Fig 1: Micrographs and NKG2D/ligands cite density determination of in situ binding affinity assay and in‐solution binding kinetics measurement by Biolayer interferometry (BLI) APhotomicrographs 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–GFlow 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).HA 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–OBLI 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.
Fig 2: Characterization of in situ force‐free kinetics of NKG2D interacting with different ligands and comparison with in‐solution affinities ADiagrammatic sketch of in situ binding kinetic assay and functionalization of RBC.BRepresentative 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, 2010). 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).FDetection 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, 2003). In‐solution affinities of proteins purified from mammalian cells were measured by BLI (Fig EV2A–G). 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.
Fig 3: Surface CD107a and phosphorylation of ERK of NK cells stimulated by immobilized NKG2D ligands A, BFlow 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, DCorresponding quantification of percentages of CD107a+ cells and pERK+ NK cells in (A) and (B).E, FPlots and Pearson correlation analysis of NKG2D ligands stimulated percentages of CD107a+ (E) and pERK+ (F) NK cells with their reciprocals of EC50 to release IFN‐γ.GIFN‐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).
Fig 4: Kinetic proofreading model analysis of Force‐dependent ligand discrimination and amplification of NKG2D in mediating NK cell’s activation 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.
Fig 5: Experimental setup of BFP for detecting the interactions between NKG2D and different ligands A, BRepresentative 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).CVar(X m) is plotted versus reciprocal of suction pressure 1/Δp and fitted by the motion blur model (Chen et al, 2008; Ju & Zhu, 2017).DThe motion‐blur corrected variance Var(X) calculated from Var(X m) is plotted versus 1/kp, which is the BFP spring constant calculated from Evans model (Chen et al, 2008; Ju & Zhu, 2017).EPhotomicrograph 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, GRepresentative force versus time curve for no adhesion (F) and force ramp (G).HForce‐dependent bond lifetimes of NKG2D and various ligands at 5 pN, 10 pN, and 15 pN.IIllustration of the retract phase (blue line, the slope of which is the loading rate) in an example event of BFP bond lifetime.J–NScatter 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).
Supplier Page from Sino Biological, Inc. for Human MICB Gene ORF cDNA clone in cloning vector