Journal: American Journal of Cancer Research

Article Title: TGF-β1 expression in regulatory NK1.1 - CD4 + NKG2D + T cells dependents on the PI3K-p85α/JNK, NF-κB and STAT3 pathways

doi:

Figure Lengend Snippet: TGF-β1 production of NK1.1-CD4+NKG2D+ cells upon various stimulations at different times. A. TGF-β1 of NK1.1-CD4+NKG2D+ cells detected by flow cytometry at 0.5-, 2-, 8-, 16-hour stimulation. B. Statistical analysis of TGF-β1 variations at different time points. C. TGF-β1 transcription of NK1.1-CD4+NKG2D+ cells detected by real-time PCR. D. Concentration of TGF-β1 in supernatants of NK1.1-CD4+NKG2D+ cells upon indicated stimulations measured by ELISA. Each experiment was repeated at least thrice. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significance.

Article Snippet: Splenic NK1.1 - CD4 + T cells were enriched from splenic single cell suspension by mouse CD4 + T lymphocyte enrichment set (BD Biosciences) following the manufacturer’s instructions, and then NKG2D + T cells were isolated by indirectly labeled the cells with PE-conjugated anti-mouse NKG2D antibody (CX5) and anti-PE MicroBeads (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany).

Techniques: Flow Cytometry, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Cancer Research

Article Title: TGF-β1 expression in regulatory NK1.1 - CD4 + NKG2D + T cells dependents on the PI3K-p85α/JNK, NF-κB and STAT3 pathways

doi:

Figure Lengend Snippet: Activation of PI3K, MAPK, STAT3 and NF-κB signaling pathways. Western blot analysis of PI3K-p85, -p110, Akt, pAkt in NK1.1-CD4+NKG2D+ cells upon different stimulations at 2-hour (A) or 8-hour (C). Statistical analysis of expression variations of PI3K-p85, -p110, Akt, pAkt at 2-hour (B) or 8-hour (D). Analysis of JNK, Erk, p38 (E, F), NF-κB, STAT3 (G, H) and their phosphorylation in NK1.1-CD4+NKG2D+ cells upon stimulations for 8 hours. Each experiment was repeated at least thrice. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significance.

Article Snippet: Splenic NK1.1 - CD4 + T cells were enriched from splenic single cell suspension by mouse CD4 + T lymphocyte enrichment set (BD Biosciences) following the manufacturer’s instructions, and then NKG2D + T cells were isolated by indirectly labeled the cells with PE-conjugated anti-mouse NKG2D antibody (CX5) and anti-PE MicroBeads (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany).

Techniques: Activation Assay, Protein-Protein interactions, Western Blot, Expressing, Phospho-proteomics

Journal: American Journal of Cancer Research

Article Title: TGF-β1 expression in regulatory NK1.1 - CD4 + NKG2D + T cells dependents on the PI3K-p85α/JNK, NF-κB and STAT3 pathways

doi:

Figure Lengend Snippet: Inhibition of PI3K/JNK/AP-1 on TGF-β1 expression. NK1.1-CD4+NKG2D+ cells were treated with the PI3K inhibitor (LY294002) under different stimulations for 8 hours (A). (B) Statistical analysis of effects of LY294002 on TGF-β1 expression. (C, D) Effects on JNK/pJNK expression by the PI3K inhibitor (LY294002). Effects of JNK inhibitor (E, F), Erk or p38 inhibitor (G), and AP-1 inhibitor (H, I) on TGF-β1 expression of NK1.1-CD4+NKG2D+ cells. Each experiment was repeated at least thrice. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significance.

Article Snippet: Splenic NK1.1 - CD4 + T cells were enriched from splenic single cell suspension by mouse CD4 + T lymphocyte enrichment set (BD Biosciences) following the manufacturer’s instructions, and then NKG2D + T cells were isolated by indirectly labeled the cells with PE-conjugated anti-mouse NKG2D antibody (CX5) and anti-PE MicroBeads (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany).

Techniques: Inhibition, Expressing

Journal: American Journal of Cancer Research

Article Title: TGF-β1 expression in regulatory NK1.1 - CD4 + NKG2D + T cells dependents on the PI3K-p85α/JNK, NF-κB and STAT3 pathways

doi:

Figure Lengend Snippet: Inhibition of NF-κB on TGF-β1 expression. Effects of Bortezomib (NF-κB inhibitor) on TGF-β1 expression (A, B). Effects of LY294002 on NF-κB p65 activation in NK1.1-CD4+NKG2D+ cells for 8 hours (C, D). Each experiment was repeated at least thrice. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significance.

Article Snippet: Splenic NK1.1 - CD4 + T cells were enriched from splenic single cell suspension by mouse CD4 + T lymphocyte enrichment set (BD Biosciences) following the manufacturer’s instructions, and then NKG2D + T cells were isolated by indirectly labeled the cells with PE-conjugated anti-mouse NKG2D antibody (CX5) and anti-PE MicroBeads (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany).

Techniques: Inhibition, Expressing, Activation Assay

Journal: American Journal of Cancer Research

Article Title: TGF-β1 expression in regulatory NK1.1 - CD4 + NKG2D + T cells dependents on the PI3K-p85α/JNK, NF-κB and STAT3 pathways

doi:

Figure Lengend Snippet: Inhibition of STAT3 on TGF-β1 expression. Effects of STAT3 inhibitors on TGF-β1 expression in NK1.1-CD4+NKG2D+ cells for 8 hours (A). (B, C) Effects of LY294002 on STAT3-pY705 phosphorylation. (D, E) Effects of Bortezomib on STAT3-pY705 phosphorylation. (F) The predicted binding sites of the TGF-β promoter by STAT3. (G) STAT3 engagement measured by a ChIP assay. Each experiment was repeated at least thrice. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significance.

Article Snippet: Splenic NK1.1 - CD4 + T cells were enriched from splenic single cell suspension by mouse CD4 + T lymphocyte enrichment set (BD Biosciences) following the manufacturer’s instructions, and then NKG2D + T cells were isolated by indirectly labeled the cells with PE-conjugated anti-mouse NKG2D antibody (CX5) and anti-PE MicroBeads (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany).

Techniques: Inhibition, Expressing, Phospho-proteomics, Binding Assay

Journal: American Journal of Cancer Research

Article Title: TGF-β1 expression in regulatory NK1.1 - CD4 + NKG2D + T cells dependents on the PI3K-p85α/JNK, NF-κB and STAT3 pathways

doi:

Figure Lengend Snippet: Diagram of the TGF-β1 transcription in NK1.1-CD4+NKG2D+ cells regulated by AP-1, NF-κB, and STAT3.

Article Snippet: Splenic NK1.1 - CD4 + T cells were enriched from splenic single cell suspension by mouse CD4 + T lymphocyte enrichment set (BD Biosciences) following the manufacturer’s instructions, and then NKG2D + T cells were isolated by indirectly labeled the cells with PE-conjugated anti-mouse NKG2D antibody (CX5) and anti-PE MicroBeads (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany).

Techniques:

Journal: Frontiers in Immunology

Article Title: Orthotopic and Heterotopic Murine Models of Pancreatic Cancer Exhibit Different Immunological Microenvironments and Different Responses to Immunotherapy

doi: 10.3389/fimmu.2022.863346

Figure Lengend Snippet: NKG2D CAR-T rescue impaired CAR-T cell activity via eliminating MDSCs. (A) Schematic diagram of NKG2D-CAR (Upper) and flow cytometry assay of transfection efficiency (Down). (B) Cytotoxicity assay of NKG2D CAR-T to Rae1 positive CHO cell at indicated E:T ratio. E:T, effector-to-target ratio. (n=6 compared with Control CAR-T group, ***P<0.001). (C) Cytotoxicity assay of NKG2D CAR-T to murine MDSCs at E:T=1:1. MDSCs were pre-labeled by CSFE. CSFE + 7AAD + cells indicate specific killed MDSCs. (D) Survival of orthotopic tumor recipients after NKG2D CAR-T transfer. The green arrows indicate NKG2D CAR-T cells administration. (E) Representative IHC images of Gr1 staining in orthotopic tumor 2 days after NKG2D CAR-T transfer. Scale bar=50μm. (F) Schematic outline of experimental approaches and analyses. (G) Survival of orthotopic tumor recipients after combination of NKG2D CAR-T and αMUC1 CAR-T. The green arrows indicate NKG2D CAR-T cells administration; the orange arrows indicate αMUC1 CAR-T cells administration. (***P<0.001). (H) Schematic diagram of bicistronic CAR (Upper) and flow cytometry assay of transfection efficiency (Down). (I) Survival of orthotopic tumor recipients after bicistronic CAR-T transfer. The orange arrow indicates CAR-T cells administration (***P<0.001).

Article Snippet: The extracellular (EC) and transmembrane (TM) regions of mice NKG2D (NM_001083322.2) were cloned form commercial plasmid (SinoBiological, MG57340). scFv sequence of CD19 was from patent US20210395364A1 in VL-(G 4 S) 3 -VH form. scFv sequence of MUC1 was from patent US10239950B2 in VL-(G 4 S) 3 -VH form.

Techniques: Activity Assay, Flow Cytometry, Transfection, Cytotoxicity Assay, Labeling, Staining

Journal: Nature Communications

Article Title: Immunoprophylactic and immunotherapeutic control of hormone receptor-positive breast cancer

doi: 10.1038/s41467-020-17644-0

Figure Lengend Snippet: a , b Tumor-free survival (TFS), overall survival (OS) and time-to-death (TTD) of WT C57BL/6 mice ( a , b ) and Rag2 − / − Il2rg − / − mice ( a ) or Ifng − / − mice ( b ) subjected to M/D-driven oncogenesis. Number of mice, hazard ratio (HR) and p values (two-sided log-rank) are reported. c – e . Variations in TFS ( c ), OS ( d ) and TTD ( e ) imposed to M/D-driven oncogenesis in mice by the indicated genotype or immunomodulatory interventions. Results are means ± SEM plus individual data points. Number of mice, HR and p values (two-sided log-rank and one way-ANOVA plus Fisher LSD, calculated with respect to individual control experiments) are reported. Green dots indicate mice that were free of disease ( c ) and alive ( d ) at the end of the experiment. f – h TFS and OS of WT C57BL/6 ( f – h ) mice and Rag2 − / − BALB/c mice ( f ), C57BL/6 mice receiving NKG2D-depleting antibodies ( g ), or C57BL/6 mice receiving CD4- and CD8-depleting antibodies ( h ). Number of mice, HR and p values (two-sided log-rank) are reported.

Article Snippet: All antibodies blocking or neutralizing specific immune cell populations, cytokines or cytokine receptors and their corresponding isotype controls were acquired from BioXCell (West Lebanon): CD4 (clone GK1.5, #BE0003-1), CD8α (clone 2.43, #BE0061), NKG2D (clone HMG2D, #BE0111), NK1.1 (clone PK136, #BE0036), PD-1 (clone RMP1-14, #BE0146), IFNγ (clone R4-6A2, #BE0054), IFNAR1 (clone MAR1-5A3, #BE0241), IL-17A (clone 17F3, #BE0173).

Techniques: Control

Journal: iScience

Article Title: Hepatic iNKT cells facilitate colorectal cancer metastasis by inducing a fibrotic niche in the liver

doi: 10.1016/j.isci.2025.112364

Figure Lengend Snippet:

Article Snippet: Rat Monoclonal NKG2D (CX5) , BioXCell , BE0334; RRID: AB_2894754.

Techniques: Recombinant, Membrane, Infection, Transfection, Plasmid Preparation, Microscopy, In Vivo, SYBR Green Assay, Amplification, Multiplexing, Staining, Reverse Transcription, RNA Sequencing, Software, Injection, Control, Ointment, Imaging

Journal: European journal of immunology

Article Title: Inhibition of NKG2D receptor function by antibody therapy attenuates transfer-induced colitis in SCID mice.

doi: 10.1002/eji.200636473

Figure Lengend Snippet: Figure 1. (A-C) Representative FACS plots of NKG2D, CD4 and CD49b expression gated on live CD45.2+ cells. (A) BALB/c donor mice. NKG2D is expressed on CD4–CD49b+ cells, but not on CD4+ cells. (B) Colitic SCID mice 3 weeks after reconstitution. NKG2D is expressed on a sub-population of CD4+ cells in addition to the CD4– cells. (C) Left: H60-transfected Ba/F3 cells stained with murine NKG2D-Fc or human GHR-Fc fusion. Low pH disrupted the binding of mNKG2D to H60. Right: Blood cells from reconstituted, colitic SCID mice stained as in (A). No significant difference in NKG2D staining before or after acid stripping. (D) Left: Percentage CD4+NKG2D+ cells in blood from BALB/c mice and reconstituted SCID mice 2–5 weeks after reconstitution. Weeks 2–3, untreated mice; weeks 4–5, animals treated with CX5 or cIg from week 2 to 4 or from week 3 to 5. Right graph: Percentage NKG2D+CD4+ cells in the mLN from BALB/c and colitic mice and the LPL from colitic mice. (E) NKG2D and HPRT RT-PCR on purified cell subsets of CD4+CD25– T cells and on CD4+ cells purified from mLN and spleen of colitic mice (20, 25, and 30 cycles of amplification). Spleen cells stimulated with Con A were used as positive controls.

Article Snippet: Nonspecific binding was blocked by incubation with TBS containing 3% skimmed milk (DifcoTM, BD Bioscience) and 10% goat serum (Dako a/s), followed by incubation with either rat anti-mouse CD4 (clone L3T4, BD PharMingen) or rat anti-mouse NKG2D (clone 191004, R&D Systems).

Techniques: Expressing, Transfection, Staining, Binding Assay, Stripping Membranes, Reverse Transcription Polymerase Chain Reaction, Purification, Amplification

Journal: European journal of immunology

Article Title: Inhibition of NKG2D receptor function by antibody therapy attenuates transfer-induced colitis in SCID mice.

doi: 10.1002/eji.200636473

Figure Lengend Snippet: Figure 2. Immunostaining for CD4 and NKG2D in frozen sections of colonic tissue from un-reconstituted controls and reconstituted colitic mice. (A) Massive infiltration with CD4+

Article Snippet: Nonspecific binding was blocked by incubation with TBS containing 3% skimmed milk (DifcoTM, BD Bioscience) and 10% goat serum (Dako a/s), followed by incubation with either rat anti-mouse CD4 (clone L3T4, BD PharMingen) or rat anti-mouse NKG2D (clone 191004, R&D Systems).

Techniques: Immunostaining

Journal: PLoS ONE

Article Title: Macrophages Help NK Cells to Attack Tumor Cells by Stimulatory NKG2D Ligand but Protect Themselves from NK Killing by Inhibitory Ligand Qa-1

doi: 10.1371/journal.pone.0036928

Figure Lengend Snippet: Sequences of primer used for real-time PCR assay.

Article Snippet: The antibodies used were as followed: carboxyfluorescein-conjugated RAE-1, FITC-conjugated MULT-1, PE-conjugated H60 (R&D Systems), anti-mouse NKG2D mAb (R&D System), PE-conjugated Ab to CD68, CD69 or IFN-γ (R&D System), FITC-conjugated affinity-purified goat anti-rat IgG (ZSGB-BIO, China) or isotype controls (eBioscience, San Diego, CA, USA).

Techniques: Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Macrophages Help NK Cells to Attack Tumor Cells by Stimulatory NKG2D Ligand but Protect Themselves from NK Killing by Inhibitory Ligand Qa-1

doi: 10.1371/journal.pone.0036928

Figure Lengend Snippet: Freshly isolated splenic NK cells were co-cultured with RAW264.7 cells that were treated with 0 or 100 µg/ml of poly I:C for 24 h. NK cells were then isolated and NKG2D expression was determined by RT-PCR and flow cytometry (A). Data are expressed as the fold change in mRNA expression normalized to untreated cells or as the percentage of positively-stained cells. * p<0.05 versus untreated cells using the paired Student’s test. The isolated NK cells from co-culture with macrophages were co-incubated with saturating amounts of anti-NKG2D mAb or isotype control, washed and used as effector cells against YAC-1 targets (B). The expression of CD69 (C), cytotoxic effector molecules FasL, TRAIL, and perforin (D), and seceretion of IFN-γ (E) were determined by flow cytometry, RT-PCR, and ELISA separately. Data are expressed as the mean ± SD from at least three separate experiments. *p<0.05, **p<0.01 compared with poly I:C-untreated RAW264.7 group; # p<0.05, ## p<0.01 compared with isotype control group.

Article Snippet: The antibodies used were as followed: carboxyfluorescein-conjugated RAE-1, FITC-conjugated MULT-1, PE-conjugated H60 (R&D Systems), anti-mouse NKG2D mAb (R&D System), PE-conjugated Ab to CD68, CD69 or IFN-γ (R&D System), FITC-conjugated affinity-purified goat anti-rat IgG (ZSGB-BIO, China) or isotype controls (eBioscience, San Diego, CA, USA).

Techniques: Isolation, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Staining, Co-Culture Assay, Incubation, Control, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Macrophages Help NK Cells to Attack Tumor Cells by Stimulatory NKG2D Ligand but Protect Themselves from NK Killing by Inhibitory Ligand Qa-1

doi: 10.1371/journal.pone.0036928

Figure Lengend Snippet: Murine macrophage RAW264.7 cells were cultured for 24 h with 0, 10, 20 or 100 µg/ml poly I:C and analyzed by RT-PCR or FACS. The data are expressed as fold induction of RAE-1, H60 or MULT-1 mRNA (A) or the percentage of positive RAE-1 staining using monoclonal pan-RAE-1 antibodies (B). Resident peritoneal macrophages from BALB/c or C57BL/6 mice were treated with either 0 or 100 µg/ml poly I:C for 24 h and NKG2D ligand transcripts and surface expression were analyzed by real-time PCR (C) or FACS (B). Data shown are expressed as the mean ± SD from at least three separate experiments. *p<0.05, **p<0.01 compared with untreated cells.

Article Snippet: The antibodies used were as followed: carboxyfluorescein-conjugated RAE-1, FITC-conjugated MULT-1, PE-conjugated H60 (R&D Systems), anti-mouse NKG2D mAb (R&D System), PE-conjugated Ab to CD68, CD69 or IFN-γ (R&D System), FITC-conjugated affinity-purified goat anti-rat IgG (ZSGB-BIO, China) or isotype controls (eBioscience, San Diego, CA, USA).

Techniques: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Staining, Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Macrophages Help NK Cells to Attack Tumor Cells by Stimulatory NKG2D Ligand but Protect Themselves from NK Killing by Inhibitory Ligand Qa-1

doi: 10.1371/journal.pone.0036928

Figure Lengend Snippet: Expression of TLR3 and TLR4 by RAW264.7 cells stimulated with 100 µg/ml poly I:C for 24 h was analyzed by RT-PCR (A). TLR3 mRNA levels of RAW264.7 cells were measured following incubation in media only or following treatment with si-TLR3 or siRNA control (si-control) for 24 h (B). The data are expressed as the folds ± SD from at least three separate experiments. *p<0.05, **p<0.01 compared with the untreated group. RAW264.7 cells first transfected with si-TLR3 or si-control were stimulated with 0 or 100 µg/ml poly I:C for 24 h and the transcripts of the NKG2D ligands RAE-1, H60, and MULT-1 were detected by RT-PCR (C). The data are expressed as the mean induction folds ± SD over untreated cells from at least three separate experiments. *p<0.05, compared with si-control group.

Article Snippet: The antibodies used were as followed: carboxyfluorescein-conjugated RAE-1, FITC-conjugated MULT-1, PE-conjugated H60 (R&D Systems), anti-mouse NKG2D mAb (R&D System), PE-conjugated Ab to CD68, CD69 or IFN-γ (R&D System), FITC-conjugated affinity-purified goat anti-rat IgG (ZSGB-BIO, China) or isotype controls (eBioscience, San Diego, CA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Control, Transfection

Journal: PLoS ONE

Article Title: Macrophages Help NK Cells to Attack Tumor Cells by Stimulatory NKG2D Ligand but Protect Themselves from NK Killing by Inhibitory Ligand Qa-1

doi: 10.1371/journal.pone.0036928

Figure Lengend Snippet: Changes in mRNA and protein levels of the NKG2A ligands Qa-1a and Qa-1b (A,B), and the NKG2D ligands RAE-1, H60, and MULT-1 (C) in YAC-1 cells, macrophages (Mφ) or RAW264.7 cells were detected by RT-PCR or FACS. Data are expressed as relative expression from at least three separate experiments. *p<0.05, **p<0.01 versus YAC-1 mRNA levels using the paired Student’s test. RAW264.7 cells first stimulated with 100 µg/ml poly I:C for 24 h and then co-incubated with saturating amounts of anti-Qa-1 mAb or isotype control or transfected with si-Qa-1 or si-control, were then used as target cells. The cytolytic activity of NK cells stimulated with poly I:C-treated macrophages was assessed at various E:T rations (D). Data are expressed as the mean ± SD from at least three separate experiments. *p<0.05, **p<0.01 compared with the isotype control or si-control group.

Article Snippet: The antibodies used were as followed: carboxyfluorescein-conjugated RAE-1, FITC-conjugated MULT-1, PE-conjugated H60 (R&D Systems), anti-mouse NKG2D mAb (R&D System), PE-conjugated Ab to CD68, CD69 or IFN-γ (R&D System), FITC-conjugated affinity-purified goat anti-rat IgG (ZSGB-BIO, China) or isotype controls (eBioscience, San Diego, CA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation, Control, Transfection, Activity Assay

Journal: PLoS ONE

Article Title: Macrophages Help NK Cells to Attack Tumor Cells by Stimulatory NKG2D Ligand but Protect Themselves from NK Killing by Inhibitory Ligand Qa-1

doi: 10.1371/journal.pone.0036928

Figure Lengend Snippet: The secretion levels of IL-12, IL-15, IL-18, IFN-β, or IFN-γ in the supernatants of RAW264.7 cells (A) or resident peritoneal macrophages from BALB/c mice (B) that were cultured with 0 or 100 µg/ml poly I:C for 24 h were analyzed by ELISA. The data are expressed as the mean ± SD from at least three separate experiments. *p<0.05, **p<0.01 compared with the untreated RAW264.7 group. NK cells were incubated with poly I:C-pretreated RAW264.7 cells in the presence of 10 µg/ml anti-IL-15 mAb, anti-IFN-β mAb or isotype control separately for another 12 h, and the expression of NKG2D were determined by flow cytometry (C). Levels of IFN-γ in the supernatants were determined by ELISA (D). NK cell-mediated lysis of YAC-1 cells was assessed by first culturing NK cells with poly I:C-treated RAW264.7 cells (Mφ) in the presence or absence of anti-IL-15 mAb, anti-IFN-β mAb or isotype control, respectively, and then NK cells were isolated and added to target cells (E). Data are expressed as the mean ± SD from at least three separate experiments. *p<0.05 compared with the isotype control group.

Article Snippet: The antibodies used were as followed: carboxyfluorescein-conjugated RAE-1, FITC-conjugated MULT-1, PE-conjugated H60 (R&D Systems), anti-mouse NKG2D mAb (R&D System), PE-conjugated Ab to CD68, CD69 or IFN-γ (R&D System), FITC-conjugated affinity-purified goat anti-rat IgG (ZSGB-BIO, China) or isotype controls (eBioscience, San Diego, CA, USA).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Control, Expressing, Flow Cytometry, Lysis, Isolation

Journal: PLoS ONE

Article Title: Macrophages Help NK Cells to Attack Tumor Cells by Stimulatory NKG2D Ligand but Protect Themselves from NK Killing by Inhibitory Ligand Qa-1

doi: 10.1371/journal.pone.0036928

Figure Lengend Snippet: Freshly isolated murine NK cells were co-cultured with poly I:C-stimulated primary macrophages separated by a transwell for 24 h. Then NK cells were stained with antibodies for cell-surface CD69 and NKG2D, and for intracellular IFN-γ. The numbers of CD69- or NKG2D-positive as well as IFN-γ-secreting NK cells were determined by flow cytometry. Data are expressed as the percentage of positively-stained cells. The data are representatives from at least three separate experiments.

Article Snippet: The antibodies used were as followed: carboxyfluorescein-conjugated RAE-1, FITC-conjugated MULT-1, PE-conjugated H60 (R&D Systems), anti-mouse NKG2D mAb (R&D System), PE-conjugated Ab to CD68, CD69 or IFN-γ (R&D System), FITC-conjugated affinity-purified goat anti-rat IgG (ZSGB-BIO, China) or isotype controls (eBioscience, San Diego, CA, USA).

Techniques: Isolation, Cell Culture, Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: Macrophages Help NK Cells to Attack Tumor Cells by Stimulatory NKG2D Ligand but Protect Themselves from NK Killing by Inhibitory Ligand Qa-1

doi: 10.1371/journal.pone.0036928

Figure Lengend Snippet: As a response to stimulation from TLR3 ligand poly I:C, macrophages up-express surface RAE-1 (NKG2D ligand) and produce soluble IL-12, IL-15, and IFN-β. RAE-1 on surface of macrophages and their cytokines will strongly activate NK cells to produce IFN-γ and up-expression of NKG2D. IFN-γ produced by NK cells will stimulate macrophages in feedback. NK cells after activation will directly attack tumor cells by recognizing tumor’s RAE-1 and indirectly arrest tumor growth by secreting IFN-γ. At the same time, poly I:C-induced up-regulation of NKG2A ligand Qa-1 protects macrophages themselves from cytolysis by NK cells in order to keep the positive feedback loop in the anti-tumor immune response.

Article Snippet: The antibodies used were as followed: carboxyfluorescein-conjugated RAE-1, FITC-conjugated MULT-1, PE-conjugated H60 (R&D Systems), anti-mouse NKG2D mAb (R&D System), PE-conjugated Ab to CD68, CD69 or IFN-γ (R&D System), FITC-conjugated affinity-purified goat anti-rat IgG (ZSGB-BIO, China) or isotype controls (eBioscience, San Diego, CA, USA).

Techniques: Expressing, Produced, Activation 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 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, 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, 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

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

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