contrast,there was no increase in cytotoxicity against nontransformed blood and mesenchymal cells. NKG2D blockadeabrogated gains in cytotoxicity to cancer cells. Receptor stimulation triggered signal transduction, secretion ofIFN-g , GM-CSF, IL-13, MIP-1a, MIP-1b, CCL5, and TNF-a, and massive release of cytotoxic granules, whichpersisted after 48 hours of continuous stimulation. NKG2D-DAP10-CD3z–expressing NK cells had considerableantitumor activity in a mouse model of osteosarcoma, whereas activated NK cells were ineffective. Thus, thecytotoxic potential of NK cells against a wide spectrum of tumor subtypes could be markedly enhanced byexpression of NKG2D-DAP10-CD3z receptors. The development of an electroporationmethod that permits rapidexpression of the receptor in a large number of human NK cells facilitates clinical translation of this NK-basedstrategy for a generalized cellular therapy that may be useful to treat a wide range of cancers. Cancer Res; 73(6);1777–86. �2012 AACR.

IntroductionNatural killer (NK) cells can recognize tumor cells as targets,

a function that suggests possibilities for NK cell therapy ofcancer (1). The capacity of NK cells to kill tumor cells dependson the combined effect of suppressive and stimulatory signalsdelivered through surface receptors. Inhibitory signals resultfrom the interaction betweenNK inhibitory receptors andHLAmolecules on potential target cells, whereas engagement ofactivating receptors by ligands expressed predominantly byvirally infected and tumor cells provoke signals that ultimatelycause target cell killing (1).A key receptor for NK cell activation is NK Group 2 member

D (NKG2D), a type II transmembrane-anchored C-type lectin-like protein expressed in all NK cells and in some T-cell subsets(2–4). NKG2D has multiple ligands including MHC class I

chain-related A (MICA), MICB, and several UL-16-bindingproteins (ULBP), which are preferentially expressed after cel-lular stress, infection, or DNA damage (3, 5). There is strongevidence in vitro and in animal models for an important role ofNKG2D in NK cell-mediated antitumor activity (1, 4, 6–13).NKG2D is associated with DNAX-activating protein 10(DAP10), which promotes and stabilizes its surface membraneexpression (14–18). NKG2D lacks a signaling motif in itscytoplasmic domain and signal transduction upon ligationoccurs via the phosphorylation of DAP10, which recruitsdownstream signaling effectormolecules and, ultimately, cyto-toxicity (14, 19).

NK cells have shown promise for immunotherapy of cancer(20–23). We reasoned that supraphysiologic activating signalsshould enhance NK cell antitumor capacity and hence theirtherapeutic usefulness. To test this idea, we designed a con-struct encoding a chimeric receptor containing NKG2D,DAP10, and CD3z (another signaling molecule known totrigger cytotoxicity in NK cells; refs. 24, 25), and expressed itinto activated NK cells. We then examined their signalingprofile and anticancer potential in vitro and in vivo.

Materials and MethodsTumor cell lines

The human B-lineage acute lymphoblastic leukemia (ALL)cell lines OP-1 and REH, and the T-lineage ALL cell lines CEM-C7, Jurkat and MOLT-4 were from the St. Jude Children'sResearch Hospital tissue repository; their cell marker profilewas periodically tested by flow cytometry to ensure thatno changes had occurred. U-2 OS, HOS, and MG-63

Authors' Affiliations: 1Department of Pediatrics, 2Department of Surgery,National University of Singapore; and 3Singapore Immunology Network,A�STAR, Singapore

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

Current address for Y.-H. Chang: Faculty of Medicine, School of Medicine,National Yang-Ming University, Taipei, Taiwan.

Corresponding Author: Dario Campana, Department of Pediatrics, YongLoo Lin School of Medicine, National University of Singapore, Centre forTranslational Medicine, 14 Medical Drive, Level 9 South, Singapore117599. Phone: 65-6601 2666; Fax: 65-6779 7486; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-12-3558

�2012 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 1777

on May 27, 2020. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst January 9, 2013; DOI: 10.1158/0008-5472.CAN-12-3558

http://cancerres.aacrjournals.org/

(osteosarcoma), DU 145, PC-3, and LNCaP (prostate carcino-ma), Km12L4 (colon carcinoma), SNU1 (gastric carcinoma),SW900 (lung squamous cell carcinoma), HepG2 (hepatocellu-lar carcinoma), and MCF7 (breast carcinoma) were from theAmerican Type Culture Collection. The rhabdomyosarcomacell lines RH18, RH36, TE-32, and the neuroblastoma cell lineSKNSH were provided by Dr. Peter Houghton (NationwideChildren's Hospital, Columbus, OH); RH30 (rhabdomyosarco-ma) was from the St. Jude Children's Research Hospital tissuerepository (11). These cell lines were characterized by theproviders for molecular and/or gene expression features. Celllines were expanded after receipt, cryopreserved and cellsfor experiments were obtained from recently thawed vials.Human mesenchymal cells were developed in our laboratory(26). RPMI-1640 (Invitrogen) with 10% FBS (Atlanta Biologi-cals) and antibiotics, was used to maintain all cell lines exceptU-2 OS, HOS, and MG-63, which were maintained in DMEM(Cellgro).

For the visualization of tumor cells in immunodeficientmice, U-2 OS cells were transduced with a murine stem cellvirus (MSCV)-internal ribosome entry site (IRES)-green fluo-rescent protein (GFP) retroviral vector (from the St. JudeVector Development and Production Shared Resource) con-taining the firefly luciferase gene and selected for their expres-sion of GFP with a FACSAria cell sorter (BD Biosciences).

Human NK cell expansionPeripheral blood samples were obtained from healthy adult

donors. Mononuclear cells collected by centrifugation on aLymphoprep density step (Nycomed) were washed twice inRPMI-1640. To expand CD56þ CD3� NK cells, we coculturedperipheral blood mononuclear cells and the genetically mod-ified K562-mb15-41BBL cell line made in our laboratory, aspreviously described (25, 27). In brief, peripheral blood mono-nuclear cells (1.5� 106)were cultured in a 24-well tissue cultureplate with 1 � 106 K562-mb15-41BBL cells in RPMI-1640medium containing and 10% FBS and 10 IU/mL human IL-2(National Cancer Institute BRB Preclinical Repository). Every 2days the tissue culture medium was exchanged with freshmedium and IL-2. After 7 days of coculture, residual T cellswere removed using Dynabeads CD3 (Invitrogen), producingcell populations containing >95% CD56þ CD3� NK cells.

PlasmidsThe pMSCV-IRES-GFP, pEQ-PAM3(-E), and pRDF were

obtained from the St. Jude Vector Development and Produc-tion Shared Resource (28). The cDNA encoding NKG2D,DAP10, and the intracellular domain of CD3z were subclonedby PCR using cDNA derived from human expanded NK cells asa template. Expression cassettes were subcloned into EcoRIsites ofMSCV vector. BecauseNKG2D andCD3z are type II andtype I proteins, respectively, we removed the ATG initiationcodon of NKG2D and added anATG start codon to the cDNA ofthe intracellular domain of CD3z to prepare a constructcontaining both proteins. NKG2D and CD3z were then assem-bled using splicing by overlapping extension by PCR (SOE-PCR). We then replaced GFP in the vector with DAP10 (con-taining a FLAG-tag) between the NcoI and NotI sites; 1

nucleotide was then removed from the NcoI site to makeDAP10 in frame. The procedures used for virus production,gene transduction, mRNA electroporation, and analysis ofchimeric receptor are described in Supplementary Methods.

Cytotoxicity and degranulation assaysTarget cells were suspended in RPMI-1640 with 10% FBS,

labeled with calcein AM (Sigma), and plated into 96-well flatbottom plates (Costar). Expanded NK cells, suspended inRPMI-1640 with 10% FBS and 50 IU/mL IL-2 were then addedat various E:T ratios as indicated in Results, and coculturedwith target cells for 4 hours. Cells were then stained withpropridium iodide and cytotoxicity was measured by flowcytometry using FACScan or Accuri flow cytometers (BectonDickinson), enumerating the number of viable target cells(calcein AM-positive, propidium-iodide negative, and lightscattering properties of viable cells; ref. 27). For adherent celllines, the plates were placed in an incubator for at least 4 hoursto allow for cell attachment before adding NK cells. At the endof the cultures, cells were detached using trypsin plus EDTA. Insome experiments, NK cells were incubated with anti-NKG2D(clone 149810; R&D), anti-CD56 (BDBiosciences) or an isotype-matched nonreactive antibody for 10minutes before coculture.

We directly tested NK cell degranulation after NKG2Dstimulation with an anti-NKG2D antibody. NK cells (1 � 105)were plated into each well of a 96-well flat bottom plateand incubated with anti-Biotin MACSiBeads (Miltenyi Biotec)coated with biotin-conjugated anti-NKG2D antibody (clone1D11; eBioscience; 10 beads for 1 NK cell) for 4 hours at 37�C.Anti-human CD107a antibody conjugated to phycoerythrin (BDBiosciences) was added at the beginning of the cultures and 1hour later GolgiStop (0.15 mL; BD Biosciences) was added. Thecells were stained with anti-human CD56 conjugated to fluo-rescein isothiocyanate (BD Biosciences) and analyzed by flowcytometry.

Expression of NKG2D ligands, phospho-protein analysis,and measurement of cytokine levels

Surface expression of NKG2D ligands was evaluated bystaining with human recombinant NKG2D/Fc chimera (R&D),PE-conjugated goat anti-human IgGFc (g ; Fisher Scientific),MIC A/B (6D4, BD Biosciences), ULBP-1 (R&D) and ULBP-2(R&D) and ULBP-3 (R&D).

For phosphoprotein analysis, we culturedmock- and NKG2D-DAP10-CD3z–transduced expanded NK cells (8 � 106) withor without anti-NKG2D antibody and beads as describedabove. After 1 hour of stimulation, cell lysates were pre-pared using a lysis buffer containing 20 mmol/L 3-(N-mor-pholino) propanesulfonic acid, 2 mmol/L EGTA, 5 mmol/LEDTA, 30 mmol/L sodium fluoride, 60 mmol/L b-glycero-phosphate, 20 mmol/L sodium pyrophosphate, 1 mmol/Lsodium orthovanadate, 1% Triton X-100, Complete Miniprotease inhibitor cocktail (Roche), and 1 mmol/L dithio-threitol. After sonication, lysates were frozen at �80�C andshipped in dry ice to Kinexus for Kinex Antibody Microarrayanalysis. To measure cytokine/chemokine production, wecultured mock- and NKG2D-DAP10-CD3z expanded NK cells(1 � 105 in 200 mL/well of a 96-well plate) with or without

Chang et al.

Cancer Res; 73(6) March 15, 2013 Cancer Research1778




anti-NKG2D antibody and beads. Supernatants (120 mL) werecollected after 4, 8, and 16 hours and analyzed using theLuminex human cytokine/chemokine panel I (41 human cyto-kines/chemokines; Merck Millipore).

Murine modelsU-2 OS cells expressing luciferase were injected intraperito-

neally in NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NOD/scid IL2RG-null) mice (Jackson Laboratory; 2� 105 per mouse; ref. 11). NKcells from healthy donors were expanded for 7 days, trans-ducedwith theMSCV vector containing either GFP or NKG2D-DAP10-CD3z, suspended in RPMI-1640 plus 10% FBS (3 � 106

cells per mouse) and then injected intraperitoneally 7 daysafter U-2 OS injection. A single injection of NK cells was giventogetherwith intraperitoneal injections of IL-2 (20,000 IU each)for 4 days. As a control, a group of mice received tissue culturemedium instead of NK cells. U-2 OS engraftment and progres-sion was evaluated using a Xenogen IVIS-200 system (CaliperLife Sciences), with imaging beginning 5 minutes after intra-peritoneal injection of an aqueous solution of D-luciferinpotassium salt (3 mg/mouse). Photons emitted from lucifer-ase-expression cells were quantified using the Living Image 3.0software program. The studies were approved by the St JudeAnimal Care and Use Committee.

ResultsChimeric receptor design and expression in expandedNK cellsWe expanded human NK cells from peripheral blood

mononuclear cells, prepared a cDNA library and cloned thegenes encoding NKG2D, DAP10, and CD3z. We then insertedthe construct containing the 3 genes into a MSCV retroviralvector and used it to transduce expanded activated NK cells(Fig. 1A).We first determined whether retroviral transduction of

the construct resulted in gains of NKG2D expression ascompared with cells transduced with an MSCV vector con-taining only GFP. In experiments with expanded NK cellsfrom 21 donors (>98% CD56þ CD3- after T-cell depletion),median percentage of GFP-positive cells after transductionwith the GFP vector (mock) was 80% (range 67–96%).Transduction with the NKG2D-DAP10-CD3z construct inNK cells from the same donors resulted in a marked increasein NKG2D expression (P < 0.0001; Fig. 1B). We compared theresults of NKG2D-DAP10-CD3z transduction to thoseobtained after transduction of a NKG2D-CD3z lackingDAP10 in experiments with NK cells from 6 donors. Asshown in Fig. 1C, NKG2D expression was consistently higherwhen DAP10 was present in the construct (P ¼ 0.0027), inagreement with previous reports indicating that DAP10supports NKG2D expression (14–17).To ensure that all components of the receptor were

expressed, we used a construct containing DAP10 with aFLAG-tag. As shown in Fig. 1D, NK cells transduced withNKG2D-DAP10-CD3z expressed DAP10. By Western blottingwith an antibody detecting phospho- (pY83)-CD3z, we showedthat these cells expressed a chimeric protein containing CD3zin addition to endogenous CD3z (Fig. 1E). Thus, the 3 compo-

nents of the NKG2D-DAP10-CD3z receptor can be effectivelyexpressed in human NK cells.

NKG2D-DAP10-CD3z receptors increase the antitumorcytotoxicity of activated NK cells

NK cells expanded and activated after coculture with theK562-mb15-41BBL cell line exert cytotoxicity, which is muchhigher than that of primary or IL-2-stimulated NK cells (11, 27).We determined whether expression of NKG2D-DAP10-CD3zreceptors in these cells could further improve their antitumorcytotoxicity. For this purpose, we targeted a broad panel oftumor cell lines originating fromT-cell ALL (CEM-C7,MOLT-4,Jurkat) and B-cell ALL (REH, OP-1), osteosarcoma (U-2 OS,MG-36, HOS), prostate carcinoma (DU 145, PC-3, LNCaP),rhabdomyosarcoma (RH18, RH30, TE32, RH36), neuroblasto-ma (SKNSH), Ewing sarcoma (TC71), colon carcinoma(Km12L4), gastric carcinoma (SNU1), lung squamous cellcarcinoma (SW900), hepatoma (HepG2), and breast carcinoma(MCF7). We conducted 4-hour cytotoxicity assays with NKcells expanded from 14 donors at 1:1 or 1: 2 effector:target (E:T)ratios for a total of 65 experiments. For each cell line, we firstdetermined the E:T ratio that would produce sub-maximallevels of cytotoxicity and then tested the gains produced bytransducing NK cells with NKG2D-DAP10-CD3z; cells from thesame donors transduced with a vector containing GFP alonewere used as a control. As shown in Fig. 2A and B, expression ofthe NKG2D-DAP10-CD3z receptor significantly increasedoverall cytotoxicity against both leukemic and solid tumorcell lines (P < 0.0001). Gains in cytotoxicity were particularlyevident in the ALL cell lines REH, MOLT4, and CEM-C7, in theosteosarcoma cell lines U-2 OS, MG-36, HOS, in the prostatecarcinoma cell lines DU 145 and PC-3, and in the rhabdomyo-sarcoma cell line RH36 (Fig. 2C). In contrast, the B-lineage ALLcell line OP-1 remained relatively refractory to NK cells despiteNKG2D-DAP10-CD3z receptor expression (Fig. 2A).

Wedeterminedwhether expression ofNKG2D-DAP10-CD3zreceptors also increased the cytotoxicity of expanded NK cellsagainst nontransformed cells, such as allogeneic peripheralblood mononuclear cells and bone marrow-derived mesen-chymal cells. As shown in Fig. 2D, cytotoxicity remained below20% at 1:1 ratio, regardless of whether NK cells were trans-duced with the receptor or with GFP (Fig. 2D). These resultsindicate that expression ofNKG2D-DAP10-CD3z receptors canmarkedly enhance NK cell cytotoxicity against cancer cellswithout significantly increasing their activity against nontu-mor cells.

NK cytotoxicity is triggered by ligation of NKG2D-DAP10-CD3z receptors

We analyzed the relation between NKG2D-DAP10-CD3z–mediated cytotoxicity and expression of NKG2D ligands ontarget cells. To this end, we used a human recombinantNKG2D/Ig Fc reagent to measure the collective expression ofall NKG2D ligands. The cell line OP-1 did not show any labelingwith NKG2D/Ig Fc, and also gave a negative staining withantibodies to MICA/B, ULBP-1, ULBP-2, and ULBP-3, thusexplaining its resistance toNK cell killing regardless of whetherthese expressedNKG2D-DAP10-CD3z or not. All the remaining

NKG2D Receptor Enhances NK Cell Killing of Tumors

www.aacrjournals.org Cancer Res; 73(6) March 15, 2013 1779




cell lines studiedwere labeled byNKG2D/Ig Fc butwe found nosignificant relation between level of overall NKG2D ligandexpression and NKG2D-DAP10-CD3z receptor-mediated cyto-toxicity (Fig. 3A). Nontransformed bone marrow-derived mes-enchymal cells and peripheral blood monocytes had a rela-tively weak staining with NKG2D/Ig Fc, and most peripheralblood lymphocytes had no staining at all.

To ascertain whether the increase in cytotoxicity pro-duced by transduction of the NKG2D-DAP10-CD3z receptorwas directly related to receptor stimulation, we used ananti-NKG2D blocking antibody (clone 149810; ref. 11). Inexperiments with the U-2 OS osteosarcoma cell line, pre-incubation of NK cells with the antibody markedly inhibitedNK cytotoxicity and abrogated the gains achieved byNKG2D-DAP10-CD3z receptor transduction (Fig. 3B). Con-versely, direct stimulation of the receptor by an anti-NKG2Dagonistic antibody (clone 1D11; ref. 29) provoked massivelysosomal granule exocytosis, as detected by CD107a expres-sion (30); this was significantly higher than that achieved byNKG2D stimulation of mock-transduced NK cells (P <0.001; Fig. 3C and D).

Engagement of NKG2D-DAP10-CD3z triggers signaltransduction, cytokine secretion, and sustainedstimulation

To further understand the signaling properties of NKG2D-DAP10-CD3z and the differences from the signals triggeredby endogenous NKG2D, we stimulated mock- and NKG2D-DAP10-CD3z–transduced activated NK cells with the anti-NKG2D agonistic antibody for 1 hour and analyzed cell lysateswith the Kinex antibody microarray, which contains 809 anti-phosphoprotein antibodies. As shown in Fig. 4A, the phospho-protein profile of NKG2D-DAP10-CD3z–expressing NK cellswas substantially different from that of mock-transduced NKcells. Particularly prominent after NKG2D-DAP10-CD3z stim-ulation was the phosphorylation of the CREB1 transcriptionfactor, known to promote activation and proliferation of Tand B cells (31), of TBK1, a serine-threonine protein kinase andNF-kB activator with prosurvival roles (32), and of ACK1, atyrosine-protein and serine/threonine-protein kinase, whichregulates AKT (33), a key effector of DAP10 signaling (34).

To determine whether NKG2D-DAP10-CD3z–signalingresulted in an increased cytokine/chemokine secretion, we

Figure 1. NKG2D-DAP10-CD3z receptor design and expression. A, schematic representation of the NKG2D-DAP10-CD3z receptor and retroviral vectorconstruct. B, mean fluorescence intensity (MFI) of NKG2D expression in expanded NK cells from 21 donors transduced with a vector containingGFP only (Mock) or a vector containing the NKG2D-DAP10-CD3z receptor construct; horizontal lines indicate median values. To measure levels ofNKG2D, we used an anti-NKG2D antibody conjugated to PerCP, which in preliminary experiments gave a weaker signal and allowed better detection ofdifferences in NKG2D expression. C, MFI of NKG2D expression in expanded NK cells from six donors transduced with either a NKG2D-CD3z or aNKG2D-DAP10-CD3z construct. D, flow cytometry dot plots illustrate expression of NKG2D and DAP10 (detected with an anti-FLAG antibody) in mock- andNKG2D-DAP10-CD3z–transduced NK cells. E, mock- and NKG2D-DAP10-CD3z (NDC)–transduced NK cells were incubated with 0.1 mmol/L sodiumorthovanadate and 0.034% H2O2 at 37�C for 10 minutes before cell lysate preparation under reducing and nonreducing conditions and Western blotting.An anti-human CD3z phospho (pY83) monoclonal antibody (clone EP776(2)Y; Epitomics) followed by a goat anti-rabbit IgG horseradish peroxidase-conjugated second antibody was used to detect endogenous and chimeric phospho-CD3z proteins.

Chang et al.





stimulated receptor- or mock-transduced NK cells from 3donors with the biotin-labeled anti-NKG2D agonistic antibodyand anti-biotin beads and measured cytokine/chemokinelevels in the supernatants after 4, 8, and 16 hours. As shownin Fig. 4B and Supplementary Fig. S1, engagement of NKG2D-DAP10-CD3z caused a marked increase in IFN-g , GM-CSF, IL-13, MIP-1a, MIP-1b, CCL5, and TNF-a production (P < 0.01 by2-way ANOVA for all comparisons). For these 7 factors, levelswere also significantly higher when NKG2D-stimulated cells(either mock- or NKG2D-DAP10-CD3z–transduced) werecompared with the same cells cultured without antibody(Supplementary Table S1). Levels of the other cytokines/che-mokines measured [IL-1a, IL-1b, IL-1ra, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-15, IL-17,sCD40L, EGF, eotaxin, FGF-2, Flt-3 ligand, fractalkine, G-CSF,GRO, IFN-a2, IP-10, MCP-1, MCP-3, MDC, PDGF-AA, PDGF-BB, TGFa, TNF-b, and VEGF] were not significantly differentbetween mock- and NKG2D-DAP10-CD3z–transduced NKcells, regardless of NKG2D stimulation (SupplementaryTable S1).To further explore themechanisms underlying the enhance-

ment of cytotoxicity triggered by the NKG2D-DAP10-CD3zreceptors, we conducted immunofluorescence imaging studiesusing the U-2 OS cell line as a target. In experiments with NK

cells from 3 donors, those expressing the NKG2D-DAP10-CD3zreceptors produced clear increases in target cell apoptosiswhen compared with mock-transfected cells (11.7 � 2.9 apo-ptotic cells/0.07 mm2 vs. 3.3 � 0.6 apoptotic cells/0.07 mm2;P ¼ 0.033; Supplementary Movie). These gains could not beattributed to an increase in cell speed movement or cell trackdisplacement length, which were similar for receptor- andmock-transduced NK cells: 0.027 � 0.01 mm/s versus 0.027� 0.01 mm/s, and 18.1 � 10.1 mm versus 17.5 � 6.7 mm,respectively.

Continuous stimulation via NKG2D ligation may result ina hyporesponsive status (1). To test the anergy-inducingpotential of NKG2D-DAP10-CD3z signaling as comparedwith that of endogenous NKG2D, we cultured mock- andNKG2D-DAP10-CD3z–transduced NK cells with the anti-NKG2D agonistic antibody and monitored exocytosis of lyticgranules with CD107a staining over 48 hours. Mock-trans-duced NK cells were unable to degranulate after 24 or 48hours of stimulation. By contrast, a substantial proportion ofNKG2D-DAP10-CD3z–transduced NK cells were CD107a-positive 24 and 48 hours after continuous NKG2D ligation(Fig. 4C). Hence, NK cells bearing the receptor are capable ofexerting cytotoxicity even after prolonged engagement ofNKG2D.

Figure 2. Expression of NKG2D-DAP10-CD3z receptors increases tumor cell killing by activated NK cells. A, percentage of cytotoxicity of mock- andNKG2D-DAP10-CD3z–transducedNKcells against leukemia cell lines (CEM-C7,MOLT-4, Jurkat, REH, andOP-1), and solid tumor-derived cell lines (U-2OS,MG-36,HOS, DU 145, PC-3, LNCaP, RH18, RH30, TE32, RH36, SKNSH, TC71, Km12L4, SNU1, SW900, HepG2, and MCF7). A total of 65 experiments wereconducted using NK cells expanded from 14 donors at an E:T of 1:1 or 1:2; cell killing was measured after 4 hours of coculture. B, flow cytometric dotplots illustrate the assay used tomeasure cell killing. Resultswith one leukemia cell line (REH, top row) andoneosteosarcomacell line (U-2OS, bottom row) areshown. Tumor cells were either cultured alone (left), with mock-transduced NK cells (middle), or with NK cells transduced with the NKG2D-DAP10-CD3zreceptor. Residual viable target cells are in the bottom right region of each panel. C, percentage of cytotoxicity of mock- and NKG2D-DAP10-CD3z–transduced NK cells against selected tumor cell lines. D, percentage of cytotoxicity of mock- and NKG2D-DAP10-CD3z–transduced NK cells from threedonors against nontransformed peripheral blood mononucleated cells (PBMC) and bone-marrow-derived mesenchymal stromal cells (MSC); P > 0.05.






Cytotoxicity of NK cells expressing NKG2D-DAP10-CD3zin xenografts

To compare the antitumor capacity of NK cells ex-pressing NKG2D-DAP10-CD3z to that of mock-transducedcells in vivo, we generated a xenograft model of osteo-sarcoma by injecting luciferase-labeled U-2 OS cells(2 � 105) intraperitoneally in 12 immunodeficient (NOD/scid-IL2Rgnull) mice (Fig. 5). In 4 mice without treatment,U-2 OS tumors progressively expanded. Another 4 micewere injected with 2 � 105 U-2 OS intraperitoneally andthen a single intraperitoneal injection of mock-transducedactivated NK cells (3 � 106) 7 days later, followed by 4daily IL-2 intraperitoneal injection; U-2 OS tumors in thisgroup also expanded. A third group of 4 mice was injectedwith an identical number of U-2 OS intraperitoneallyand a single intraperitoneal injection of NK cells trans-duced with the NKG2D-DAP10-CD3z construct (3 � 106),followed by 4 daily IL-2 intraperitoneal injection. Sevendays after the NK cells were injected, the average signalintensity decreased dramatically and overall tumor burdenremained significantly lower to that measured in micetreated with mock-transduced NK cells (P ¼ 0.0028 by2-way ANOVA; Fig. 5).

Expression of NKG2D-DAP10-CD3z by electroporationAlthough effective, gene expression by retroviral trans-

duction presents considerable practical constraints forlarge-scale clinical application. We previously found thatelectroporation of mRNA results in highly efficient expres-sion of functional receptors in NK cells, and that this methodcan be adapted to a clinical grade protocol for geneticengineering of large cell numbers (35). To determine wheth-er the NKG2D-DAP10-CD3z receptor could be expressedby this method, we produced mRNA encoding NKG2D-CD3zand DAP10, electroporated them into expanded NK cells,and determined NKG2D expression 24 hours later. As shownin Fig. 6A, electroporation resulted in high NKG2D expres-sion. NK cells electroporated with the receptor weremarkedly more cytotoxic against the U-2 OS cell line thanmock-electroporated NK cells (Fig. 6B).

DiscussionThe NKG2D activating receptor is central to the capacity of

NK cells to sense cellular stress and lyse virally infected andtumor cells (1, 4, 6, 7, 9–13). In this study, we found thatexpression of an activating receptor with the binding

Figure 3. Relation between NKG2D-DAP10-CD3z ligation and increased cytotoxicity. A, relation between levels of NKG2D ligand (NKG2DL) expression andthe increase in cytotoxicity caused by NKG2D-DAP10-CD3z receptor expression. Mean fluorescence intensity (MFI) of NKG2DL expression afterstaining cells with a human recombinant NKG2D/Ig Fc is shown on the y axis. Cytotoxicities obtained with mock- and NKG2D-DAP10-CD3z–transduced NKcells (from threeormore donors)were compared for eachcell line. Themedian gain in cytotoxicity value of 43%wasused to divide the cell lines into twogroups(P > 0.05). B, pre-incubation of NK cells with an inhibitory anti-NKG2D antibody (clone 149810; R&D) abrogated the gains in cytotoxicity produced by theexpression of NKG2D-DAP10-CD3z. Mock- and NKG2D-DAP10-CD3z–transduced NK cells were incubated with anti-NKG2D, anti-CD56, or an isotype-matched nonreactive antibody for 10 minutes; 4-hour cytotoxicity against the U-2 OS cell line at 1:1 ratio was tested. Bars represent mean (�SD) of triplicatemeasurements. C, incubation of NK cells with a biotin-conjugated anti-NKG2D agonistic antibody (clone 1D11; eBioscience) and anti-biotin beads(MACSiBeads; Miltenyi Biotec) induced degranulation, which was significantly higher in NK cells expressing NKG2D-DAP10-CD3z. Percentage of CD56þcells from six donors expressing CD107a after 4 hours of anti-NKG2D stimulation is shown. D, flow cytometric dot plots illustrating CD107a expression onmock- or NKG2D-DAP10-CD3z–transduced CD56þ cells.

Chang et al.





specificity of NKG2D and the combined signaling capacities ofDAP10 and CD3z could considerably enhance the cytotoxicityof activated NK cells against leukemias and solid tumors. Thecytotoxicity of NK cells expressing NKG2D-DAP10-CD3zreceptors was directly triggered by engagement of NKG2D;receptor expression did not significantly increase cytotoxicityagainst nontransformed cells with low or no NKG2D ligandexpression, or against leukemic cells lacking NKG2D ligands.Although most of our experiments relied on retroviral trans-fection of the receptor, we also developed a method to effi-ciently express it by electroporation, thus greatly facilitating itsclinical application for cell therapy of cancer (35).The configuration of our receptor allows for signal trans-

duction by both DAP10 and CD3z and differs from the typicalchimeric-antigen receptors, which contain only 1 signalingmolecule, or a stimulatory plus a costimulatory molecule intandem (36). In line with previous reports indicating thatDAP10 promotes NKG2D expression on the surfacemembrane(14–17), we found that expression of the NKG2D-CD3z con-struct was significantly improved by concomitant expressionof DAP10. Other investigators reported that a receptor cou-pling NKG2D and CD3z could be expressed in T lymphocytes

and enhanced their cytotoxicity against lymphoma (37), mye-loma (38), ovarian cancer (39), and Ewing's sarcoma cells (40).Whether expression of DAP10 would increase NKG2D-CD3zexpression also in T lymphocytes and/or increase their cyto-toxicity remains to be determined.

NKG2D ligands are widely expressed among cancer cells (41,42). Indeed, NKG2D-DAP10-CD3z–receptor signaling aug-mented the cytotoxicity of activated NK cells against a widespectrum of tumor cell targets. However, there was consider-ably heterogeneity in the degree of response, with cell linesderived from ALL, osteosarcoma, prostate carcinoma andrhabdomyosarcomamost prominently revealing the enhancedcytotoxicity caused by the receptor. We suggest that thesetumor types should have priority for inclusion in clinicaltrials of this approach. The magnitude of the increase that weobserved (more than twice cells killed within 4 hours in somecases) is particularly noteworthy considering that the NK cellsincluded in our studies were activated and can exert cytotoxi-cities that are already significantly higher than those of primaryand IL-2 activated NK cells (27). Thus, the cytotoxic capacity ofactivated NK cells is not maximal and can be further enhancedby boosting activating signals. The gains in NK-mediated

Figure 4. NKG2D-DAP10-CD3z signaling and its cellular consequences. A, mock- and NKG2D-DAP10-CD3z–transduced NK cells were incubated with abiotin-conjugated anti-NKG2D agonistic antibody (clone 1D11; eBioscience) and anti-biotin beads (MACSiBeads; Miltenyi Biotec) for 1 hour and cell lysateswere analyzed by Kinex Antibody Microarray (Kinexus). Of 809 antiphosphoprotein antibodies tested, shown are those whose signals had a Z-ratio � 1and a % error range � 50. Bars indicate percentage signal change in NK cells expressing NKG2D-DAP10-CD3z as compared with the normalized intensityin mock-transduced NK cells. B, mock- and NKG2D-DAP10-CD3z–transduced NK cells from 3 donors were incubated with a biotin-conjugatedanti-NKG2D agonistic antibody (clone 1D11; eBioscience) and anti-biotin beads (MACSiBeads; Miltenyi Biotec). Concentration of IFN-g and GM-CSF in thesupernatants collected 4, 8, and 16 hours after initiation of stimulation was measured by Luminex (Merck Millipore). Data of the remaining cytokines/chemokinesmeasured is in Supplementary Fig. S1 and Table S1. C, degranulation inmock- andNKG2D-DAP10-CD3z–transducedNK cells after continuousstimulation with anti-NKG2D. NK cells were incubated with anti-NKG2D and beads as described in A. After 4, 24, and 48 hours, expression of CD107a inCD56þ cells was measured by flow cytometry. Results from experiments with NK cells from two donors are shown.






antitumor activity were also evident in experiments withimmunodeficient mice engrafted with osteosarcoma cells,where NK cells expressing NKG2D-DAP10-CD3z receptorsproduced marked tumor reductions while mock-transducedactivated NK cells were ineffective. Although the possibilitythat tumor cell subsets can escape NKG2D-DAP10-CD3z–mediated cytotoxicity cannot be ruled out, we think that thefailure of NK cells to completely eradicate the tumor was mostlikely due to the fact NK cells were infused only once, and thatIL-2 administration (which is critical for the survival andexpansion of the activated NK cells inmice; ref. 27) was limitedto 4 days.

In our study, there was no clear relation between levels ofNKG2D-ligand expression and susceptibility to NKG2D-DAP10-CD3z–bearing NK cells, suggesting that other signalingactivating or inhibitory signal interactions may influence thedegree of cell killing. It has also been shown that the pattern of

NKG2D-ligand partitioning in the target cell membrane, andthe degree of ligand shedding can play a role in triggeringcytotoxicity (43–45). Gains in cytotoxicity brought about byNKG2D-DAP10-CD3z–receptor expressionwere dependent onits signaling, as an antagonist anti-NKG2D antibody abrogatedthem. It is thought that persistent stimulation of NK cells mayresult in suppression of NK cell cytotoxic function (46, 47).Indeed, mock-transduced NK cells were unable to degranulateafter 24 hours of continuous stimulation. However, a consid-erable proportion of NK cells expressing NKG2D-DAP10-CD3zreceptors were CD107a positive even after 48 hours of stim-ulation, indicating that the combined DAP10 and CD3z signalsdo not accelerate the occurrence of hyporesponsiveness; on thecontrary, they significantly prolong NK cell function. TheNKG2D receptor has been shown to contribute to autoimmu-nity but pathologic responses against normal tissues could beattributed to the fraction of CD8 T lymphocytes expressing this

Figure 5. Antitumor capacity of NKG2D-DAP10-CD3z–transduced NK cells in a xenograft model of osteosarcoma. Luciferase-labeled U-2 OS cells(2 � 105) were injected intraperitoneally in 12 immunodeficient (NOD/scid-IL2Rgnull) mice. Control mice (No NK; n ¼ 4) received no treatment (top row);the remaining 8 mice received a single intraperitoneal injection of either mock-transduced (Mock, middle row) or NKG2D-DAP10-CD3z–transduced3 � 106 NK cells (NKG2D-DAP10-CD3z, bottom row), followed by four daily IL-2 intraperitoneal injection. Photoluminescence signals were measuredat weekly intervals with a Xenogen IVIS-200 system (Caliper Life Sciences), with imaging beginning 5 minutes after intraperitoneal injection of anaqueous solution of D-luciferin potassium salt (3 mg/mouse). Right graph shows mean (�SD) measurements of photons/second quantified using theLiving Image 3.0 software program (analyzed by two-way ANOVA).

Figure 6. Expression of NKG2D-DAP10-CD3z by electroporation.A, flow cytometric analysis ofNKG2D expression in activatedCD56þ CD3- NK cells 24 hoursafter electroporation with NKG2D-CD3z and DAP10 mRNA (NKG2D-DAP10-CD3z) or no mRNA (mock).B, killing of U-2 OS cells afterfour-hour coculture with NK cellselectroporated with NKG2D-CD3zand DAP10 mRNA or mock-electroporated at the indicated E:Tratios. Each symbol correspondsto mean (�SD) of three cocultures;P value at each E:T ratio by t-test isshown.

Chang et al.





receptor (48). We found that expression of our receptor did notsignificantly increase cytotoxicity against nontransformedperipheral blood lymphocytes or bone marrow-derived mes-enchymal cells. For clinical application, this potential problemshould be prevented by careful depletion of T cells from the NKcell product together with transient expression of the receptorby electroporation.It is well established that donor NK cell alloreactivity sup-

presses leukemia relapse after allogeneic hematopoietic stemcell transplantation (20, 21). Infusion of NK cells in a non-transplant setting has shown promise in some studies (22, 23),and hence this approach is being actively pursued at severalcenters using either freshly purified or activated NK cells. Themethod that we described here offers a newway to increase theantitumor efficacy of NK cell therapy and to widen its appli-cation. Stimulation via the NKG2D-DAP10-CD3z receptor alsoresulted in a marked increase in cytokine/chemokine secre-tion. Thus, NK-derived GM-CSF, IFN-g , and TNF-a promotemonocyte differentiation, macrophage activation and dendrit-ic cell maturation (1, 49, 50). Whether these cellular effectswould amplify the antitumor response in vivo is unclear butthey should be important during immune responses againstpathogens, suggesting that infusion of NKG2D-DAP10-CD3z-NK cells should also be tested in the setting of infectiousdiseases.

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

Authors' ContributionsConception and design: Y.-H. Chang, D. CampanaDevelopment of methodology: Y.-H. Chang, K. Mimura, K. Kono, D. CampanaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y.-H. Chang, J. Connolly, N. Shimasaki, K. MimuraAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y.-H. Chang, K. Mimura, K. Kono, D. CampanaWriting, review, and/or revision of themanuscript: Y.-H. Chang, K. Kono, D.CampanaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): Y.-H. Chang, N. ShimasakiStudy supervision: D. Campana

AcknowledgmentsWe thank Elaine Coustan-Smith for help with the analysis of chimeric receptor

expression, Veonice Au Bijin for the cytokine/chemokine studies, Wang Yilin forhelp with cell imaging studies, and Stephan Gasser for valuable discussions.

Grant SupportThis work was supported by the American Lebanese Syrian Associated

Charities (ALSAC) and by a Singapore Translational Research InvestigatorAward from the National Medical Research Council of Singapore.

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

Received September 13, 2012; revised December 1, 2012; accepted December17, 2012; published OnlineFirst January 10, 2013.

References1. Vivier E, Raulet DH, Moretta A, Caligiuri MA, Zitvogel L, Lanier LL, et al.

Innate or adaptive immunity? The example of natural killer cells.Science 2011;331:44–9.

2. Ho EL, Heusel JW, Brown MG, Matsumoto K, Scalzo AA, YokoyamaWM. Murine NKG2D and Cd94 are clustered within the natural killercomplex and are expressed independently in natural killer cells. ProcNatl Acad Sci U S A 1998;95:6320–5.

3. Bauer S, Groh V,Wu J, Steinle A, Phillips JH, Lanier LL, et al. ActivationofNKcells andTcells byNKG2D, a receptor for stress-inducibleMICA.Science 1999;285:727–9.

4. Champsaur M, Lanier LL. Effect of NKG2D ligand expression on hostimmune responses. Immunol Rev 2010;235:267–85.

5. Gasser S, Orsulic S, Brown EJ, Raulet DH. The DNA damage pathwayregulates innate immune system ligands of the NKG2D receptor.Nature 2005;436:1186–90.

6. Smyth MJ, Swann J, Cretney E, Zerafa N, Yokoyama WM, HayakawaY. NKG2D function protects the host from tumor initiation. J Exp Med2005;202:583–8.

7. Routes JM, Ryan S, Morris K, Takaki R, Cerwenka A, Lanier LL.Adenovirus serotype 5 E1A sensitizes tumor cells to NKG2D-depen-dent NK cell lysis and tumor rejection. J Exp Med 2005;202:1477–82.

8. Stern-Ginossar N, Gur C, Biton M, Horwitz E, Elboim M, Stanietsky N,et al. Human microRNAs regulate stress-induced immune responsesmediated by the receptor NKG2D. Nat Immunol 2008;9:1065–73.

9. Karimi M, Cao TM, Baker JA, Verneris MR, Soares L, Negrin RS.Silencing human NKG2D, DAP10, and DAP12 reduces cytotoxicityof activated CD8þ T cells and NK cells. J Immunol 2005;175:7819–28.

10. Guerra N, Tan YX, Joncker NT, Choy A, Gallardo F, Xiong N, et al.NKG2D-deficient mice are defective in tumor surveillance in models ofspontaneous malignancy. Immunity 2008;28:571–80.

11. Cho D, Shook DR, Shimasaki N, Chang YH, Fujisaki H, Campana D.Cytotoxicity of activated natural killer cells against pediatric solidtumors. Clin Cancer Res 2010;16:3901–9.

12. Raulet DH. Roles of the NKG2D immunoreceptor and its ligands. NatRev Immunol 2003;3:781–90.

13. Bryceson YT, Ljunggren HG. Tumor cell recognition by the NK cellactivating receptor NKG2D. Eur J Immunol 2008;38:2957–61.

14. Wu J, Song Y, Bakker AB, Bauer S, Spies T, Lanier LL, et al. Anactivating immunoreceptor complex formed by NKG2D and DAP10.Science 1999;285:730–2.

15. Diefenbach A, Tomasello E, Lucas M, Jamieson AM, Hsia JK, Vivier E,et al. Selective associations with signaling proteins determine stimu-latory versus costimulatory activity of NKG2D. Nat Immunol2002;3:1142–9.

16. Garrity D, Call ME, Feng J, Wucherpfennig KW. The activating NKG2Dreceptor assembles in the membrane with two signaling dimers into ahexameric structure. Proc Natl Acad Sci U S A 2005;102:7641–6.

17. Horng T, Bezbradica JS, Medzhitov R. NKG2D signaling is coupled tothe interleukin 15 receptor signaling pathway. Nat Immunol 2007;8:1345–52.

18. Park YP, Choi SC, Kiesler P, Gil-Krzewska A, Borrego F, Weck J, et al.Complex regulation of human NKG2D-DAP10 cell surface expression:opposing roles of the gamma c cytokines and TGF-beta1. Blood2011;118:3019–27.

19. Upshaw JL, Arneson LN, Schoon RA, Dick CJ, Billadeau DD, LeibsonPJ. NKG2D-mediated signaling requires a DAP10-bound Grb2-Vav1intermediate and phosphatidylinositol-3-kinase in human natural killercells. Nat Immunol 2006;7:524–32.

20. Ruggeri L, Capanni M, Urbani E, Perruccio K, Shlomchik WD, TostiA, et al. Effectiveness of donor natural killer cell alloreactivityin mismatched hematopoietic transplants. Science 2002;295:2097–100.

21. Cooley S, Weisdorf DJ, Guethlein LA, Klein JP, Wang T, Le CT, et al.Donor selection for natural killer cell receptor genes leads to superiorsurvival after unrelated transplantation for acute myelogenous leuke-mia. Blood 2010;116:2411–9.

22. Miller JS, Soignier Y, Panoskaltsis-Mortari A, McNearney SA, Yun GH,FautschSK, et al. Successful adoptive transfer and in vivoexpansion ofhuman haploidentical NK cells in cancer patients. Blood 2005;105:3051–7.






23. Rubnitz JE, Inaba H, Ribeiro RC, Pounds S, Rooney B, Bell T, et al.NKAML: a pilot study to determine the safety and feasibility of hap-loidentical natural killer cell transplantation in childhood acute myeloidleukemia. J Clin Oncol 2010;28:955–9.

24. Andre P, Castriconi R, Espeli M, Anfossi N, Juarez T, Hue S, et al.Comparative analysis of human NK cell activation induced byNKG2D and natural cytotoxicity receptors. Eur J Immunol 2004;34:961–71.

25. Imai C, Iwamoto S, Campana D. Genetic modification of primarynatural killer cells overcomes inhibitory signals and induces specifickilling of leukemic cells. Blood 2005;106:376–83.

26. Mihara K, Imai C, Coustan-Smith E, Dome JS, Dominici M, Vanin E,et al. Development and functional characterization of human bonemarrow mesenchymal cells immortalized by enforced expression oftelomerase. Br J Haematol 2003;120:846–9.

27. Fujisaki H, Kakuda H, Shimasaki N, Imai C, Ma J, Lockey T, et al.Expansion of highly cytotoxic human natural killer cells for cancer celltherapy. Cancer Res 2009;69:4010–7.

28. Imai C, Mihara K, Andreansky M, Nicholson IC, Pui CH, Campana D.Chimeric receptors with 4-1BB signaling capacity provoke potentcytotoxicity against acute lymphoblastic leukemia. Leukemia 2004;18:676–84.

29. Barber A, Sentman CL. NKG2D receptor regulates human effector T-cell cytokine production. Blood 2011;117:6571–81.

30. Betts MR, Brenchley JM, Price DA, De Rosa SC, Douek DC, RoedererM, et al. Sensitive and viable identification of antigen-specific CD8þ Tcells by a flow cytometric assay for degranulation. J ImmunolMethods2003;281:65–78.

31. Wen AY, Sakamoto KM, Miller LS. The role of the transcription factorCREB in immune function. J Immunol 2010;185:6413–9.

32. Baldwin AS. Regulation of cell death and autophagy by IKK and NF-kappaB: critical mechanisms in immune function and cancer. ImmunolRev 2012;246:327–45.

33. Mahajan K, Mahajan NP. Shepherding AKT and androgen receptor byAck1 tyrosine kinase. J Cell Physiol 2010;224:327–33.

34. ChangC, Dietrich J, Harpur AG, Lindquist JA, Haude A, Loke YW, et al.Cutting edge: KAP10, a novel transmembrane adapter protein genet-ically linked to DAP12 but with unique signaling properties. J Immunol1999;163:4651–4.

35. Shimasaki N, Fujisaki H, Cho D, Masselli M, Lockey T, Eldridge P,et al. A clinically adaptable method to enhance the cytotoxicity ofnatural killer cells against B-cell malignancies. Cytotherapy2012;14:830–40.

36. Kohn DB, Dotti G, Brentjens R, Savoldo B, JensenM, Cooper LJ, et al.CARs on track in the clinic. Mol Ther 2011;19:432–8.

37. Zhang T, Barber A, Sentman CL. Chimeric NKG2D modified T cellsinhibit systemic T-cell lymphoma growth in a manner involvingmultiple cytokines and cytotoxic pathways. Cancer Res 2007;67:11029–36.

38. Barber A, Zhang T,Megli CJ,Wu J,MeehanKR, SentmanCL. ChimericNKG2D receptor-expressing T cells as an immunotherapy for multiplemyeloma. Exp Hematol 2008;36:1318–28.

39. Barber A, Rynda A, Sentman CL. Chimeric NKG2D expressing T cellseliminate immunosuppression and activate immunity within the ovar-ian tumor microenvironment. J Immunol 2009;183:6939–47.

40. Lehner M, Gotz G, Proff J, Schaft N, Dorrie J, Full F, et al. Redirecting Tcells to Ewing's sarcoma family of tumors by a chimeric NKG2Dreceptor expressed by lentiviral transduction or mRNA transfection.PloS One 2012;7:e31210.

41. Groh V, Rhinehart R, Secrist H, Bauer S, Grabstein KH, Spies T. Broadtumor-associated expression and recognition by tumor-derived gam-ma delta T cells of MICA and MICB. Proc Natl Acad Sci U S A1999;96:6879–84.

42. PendeD, Rivera P,Marcenaro S, ChangCC, Biassoni R, Conte R, et al.Major histocompatibility complex class I-related chain A and UL16-binding protein expression on tumor cell lines of different histotypes:analysis of tumor susceptibility to NKG2D-dependent natural killer cellcytotoxicity. Cancer Res 2002;62:6178–86.

43. Martinez E, Brzostowski JA, Long EO, Gross CC. Cutting edge:NKG2D-dependent cytotoxicity is controlled by ligand distribution inthe target cell membrane. J Immunol 2011;186:5538–42.

44. SalihHR,RammenseeHG,Steinle A.Cutting edge: down-regulation ofMICA on human tumors by proteolytic shedding. J Immunol2002;169:4098–102.

45. Aguera-Gonzalez S, Gross CC, Fernandez-Messina L, Ashiru O,Esteso G, Hang HC, et al. Palmitoylation of MICA, a ligand for NKG2D,mediates its recruitment tomembranemicrodomains and promotes itsshedding. Eur J Immunol 2011;41:3667–76.

46. Coudert JD, Zimmer J, Tomasello E, Cebecauer M, Colonna M, VivierE, et al. Altered NKG2D function in NK cells induced by chronicexposure to NKG2D ligand-expressing tumor cells. Blood 2005;106:1711–7.

47. OppenheimDE, Roberts SJ, Clarke SL, Filler R, Lewis JM, Tigelaar RE,et al. Sustained localizedexpressionof ligand for the activatingNKG2Dreceptor impairs natural cytotoxicity in vivo and reduces tumor immu-nosurveillance. Nat Immunol 2005;6:928–37.

48. Markiewicz MA, Wise EL, Buchwald ZS, Pinto AK, Zafirova B, Polic B,et al. RAE1epsilon ligand expressed on pancreatic islets recruitsNKG2D receptor-expressing cytotoxic T cells independent of T cellreceptor recognition. Immunity 2012;36:132–41.

49. Goldszmid RS, Caspar P, Rivollier A,White S, Dzutsev A, Hieny S, et al.NK cell-derived interferon-gamma orchestrates cellular dynamics andthe differentiation of monocytes into dendritic cells at the site ofinfection. Immunity 2012;36:1047–59.

50. Spear P, Barber A, Rynda-Apple A, Sentman CL. Chimeric antigenreceptor T cells shape myeloid cell function within the tumor micro-environment through IFN-gamma and GM-CSF. J Immunol 2012;188:6389–98.

Chang et al.





2013;73:1777-1786. Published OnlineFirst January 9, 2013.Cancer Res Yu-Hsiang Chang, John Connolly, Noriko Shimasaki, et al. Killer Cell Activation and Killing of Tumor CellsA Chimeric Receptor with NKG2D Specificity Enhances Natural

Updated version

10.1158/0008-5472.CAN-12-3558doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2013/01/09/0008-5472.CAN-12-3558.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/73/6/1777.full#ref-list-1

This article cites 50 articles, 27 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/73/6/1777.full#related-urls

This article has been cited by 13 HighWire-hosted articles. Access the articles at:

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

Subscriptions

Reprints and

[email protected]

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

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/73/6/1777To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-12-3558

http://cancerres.aacrjournals.org/content/suppl/2013/01/09/0008-5472.CAN-12-3558.DC1

http://cancerres.aacrjournals.org/content/73/6/1777.full#ref-list-1

http://cancerres.aacrjournals.org/content/73/6/1777.full#related-urls

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

mailto:[email protected]

http://cancerres.aacrjournals.org/content/73/6/1777


Documents

A Chimeric Receptor with NKG2D Speciﬁcity Enhances Natural Killer Cell Activation ... · Microenvironment and Immunology A Chimeric Receptor with NKG2D Speciﬁcity Enhances Natural