Article Figures & Data
Figures
- 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 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 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 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 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 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.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Methods, Figure Legends - PDF file - 298K, SF1. Cytokine/chemokine secretion of mock- and NKG2D-DAP10-CD3-transduced NK cells from 3 donors after incubation with a biotin-conjugated anti-NKG2D agonistic antibody (clone 1D11; eBioscience) and anti-biotin beads (MACSiBeads; Miltenyi Biotec) (see also Fig. 4 and Supplementary Table S1). Supplemental Movie. Live cell confocal photography of mock- (left panel) and NKG2D-DAP10-CD3-transduced NK cells (right panel) co-cultured U2 OS osteosarcoma cells. NK cells were labeled with PKH26 (Sigma) before culture and appear red. Microscopy was performed with a Nikon TE2000E2 microscope equipped with a Nikon C1Si confocal using 488nm and 561nm DPSS lasers for excitation. Temperature was maintained at 37oC and CO2 at 5% using an environmental control chamber. Images were acquired with a Nikon 40x 1.3 NA DIC objective every 30s for 2hr using Nikon EZC1 software. Imaris (Bitplane Scientific Software) was used to analyze speed and displacement of NK cells. Supplementary Table S1. Cytokine/chemokine secretion (mean � SD pg/mL) after stimulation of mock- or NKG2D-DAP10-CD3-transduced NK cells with anti-NKG2Da.
- Supplementary Movie 1 - WMV file - 3260K, Live cell confocal photography of mock- (left panel) and NKG2D-DAP10-CD3ξ-transduced NK cells (right panel) co-cultured U2 OS osteosarcoma cells. NK cells were labeled with PKH26 (Sigma) before culture and appear red. Microscopy was performed with a Nikon TE2000E2 microscope equipped with a Nikon C1Si confocal using 488nm and 561nm DPSS lasers for excitation. Temperature was maintained at 37oC and CO2 at 5% using an environmental control chamber. Images were acquired with a Nikon 40x 1.3 NA DIC objective every 30s for 2hr using Nikon EZC1 software. Imaris (Bitplane Scientific Software) was used to analyze speed and displacement of NK cells.
Volume 73, Issue 6
