Journal: bioRxiv

Article Title: γδ T cells are effectors of immune checkpoint blockade in mismatch repair-deficient colon cancers with antigen presentation defects

doi: 10.1101/2021.10.14.464229

Figure Lengend Snippet: a. Flow cytometry gating strategy on PDTO cells for analysis of surface staining. Selected cells were gated on single, live cells before quantification of staining signal. b. Histogram representation and count for surface staining of MHC-I, PD-L1, and β2m expression on two PDTO lines B2MWT and B2MKO after IFNγ pre-stimulation. Staining with isotype antibodies for each fluorochrome (PE, APC and FITC) were included as negative control. c. Flow cytometry gating strategy on γδ T cell samples for analysis of intracellular staining to test antitumor reactivity upon PDTO stimulation. Lymphocyte population was further gated on single cells, live and CD3+ cells, γδ TCR+ cells and CD8+ as well as CD8–CD4– cells. Reactivity of the sample was based on IFNγ+ cells of the selected population. d. Histogram representation and count for surface staining of NKG2D ligands MICA/B, ULBP1, ULBP2/5/6, ULBP3, and ULBP4 on two PDTO lines B2MWT and B2MKO after IFNγ pre-stimulation. e. Flow cytometry gating strategy on γδ T cell samples for analysis of intracellular staining after stimulation with PDTOs in the presence of NKG2D ligand blocking. Lymphocyte population was further gated on single cells, live and CD3+ cells, followed by γδ TCR+ and CD8+ as well as CD8– cells. Reactivity of final population was based on IFNγ+ or CD107a+ cells.

Article Snippet: Briefly, cells were incubated with human Fc receptor block (BioLegend) and stained with the different cell surface antibodies (1:10 anti-CD112-PE [clone R2.525, BD Biosciences], 1:10 anti-CD155-PE [clone 300907, R&D Systems], 1:50 anti-CD277/BTN3A1-PE [clone BT3.1, Miltenyi], 1:100 anti-HLA-A,B,C-FITC [clone W6/32, eBioscience], 1:20 anti-HLA-E­BV421 [clone 3D12, BioLegend], 1:20 anti-HLA-G-APC [clone 87G, BioLegend], 1:300 anti-MICA/B-PE [clone 6D4, BioLegend], 1:10 anti-ULBP1-PE [clone 170818, R&D Systems], 1:20 anti-ULBP2/5/6-PE [clone 165903, R&D Systems], 1:20 anti-ULBP3-PE [clone 166510, R&D Systems], or 1:20 anti-ULBP4-PE [clone 709116, R&D Systems] for 45 min at 4°C.

Techniques: Flow Cytometry, Staining, Expressing, Negative Control, Blocking Assay

Journal: Frontiers in Immunology

Article Title: Low-Dose Gemcitabine Treatment Enhances Immunogenicity and Natural Killer Cell-Driven Tumor Immunity in Lung Cancer

doi: 10.3389/fimmu.2020.00331

Figure Lengend Snippet: List of primers used in qRT-PCR.

Article Snippet: Anti-human ULBP1, ULBP2/5/6, and ULBP3 antibody were purchased from R&D Systems.

Techniques:

Journal: Frontiers in Immunology

Article Title: Low-Dose Gemcitabine Treatment Enhances Immunogenicity and Natural Killer Cell-Driven Tumor Immunity in Lung Cancer

doi: 10.3389/fimmu.2020.00331

Figure Lengend Snippet: Gemcitabine up-regulates NKG2D ligands in lung cancer cells. (A) qRT-PCR of H60, Raet-1 , and Ulbp1 mRNA in LLC cells. (B) qRT-PCR of major histocompatibility complex class I polypeptide-related sequence A ( MICA ), MICB , and UL16 binding protein ( ULBP ) 1-6 mRNA in A549 cells. Data are the fold-change of mRNA expression in gemcitabine- and cisplatin-treated cells relative to untreated cells. (C) Surface expression of MICA/B, ULBP1, ULBP2/5/6, and ULBP3 was measured by flow cytometry on A549 cells treated with vehicle control (DMSO), gemcitabine or cisplatin. Data are representative of three independent experiments. One-way analysis of variance (ANOVA) was used. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Anti-human ULBP1, ULBP2/5/6, and ULBP3 antibody were purchased from R&D Systems.

Techniques: Quantitative RT-PCR, Immunopeptidomics, Sequencing, Binding Assay, Expressing, Flow Cytometry, Control

Journal: The EMBO Journal

Article Title: NKG2D discriminates diverse ligands through selectively mechano‐regulated ligand conformational changes

doi: 10.15252/embj.2021107739

Figure Lengend Snippet: A Diagrammatic sketch of in situ binding kinetic assay and functionalization of RBC. B Representative adhesion frequency ( P a ) versus contact duration ( t c ) curves for NKG2D expressing NK cells ( n ≥ 3) in contact with RBCs ( n ≥ 3) coated with a ligand (MICA in red, MICB in orange, ULBP1 in green, or ULBP3 in blue) at different contact durations, fitted by a non‐linear in situ binding‐kinetic model (Huang et al , ). Site densities of NKG2D ( m r ) and its ligands ( m l ) are indicated. C–E In situ force‐free affinities (C), on‐rates (D), and off‐rates (E) of NKG2D binding with indicated ligands from mammalian cells. The in situ force‐free kinetics were obtained from fittings with an in situ binding‐kinetic model in (B). F Detection range comparison in affinity measurement of NKG2D and indicated ligands between in situ and in‐solution assay. Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1. Data information: In‐solution affinities of proteins purified from E. coli were from previous study (McFarland & Strong, ). In‐solution affinities of proteins purified from mammalian cells were measured by BLI (Fig ). Every dot in (C–E) represents one independent binding experiment. Error bars in (B‐E) are mean ± SEM for at least three independent biological experiments where * P < 0.05, ** P < 0.01 (two‐tailed unpaired t ‐test). Source data are available online for this figure.

Article Snippet: To measure the site densities of NKG2D receptor, NK cells were incubated with PE‐labeled mouse anti‐human NKG2D monoclonal antibody 5C6 (12‐5879‐42, eBioscience, USA) or isotype control at 2.5 μg/ml in 100 μl of FACS Buffer (DMEM, 5 mM EDTA and 1% BSA) at RT for 30 min. To measure the site densities of NKG2D ligands linked on the surfaces of RBCs via biotin‐streptavidin coupling, NKG2D ligand‐coated RBCs were incubated with monoclonal antibodies of PE‐conjugated NKG2D ligands (mouse anti‐human MICA antibody, 12302‐MM04‐P, Sino Biological Inc., China; mouse anti‐human MICB antibody, 10759‐MM12‐P, Sino Biological Inc., China; mouse anti‐human ULBP1 antibody, FAB1380P, R&D Systems, USA; mouse anti‐human ULBP3 antibody, FAB1380P, R&D Systems, USA) or isotype controls according to the manufacturer’s instructions in 100 μl of FACS Buffer (DMEM, 5 mM EDTA, and 1% BSA) at RT for 30 min. NK cells or RBCs incubated with corresponding antibodies were analyzed by a flow cytometer (CytoFLEX S, Beckman Coulter, USA) together with Quantibrite (340495.0, BD Biosciences, USA).

Techniques: In Situ, Binding Assay, Kinetic Assay, Expressing, Comparison, Purification, Two Tailed Test

Journal: The EMBO Journal

Article Title: NKG2D discriminates diverse ligands through selectively mechano‐regulated ligand conformational changes

doi: 10.15252/embj.2021107739

Figure Lengend Snippet: A Photomicrographs of micropipette adhesion frequency assay in which NK cell controlled by a micropipette approach, contact, detach with RBC with/without adhesion as marked. Scale bars in (A) represent 5 μm. B–G Flow cytometry analysis of NKG2D and ligands by specific antibodies along with four standard calibration beads (Gray histogram means isotype control, histogram of other colors means sample). NKG2D ligands purified from 293F cells were biotinylated linked to the membrane of SA‐coated human RBC cells. MICA‐linked (B), MICB‐linked (C), ULBP1‐linked (D), and ULBP3‐linked (E) RBC cells were incubated with PE‐labeled primary monoclonal antibodies and analyzed by flow cytometry. NK cells were incubated with PE‐labeled primary mAb of NKG2D and analyzed by flow cytometry (F). PE standard calibration beads were analyzed along with the isotype control for nonspecific binding (G). H A calibration curve of log of PE molecules/bead (provided by the manufacturer) versus log of measured fluorescence intensity PE‐A was plotted based on data of four standard beads (filled circles). The site density of MICA on RBC was calculated by comparing the log of fluorescence intensity of the sample (open square) with the calibration curve after subtracting negative control fluorescence intensity. I–O BLI binding curves of NKG2D receptor at serious concentrations with immobilized MICA (I), MICB (J), ULBP1 (K), and ULBP3 (L) and the corresponding binding affinities (M), on‐rates (N), and off‐rates (O) derived from BLI experiments. Concentrations of NKG2D were 200, 100, 50, 25 and 12.5 nM labeled from dark color to light color. Error bars in (M–O) represent mean ± SEM for biological triplicate experiments.

Article Snippet: To measure the site densities of NKG2D receptor, NK cells were incubated with PE‐labeled mouse anti‐human NKG2D monoclonal antibody 5C6 (12‐5879‐42, eBioscience, USA) or isotype control at 2.5 μg/ml in 100 μl of FACS Buffer (DMEM, 5 mM EDTA and 1% BSA) at RT for 30 min. To measure the site densities of NKG2D ligands linked on the surfaces of RBCs via biotin‐streptavidin coupling, NKG2D ligand‐coated RBCs were incubated with monoclonal antibodies of PE‐conjugated NKG2D ligands (mouse anti‐human MICA antibody, 12302‐MM04‐P, Sino Biological Inc., China; mouse anti‐human MICB antibody, 10759‐MM12‐P, Sino Biological Inc., China; mouse anti‐human ULBP1 antibody, FAB1380P, R&D Systems, USA; mouse anti‐human ULBP3 antibody, FAB1380P, R&D Systems, USA) or isotype controls according to the manufacturer’s instructions in 100 μl of FACS Buffer (DMEM, 5 mM EDTA, and 1% BSA) at RT for 30 min. NK cells or RBCs incubated with corresponding antibodies were analyzed by a flow cytometer (CytoFLEX S, Beckman Coulter, USA) together with Quantibrite (340495.0, BD Biosciences, USA).

Techniques: Flow Cytometry, Control, Purification, Membrane, Incubation, Labeling, Bioprocessing, Binding Assay, Fluorescence, Negative Control, Derivative Assay

Journal: The EMBO Journal

Article Title: NKG2D discriminates diverse ligands through selectively mechano‐regulated ligand conformational changes

doi: 10.15252/embj.2021107739

Figure Lengend Snippet: A–C Schematic diagram (A) of IFN‐γ release assay and IFN‐γ production (B) by human peripheral NK cells stimulated with plate‐coated NKG2D ligands (MICA in red, MICB in green and ULBP3 in blue) assessed by Cytometric Bead Array, of which are their half‐maximal effective concentration (EC 50 ) (C). D–F Plots of reciprocals of EC 50 versus the effective in situ affinities ( A c K a ) (D), the effective in situ on‐rate ( A c K on ) (E) and in situ off‐rate ( k off ) (F). Data information: The IFN‐γ release assay in (B) was one representative experiment of three total independent experiments. Data points in (B) and bars in (C) represent mean values. Error bars in (B) and (C) represent mean ± SEM. *** P < 0.001, **** P < 0.0001 (two‐tailed unpaired t ‐test). Source data are available online for this figure.

Article Snippet: To measure the site densities of NKG2D receptor, NK cells were incubated with PE‐labeled mouse anti‐human NKG2D monoclonal antibody 5C6 (12‐5879‐42, eBioscience, USA) or isotype control at 2.5 μg/ml in 100 μl of FACS Buffer (DMEM, 5 mM EDTA and 1% BSA) at RT for 30 min. To measure the site densities of NKG2D ligands linked on the surfaces of RBCs via biotin‐streptavidin coupling, NKG2D ligand‐coated RBCs were incubated with monoclonal antibodies of PE‐conjugated NKG2D ligands (mouse anti‐human MICA antibody, 12302‐MM04‐P, Sino Biological Inc., China; mouse anti‐human MICB antibody, 10759‐MM12‐P, Sino Biological Inc., China; mouse anti‐human ULBP1 antibody, FAB1380P, R&D Systems, USA; mouse anti‐human ULBP3 antibody, FAB1380P, R&D Systems, USA) or isotype controls according to the manufacturer’s instructions in 100 μl of FACS Buffer (DMEM, 5 mM EDTA, and 1% BSA) at RT for 30 min. NK cells or RBCs incubated with corresponding antibodies were analyzed by a flow cytometer (CytoFLEX S, Beckman Coulter, USA) together with Quantibrite (340495.0, BD Biosciences, USA).

Techniques: Release Assay, Concentration Assay, In Situ, Two Tailed Test

Journal: The EMBO Journal

Article Title: NKG2D discriminates diverse ligands through selectively mechano‐regulated ligand conformational changes

doi: 10.15252/embj.2021107739

Figure Lengend Snippet: A, B Flow cytometry analysis of the percentages of CD107a + (A) cells pERK + cells (B) under stimulation of different NKG2D ligands (MICA in red, MICB in orange, ULBLP3 in blue) compared with SA negative control (gray). C, D Corresponding quantification of percentages of CD107a + cells and pERK + NK cells in (A) and (B). E, F Plots and Pearson correlation analysis of NKG2D ligands stimulated percentages of CD107a + (E) and pERK + (F) NK cells with their reciprocals of EC 50 to release IFN‐γ. G IFN‐γ release (one representative experiment of total three independent biological experiments) of periphery human NK cells under the stimulation of soluble NKG2D ligands at 100 nM. Data information: Every dot in (C) and (D) represents one independent biological experiment. Data are mean ± SEM for biological triplicate experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (two‐tailed unpaired t ‐test).

Article Snippet: To measure the site densities of NKG2D receptor, NK cells were incubated with PE‐labeled mouse anti‐human NKG2D monoclonal antibody 5C6 (12‐5879‐42, eBioscience, USA) or isotype control at 2.5 μg/ml in 100 μl of FACS Buffer (DMEM, 5 mM EDTA and 1% BSA) at RT for 30 min. To measure the site densities of NKG2D ligands linked on the surfaces of RBCs via biotin‐streptavidin coupling, NKG2D ligand‐coated RBCs were incubated with monoclonal antibodies of PE‐conjugated NKG2D ligands (mouse anti‐human MICA antibody, 12302‐MM04‐P, Sino Biological Inc., China; mouse anti‐human MICB antibody, 10759‐MM12‐P, Sino Biological Inc., China; mouse anti‐human ULBP1 antibody, FAB1380P, R&D Systems, USA; mouse anti‐human ULBP3 antibody, FAB1380P, R&D Systems, USA) or isotype controls according to the manufacturer’s instructions in 100 μl of FACS Buffer (DMEM, 5 mM EDTA, and 1% BSA) at RT for 30 min. NK cells or RBCs incubated with corresponding antibodies were analyzed by a flow cytometer (CytoFLEX S, Beckman Coulter, USA) together with Quantibrite (340495.0, BD Biosciences, USA).

Techniques: Flow Cytometry, Negative Control, Two Tailed Test

Journal: The EMBO Journal

Article Title: NKG2D discriminates diverse ligands through selectively mechano‐regulated ligand conformational changes

doi: 10.15252/embj.2021107739

Figure Lengend Snippet: A, B Representative raw (black) and low‐frequency drift corrected (red) tracked displacements ( X m ) (A) and corresponding histograms and Gaussian fits (B) of BFP. The corrected variance Var( X m ) was obtained from (A). C Var( X m ) is plotted versus reciprocal of suction pressure 1/Δ p and fitted by the motion blur model (Chen et al , ; Ju & Zhu, ). D The motion‐blur corrected variance Var(X) calculated from Var( X m ) is plotted versus 1/ k p, which is the BFP spring constant calculated from Evans model (Chen et al , ; Ju & Zhu, ). E Photomicrograph of BFP. An NK cell and an RBC with a probe bead attached to its apex were aspirated by two opposing micropipettes respectively. The Region of Interest (ROI) for tracking the edge of the probe bead as shown in dashed lines. F, G Representative force versus time curve for no adhesion (F) and force ramp (G). H Force‐dependent bond lifetimes of NKG2D and various ligands at 5 pN, 10 pN, and 15 pN. I Illustration of the retract phase (blue line, the slope of which is the loading rate) in an example event of BFP bond lifetime. J–N Scatter plot of loading rates (J) of NKG2D interacting with MICA (red, n = 1,499 bond lifetimes), MICB (orange, n = 1,847 bond lifetimes), ULBPL1 (green, n = 530 bond lifetimes), and ULBP3 (blue, n = 1,234 bond lifetimes) and their respective distributions and descriptive statistics for MICA (K), MICB (L) ULBP1 (M), and ULBP3 (N) interacting with NKG2D. The bond lifetimes are from at least 19 NK cell‐bead pairs of at least four independent biological experiments. Data information: Every dot in (H) represents one bond lifetime of NKG2D binding with corresponding ligand from at least 19 NK cell‐bead pairs in at least 4 independent biological experiments. The scale bar in the picture represents 5 μm. Error bars in (C, D, H, and J) represent mean ± SEM for at least three independent biological experiments. P = 0.1410 between groups (one‐way ANOVA) in (J).

Article Snippet: To measure the site densities of NKG2D receptor, NK cells were incubated with PE‐labeled mouse anti‐human NKG2D monoclonal antibody 5C6 (12‐5879‐42, eBioscience, USA) or isotype control at 2.5 μg/ml in 100 μl of FACS Buffer (DMEM, 5 mM EDTA and 1% BSA) at RT for 30 min. To measure the site densities of NKG2D ligands linked on the surfaces of RBCs via biotin‐streptavidin coupling, NKG2D ligand‐coated RBCs were incubated with monoclonal antibodies of PE‐conjugated NKG2D ligands (mouse anti‐human MICA antibody, 12302‐MM04‐P, Sino Biological Inc., China; mouse anti‐human MICB antibody, 10759‐MM12‐P, Sino Biological Inc., China; mouse anti‐human ULBP1 antibody, FAB1380P, R&D Systems, USA; mouse anti‐human ULBP3 antibody, FAB1380P, R&D Systems, USA) or isotype controls according to the manufacturer’s instructions in 100 μl of FACS Buffer (DMEM, 5 mM EDTA, and 1% BSA) at RT for 30 min. NK cells or RBCs incubated with corresponding antibodies were analyzed by a flow cytometer (CytoFLEX S, Beckman Coulter, USA) together with Quantibrite (340495.0, BD Biosciences, USA).

Techniques: Binding Assay

Journal: The EMBO Journal

Article Title: NKG2D discriminates diverse ligands through selectively mechano‐regulated ligand conformational changes

doi: 10.15252/embj.2021107739

Figure Lengend Snippet: A, B Schematic diagram (A) and experimental setup (B) of BFP assay to characterize force‐dependent dissociation kinetics of NKG2D binding with different ligands. C Verification of the binding specificity of NKG2D with different ligands. D Representative force versus time curve for measuring single NKG2D‐ligand bond lifetime. E, F Force‐dependent bond lifetimes of NKG2D and various ligands at 5 pN, 10 pN and 15 pN (E) and full force spectrum (F). G Ratios of average bond lifetimes for NKG2D/MICA to that of NKG2D and other ligands. Data information: Every dot in (C) represents the adhesion frequency of one cell‐bead pair and the experimental data came from at least two to three repeated trials. Bond lifetimes in (E) and (F) (in which n = 1,505 for MICA, n = 1,852 for MICB, n = 531 for ULBP1, n = 1,241 for ULBP3) of all ligands with NKG2D came from at least 19 NK cell‐bead pairs of at least four independent biological experiments. Horizontal lines in (C), bars in (E), and data points in (F) represent mean values. Error bars in (C), (E), and (F) represent mean ± SEM. Source data are available online for this figure.

Article Snippet: To measure the site densities of NKG2D receptor, NK cells were incubated with PE‐labeled mouse anti‐human NKG2D monoclonal antibody 5C6 (12‐5879‐42, eBioscience, USA) or isotype control at 2.5 μg/ml in 100 μl of FACS Buffer (DMEM, 5 mM EDTA and 1% BSA) at RT for 30 min. To measure the site densities of NKG2D ligands linked on the surfaces of RBCs via biotin‐streptavidin coupling, NKG2D ligand‐coated RBCs were incubated with monoclonal antibodies of PE‐conjugated NKG2D ligands (mouse anti‐human MICA antibody, 12302‐MM04‐P, Sino Biological Inc., China; mouse anti‐human MICB antibody, 10759‐MM12‐P, Sino Biological Inc., China; mouse anti‐human ULBP1 antibody, FAB1380P, R&D Systems, USA; mouse anti‐human ULBP3 antibody, FAB1380P, R&D Systems, USA) or isotype controls according to the manufacturer’s instructions in 100 μl of FACS Buffer (DMEM, 5 mM EDTA, and 1% BSA) at RT for 30 min. NK cells or RBCs incubated with corresponding antibodies were analyzed by a flow cytometer (CytoFLEX S, Beckman Coulter, USA) together with Quantibrite (340495.0, BD Biosciences, USA).

Techniques: Binding Assay

Journal: The EMBO Journal

Article Title: NKG2D discriminates diverse ligands through selectively mechano‐regulated ligand conformational changes

doi: 10.15252/embj.2021107739

Figure Lengend Snippet: A, B SMD snapshots of NKG2D dissociation with MICA (A) and ULBP3 (B) in the presence of force (directions are indicated by black arrows). C The force versus extension curves from the simulations shown in (A) and (B). Occurrence of the sudden extension changes are indicated in the shaded area and time points correspond with the snapshots in (A) and (B) are marked. D, E Zoomed‐in binding interfaces of NKG2D with MICA (D) or ULBP3 (E), corresponding with the configuration (shown as gray dashed box in (A) and (B), respectively). F Distance versus time curves for force‐induced H‐bond formation between indicated residues within NKG2D‐MICA binding interfaces. The dashed red lines represent H‐bonds whose distances are < 3.5 Å. Source data are available online for this figure.

Article Snippet: To measure the site densities of NKG2D receptor, NK cells were incubated with PE‐labeled mouse anti‐human NKG2D monoclonal antibody 5C6 (12‐5879‐42, eBioscience, USA) or isotype control at 2.5 μg/ml in 100 μl of FACS Buffer (DMEM, 5 mM EDTA and 1% BSA) at RT for 30 min. To measure the site densities of NKG2D ligands linked on the surfaces of RBCs via biotin‐streptavidin coupling, NKG2D ligand‐coated RBCs were incubated with monoclonal antibodies of PE‐conjugated NKG2D ligands (mouse anti‐human MICA antibody, 12302‐MM04‐P, Sino Biological Inc., China; mouse anti‐human MICB antibody, 10759‐MM12‐P, Sino Biological Inc., China; mouse anti‐human ULBP1 antibody, FAB1380P, R&D Systems, USA; mouse anti‐human ULBP3 antibody, FAB1380P, R&D Systems, USA) or isotype controls according to the manufacturer’s instructions in 100 μl of FACS Buffer (DMEM, 5 mM EDTA, and 1% BSA) at RT for 30 min. NK cells or RBCs incubated with corresponding antibodies were analyzed by a flow cytometer (CytoFLEX S, Beckman Coulter, USA) together with Quantibrite (340495.0, BD Biosciences, USA).

Techniques: Binding Assay

Journal: The EMBO Journal

Article Title: NKG2D discriminates diverse ligands through selectively mechano‐regulated ligand conformational changes

doi: 10.15252/embj.2021107739

Figure Lengend Snippet: A–D Distance versus time curves for force‐induced binding residues within NKG2D and loop1 (A) and loop2 (B) in α1 domain of MICA and loop1 (C) and loop2 (D) in α1 domain of ULBP3. A‐D showed the minimum distances between NKG2D and two loops in α1 domain of MICA or ULBP3 during dissociation under mechanical force. E–I SMD snapshots of NKG2D dissociation with MICA 3A (E) and TAT mutants (F) in the absence or presence of force (directions are indicated by black arrows), their force versus extension curves (G), and their respective zoomed‐in (from gray dashed box and purple dashed box in (E) and (F), respectively) binding interfaces of NKG2D with MICA 3A (H) or TAT mutant (I). Extension transition points are indicated by circles in (G). J, K Distance versus time curves for force‐induced binding residues within NKG2D‐MICA residue 15 of MICA 3A (J) or TAT mutant (K) binding interfaces. E‐K showed that MICA mutants weaken stability of the intermediate states during NKG2D dissociation with MICA 3A mutant and MICA TAT mutant. The similar intermediate states are found for NKG2D dissociation with MICA 3A and MICA TAT mutants compared to MICA WT; however, there is only one new H‐bond formation between NKG2D K186 and backbone oxygen atom of MICA mutants, compared to 2–3 H‐bonds for MICA WT.

Article Snippet: To measure the site densities of NKG2D receptor, NK cells were incubated with PE‐labeled mouse anti‐human NKG2D monoclonal antibody 5C6 (12‐5879‐42, eBioscience, USA) or isotype control at 2.5 μg/ml in 100 μl of FACS Buffer (DMEM, 5 mM EDTA and 1% BSA) at RT for 30 min. To measure the site densities of NKG2D ligands linked on the surfaces of RBCs via biotin‐streptavidin coupling, NKG2D ligand‐coated RBCs were incubated with monoclonal antibodies of PE‐conjugated NKG2D ligands (mouse anti‐human MICA antibody, 12302‐MM04‐P, Sino Biological Inc., China; mouse anti‐human MICB antibody, 10759‐MM12‐P, Sino Biological Inc., China; mouse anti‐human ULBP1 antibody, FAB1380P, R&D Systems, USA; mouse anti‐human ULBP3 antibody, FAB1380P, R&D Systems, USA) or isotype controls according to the manufacturer’s instructions in 100 μl of FACS Buffer (DMEM, 5 mM EDTA, and 1% BSA) at RT for 30 min. NK cells or RBCs incubated with corresponding antibodies were analyzed by a flow cytometer (CytoFLEX S, Beckman Coulter, USA) together with Quantibrite (340495.0, BD Biosciences, USA).

Techniques: Binding Assay, Mutagenesis, Residue

Journal: The EMBO Journal

Article Title: NKG2D discriminates diverse ligands through selectively mechano‐regulated ligand conformational changes

doi: 10.15252/embj.2021107739

Figure Lengend Snippet: A, B Abolishments of force‐induced binding residues by 3A mutations (MICA 3A, n = 526) and TAT mutations (MICA TAT, n = 580) impairs NKG2D’s catch bond with MICA WT ( n = 1,505) (A) and IFN‐γ release (B). C–E EC 50 (C) and IFN‐γ release of NK cells stimulated by indicated MICA mutants at a concentration of 11 nM (D) and 22 nM (E). Data information: Bond lifetimes in (A) of MICA WT and mutants with NKG2D came from at least 21 pairs of cells and beads of at least three repeated experiments. IFN‐γ release of NK cells in (B) was one representative experiment of three total independent experiments. Data points in (A) and (B), horizontal lines in (C), bars in (E), and data point in (F) represent mean values. Error bars in (A–E) represent mean ± SEM for biological triplicate experiments. ** P < 0.01, *** P < 0.001, **** P < 0.0001 (two‐tailed unpaired t ‐test). Source data are available online for this figure.

Article Snippet: To measure the site densities of NKG2D receptor, NK cells were incubated with PE‐labeled mouse anti‐human NKG2D monoclonal antibody 5C6 (12‐5879‐42, eBioscience, USA) or isotype control at 2.5 μg/ml in 100 μl of FACS Buffer (DMEM, 5 mM EDTA and 1% BSA) at RT for 30 min. To measure the site densities of NKG2D ligands linked on the surfaces of RBCs via biotin‐streptavidin coupling, NKG2D ligand‐coated RBCs were incubated with monoclonal antibodies of PE‐conjugated NKG2D ligands (mouse anti‐human MICA antibody, 12302‐MM04‐P, Sino Biological Inc., China; mouse anti‐human MICB antibody, 10759‐MM12‐P, Sino Biological Inc., China; mouse anti‐human ULBP1 antibody, FAB1380P, R&D Systems, USA; mouse anti‐human ULBP3 antibody, FAB1380P, R&D Systems, USA) or isotype controls according to the manufacturer’s instructions in 100 μl of FACS Buffer (DMEM, 5 mM EDTA, and 1% BSA) at RT for 30 min. NK cells or RBCs incubated with corresponding antibodies were analyzed by a flow cytometer (CytoFLEX S, Beckman Coulter, USA) together with Quantibrite (340495.0, BD Biosciences, USA).

Techniques: Binding Assay, Concentration Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: NKG2D discriminates diverse ligands through selectively mechano‐regulated ligand conformational changes

doi: 10.15252/embj.2021107739

Figure Lengend Snippet: A–C Plots and Pearson correlation analysis of reciprocals of EC 50 versus the force‐dependent in situ k off at 5 pN (A), 10 pN (B) 15pN (C). D Heatmap of force‐dependent in situ affinities A c K a of NKG2D binding with different ligands. E–G Plots and Pearson correlation analysis of reciprocals of EC 50 versus the force‐dependent effective in situ affinities A c K a at 5 pN (E), 10 pN (F), and 15 pN (G). H Comparison of corresponding Pearson coefficients between all NKG2D ligands in situ binding kinetics and ligand‐induced NK functions. I Detection range comparison in force‐dependent affinity of NKG2D and indicated ligands under difference force. Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1. Source data are available online for this figure.

Article Snippet: To measure the site densities of NKG2D receptor, NK cells were incubated with PE‐labeled mouse anti‐human NKG2D monoclonal antibody 5C6 (12‐5879‐42, eBioscience, USA) or isotype control at 2.5 μg/ml in 100 μl of FACS Buffer (DMEM, 5 mM EDTA and 1% BSA) at RT for 30 min. To measure the site densities of NKG2D ligands linked on the surfaces of RBCs via biotin‐streptavidin coupling, NKG2D ligand‐coated RBCs were incubated with monoclonal antibodies of PE‐conjugated NKG2D ligands (mouse anti‐human MICA antibody, 12302‐MM04‐P, Sino Biological Inc., China; mouse anti‐human MICB antibody, 10759‐MM12‐P, Sino Biological Inc., China; mouse anti‐human ULBP1 antibody, FAB1380P, R&D Systems, USA; mouse anti‐human ULBP3 antibody, FAB1380P, R&D Systems, USA) or isotype controls according to the manufacturer’s instructions in 100 μl of FACS Buffer (DMEM, 5 mM EDTA, and 1% BSA) at RT for 30 min. NK cells or RBCs incubated with corresponding antibodies were analyzed by a flow cytometer (CytoFLEX S, Beckman Coulter, USA) together with Quantibrite (340495.0, BD Biosciences, USA).

Techniques: In Situ, Binding Assay, Comparison

Journal: The EMBO Journal

Article Title: NKG2D discriminates diverse ligands through selectively mechano‐regulated ligand conformational changes

doi: 10.15252/embj.2021107739

Figure Lengend Snippet: Schematic diagram of NKG2D‐ligand combination and dissociation model. NKG2D binds and dissociates with multiple ligands at different initial on‐rates ( k on‐initial ) and off‐rates ( k off ). Ubiquitous mechanical forces in vivo regulate the disassociation of NKG2D and ligands. Probabilities of productive signals of different ligands binding with NKG2D when at forces of 0 pN, 5 pN, 10 pN, and 15 pN, respectively. Probabilities of output signals at 300s varied with force‐dependent off‐rates of the three ligands, MICA, MICB, and ULBP3. Off‐rates at different mechanical forces measured by experiments had been marked by specific symbols: pentagram, square, circle, and right‐pointing triangle represented off‐rates at 0 pN, 5 pN, 10 pN, and 15 pN, respectively. Contour plots showed probabilities of output signals at 300s produced by continuously variable initial on‐rates and force‐dependent off‐rates.

Article Snippet: To measure the site densities of NKG2D receptor, NK cells were incubated with PE‐labeled mouse anti‐human NKG2D monoclonal antibody 5C6 (12‐5879‐42, eBioscience, USA) or isotype control at 2.5 μg/ml in 100 μl of FACS Buffer (DMEM, 5 mM EDTA and 1% BSA) at RT for 30 min. To measure the site densities of NKG2D ligands linked on the surfaces of RBCs via biotin‐streptavidin coupling, NKG2D ligand‐coated RBCs were incubated with monoclonal antibodies of PE‐conjugated NKG2D ligands (mouse anti‐human MICA antibody, 12302‐MM04‐P, Sino Biological Inc., China; mouse anti‐human MICB antibody, 10759‐MM12‐P, Sino Biological Inc., China; mouse anti‐human ULBP1 antibody, FAB1380P, R&D Systems, USA; mouse anti‐human ULBP3 antibody, FAB1380P, R&D Systems, USA) or isotype controls according to the manufacturer’s instructions in 100 μl of FACS Buffer (DMEM, 5 mM EDTA, and 1% BSA) at RT for 30 min. NK cells or RBCs incubated with corresponding antibodies were analyzed by a flow cytometer (CytoFLEX S, Beckman Coulter, USA) together with Quantibrite (340495.0, BD Biosciences, USA).

Techniques: In Vivo, Binding Assay, Produced

Journal: Clinical Cancer Research

Article Title: NKG2D Ligand Expression in Human Colorectal Cancer Reveals Associations with Prognosis and Evidence for Immunoediting

doi: 10.1158/1078-0432.ccr-09-0991

Figure Lengend Snippet: Fig. 2. Typical staining for MIC, RAET1E, RAET1G, ULBP1, ULBP2, and ULBP3. The photographs presented are from representative tissue microarray cores showing high (A) and low (B) expression at ×100 magnification. Bar, 0 to 500 μm in 100-μm increments.

Article Snippet: Anti-MIC monoclonal antibody SR99 (a kind gift from Dr. Sophie Caillat-Zucman, Institut National de la Santé et de la Recherche Médicale, Unité 561, Hôpital Saint Vincent de Paul, Paris, France), anti-ULBP1 goat polyclonal antibody (R&D Systems, Inc.), antiULBP2 monoclonal antibody M311 (a kind gift from Dr. David Cosman, Amgen, Seattle, WA), anti-ULBP3 goat polyclonal antibody (R&D Systems), anti-RAET1E rabbit polyclonal (this study), anti-RAET1G rabbit polyclonal (39), anti-CD16 mouse monoclonal antibody, and clone DJ130c (AbD Serotec).

Techniques: Staining, Microarray, Expressing

Journal: Oncotarget

Article Title: Increasing TIMP3 expression by hypomethylating agents diminishes soluble MICA, MICB and ULBP2 shedding in acute myeloid leukemia, facilitating NK cell-mediated immune recognition

doi: 10.18632/oncotarget.16657

Figure Lengend Snippet: ( A ) AML cell lines (KG1a and NB4 cells) were treated with DMSO (Ctrl), DAC (1 and 5 μM) or AZA (1 and 5 μM) for 48 hours. Soluble levels of MICA, MICB, ULBP1, ULBP2 and ULBP3 ligands were quantified by sandwich ELISA. Each bar represents the mean ± SEM of at least four independent experiments. * p < 0.05 and * p < 0.001. ( B ) KG1a and NB4 cells were treated with 5 μM of DAC for 48 hours. Before and after treatment, cell surface expression of NKG2DL was analyzed by flow cytometry using monoclonal antibodies specific to each NKG2DL (MICA, MICB, ULBP1, ULBP2 and ULBP3), followed by incubation with FITC-conjugated goat anti-mouse IgG. The white histograms represent the isotype control antibody and shaded grey histograms show the NKG2DL expression. Dotted vertical lines indicate mean fluorescence intensity value in untreated samples.

Article Snippet: After washes, a specific biotinylated polyclonal antibody for each NKG2DL (0.4 μg/ml) (MICA Ref: BAF1300,, MICB Ref: BAF1599, ULBP1 Ref: BAF1380, ULBP2 Ref: BAF1298, and ULBP3 Ref: BAF1517, all from R&D Systems) was added for 2 hours at RT.

Techniques: Sandwich ELISA, Expressing, Flow Cytometry, Bioprocessing, Incubation, Control, Fluorescence

Journal: Oncotarget

Article Title: Increasing TIMP3 expression by hypomethylating agents diminishes soluble MICA, MICB and ULBP2 shedding in acute myeloid leukemia, facilitating NK cell-mediated immune recognition

doi: 10.18632/oncotarget.16657

Figure Lengend Snippet: ( A ) NKL cells were co-cultured with cell-free supernatants (sn) obtained from KG1a and NB4 cells untreated (sn-DMSO) or treated with 1 μM DAC for 48 hours (sn-DAC). NKL cells grow in culture medium were considered as a control (Ctrl). NKG2D expression was analyzed by flow cytometry and represented as mean fluorescence intensity (MFI). Each bar represents the mean ± SEM of three independent experiments. * versus control and p < 0.01; # versus sn-DMSO and p < 0.05. ( B ) NKL cells were co-cultured with K562 cells at the indicated E:T ratio in a cell lysis assay, in the absence (Ctrl) or presence of cellular supernatant derived from KG1a (left panel) and NB4 (middle panel) cells previously treated with DMSO (sn-DMSO) or 1 μM DAC (sn-DAC) for 48 hours. Specificity of the NKG2D-NKG2DL interaction was corroborated using an anti-NKG2D blocking mAb and the effect of DAC was assayed to analyze the non-specific effects on the lytic capacity of NKL cells (right panel). Measurements were made in duplicate and the mean ± SEM of the two independent experiments are shown. * versus control and p < 0.05; * versus control and p < 0.01; # versus sn-DMSO and p < 0.05.

Article Snippet: After washes, a specific biotinylated polyclonal antibody for each NKG2DL (0.4 μg/ml) (MICA Ref: BAF1300,, MICB Ref: BAF1599, ULBP1 Ref: BAF1380, ULBP2 Ref: BAF1298, and ULBP3 Ref: BAF1517, all from R&D Systems) was added for 2 hours at RT.

Techniques: Cell Culture, Control, Expressing, Flow Cytometry, Fluorescence, Lysis, Derivative Assay, Blocking Assay

Journal: Oncotarget

Article Title: Increasing TIMP3 expression by hypomethylating agents diminishes soluble MICA, MICB and ULBP2 shedding in acute myeloid leukemia, facilitating NK cell-mediated immune recognition

doi: 10.18632/oncotarget.16657

Figure Lengend Snippet: ( A ) KG1a and NB4 cells were treated with inhibitors specific to ADAM17 (10 μM GW280264X) and ADAM10 (50 μM of GI254023X) for 48 hours. Levels of soluble NKG2DL (sMICA/B and sULBPs1-3) were quantified by sandwich ELISA. Values are the mean ± SEM of at least three independent experiments. * p < 0.05 and * p < 0.01. ( B ) KG1a and NB4 cells were treated with DMSO (Ctrl) or DAC (1 μM) for 48 hours. After treatment, cell surface expression of ADAM17 was analyzed by flow cytometry using anti-human ADAM17 monoclonal antibody, followed by incubation with FITC-conjugated goat anti-mouse IgG. The white histograms represent the isotype control antibody and the shaded grey histograms show the ADAM17 expression. ( C ) ADAM17 activity in KG1a and NB4 cells was measured in whole-cell lysates after treatment with DMSO (Ctrl) or DAC (5 μM) for 48 hours. Data are expressed as relative fluorescence units (RLU) at Ex/Em=490/520 nm absorbance normalized with respect to micrograms of total protein (RLU/μg). Values are the mean ± SEM of three independent experiments. * p < 0.05 and * p < 0.01.

Article Snippet: After washes, a specific biotinylated polyclonal antibody for each NKG2DL (0.4 μg/ml) (MICA Ref: BAF1300,, MICB Ref: BAF1599, ULBP1 Ref: BAF1380, ULBP2 Ref: BAF1298, and ULBP3 Ref: BAF1517, all from R&D Systems) was added for 2 hours at RT.

Techniques: Sandwich ELISA, Expressing, Flow Cytometry, Incubation, Control, Activity Assay, Fluorescence

Journal: Oncotarget

Article Title: Increasing TIMP3 expression by hypomethylating agents diminishes soluble MICA, MICB and ULBP2 shedding in acute myeloid leukemia, facilitating NK cell-mediated immune recognition

doi: 10.18632/oncotarget.16657

Figure Lengend Snippet: ( A ) KG1a and NB4 cells were treated with DMSO (Ctrl) or DAC (0.25, 0.5 and 1 μM) for 48 hours, and TIMP3 expression was analyzed by qRT-PCR. Each bar represents the relative expression of TIMP3 normalized with respect to the reference gene (GAPDH), using the 2 −ΔCt method. MICA transcription levels in the KG1a cell line untreated (DMSO, Ctrl) or treated with DAC at different concentrations were used as a positive control. Results are summarized as the mean ± SEM of five independent experiments. * p < 0.05 and * p < 0.01. ( B ) TIMP3 protein levels were evaluated by western blot in KG1a and NB4 cells after treatment with DMSO (Ctrl) or DAC (1 μM or 5 μM) for 48 hours. * p < 0.05. ( C ) The TIMP3 methylation pattern was quantified by pyrosequencing in AML cell lines (KG1a and NB4 cells) before and after treatment with 1 μM or 5 μM DAC. Pie charts show the average percentage of methylation for the CpGs analyzed in the TIMP3 gene. ( D ) TIMP3 expression was inhibited by transfection of KG1a cells previously treated with DAC (1 μM) with a TIMP3-specific siRNA or nonspecific scramble siRNA (200 nM). * p < 0.05 ( E ) Soluble NKG2DL were quantified by sandwich ELISA after TIMP3 inhibition. Values shown are the mean ± SEM of three independent experiments. * versus control and p < 0.05; * versus control and p < 0.01 # versus nonspecific scramble siRNA and p < 0.05.

Article Snippet: After washes, a specific biotinylated polyclonal antibody for each NKG2DL (0.4 μg/ml) (MICA Ref: BAF1300,, MICB Ref: BAF1599, ULBP1 Ref: BAF1380, ULBP2 Ref: BAF1298, and ULBP3 Ref: BAF1517, all from R&D Systems) was added for 2 hours at RT.

Techniques: Expressing, Quantitative RT-PCR, Positive Control, Western Blot, Methylation, Transfection, Sandwich ELISA, Inhibition, Control

Journal: Oncotarget

Article Title: Increasing TIMP3 expression by hypomethylating agents diminishes soluble MICA, MICB and ULBP2 shedding in acute myeloid leukemia, facilitating NK cell-mediated immune recognition

doi: 10.18632/oncotarget.16657

Figure Lengend Snippet: ( A ) Soluble NKG2DL were quantified by sandwich ELISA in sera from twelve AML patients before and after Vidaza ® treatment. Lines represent the levels of each sNKG2DL (ng/mL) before and after treatment of each individual AML patient. ( B ) Expression of NKG2DL on the cell surface of blasts from five AML patients before and after Vidaza ® treatment (left panel). The right panel shows dot plots of NKG2DL expression on the cell surface of blats from a representative patient. Numbers included in the figure quadrants indicate the percentage of positive cells for each NKG2DL.

Article Snippet: After washes, a specific biotinylated polyclonal antibody for each NKG2DL (0.4 μg/ml) (MICA Ref: BAF1300,, MICB Ref: BAF1599, ULBP1 Ref: BAF1380, ULBP2 Ref: BAF1298, and ULBP3 Ref: BAF1517, all from R&D Systems) was added for 2 hours at RT.

Techniques: Sandwich ELISA, Expressing

Journal: International journal of cancer

Article Title: Expression of the ULBP ligands for NKG2D by B-NHL cells plays an important role in determining their susceptibility to rituximab-induced ADCC.

doi: 10.1002/ijc.24351

Figure Lengend Snippet: FIGURE 2 – Correlations between the expression of MHC class I or total NKG2D ligand and rituximab-induced ADCC in B-NHL cell lines. ADCC (upper panels) or CDC (lower panels) in the presence of 10 lg/ml rituximab against 30 different CD201 B-NHL cell lines determined by 51Cr release assays. Y-axis: % lysis. X-axis: MFI ratio of MHC class I (left panels) and total NKG2D ligand (right panels). Each dot plot in each panel represents 1 cell line in the same manner as in Figure 1. The coefficients and P values assessed by Spearman rank correlation coefficient testing are indicated in each panel.

Article Snippet: Mouse mAbs to human UL16-binding protein (ULBP)1 (clone: 170818, IgG2a), ULBP2 (165903, IgG2a), ULBP3 (166510, IgG2a), Allophycocyanin (APC)-conjugated antihuman major histocompatibility complex (MHC) class I chain-related genes A (MICA) (159227, IgG2b), MHC class I chain-related genes B (MICB) (236511, IgG2b), Phycoerythrin (PE)-conjugated antihuman NKG2D (149810, IgG1) and appropriate isotype-matched control mAbs were purchased from R&D Systems, (Minneapolis, MN).

Techniques: Expressing, Lysis

Journal: International journal of cancer

Article Title: Expression of the ULBP ligands for NKG2D by B-NHL cells plays an important role in determining their susceptibility to rituximab-induced ADCC.

doi: 10.1002/ijc.24351

Figure Lengend Snippet: FIGURE 3 – Correlations between the expression of the NKG2D ligand, ULBP1–3, or MICA/B and rituximab-induced ADCC in B-NHL cell lines. ADCC in the presence of 10 lg/ml rituximab against 30 different CD201 B-NHL cell lines determined by 51Cr release assays. Y-axis: % lysis. X-axis: MFI ratio of ULBP1 (upper left panel), ULBP2 (upper middle panel), ULBP3 (upper right panel), MICA (lower left panel), and MICB (lower right panel). Each dot plot in each panel represents 1 cell line in the same manner as in Figure 1. The coefficients and p values assessed by Spearman rank correlation coefficient testing are indicated in each panel.

Article Snippet: Mouse mAbs to human UL16-binding protein (ULBP)1 (clone: 170818, IgG2a), ULBP2 (165903, IgG2a), ULBP3 (166510, IgG2a), Allophycocyanin (APC)-conjugated antihuman major histocompatibility complex (MHC) class I chain-related genes A (MICA) (159227, IgG2b), MHC class I chain-related genes B (MICB) (236511, IgG2b), Phycoerythrin (PE)-conjugated antihuman NKG2D (149810, IgG1) and appropriate isotype-matched control mAbs were purchased from R&D Systems, (Minneapolis, MN).

Techniques: Expressing, Lysis

Journal: Cancer Medicine

Article Title: A signature based on NKG2D ligands to predict the recurrence of hepatocellular carcinoma after radical resection

doi: 10.1002/cam4.5318

Figure Lengend Snippet: The primer sequences used for QRT‐PCR

Article Snippet: Antibodies used in IHC were as follows: goat anti‐human ULBP‐2/5/6 (Invitrogen Cat# PA5‐47118, 1:500 dilution), rabbit anti‐human MICA/MICB (Abcam Cat# ab224702, 1:50 dilution), rabbit anti‐human ULBP1 (Abcam Cat# ab238331, 1:1000 dilution), rabbit anti‐human ULBP3 (Novus Cat# nbp2‐31,866, 1:1000 dilution), and mouse anti‐human ULBP4 (Santa Cruz Cat# sc‐390,784, 1:50 dilution).

Techniques: Sequencing