Natural killer (NK) cells rely on surface receptors to distinguish healthy cells from cancer cells. We designed a receptor termed NKG2D-DAP10-CD3ζ that is composed of the NK cell activating molecule NKG2D plus 2 key signaling molecules, DAP10 and CD3ζ, and evaluated its capacity to promote cancer cell killing. Retroviral transduction of NKG2D-DAP10-CD3ζ markedly increased NKG2D surface expression in NK cells, which became consistently 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 blockade abrogated gains in cytotoxicity to cancer cells. Receptor stimulation triggered signal transduction, secretion of IFN-γ, GM-CSF, IL-13, MIP-1α, MIP-1β, CCL5, and TNF-α, and massive release of cytotoxic granules, which persisted after 48 hours of continuous stimulation. NKG2D-DAP10-CD3ζ–expressing NK cells had considerable antitumor activity in a mouse model of osteosarcoma, whereas activated NK cells were ineffective. Thus, the cytotoxic potential of NK cells against a wide spectrum of tumor subtypes could be markedly enhanced by expression of NKG2D-DAP10-CD3ζ receptors. The development of an electroporation method that permits rapid expression of the receptor in a large number of human NK cells facilitates clinical translation of this NK-based strategy for a generalized cellular therapy that may be useful to treat a wide range of cancers. Cancer Res; 73(6); 1777–86. ©2012 AACR.

Natural killer (NK) cells can recognize tumor cells as targets, a function that suggests possibilities for NK cell therapy of cancer (1). The capacity of NK cells to kill tumor cells depends on the combined effect of suppressive and stimulatory signals delivered through surface receptors. Inhibitory signals result from the interaction between NK inhibitory receptors and HLA molecules on potential target cells, whereas engagement of activating receptors by ligands expressed predominantly by virally infected and tumor cells provoke signals that ultimately cause 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-binding proteins (ULBP), which are preferentially expressed after cellular stress, infection, or DNA damage (3, 5). There is strong evidence in vitro and in animal models for an important role of NKG2D 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 membrane expression (14–18). NKG2D lacks a signaling motif in its cytoplasmic domain and signal transduction upon ligation occurs via the phosphorylation of DAP10, which recruits downstream signaling effector molecules and, ultimately, cytotoxicity (14, 19).

NK cells have shown promise for immunotherapy of cancer (20–23). We reasoned that supraphysiologic activating signals should enhance NK cell antitumor capacity and hence their therapeutic usefulness. To test this idea, we designed a construct encoding a chimeric receptor containing NKG2D, DAP10, and CD3ζ (another signaling molecule known to trigger cytotoxicity in NK cells; refs. 24, 25), and expressed it into activated NK cells. We then examined their signaling profile and anticancer potential in vitro and in vivo.

Tumor 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's Research Hospital tissue repository; their cell marker profile was periodically tested by flow cytometry to ensure that no changes had occurred. U-2 OS, HOS, and MG-63 (osteosarcoma), DU 145, PC-3, and LNCaP (prostate carcinoma), Km12L4 (colon carcinoma), SNU1 (gastric carcinoma), SW900 (lung squamous cell carcinoma), HepG2 (hepatocellular carcinoma), and MCF7 (breast carcinoma) were from the American Type Culture Collection. The rhabdomyosarcoma cell lines RH18, RH36, TE-32, and the neuroblastoma cell line SKNSH were provided by Dr. Peter Houghton (Nationwide Children's Hospital, Columbus, OH); RH30 (rhabdomyosarcoma) was from the St. Jude Children's Research Hospital tissue repository (11). These cell lines were characterized by the providers for molecular and/or gene expression features. Cell lines were expanded after receipt, cryopreserved and cells for experiments were obtained from recently thawed vials. Human mesenchymal cells were developed in our laboratory (26). RPMI-1640 (Invitrogen) with 10% FBS (Atlanta Biologicals) and antibiotics, was used to maintain all cell lines except U-2 OS, HOS, and MG-63, which were maintained in DMEM (Cellgro).

For the visualization of tumor cells in immunodeficient mice, U-2 OS cells were transduced with a murine stem cell virus (MSCV)-internal ribosome entry site (IRES)-green fluorescent protein (GFP) retroviral vector (from the St. Jude Vector Development and Production Shared Resource) containing the firefly luciferase gene and selected for their expression of GFP with a FACSAria cell sorter (BD Biosciences).

Human NK cell expansion

Peripheral blood samples were obtained from healthy adult donors. Mononuclear cells collected by centrifugation on a Lymphoprep density step (Nycomed) were washed twice in RPMI-1640. To expand CD56+ CD3− NK cells, we cocultured peripheral blood mononuclear cells and the genetically modified K562-mb15-41BBL cell line made in our laboratory, as previously described (25, 27). In brief, peripheral blood mononuclear cells (1.5 × 106) were cultured in a 24-well tissue culture plate with 1 × 106 K562-mb15-41BBL cells in RPMI-1640 medium containing and 10% FBS and 10 IU/mL human IL-2 (National Cancer Institute BRB Preclinical Repository). Every 2 days the tissue culture medium was exchanged with fresh medium and IL-2. After 7 days of coculture, residual T cells were removed using Dynabeads CD3 (Invitrogen), producing cell populations containing >95% CD56+ CD3− NK cells.

Plasmids

The pMSCV-IRES-GFP, pEQ-PAM3(-E), and pRDF were obtained from the St. Jude Vector Development and Production Shared Resource (28). The cDNA encoding NKG2D, DAP10, and the intracellular domain of CD3ζ were subcloned by PCR using cDNA derived from human expanded NK cells as a template. Expression cassettes were subcloned into EcoRI sites of MSCV vector. Because NKG2D and CD3ζ are type II and type I proteins, respectively, we removed the ATG initiation codon of NKG2D and added an ATG start codon to the cDNA of the intracellular domain of CD3ζ to prepare a construct containing both proteins. NKG2D and CD3ζ were then assembled using splicing by overlapping extension by PCR (SOE-PCR). We then replaced GFP in the vector with DAP10 (containing a FLAG-tag) between the NcoI and NotI sites; 1 nucleotide was then removed from the NcoI site to make DAP10 in frame. The procedures used for virus production, gene transduction, mRNA electroporation, and analysis of chimeric receptor are described in Supplementary Methods.

Cytotoxicity and degranulation assays

Target cells were suspended in RPMI-1640 with 10% FBS, labeled with calcein AM (Sigma), and plated into 96-well flat bottom plates (Costar). Expanded NK cells, suspended in RPMI-1640 with 10% FBS and 50 IU/mL IL-2 were then added at various E:T ratios as indicated in Results, and cocultured with target cells for 4 hours. Cells were then stained with propridium iodide and cytotoxicity was measured by flow cytometry using FACScan or Accuri flow cytometers (Becton Dickinson), enumerating the number of viable target cells (calcein AM-positive, propidium-iodide negative, and light scattering properties of viable cells; ref. 27). For adherent cell lines, the plates were placed in an incubator for at least 4 hours to allow for cell attachment before adding NK cells. At the end of the cultures, cells were detached using trypsin plus EDTA. In some experiments, NK cells were incubated with anti-NKG2D (clone 149810; R&D), anti-CD56 (BD Biosciences) or an isotype-matched nonreactive antibody for 10 minutes before coculture.

We directly tested NK cell degranulation after NKG2D stimulation with an anti-NKG2D antibody. NK cells (1 × 105) were plated into each well of a 96-well flat bottom plate and incubated with anti-Biotin MACSiBeads (Miltenyi Biotec) coated with biotin-conjugated anti-NKG2D antibody (clone 1D11; eBioscience; 10 beads for 1 NK cell) for 4 hours at 37°C. Anti-human CD107a antibody conjugated to phycoerythrin (BD Biosciences) was added at the beginning of the cultures and 1 hour later GolgiStop (0.15 μL; BD Biosciences) was added. The cells were stained with anti-human CD56 conjugated to fluorescein isothiocyanate (BD Biosciences) and analyzed by flow cytometry.

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

Surface expression of NKG2D ligands was evaluated by staining with human recombinant NKG2D/Fc chimera (R&D), PE-conjugated goat anti-human IgGFc (γ; 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 cultured mock- and NKG2D-DAP10-CD3ζ–transduced expanded NK cells (8 × 106) with or without anti-NKG2D antibody and beads as described above. After 1 hour of stimulation, cell lysates were prepared using a lysis buffer containing 20 mmol/L 3-(N-morpholino) propanesulfonic acid, 2 mmol/L EGTA, 5 mmol/L EDTA, 30 mmol/L sodium fluoride, 60 mmol/L β-glycerophosphate, 20 mmol/L sodium pyrophosphate, 1 mmol/L sodium orthovanadate, 1% Triton X-100, Complete Mini protease inhibitor cocktail (Roche), and 1 mmol/L dithiothreitol. After sonication, lysates were frozen at −80°C and shipped in dry ice to Kinexus for Kinex Antibody Microarray analysis. To measure cytokine/chemokine production, we cultured mock- and NKG2D-DAP10-CD3ζ expanded NK cells (1 × 105 in 200 μL/well of a 96-well plate) with or without anti-NKG2D antibody and beads. Supernatants (120 μL) were collected after 4, 8, and 16 hours and analyzed using the Luminex human cytokine/chemokine panel I (41 human cytokines/chemokines; Merck Millipore).

Murine models

U-2 OS cells expressing luciferase were injected intraperitoneally in NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NOD/scid IL2RGnull) mice (Jackson Laboratory; 2 × 105 per mouse; ref. 11). NK cells from healthy donors were expanded for 7 days, transduced with the MSCV vector containing either GFP or NKG2D-DAP10-CD3ζ, suspended in RPMI-1640 plus 10% FBS (3 × 106 cells per mouse) and then injected intraperitoneally 7 days after U-2 OS injection. A single injection of NK cells was given together with intraperitoneal injections of IL-2 (20,000 IU each) for 4 days. As a control, a group of mice received tissue culture medium instead of NK cells. U-2 OS engraftment and progression was evaluated using a Xenogen IVIS-200 system (Caliper Life Sciences), with imaging beginning 5 minutes after intraperitoneal injection of an aqueous solution of d-luciferin potassium salt (3 mg/mouse). Photons emitted from luciferase-expression cells were quantified using the Living Image 3.0 software program. The studies were approved by the St Jude Animal Care and Use Committee.

Chimeric receptor design and expression in expanded NK cells

We expanded human NK cells from peripheral blood mononuclear cells, prepared a cDNA library and cloned the genes encoding NKG2D, DAP10, and CD3ζ. We then inserted the construct containing the 3 genes into a MSCV retroviral vector and used it to transduce expanded activated NK cells (Fig. 1A).

Figure 1.

NKG2D-DAP10-CD3ζ receptor design and expression. A, schematic representation of the NKG2D-DAP10-CD3ζ receptor and retroviral vector construct. B, mean fluorescence intensity (MFI) of NKG2D expression in expanded NK cells from 21 donors transduced with a vector containing GFP only (Mock) or a vector containing the NKG2D-DAP10-CD3ζ receptor construct; horizontal lines indicate median values. To measure levels of NKG2D, we used an anti-NKG2D antibody conjugated to PerCP, which in preliminary experiments gave a weaker signal and allowed better detection of differences in NKG2D expression. C, MFI of NKG2D expression in expanded NK cells from six donors transduced with either a NKG2D-CD3ζ or a NKG2D-DAP10-CD3ζ construct. D, flow cytometry dot plots illustrate expression of NKG2D and DAP10 (detected with an anti-FLAG antibody) in mock- and NKG2D-DAP10-CD3ζ–transduced NK cells. E, mock- and NKG2D-DAP10-CD3ζ (NDC)–transduced NK cells were incubated with 0.1 μmol/L sodium orthovanadate 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 CD3ζ 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-CD3ζ proteins.

Figure 1.

NKG2D-DAP10-CD3ζ receptor design and expression. A, schematic representation of the NKG2D-DAP10-CD3ζ receptor and retroviral vector construct. B, mean fluorescence intensity (MFI) of NKG2D expression in expanded NK cells from 21 donors transduced with a vector containing GFP only (Mock) or a vector containing the NKG2D-DAP10-CD3ζ receptor construct; horizontal lines indicate median values. To measure levels of NKG2D, we used an anti-NKG2D antibody conjugated to PerCP, which in preliminary experiments gave a weaker signal and allowed better detection of differences in NKG2D expression. C, MFI of NKG2D expression in expanded NK cells from six donors transduced with either a NKG2D-CD3ζ or a NKG2D-DAP10-CD3ζ construct. D, flow cytometry dot plots illustrate expression of NKG2D and DAP10 (detected with an anti-FLAG antibody) in mock- and NKG2D-DAP10-CD3ζ–transduced NK cells. E, mock- and NKG2D-DAP10-CD3ζ (NDC)–transduced NK cells were incubated with 0.1 μmol/L sodium orthovanadate 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 CD3ζ 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-CD3ζ proteins.

Close modal

We first determined whether retroviral transduction of the construct resulted in gains of NKG2D expression as compared with cells transduced with an MSCV vector containing only GFP. In experiments with expanded NK cells from 21 donors (>98% CD56+ CD3- after T-cell depletion), median percentage of GFP-positive cells after transduction with the GFP vector (mock) was 80% (range 67–96%). Transduction with the NKG2D-DAP10-CD3ζ construct in NK cells from the same donors resulted in a marked increase in NKG2D expression (P < 0.0001; Fig. 1B). We compared the results of NKG2D-DAP10-CD3ζ transduction to those obtained after transduction of a NKG2D-CD3ζ lacking DAP10 in experiments with NK cells from 6 donors. As shown in Fig. 1C, NKG2D expression was consistently higher when DAP10 was present in the construct (P = 0.0027), in agreement with previous reports indicating that DAP10 supports NKG2D expression (14–17).

To ensure that all components of the receptor were expressed, we used a construct containing DAP10 with a FLAG-tag. As shown in Fig. 1D, NK cells transduced with NKG2D-DAP10-CD3ζ expressed DAP10. By Western blotting with an antibody detecting phospho- (pY83)-CD3ζ, we showed that these cells expressed a chimeric protein containing CD3ζ in addition to endogenous CD3ζ (Fig. 1E). Thus, the 3 components of the NKG2D-DAP10-CD3ζ receptor can be effectively expressed in human NK cells.

NKG2D-DAP10-CD3ζ receptors increase the antitumor cytotoxicity of activated NK cells

NK cells expanded and activated after coculture with the K562-mb15-41BBL cell line exert cytotoxicity, which is much higher than that of primary or IL-2-stimulated NK cells (11, 27). We determined whether expression of NKG2D-DAP10-CD3ζ receptors in these cells could further improve their antitumor cytotoxicity. For this purpose, we targeted a broad panel of tumor cell lines originating from T-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), neuroblastoma (SKNSH), Ewing sarcoma (TC71), colon carcinoma (Km12L4), gastric carcinoma (SNU1), lung squamous cell carcinoma (SW900), hepatoma (HepG2), and breast carcinoma (MCF7). We conducted 4-hour cytotoxicity assays with NK cells 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 first determined the E:T ratio that would produce sub-maximal levels of cytotoxicity and then tested the gains produced by transducing NK cells with NKG2D-DAP10-CD3ζ; cells from the same donors transduced with a vector containing GFP alone were used as a control. As shown in Fig. 2A and B, expression of the NKG2D-DAP10-CD3ζ receptor significantly increased overall cytotoxicity against both leukemic and solid tumor cell lines (P < 0.0001). Gains in cytotoxicity were particularly evident in the ALL cell lines REH, MOLT4, and CEM-C7, in the osteosarcoma cell lines U-2 OS, MG-36, HOS, in the prostate carcinoma cell lines DU 145 and PC-3, and in the rhabdomyosarcoma cell line RH36 (Fig. 2C). In contrast, the B-lineage ALL cell line OP-1 remained relatively refractory to NK cells despite NKG2D-DAP10-CD3ζ receptor expression (Fig. 2A).

Figure 2.

Expression of NKG2D-DAP10-CD3ζ receptors increases tumor cell killing by activated NK cells. A, percentage of cytotoxicity of mock- and NKG2D-DAP10-CD3ζ–transduced NK cells against leukemia cell lines (CEM-C7, MOLT-4, Jurkat, REH, and OP-1), and solid tumor-derived cell lines (U-2 OS, 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 were conducted 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 dot plots illustrate the assay used to measure cell killing. Results with one leukemia cell line (REH, top row) and one osteosarcoma cell line (U-2 OS, bottom row) are shown. Tumor cells were either cultured alone (left), with mock-transduced NK cells (middle), or with NK cells transduced with the NKG2D-DAP10-CD3ζ receptor. Residual viable target cells are in the bottom right region of each panel. C, percentage of cytotoxicity of mock- and NKG2D-DAP10-CD3ζ–transduced NK cells against selected tumor cell lines. D, percentage of cytotoxicity of mock- and NKG2D-DAP10-CD3ζ–transduced NK cells from three donors against nontransformed peripheral blood mononucleated cells (PBMC) and bone-marrow-derived mesenchymal stromal cells (MSC); P > 0.05.

Figure 2.

Expression of NKG2D-DAP10-CD3ζ receptors increases tumor cell killing by activated NK cells. A, percentage of cytotoxicity of mock- and NKG2D-DAP10-CD3ζ–transduced NK cells against leukemia cell lines (CEM-C7, MOLT-4, Jurkat, REH, and OP-1), and solid tumor-derived cell lines (U-2 OS, 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 were conducted 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 dot plots illustrate the assay used to measure cell killing. Results with one leukemia cell line (REH, top row) and one osteosarcoma cell line (U-2 OS, bottom row) are shown. Tumor cells were either cultured alone (left), with mock-transduced NK cells (middle), or with NK cells transduced with the NKG2D-DAP10-CD3ζ receptor. Residual viable target cells are in the bottom right region of each panel. C, percentage of cytotoxicity of mock- and NKG2D-DAP10-CD3ζ–transduced NK cells against selected tumor cell lines. D, percentage of cytotoxicity of mock- and NKG2D-DAP10-CD3ζ–transduced NK cells from three donors against nontransformed peripheral blood mononucleated cells (PBMC) and bone-marrow-derived mesenchymal stromal cells (MSC); P > 0.05.

Close modal

We determined whether expression of NKG2D-DAP10-CD3ζ receptors also increased the cytotoxicity of expanded NK cells against nontransformed cells, such as allogeneic peripheral blood mononuclear cells and bone marrow-derived mesenchymal cells. As shown in Fig. 2D, cytotoxicity remained below 20% at 1:1 ratio, regardless of whether NK cells were transduced with the receptor or with GFP (Fig. 2D). These results indicate that expression of NKG2D-DAP10-CD3ζ receptors can markedly enhance NK cell cytotoxicity against cancer cells without significantly increasing their activity against nontumor cells.

NK cytotoxicity is triggered by ligation of NKG2D-DAP10-CD3ζ receptors

We analyzed the relation between NKG2D-DAP10-CD3ζ–mediated cytotoxicity and expression of NKG2D ligands on target cells. To this end, we used a human recombinant NKG2D/Ig Fc reagent to measure the collective expression of all NKG2D ligands. The cell line OP-1 did not show any labeling with NKG2D/Ig Fc, and also gave a negative staining with antibodies to MICA/B, ULBP-1, ULBP-2, and ULBP-3, thus explaining its resistance to NK cell killing regardless of whether these expressed NKG2D-DAP10-CD3ζ or not. All the remaining cell lines studied were labeled by NKG2D/Ig Fc but we found no significant relation between level of overall NKG2D ligand expression and NKG2D-DAP10-CD3ζ receptor-mediated cytotoxicity (Fig. 3A). Nontransformed bone marrow-derived mesenchymal cells and peripheral blood monocytes had a relatively weak staining with NKG2D/Ig Fc, and most peripheral blood lymphocytes had no staining at all.

Figure 3.

Relation between NKG2D-DAP10-CD3ζ ligation and increased cytotoxicity. A, relation between levels of NKG2D ligand (NKG2DL) expression and the increase in cytotoxicity caused by NKG2D-DAP10-CD3ζ receptor expression. Mean fluorescence intensity (MFI) of NKG2DL expression after staining cells with a human recombinant NKG2D/Ig Fc is shown on the y axis. Cytotoxicities obtained with mock- and NKG2D-DAP10-CD3ζ–transduced NK cells (from three or more donors) were compared for each cell line. The median gain in cytotoxicity value of 43% was used to divide the cell lines into two groups (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 the expression of NKG2D-DAP10-CD3ζ. Mock- and NKG2D-DAP10-CD3ζ–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 triplicate measurements. 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-CD3ζ. Percentage of CD56+ cells from six donors expressing CD107a after 4 hours of anti-NKG2D stimulation is shown. D, flow cytometric dot plots illustrating CD107a expression on mock- or NKG2D-DAP10-CD3ζ–transduced CD56+ cells.

Figure 3.

Relation between NKG2D-DAP10-CD3ζ ligation and increased cytotoxicity. A, relation between levels of NKG2D ligand (NKG2DL) expression and the increase in cytotoxicity caused by NKG2D-DAP10-CD3ζ receptor expression. Mean fluorescence intensity (MFI) of NKG2DL expression after staining cells with a human recombinant NKG2D/Ig Fc is shown on the y axis. Cytotoxicities obtained with mock- and NKG2D-DAP10-CD3ζ–transduced NK cells (from three or more donors) were compared for each cell line. The median gain in cytotoxicity value of 43% was used to divide the cell lines into two groups (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 the expression of NKG2D-DAP10-CD3ζ. Mock- and NKG2D-DAP10-CD3ζ–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 triplicate measurements. 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-CD3ζ. Percentage of CD56+ cells from six donors expressing CD107a after 4 hours of anti-NKG2D stimulation is shown. D, flow cytometric dot plots illustrating CD107a expression on mock- or NKG2D-DAP10-CD3ζ–transduced CD56+ cells.

Close modal

To ascertain whether the increase in cytotoxicity produced by transduction of the NKG2D-DAP10-CD3ζ receptor was directly related to receptor stimulation, we used an anti-NKG2D blocking antibody (clone 149810; ref. 11). In experiments with the U-2 OS osteosarcoma cell line, preincubation of NK cells with the antibody markedly inhibited NK cytotoxicity and abrogated the gains achieved by NKG2D-DAP10-CD3ζ receptor transduction (Fig. 3B). Conversely, direct stimulation of the receptor by an anti-NKG2D agonistic antibody (clone 1D11; ref. 29) provoked massive lysosomal granule exocytosis, as detected by CD107a expression (30); this was significantly higher than that achieved by NKG2D stimulation of mock-transduced NK cells (P < 0.001; Fig. 3C and D).

Engagement of NKG2D-DAP10-CD3ζ triggers signal transduction, cytokine secretion, and sustained stimulation

To further understand the signaling properties of NKG2D-DAP10-CD3ζ and the differences from the signals triggered by endogenous NKG2D, we stimulated mock- and NKG2D-DAP10-CD3ζ–transduced activated NK cells with the anti-NKG2D agonistic antibody for 1 hour and analyzed cell lysates with the Kinex antibody microarray, which contains 809 antiphosphoprotein antibodies. As shown in Fig. 4A, the phosphoprotein profile of NKG2D-DAP10-CD3ζ–expressing NK cells was substantially different from that of mock-transduced NK cells. Particularly prominent after NKG2D-DAP10-CD3ζ stimulation was the phosphorylation of the CREB1 transcription factor, known to promote activation and proliferation of T and B cells (31), of TBK1, a serine-threonine protein kinase and NF-κB activator with prosurvival roles (32), and of ACK1, a tyrosine-protein and serine/threonine-protein kinase, which regulates AKT (33), a key effector of DAP10 signaling (34).

Figure 4.

NKG2D-DAP10-CD3ζ signaling and its cellular consequences. A, mock- and NKG2D-DAP10-CD3ζ–transduced NK cells were incubated with a biotin-conjugated anti-NKG2D agonistic antibody (clone 1D11; eBioscience) and anti-biotin beads (MACSiBeads; Miltenyi Biotec) for 1 hour and cell lysates were analyzed by Kinex Antibody Microarray (Kinexus). Of 809 antiphosphoprotein antibodies tested, shown are those whose signals had a Z-ratio ≥ 1 and a % error range ≤ 50. Bars indicate percentage signal change in NK cells expressing NKG2D-DAP10-CD3ζ as compared with the normalized intensity in mock-transduced NK cells. B, mock- and NKG2D-DAP10-CD3ζ–transduced NK cells from 3 donors were incubated with a biotin-conjugated anti-NKG2D agonistic antibody (clone 1D11; eBioscience) and anti-biotin beads (MACSiBeads; Miltenyi Biotec). Concentration of IFN-γ and GM-CSF in the supernatants collected 4, 8, and 16 hours after initiation of stimulation was measured by Luminex (Merck Millipore). Data of the remaining cytokines/chemokines measured is in Supplementary Fig. S1 and Table S1. C, degranulation in mock- and NKG2D-DAP10-CD3ζ–transduced NK cells after continuous stimulation 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 in CD56+ cells was measured by flow cytometry. Results from experiments with NK cells from two donors are shown.

Figure 4.

NKG2D-DAP10-CD3ζ signaling and its cellular consequences. A, mock- and NKG2D-DAP10-CD3ζ–transduced NK cells were incubated with a biotin-conjugated anti-NKG2D agonistic antibody (clone 1D11; eBioscience) and anti-biotin beads (MACSiBeads; Miltenyi Biotec) for 1 hour and cell lysates were analyzed by Kinex Antibody Microarray (Kinexus). Of 809 antiphosphoprotein antibodies tested, shown are those whose signals had a Z-ratio ≥ 1 and a % error range ≤ 50. Bars indicate percentage signal change in NK cells expressing NKG2D-DAP10-CD3ζ as compared with the normalized intensity in mock-transduced NK cells. B, mock- and NKG2D-DAP10-CD3ζ–transduced NK cells from 3 donors were incubated with a biotin-conjugated anti-NKG2D agonistic antibody (clone 1D11; eBioscience) and anti-biotin beads (MACSiBeads; Miltenyi Biotec). Concentration of IFN-γ and GM-CSF in the supernatants collected 4, 8, and 16 hours after initiation of stimulation was measured by Luminex (Merck Millipore). Data of the remaining cytokines/chemokines measured is in Supplementary Fig. S1 and Table S1. C, degranulation in mock- and NKG2D-DAP10-CD3ζ–transduced NK cells after continuous stimulation 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 in CD56+ cells was measured by flow cytometry. Results from experiments with NK cells from two donors are shown.

Close modal

To determine whether NKG2D-DAP10-CD3ζ–signaling resulted in an increased cytokine/chemokine secretion, we stimulated receptor- or mock-transduced NK cells from 3 donors with the biotin-labeled anti-NKG2D agonistic antibody and anti-biotin beads and measured cytokine/chemokine levels in the supernatants after 4, 8, and 16 hours. As shown in Fig. 4B and Supplementary Fig. S1, engagement of NKG2D-DAP10-CD3ζ caused a marked increase in IFN-γ, GM-CSF, IL-13, MIP-1α, MIP-1β, CCL5, and TNF-α production (P < 0.01 by 2-way ANOVA for all comparisons). For these 7 factors, levels were also significantly higher when NKG2D-stimulated cells (either mock- or NKG2D-DAP10-CD3ζ–transduced) were compared with the same cells cultured without antibody (Supplementary Table S1). Levels of the other cytokines/chemokines measured [IL-1α, IL-1β, IL-1rα, 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-α2, IP-10, MCP-1, MCP-3, MDC, PDGF-AA, PDGF-BB, TGFα, TNF-β, and VEGF] were not significantly different between mock- and NKG2D-DAP10-CD3ζ–transduced NK cells, regardless of NKG2D stimulation (Supplementary Table S1).

To further explore the mechanisms underlying the enhancement of cytotoxicity triggered by the NKG2D-DAP10-CD3ζ receptors, we conducted immunofluorescence imaging studies using the U-2 OS cell line as a target. In experiments with NK cells from 3 donors, those expressing the NKG2D-DAP10-CD3ζ receptors produced clear increases in target cell apoptosis when compared with mock-transfected cells (11.7 ± 2.9 apoptotic cells/0.07 mm2 vs. 3.3 ± 0.6 apoptotic cells/0.07 mm2; P = 0.033; Supplementary Movie). These gains could not be attributed to an increase in cell speed movement or cell track displacement length, which were similar for receptor- and mock-transduced NK cells: 0.027 ± 0.01 μm/s versus 0.027 ± 0.01 μm/s, and 18.1 ± 10.1 μm versus 17.5 ± 6.7 μm, respectively.

Continuous stimulation via NKG2D ligation may result in a hyporesponsive status (1). To test the anergy-inducing potential of NKG2D-DAP10-CD3ζ signaling as compared with that of endogenous NKG2D, we cultured mock- and NKG2D-DAP10-CD3ζ–transduced NK cells with the anti-NKG2D agonistic antibody and monitored exocytosis of lytic granules with CD107a staining over 48 hours. Mock-transduced NK cells were unable to degranulate after 24 or 48 hours of stimulation. By contrast, a substantial proportion of NKG2D-DAP10-CD3ζ–transduced NK cells were CD107a-positive 24 and 48 hours after continuous NKG2D ligation (Fig. 4C). Hence, NK cells bearing the receptor are capable of exerting cytotoxicity even after prolonged engagement of NKG2D.

Cytotoxicity of NK cells expressing NKG2D-DAP10-CD3ζ in xenografts

To compare the antitumor capacity of NK cells expressing NKG2D-DAP10-CD3ζ to that of mock-transduced cells in vivo, we generated a xenograft model of osteosarcoma 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 mice were injected with 2 × 105 U-2 OS intraperitoneally and then a single intraperitoneal injection of mock-transduced activated NK cells (3 × 106) 7 days later, followed by 4 daily IL-2 intraperitoneal injection; U-2 OS tumors in this group also expanded. A third group of 4 mice was injected with an identical number of U-2 OS intraperitoneally and a single intraperitoneal injection of NK cells transduced with the NKG2D-DAP10-CD3ζ construct (3 × 106), followed by 4 daily IL-2 intraperitoneal injection. Seven days after the NK cells were injected, the average signal intensity decreased dramatically and overall tumor burden remained significantly lower to that measured in mice treated with mock-transduced NK cells (P = 0.0028 by 2-way ANOVA; Fig. 5).

Figure 5.

Antitumor capacity of NKG2D-DAP10-CD3ζ–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 = 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-CD3ζ–transduced 3 × 106 NK cells (NKG2D-DAP10-CD3ζ, bottom row), followed by four daily IL-2 intraperitoneal injection. Photoluminescence signals were measured at weekly intervals with a Xenogen IVIS-200 system (Caliper Life Sciences), with imaging beginning 5 minutes after intraperitoneal injection of an aqueous solution of d-luciferin potassium salt (3 mg/mouse). Right graph shows mean (±SD) measurements of photons/second quantified using the Living Image 3.0 software program (analyzed by two-way ANOVA).

Figure 5.

Antitumor capacity of NKG2D-DAP10-CD3ζ–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 = 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-CD3ζ–transduced 3 × 106 NK cells (NKG2D-DAP10-CD3ζ, bottom row), followed by four daily IL-2 intraperitoneal injection. Photoluminescence signals were measured at weekly intervals with a Xenogen IVIS-200 system (Caliper Life Sciences), with imaging beginning 5 minutes after intraperitoneal injection of an aqueous solution of d-luciferin potassium salt (3 mg/mouse). Right graph shows mean (±SD) measurements of photons/second quantified using the Living Image 3.0 software program (analyzed by two-way ANOVA).

Close modal

Expression of NKG2D-DAP10-CD3ζ by electroporation

Although effective, gene expression by retroviral transduction presents considerable practical constraints for large-scale clinical application. We previously found that electroporation of mRNA results in highly efficient expression of functional receptors in NK cells, and that this method can be adapted to a clinical grade protocol for genetic engineering of large cell numbers (35). To determine whether the NKG2D-DAP10-CD3ζ receptor could be expressed by this method, we produced mRNA encoding NKG2D-CD3ζ and DAP10, electroporated them into expanded NK cells, and determined NKG2D expression 24 hours later. As shown in Fig. 6A, electroporation resulted in high NKG2D expression. NK cells electroporated with the receptor were markedly more cytotoxic against the U-2 OS cell line than mock-electroporated NK cells (Fig. 6B).

Figure 6.

Expression of NKG2D-DAP10-CD3ζ by electroporation. A, flow cytometric analysis of NKG2D expression in activated CD56+ CD3- NK cells 24 hours after electroporation with NKG2D-CD3ζ and DAP10 mRNA (NKG2D-DAP10-CD3ζ) or no mRNA (mock). B, killing of U-2 OS cells after four-hour coculture with NK cells electroporated with NKG2D-CD3ζ and DAP10 mRNA or mock-electroporated at the indicated E:T ratios. Each symbol corresponds to mean (±SD) of three cocultures; P value at each E:T ratio by t-test is shown.

Figure 6.

Expression of NKG2D-DAP10-CD3ζ by electroporation. A, flow cytometric analysis of NKG2D expression in activated CD56+ CD3- NK cells 24 hours after electroporation with NKG2D-CD3ζ and DAP10 mRNA (NKG2D-DAP10-CD3ζ) or no mRNA (mock). B, killing of U-2 OS cells after four-hour coculture with NK cells electroporated with NKG2D-CD3ζ and DAP10 mRNA or mock-electroporated at the indicated E:T ratios. Each symbol corresponds to mean (±SD) of three cocultures; P value at each E:T ratio by t-test is shown.

Close modal

The NKG2D activating receptor is central to the capacity of NK cells to sense cellular stress and lyse virally infected and tumor cells (1, 4, 6, 7, 9–13). In this study, we found that expression of an activating receptor with the binding specificity of NKG2D and the combined signaling capacities of DAP10 and CD3ζ could considerably enhance the cytotoxicity of activated NK cells against leukemias and solid tumors. The cytotoxicity of NK cells expressing NKG2D-DAP10-CD3ζ receptors was directly triggered by engagement of NKG2D; receptor expression did not significantly increase cytotoxicity against nontransformed cells with low or no NKG2D ligand expression, or against leukemic cells lacking NKG2D ligands. Although most of our experiments relied on retroviral transfection of the receptor, we also developed a method to efficiently express it by electroporation, thus greatly facilitating its clinical application for cell therapy of cancer (35).

The configuration of our receptor allows for signal transduction by both DAP10 and CD3ζ and differs from the typical chimeric-antigen receptors, which contain only 1 signaling molecule, or a stimulatory plus a costimulatory molecule in tandem (36). In line with previous reports indicating that DAP10 promotes NKG2D expression on the surface membrane (14–17), we found that expression of the NKG2D-CD3ζ construct was significantly improved by concomitant expression of DAP10. Other investigators reported that a receptor coupling NKG2D and CD3ζ could be expressed in T lymphocytes and enhanced their cytotoxicity against lymphoma (37), myeloma (38), ovarian cancer (39), and Ewing's sarcoma cells (40). Whether expression of DAP10 would increase NKG2D-CD3ζ expression also in T lymphocytes and/or increase their cytotoxicity remains to be determined.

NKG2D ligands are widely expressed among cancer cells (41, 42). Indeed, NKG2D-DAP10-CD3ζ–receptor signaling augmented the cytotoxicity of activated NK cells against a wide spectrum of tumor cell targets. However, there was considerably heterogeneity in the degree of response, with cell lines derived from ALL, osteosarcoma, prostate carcinoma and rhabdomyosarcoma most prominently revealing the enhanced cytotoxicity caused by the receptor. We suggest that these tumor types should have priority for inclusion in clinical trials of this approach. The magnitude of the increase that we observed (more than twice cells killed within 4 hours in some cases) is particularly noteworthy considering that the NK cells included in our studies were activated and can exert cytotoxicities that are already significantly higher than those of primary and IL-2 activated NK cells (27). Thus, the cytotoxic capacity of activated NK cells is not maximal and can be further enhanced by boosting activating signals. The gains in NK-mediated antitumor activity were also evident in experiments with immunodeficient mice engrafted with osteosarcoma cells, where NK cells expressing NKG2D-DAP10-CD3ζ receptors produced marked tumor reductions while mock-transduced activated NK cells were ineffective. Although the possibility that tumor cell subsets can escape NKG2D-DAP10-CD3ζ–mediated cytotoxicity cannot be ruled out, we think that the failure of NK cells to completely eradicate the tumor was most likely due to the fact NK cells were infused only once, and that IL-2 administration (which is critical for the survival and expansion of the activated NK cells in mice; ref. 27) was limited to 4 days.

In our study, there was no clear relation between levels of NKG2D-ligand expression and susceptibility to NKG2D-DAP10-CD3ζ–bearing NK cells, suggesting that other signaling activating or inhibitory signal interactions may influence the degree of cell killing. It has also been shown that the pattern of NKG2D-ligand partitioning in the target cell membrane, and the degree of ligand shedding can play a role in triggering cytotoxicity (43–45). Gains in cytotoxicity brought about by NKG2D-DAP10-CD3ζ–receptor expression were dependent on its signaling, as an antagonist anti-NKG2D antibody abrogated them. It is thought that persistent stimulation of NK cells may result in suppression of NK cell cytotoxic function (46, 47). Indeed, mock-transduced NK cells were unable to degranulate after 24 hours of continuous stimulation. However, a considerable proportion of NK cells expressing NKG2D-DAP10-CD3ζ receptors were CD107a positive even after 48 hours of stimulation, indicating that the combined DAP10 and CD3ζ signals do not accelerate the occurrence of hyporesponsiveness; on the contrary, they significantly prolong NK cell function. The NKG2D receptor has been shown to contribute to autoimmunity but pathologic responses against normal tissues could be attributed to the fraction of CD8 T lymphocytes expressing this receptor (48). We found that expression of our receptor did not significantly increase cytotoxicity against nontransformed peripheral blood lymphocytes or bone marrow-derived mesenchymal cells. For clinical application, this potential problem should be prevented by careful depletion of T cells from the NK cell product together with transient expression of the receptor by electroporation.

It is well established that donor NK cell alloreactivity suppresses leukemia relapse after allogeneic hematopoietic stem cell transplantation (20, 21). Infusion of NK cells in a nontransplant setting has shown promise in some studies (22, 23), and hence this approach is being actively pursued at several centers using either freshly purified or activated NK cells. The method that we described here offers a new way to increase the antitumor efficacy of NK cell therapy and to widen its application. Stimulation via the NKG2D-DAP10-CD3ζ receptor also resulted in a marked increase in cytokine/chemokine secretion. Thus, NK-derived GM-CSF, IFN-γ, and TNF-α promote monocyte differentiation, macrophage activation and dendritic cell maturation (1, 49, 50). Whether these cellular effects would amplify the antitumor response in vivo is unclear but they should be important during immune responses against pathogens, suggesting that infusion of NKG2D-DAP10-CD3ζ-NK cells should also be tested in the setting of infectious diseases.

No potential conflicts of interest were disclosed.

Conception and design: Y.-H. Chang, D. Campana

Development of methodology: Y.-H. Chang, K. Mimura, K. Kono, D. Campana

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y.-H. Chang, J. Connolly, N. Shimasaki, K. Mimura

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y.-H. Chang, K. Mimura, K. Kono, D. Campana

Writing, review, and/or revision of the manuscript: Y.-H. Chang, K. Kono, D. Campana

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y.-H. Chang, N. Shimasaki

Study supervision: D. Campana

We thank Elaine Coustan-Smith for help with the analysis of chimeric receptor expression, Veonice Au Bijin for the cytokine/chemokine studies, Wang Yilin for help with cell imaging studies, and Stephan Gasser for valuable discussions.

This work was supported by the American Lebanese Syrian Associated Charities (ALSAC) and by a Singapore Translational Research Investigator Award from the National Medical Research Council of Singapore.

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

1.
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
,
Yokoyama
WM
. 
Murine NKG2D and Cd94 are clustered within the natural killer complex and are expressed independently in natural killer cells
.
Proc Natl Acad Sci U S A
1998
;
95
:
6320
5
.
3.
Bauer
S
,
Groh
V
,
Wu
J
,
Steinle
A
,
Phillips
JH
,
Lanier
LL
, et al
Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA
.
Science
1999
;
285
:
727
9
.
4.
Champsaur
M
,
Lanier
LL
. 
Effect of NKG2D ligand expression on host immune responses
.
Immunol Rev
2010
;
235
:
267
85
.
5.
Gasser
S
,
Orsulic
S
,
Brown
EJ
,
Raulet
DH
. 
The DNA damage pathway regulates innate immune system ligands of the NKG2D receptor
.
Nature
2005
;
436
:
1186
90
.
6.
Smyth
MJ
,
Swann
J
,
Cretney
E
,
Zerafa
N
,
Yokoyama
WM
,
Hayakawa
Y
. 
NKG2D function protects the host from tumor initiation
.
J Exp Med
2005
;
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-dependent 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 responses mediated 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 cytotoxicity of activated CD8+ 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 of spontaneous 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 solid tumors
.
Clin Cancer Res
2010
;
16
:
3901
9
.
12.
Raulet
DH
. 
Roles of the NKG2D immunoreceptor and its ligands
.
Nat Rev Immunol
2003
;
3
:
781
90
.
13.
Bryceson
YT
,
Ljunggren
HG
. 
Tumor cell recognition by the NK cell activating receptor NKG2D
.
Eur J Immunol
2008
;
38
:
2957
61
.
14.
Wu
J
,
Song
Y
,
Bakker
AB
,
Bauer
S
,
Spies
T
,
Lanier
LL
, et al
An activating 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 stimulatory versus costimulatory activity of NKG2D
.
Nat Immunol
2002
;
3
:
1142
9
.
16.
Garrity
D
,
Call
ME
,
Feng
J
,
Wucherpfennig
KW
. 
The activating NKG2D receptor assembles in the membrane with two signaling dimers into a hexameric structure
.
Proc Natl Acad Sci U S A
2005
;
102
:
7641
6
.
17.
Horng
T
,
Bezbradica
JS
,
Medzhitov
R
. 
NKG2D signaling is coupled to the 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
.
Blood
2011
;
118
:
3019
27
.
19.
Upshaw
JL
,
Arneson
LN
,
Schoon
RA
,
Dick
CJ
,
Billadeau
DD
,
Leibson
PJ
. 
NKG2D-mediated signaling requires a DAP10-bound Grb2-Vav1 intermediate and phosphatidylinositol-3-kinase in human natural killer cells
.
Nat Immunol
2006
;
7
:
524
32
.
20.
Ruggeri
L
,
Capanni
M
,
Urbani
E
,
Perruccio
K
,
Shlomchik
WD
,
Tosti
A
, et al
Effectiveness of donor natural killer cell alloreactivity in 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 superior survival after unrelated transplantation for acute myelogenous leukemia
.
Blood
2010
;
116
:
2411
9
.
22.
Miller
JS
,
Soignier
Y
,
Panoskaltsis-Mortari
A
,
McNearney
SA
,
Yun
GH
,
Fautsch
SK
, et al
Successful adoptive transfer and in vivo expansion of human 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 haploidentical natural killer cell transplantation in childhood acute myeloid leukemia
.
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 by NKG2D and natural cytotoxicity receptors
.
Eur J Immunol
2004
;
34
:
961
71
.
25.
Imai
C
,
Iwamoto
S
,
Campana
D
. 
Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing 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 bone marrow mesenchymal cells immortalized by enforced expression of telomerase
.
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 cell therapy
.
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 potent cytotoxicity 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
,
Roederer
M
, et al
Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation
.
J Immunol Methods
2003
;
281
:
65
78
.
31.
Wen
AY
,
Sakamoto
KM
,
Miller
LS
. 
The role of the transcription factor CREB 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
.
Immunol Rev
2012
;
246
:
327
45
.
33.
Mahajan
K
,
Mahajan
NP
. 
Shepherding AKT and androgen receptor by Ack1 tyrosine kinase
.
J Cell Physiol
2010
;
224
:
327
33
.
34.
Chang
C
,
Dietrich
J
,
Harpur
AG
,
Lindquist
JA
,
Haude
A
,
Loke
YW
, et al
Cutting edge: KAP10, a novel transmembrane adapter protein genetically linked to DAP12 but with unique signaling properties
.
J Immunol
1999
;
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 of natural killer cells against B-cell malignancies
.
Cytotherapy
2012
;
14
:
830
40
.
36.
Kohn
DB
,
Dotti
G
,
Brentjens
R
,
Savoldo
B
,
Jensen
M
,
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 cells inhibit systemic T-cell lymphoma growth in a manner involving multiple cytokines and cytotoxic pathways
.
Cancer Res
2007
;
67
:
11029
36
.
38.
Barber
A
,
Zhang
T
,
Megli
CJ
,
Wu
J
,
Meehan
KR
,
Sentman
CL
. 
Chimeric NKG2D receptor-expressing T cells as an immunotherapy for multiple myeloma
.
Exp Hematol
2008
;
36
:
1318
28
.
39.
Barber
A
,
Rynda
A
,
Sentman
CL
. 
Chimeric NKG2D expressing T cells eliminate immunosuppression and activate immunity within the ovarian tumor microenvironment
.
J Immunol
2009
;
183
:
6939
47
.
40.
Lehner
M
,
Gotz
G
,
Proff
J
,
Schaft
N
,
Dorrie
J
,
Full
F
, et al
Redirecting T cells to Ewing's sarcoma family of tumors by a chimeric NKG2D receptor 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
. 
Broad tumor-associated expression and recognition by tumor-derived gamma delta T cells of MICA and MICB
.
Proc Natl Acad Sci U S A
1999
;
96
:
6879
84
.
42.
Pende
D
,
Rivera
P
,
Marcenaro
S
,
Chang
CC
,
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 cell cytotoxicity
.
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 in the target cell membrane
.
J Immunol
2011
;
186
:
5538
42
.
44.
Salih
HR
,
Rammensee
HG
,
Steinle
A
. 
Cutting edge: down-regulation of MICA on human tumors by proteolytic shedding
.
J Immunol
2002
;
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 to membrane microdomains and promotes its shedding
.
Eur J Immunol
2011
;
41
:
3667
76
.
46.
Coudert
JD
,
Zimmer
J
,
Tomasello
E
,
Cebecauer
M
,
Colonna
M
,
Vivier
E
, et al
Altered NKG2D function in NK cells induced by chronic exposure to NKG2D ligand-expressing tumor cells
.
Blood
2005
;
106
:
1711
7
.
47.
Oppenheim
DE
,
Roberts
SJ
,
Clarke
SL
,
Filler
R
,
Lewis
JM
,
Tigelaar
RE
, et al
Sustained localized expression of ligand for the activating NKG2D receptor impairs natural cytotoxicity in vivo and reduces tumor immunosurveillance
.
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 recruits NKG2D receptor-expressing cytotoxic T cells independent of T cell receptor 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 and the differentiation of monocytes into dendritic cells at the site of infection
.
Immunity
2012
;
36
:
1047
59
.
50.
Spear
P
,
Barber
A
,
Rynda-Apple
A
,
Sentman
CL
. 
Chimeric antigen receptor T cells shape myeloid cell function within the tumor microenvironment through IFN-gamma and GM-CSF
.
J Immunol
2012
;
188
:
6389
98
.

Supplementary data