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, Purification, Membrane, Incubation, Labeling, 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: 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, Purification, Membrane, Incubation, Labeling, 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: Cell Reports Medicine

Article Title: Engineering human stem cell-derived islets to evade immune rejection and promote localized immune tolerance

doi: 10.1016/j.xcrm.2022.100879

Figure Lengend Snippet:

Article Snippet: Cells were then stained for 30 min on ice in blocking buffer with APC anti-human CD47 (Biolegend, 323123), APC anti-human CD324 (E-Cadherin) (Biolegend, 324107), PE anti-human CD325 (N-Cadherin) (Biolegend, 350806), PE anti-human CD112 (Nectin-2) (Biolegend, 337409), APC anti-human CD155 (PVR) (Biolegend, 337617), PE anti-human CD111 (Nectin-1) (Biolegend, 340404), APC anti-human MICA/MICB (Biolegend, 320907), AF647 anti-human PCNA (Biolegend, 307912), PE anti-human BAG6 (Abcam, ab210838), APC anti-human CD48 (Biolegend, 336713), PE anti-human CD70 (Biolegend, 355103), mouse anti-human CD113 (Nectin-3) (Millipore-Sigma, MABT63), APC anti-human ULBP1 (R&D, FAB1380A), PE anti-human ULBP2/5/6 (R&D, FAB1298P), APC anti-human CLEC2D (R&D, FAB3480A) and PE anti-human CD72 (Biolegend, 316207).

Techniques: Purification, Recombinant, Plasmid Preparation, Sequencing, Enzyme-linked Immunosorbent Assay, RNA Sequencing Assay, Software, Flow Cytometry, Microscopy

Journal: Scientific Reports

Article Title: Toll-like Receptor-4 Activation Boosts the Immunosuppressive Properties of Tumor Cells-derived Exosomes

doi: 10.1038/s41598-019-44949-y

Figure Lengend Snippet: Expression of NKG2D ligands on the surface of exosomes and modulation of NKG2D expression on NK and CD8+ T cells. Exosomes were coupled to ExoFlow beads, stained with monoclonal antibodies either against MICA/B or ULBP-1 and analyzed by FACS. ( A ) Representative plots of the FACS analyses for MICA/B staining of control (black line) and of LPS-derived exosomes (red line) are shown. The histograms represent the percentages of MICA/B- ( B ) and ULBP-1 ( C ) -positive beads bound to the exosomes released by unstimulated (CTRL, white column) o LPS-activated cells (LPS, black column). Data are shown as mean (n = 6); bars, SD; *difference with exosomes released by unstimulated tumor cells, P < 0.05. PBMCs or isolated CD16+ CD56+ NK cells were treated for 24 hours with exosomes released by unstimulated (CTRL, white column) o LPS-activated tumor cells (LPS, black column). In a separate set of experiments, exosomes were pre-treated with neutralizing anti-MICA/B antibody ( E ). Expression of NKG2D by CD8+ ( D,E ) and NK cells ( F ) was analyzed by FACS. Data are shown as mean (n = 6); bars, SD; § difference with untreated cells; *difference with cells treated with exosomes released by unstimulated tumor cells, P < 0.05, $ difference with exosomes treated cells released by LPS-activated cells, P < 0.05.

Article Snippet: Exosomes (2 × 10 9 ) were also purified by immunoaffinity Exo-Flow kit (System Biosciences) , stained with Exo-FITC provided by the kit or specific monoclonal antibodies anti-CD81 FITC (Biolegend), anti-CD63 FITC (Santa Cruz), anti-CD9 PE (eBiosciences), anti-MICA/B Alexa Fluor 488 (Invitrogen), anti-ULBP-1 APC (Invitrogen) and anti-TGFβ1 PE (eBiosciences) and analyzed by flow cytometry.

Techniques: Expressing, Staining, Derivative Assay, Isolation