hpd l1 protein  (Sino Biological)


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    Name:
    PD L1 Protein Human Recombinant Biotinylated
    Description:
    A DNA sequence encoding the human CD274 NP 054862 1 Met1 Thr239 was expressed with C terminal fused Fc region of human IgG1 The purified protein was biotinylated in vitro
    Catalog Number:
    10084-H02H-B
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    B7-H Protein Human, B7-H1 Protein Human, B7H1 Protein Human, PD-L1 Protein Human, PDCD1L1 Protein Human, PDCD1LG1 Protein Human, PDL1 Protein Human
    Host:
    HEK293 Cells
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    Structured Review

    Sino Biological hpd l1 protein
    MB is effective to activate human CD8 +  T cells Effect of MB on proliferation of human CD8 +  T cells. CFSE‐labeled human peripheral blood mononuclear cells (PBMC) were preincubated with DMSO, MB, 25 μg/ml of pembrolizumab, or 20 μg/ml of nivolumab for 1 h and then seeded in the 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 in the presence of 10 μg/ml human PD‐L1 protein. Cell proliferation was analyzed through FACS analysis of dilution of CFSE. Cytokine and cytolytic granule production in CD8 +  T cells. Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg/ml of phytohaemagglutinin for 2 days and then rested for 1 day. Cells were pretreated with DMSO, MB, 25 μg/ml of pembrolizumab (Pem), or 20 μg/ml of nivolumab (Niv) for 1 h and then seeded in a 96‐well plate precoated with 10 μg/ml aCD3/aCD28, with media supplemented with 10 μg/ml of human PD‐L1 protein. Cytokine and cytolytic granule production was determined by intracellular flow cytometry by gating on CD8 +  populations. Comparison of the efficacy of MB with PD1 inhibitors currently used in clinic to activate T cell. JP‐luc cells were stimulated with Raji‐L1 or parental Raji cells preloaded with 1 μg/ml of superantigen (SEE) in the presence of MB at indicated concentration or 25 μg/ml of pembrolizumab (Pem) or 20 μg/ml of nivolumab (Niv) for 6 h. Luciferase activity was measured by luminometer. Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. JP‐luc: Jurkat cell harboring NFAT‐luciferase transgene and overexpressing PD‐1; Raji‐L1: Raji overexpressing PD‐L1. Data information: Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. Error bars denote SEM. ** P
    A DNA sequence encoding the human CD274 NP 054862 1 Met1 Thr239 was expressed with C terminal fused Fc region of human IgG1 The purified protein was biotinylated in vitro
    https://www.bioz.com/result/hpd l1 protein/product/Sino Biological
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hpd l1 protein - by Bioz Stars, 2021-05
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    1) Product Images from "Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors"

    Article Title: Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201911571

    MB is effective to activate human CD8 +  T cells Effect of MB on proliferation of human CD8 +  T cells. CFSE‐labeled human peripheral blood mononuclear cells (PBMC) were preincubated with DMSO, MB, 25 μg/ml of pembrolizumab, or 20 μg/ml of nivolumab for 1 h and then seeded in the 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 in the presence of 10 μg/ml human PD‐L1 protein. Cell proliferation was analyzed through FACS analysis of dilution of CFSE. Cytokine and cytolytic granule production in CD8 +  T cells. Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg/ml of phytohaemagglutinin for 2 days and then rested for 1 day. Cells were pretreated with DMSO, MB, 25 μg/ml of pembrolizumab (Pem), or 20 μg/ml of nivolumab (Niv) for 1 h and then seeded in a 96‐well plate precoated with 10 μg/ml aCD3/aCD28, with media supplemented with 10 μg/ml of human PD‐L1 protein. Cytokine and cytolytic granule production was determined by intracellular flow cytometry by gating on CD8 +  populations. Comparison of the efficacy of MB with PD1 inhibitors currently used in clinic to activate T cell. JP‐luc cells were stimulated with Raji‐L1 or parental Raji cells preloaded with 1 μg/ml of superantigen (SEE) in the presence of MB at indicated concentration or 25 μg/ml of pembrolizumab (Pem) or 20 μg/ml of nivolumab (Niv) for 6 h. Luciferase activity was measured by luminometer. Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. JP‐luc: Jurkat cell harboring NFAT‐luciferase transgene and overexpressing PD‐1; Raji‐L1: Raji overexpressing PD‐L1. Data information: Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. Error bars denote SEM. ** P
    Figure Legend Snippet: MB is effective to activate human CD8 + T cells Effect of MB on proliferation of human CD8 + T cells. CFSE‐labeled human peripheral blood mononuclear cells (PBMC) were preincubated with DMSO, MB, 25 μg/ml of pembrolizumab, or 20 μg/ml of nivolumab for 1 h and then seeded in the 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 in the presence of 10 μg/ml human PD‐L1 protein. Cell proliferation was analyzed through FACS analysis of dilution of CFSE. Cytokine and cytolytic granule production in CD8 + T cells. Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg/ml of phytohaemagglutinin for 2 days and then rested for 1 day. Cells were pretreated with DMSO, MB, 25 μg/ml of pembrolizumab (Pem), or 20 μg/ml of nivolumab (Niv) for 1 h and then seeded in a 96‐well plate precoated with 10 μg/ml aCD3/aCD28, with media supplemented with 10 μg/ml of human PD‐L1 protein. Cytokine and cytolytic granule production was determined by intracellular flow cytometry by gating on CD8 + populations. Comparison of the efficacy of MB with PD1 inhibitors currently used in clinic to activate T cell. JP‐luc cells were stimulated with Raji‐L1 or parental Raji cells preloaded with 1 μg/ml of superantigen (SEE) in the presence of MB at indicated concentration or 25 μg/ml of pembrolizumab (Pem) or 20 μg/ml of nivolumab (Niv) for 6 h. Luciferase activity was measured by luminometer. Data are representative of three independent experiments and were analyzed by unpaired t ‐test. JP‐luc: Jurkat cell harboring NFAT‐luciferase transgene and overexpressing PD‐1; Raji‐L1: Raji overexpressing PD‐L1. Data information: Data are representative of three independent experiments and were analyzed by unpaired t ‐test. Error bars denote SEM. ** P

    Techniques Used: Labeling, FACS, Flow Cytometry, Concentration Assay, Luciferase, Activity Assay

    MB suppress PD‐1 signaling through blocking SHP2 recruitment by PD‐1 Confocal cell images of Jurkat cells co‐expressing PD‐1‐EGFP and SHP2‐mCherry. Jurkat cells co‐expressing PD‐1‐EGFP and SHP2‐mCherry were incubated with 10 μg/ml of human PD‐L1 protein in the presence of 100 nM and 1 μM of MB for 2 min, fixed with 4% paraformaldehyde, and stained with DAPI. Colocalization signal was examined with confocal microscope (scale bar = 2 μm). Fluorescence complementation analysis of the effect of MB on the interaction between PD‐1 and SHP2. Jurkat cells co‐expressing PD1‐C‐Luc or PD1 Y248F ‐C‐Luc and SHP2‐N‐luc were seeded in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 with media supplemented with 10 μg/ml human PD‐L1 protein in the presence of MB at indicated concentrations for 6 h followed by analysis of luciferase activity. Co‐IP analysis of impact of MB on interaction between PD‐1 and SHP2. 293T cells co‐expressing PD‐1 and SHP2‐FLAG were treated with MB at indicated concentration for 6 h. Ability of MB to block interaction between PD‐1 with SHP2 revealed through ELISA. Y248‐phosphorylated human PD‐1 ITSM peptide was coated at 4 μg/ml in 96‐well plate overnight. Recombinant human SHP2 protein incubated with the coated peptide in the presence of MB for 2 h. Bound SHP2 was measured by monitoring the activity of horse peroxidase conjugated on secondary antibody targeting SHP2. Representative Western blot showing the levels of total and phosphorylated ZAP70, PKCθ, and PLCγ1 in the lysates of stimulated CTL, inhibited by PD‐L1 in the presence of 1 μM of MB. Representative Western blot showing the levels CD28 phosphorylation. CTL was stimulated with EG7 cells (parental), EG7‐L1, and EG7‐L1 in the presence of 100 nM of MB for 2 h. Protein immunoprecipitated (IP) with anti‐pY from the lysates of the indicated CTL‐EG7 co‐culture was subjected to SDS–PAGE separation and blotted with aCD28. β‐Actin served as loading control. EG7‐L1: EG7 overexpressing PD‐L1. Phos‐tag analysis of CD28 phosphorylation in CTL. CTLs were stimulated with EG7 or EG7‐L1 in the presence of 1 μM MB. Cell lysate was then separated by SDS–PAGE containing 50 μM of Phos‐tag and blotted with aCD28 antibody. Phosphorylated CD28 (slow‐moving) species were visualized through exposure. Fluorescence complementation analysis of the effect of MB on the interaction between CD28 with P85. Jurkat or Jurkat‐PD‐1 cells were co‐transfected with CD28‐C‐Luc or CD28 Y191F ‐C‐Luc and P85‐N‐Luc. Cells were seeded in a 96‐well plate coated with 10 μg/ml of aCD3/aCD28 in the presence of human 10 μg/ml of PD‐L1 protein and 100 nM of MB for 6 h. Luciferase activity was measured as readout for CD28‐P85 interaction. Data information: Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. Error bars denote SEM. * P
    Figure Legend Snippet: MB suppress PD‐1 signaling through blocking SHP2 recruitment by PD‐1 Confocal cell images of Jurkat cells co‐expressing PD‐1‐EGFP and SHP2‐mCherry. Jurkat cells co‐expressing PD‐1‐EGFP and SHP2‐mCherry were incubated with 10 μg/ml of human PD‐L1 protein in the presence of 100 nM and 1 μM of MB for 2 min, fixed with 4% paraformaldehyde, and stained with DAPI. Colocalization signal was examined with confocal microscope (scale bar = 2 μm). Fluorescence complementation analysis of the effect of MB on the interaction between PD‐1 and SHP2. Jurkat cells co‐expressing PD1‐C‐Luc or PD1 Y248F ‐C‐Luc and SHP2‐N‐luc were seeded in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 with media supplemented with 10 μg/ml human PD‐L1 protein in the presence of MB at indicated concentrations for 6 h followed by analysis of luciferase activity. Co‐IP analysis of impact of MB on interaction between PD‐1 and SHP2. 293T cells co‐expressing PD‐1 and SHP2‐FLAG were treated with MB at indicated concentration for 6 h. Ability of MB to block interaction between PD‐1 with SHP2 revealed through ELISA. Y248‐phosphorylated human PD‐1 ITSM peptide was coated at 4 μg/ml in 96‐well plate overnight. Recombinant human SHP2 protein incubated with the coated peptide in the presence of MB for 2 h. Bound SHP2 was measured by monitoring the activity of horse peroxidase conjugated on secondary antibody targeting SHP2. Representative Western blot showing the levels of total and phosphorylated ZAP70, PKCθ, and PLCγ1 in the lysates of stimulated CTL, inhibited by PD‐L1 in the presence of 1 μM of MB. Representative Western blot showing the levels CD28 phosphorylation. CTL was stimulated with EG7 cells (parental), EG7‐L1, and EG7‐L1 in the presence of 100 nM of MB for 2 h. Protein immunoprecipitated (IP) with anti‐pY from the lysates of the indicated CTL‐EG7 co‐culture was subjected to SDS–PAGE separation and blotted with aCD28. β‐Actin served as loading control. EG7‐L1: EG7 overexpressing PD‐L1. Phos‐tag analysis of CD28 phosphorylation in CTL. CTLs were stimulated with EG7 or EG7‐L1 in the presence of 1 μM MB. Cell lysate was then separated by SDS–PAGE containing 50 μM of Phos‐tag and blotted with aCD28 antibody. Phosphorylated CD28 (slow‐moving) species were visualized through exposure. Fluorescence complementation analysis of the effect of MB on the interaction between CD28 with P85. Jurkat or Jurkat‐PD‐1 cells were co‐transfected with CD28‐C‐Luc or CD28 Y191F ‐C‐Luc and P85‐N‐Luc. Cells were seeded in a 96‐well plate coated with 10 μg/ml of aCD3/aCD28 in the presence of human 10 μg/ml of PD‐L1 protein and 100 nM of MB for 6 h. Luciferase activity was measured as readout for CD28‐P85 interaction. Data information: Data are representative of three independent experiments and were analyzed by unpaired t ‐test. Error bars denote SEM. * P

    Techniques Used: Blocking Assay, Expressing, Incubation, Staining, Microscopy, Fluorescence, Luciferase, Activity Assay, Co-Immunoprecipitation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Western Blot, Immunoprecipitation, Co-Culture Assay, SDS Page, Transfection

    MB enhanced activation and effector function of CTL Effect of PD‐1 antibody on the proliferation of OT‐I CTLs. Splenocytes from OT‐I mice were labeled with CFSE and seeded in a 96‐well plate. Media were supplemented with 10 nM of SINFEEKL peptide, 10 ng/ml of human IL‐2 and 10 μg/ml of mouse PD‐L1 protein in the presence of 10 μg/ml of PD‐1 antibody. Cell proliferation was measured by FACS. Bar graph of (Fig   EV2 A). MB enhancing production of cytokine and cytolytic granule by OT‐1 CTLs. CTLs were co‐incubated with CFSE‐labeled EG7‐L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule was determined by flow cytometry. aPD‐1 antibody served as positive control. EG7‐L1: EG7 overexpressing PD‐L1. Bar graph of IFNγ production of OT‐1 CTLs in (Fig   EV2 C). Bar graph of GZMB production of OT‐1 CTLs in (Fig   EV2 C). Data information: Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. Error bars denote SEM. * P
    Figure Legend Snippet: MB enhanced activation and effector function of CTL Effect of PD‐1 antibody on the proliferation of OT‐I CTLs. Splenocytes from OT‐I mice were labeled with CFSE and seeded in a 96‐well plate. Media were supplemented with 10 nM of SINFEEKL peptide, 10 ng/ml of human IL‐2 and 10 μg/ml of mouse PD‐L1 protein in the presence of 10 μg/ml of PD‐1 antibody. Cell proliferation was measured by FACS. Bar graph of (Fig  EV2 A). MB enhancing production of cytokine and cytolytic granule by OT‐1 CTLs. CTLs were co‐incubated with CFSE‐labeled EG7‐L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule was determined by flow cytometry. aPD‐1 antibody served as positive control. EG7‐L1: EG7 overexpressing PD‐L1. Bar graph of IFNγ production of OT‐1 CTLs in (Fig  EV2 C). Bar graph of GZMB production of OT‐1 CTLs in (Fig  EV2 C). Data information: Data are representative of three independent experiments and were analyzed by unpaired t ‐test. Error bars denote SEM. * P

    Techniques Used: Activation Assay, Mouse Assay, Labeling, FACS, Incubation, Expressing, Flow Cytometry, Positive Control

    MB enhanced activation and effector function of CTL Effect of MB on the proliferation of WT CTLs and PD‐1KO CTLs. Splenic cells were stained with 5 μM of CFSE and seeded into aCD3/aCD28‐coated 96‐well plates. 10 μg/ml of PD‐L1 was administered in media. Cell proliferation was checked by monitoring dilution of CFSE 48 h after CD3/CD28 stimulation by gating on CD8‐positive population through FACS analysis. WT: splenic cell from wild‐type C57BL/J mice. PD‐1KO: splenic cell from PD‐1 knockout mice. Statistical results of (A). FACS analysis of the effect of MB on the activation of OT‐I CTLs. OT‐I CTLs were stimulated with precoated aCD3/aCD28 and 10 μg/ml of mouse PD‐L1 protein in the presence of 100 nM MB or 10 μg/ml aPD1 (served as positive control). After 24 h, surface expression of CD25 and CD69 on OT‐1 CTLs was determined through FACS analysis. Statistical results of (C). Effect of MB on the activation status of Jurkat T cells. Luciferase activity is suppressed in JP‐luc by co‐culture with Raji‐L1 preloaded with 1 μg/ml superantigen (SEE). Treatment with MB enhanced luciferase expression (SEE‐loaded Raji (PD‐L1 negative) served as positive control). IC 50  is calculated to be 117 nM. JP‐luc: Jurkat cell harboring NFAT‐luciferase transgene and overexpressing PD‐1; Raji‐L1: Raji overexpressing PD‐L1. Impact of MB on luciferase activity of various engineered Jurkat T cells. J‐luc: Jurkat cell harboring NFAT‐luciferase transgene; J‐luc‐sgPD‐1: J‐luc cells treated with lentivirus expressing sgPD‐1/CAS9 simultaneously. qRT–PCR analysis of IL‐2 mRNA level in JP‐luc cells stimulated with precoated aCD3/aCD28 (10 μg/ml) in the presence of 10 μg/ml of PD‐L1 and MB at indicated concentrations. MB enhancing IL‐2 expression by JP‐luc stimulated with Raji‐L1 for 24 h quantified through ELISA analysis (aPD1 served as positive control). MB enhancing production of cytokine and cytolytic granule by OT‐I CTLs. CTLs were incubated with EG7‐L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule was determined by flow cytometry. aPD1 served as positive control. EG7‐L1: EG7 overexpressing PD‐L1. Statistical results of (I). Data information: Data are representative of three independent experiments. Unpaired  t ‐test; error bars denote SEM. * P
    Figure Legend Snippet: MB enhanced activation and effector function of CTL Effect of MB on the proliferation of WT CTLs and PD‐1KO CTLs. Splenic cells were stained with 5 μM of CFSE and seeded into aCD3/aCD28‐coated 96‐well plates. 10 μg/ml of PD‐L1 was administered in media. Cell proliferation was checked by monitoring dilution of CFSE 48 h after CD3/CD28 stimulation by gating on CD8‐positive population through FACS analysis. WT: splenic cell from wild‐type C57BL/J mice. PD‐1KO: splenic cell from PD‐1 knockout mice. Statistical results of (A). FACS analysis of the effect of MB on the activation of OT‐I CTLs. OT‐I CTLs were stimulated with precoated aCD3/aCD28 and 10 μg/ml of mouse PD‐L1 protein in the presence of 100 nM MB or 10 μg/ml aPD1 (served as positive control). After 24 h, surface expression of CD25 and CD69 on OT‐1 CTLs was determined through FACS analysis. Statistical results of (C). Effect of MB on the activation status of Jurkat T cells. Luciferase activity is suppressed in JP‐luc by co‐culture with Raji‐L1 preloaded with 1 μg/ml superantigen (SEE). Treatment with MB enhanced luciferase expression (SEE‐loaded Raji (PD‐L1 negative) served as positive control). IC 50 is calculated to be 117 nM. JP‐luc: Jurkat cell harboring NFAT‐luciferase transgene and overexpressing PD‐1; Raji‐L1: Raji overexpressing PD‐L1. Impact of MB on luciferase activity of various engineered Jurkat T cells. J‐luc: Jurkat cell harboring NFAT‐luciferase transgene; J‐luc‐sgPD‐1: J‐luc cells treated with lentivirus expressing sgPD‐1/CAS9 simultaneously. qRT–PCR analysis of IL‐2 mRNA level in JP‐luc cells stimulated with precoated aCD3/aCD28 (10 μg/ml) in the presence of 10 μg/ml of PD‐L1 and MB at indicated concentrations. MB enhancing IL‐2 expression by JP‐luc stimulated with Raji‐L1 for 24 h quantified through ELISA analysis (aPD1 served as positive control). MB enhancing production of cytokine and cytolytic granule by OT‐I CTLs. CTLs were incubated with EG7‐L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule was determined by flow cytometry. aPD1 served as positive control. EG7‐L1: EG7 overexpressing PD‐L1. Statistical results of (I). Data information: Data are representative of three independent experiments. Unpaired t ‐test; error bars denote SEM. * P

    Techniques Used: Activation Assay, Staining, FACS, Mouse Assay, Knock-Out, Positive Control, Expressing, Luciferase, Activity Assay, Co-Culture Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry

    2) Product Images from "Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors"

    Article Title: Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201911571

    MB is effective to activate human CD8 +  T cells Effect of MB on proliferation of human CD8 +  T cells. CFSE‐labeled human peripheral blood mononuclear cells (PBMC) were preincubated with DMSO, MB, 25 μg/ml of pembrolizumab, or 20 μg/ml of nivolumab for 1 h and then seeded in the 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 in the presence of 10 μg/ml human PD‐L1 protein. Cell proliferation was analyzed through FACS analysis of dilution of CFSE. Cytokine and cytolytic granule production in CD8 +  T cells. Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg/ml of phytohaemagglutinin for 2 days and then rested for 1 day. Cells were pretreated with DMSO, MB, 25 μg/ml of pembrolizumab (Pem), or 20 μg/ml of nivolumab (Niv) for 1 h and then seeded in a 96‐well plate precoated with 10 μg/ml aCD3/aCD28, with media supplemented with 10 μg/ml of human PD‐L1 protein. Cytokine and cytolytic granule production was determined by intracellular flow cytometry by gating on CD8 +  populations. Comparison of the efficacy of MB with PD1 inhibitors currently used in clinic to activate T cell. JP‐luc cells were stimulated with Raji‐L1 or parental Raji cells preloaded with 1 μg/ml of superantigen (SEE) in the presence of MB at indicated concentration or 25 μg/ml of pembrolizumab (Pem) or 20 μg/ml of nivolumab (Niv) for 6 h. Luciferase activity was measured by luminometer. Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. JP‐luc: Jurkat cell harboring NFAT‐luciferase transgene and overexpressing PD‐1; Raji‐L1: Raji overexpressing PD‐L1. Data information: Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. Error bars denote SEM. ** P
    Figure Legend Snippet: MB is effective to activate human CD8 + T cells Effect of MB on proliferation of human CD8 + T cells. CFSE‐labeled human peripheral blood mononuclear cells (PBMC) were preincubated with DMSO, MB, 25 μg/ml of pembrolizumab, or 20 μg/ml of nivolumab for 1 h and then seeded in the 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 in the presence of 10 μg/ml human PD‐L1 protein. Cell proliferation was analyzed through FACS analysis of dilution of CFSE. Cytokine and cytolytic granule production in CD8 + T cells. Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg/ml of phytohaemagglutinin for 2 days and then rested for 1 day. Cells were pretreated with DMSO, MB, 25 μg/ml of pembrolizumab (Pem), or 20 μg/ml of nivolumab (Niv) for 1 h and then seeded in a 96‐well plate precoated with 10 μg/ml aCD3/aCD28, with media supplemented with 10 μg/ml of human PD‐L1 protein. Cytokine and cytolytic granule production was determined by intracellular flow cytometry by gating on CD8 + populations. Comparison of the efficacy of MB with PD1 inhibitors currently used in clinic to activate T cell. JP‐luc cells were stimulated with Raji‐L1 or parental Raji cells preloaded with 1 μg/ml of superantigen (SEE) in the presence of MB at indicated concentration or 25 μg/ml of pembrolizumab (Pem) or 20 μg/ml of nivolumab (Niv) for 6 h. Luciferase activity was measured by luminometer. Data are representative of three independent experiments and were analyzed by unpaired t ‐test. JP‐luc: Jurkat cell harboring NFAT‐luciferase transgene and overexpressing PD‐1; Raji‐L1: Raji overexpressing PD‐L1. Data information: Data are representative of three independent experiments and were analyzed by unpaired t ‐test. Error bars denote SEM. ** P

    Techniques Used: Labeling, FACS, Flow Cytometry, Concentration Assay, Luciferase, Activity Assay

    MB suppress PD‐1 signaling through blocking SHP2 recruitment by PD‐1 Confocal cell images of Jurkat cells co‐expressing PD‐1‐EGFP and SHP2‐mCherry. Jurkat cells co‐expressing PD‐1‐EGFP and SHP2‐mCherry were incubated with 10 μg/ml of human PD‐L1 protein in the presence of 100 nM and 1 μM of MB for 2 min, fixed with 4% paraformaldehyde, and stained with DAPI. Colocalization signal was examined with confocal microscope (scale bar = 2 μm). Fluorescence complementation analysis of the effect of MB on the interaction between PD‐1 and SHP2. Jurkat cells co‐expressing PD1‐C‐Luc or PD1 Y248F ‐C‐Luc and SHP2‐N‐luc were seeded in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 with media supplemented with 10 μg/ml human PD‐L1 protein in the presence of MB at indicated concentrations for 6 h followed by analysis of luciferase activity. Co‐IP analysis of impact of MB on interaction between PD‐1 and SHP2. 293T cells co‐expressing PD‐1 and SHP2‐FLAG were treated with MB at indicated concentration for 6 h. Ability of MB to block interaction between PD‐1 with SHP2 revealed through ELISA. Y248‐phosphorylated human PD‐1 ITSM peptide was coated at 4 μg/ml in 96‐well plate overnight. Recombinant human SHP2 protein incubated with the coated peptide in the presence of MB for 2 h. Bound SHP2 was measured by monitoring the activity of horse peroxidase conjugated on secondary antibody targeting SHP2. Representative Western blot showing the levels of total and phosphorylated ZAP70, PKCθ, and PLCγ1 in the lysates of stimulated CTL, inhibited by PD‐L1 in the presence of 1 μM of MB. Representative Western blot showing the levels CD28 phosphorylation. CTL was stimulated with EG7 cells (parental), EG7‐L1, and EG7‐L1 in the presence of 100 nM of MB for 2 h. Protein immunoprecipitated (IP) with anti‐pY from the lysates of the indicated CTL‐EG7 co‐culture was subjected to SDS–PAGE separation and blotted with aCD28. β‐Actin served as loading control. EG7‐L1: EG7 overexpressing PD‐L1. Phos‐tag analysis of CD28 phosphorylation in CTL. CTLs were stimulated with EG7 or EG7‐L1 in the presence of 1 μM MB. Cell lysate was then separated by SDS–PAGE containing 50 μM of Phos‐tag and blotted with aCD28 antibody. Phosphorylated CD28 (slow‐moving) species were visualized through exposure. Fluorescence complementation analysis of the effect of MB on the interaction between CD28 with P85. Jurkat or Jurkat‐PD‐1 cells were co‐transfected with CD28‐C‐Luc or CD28 Y191F ‐C‐Luc and P85‐N‐Luc. Cells were seeded in a 96‐well plate coated with 10 μg/ml of aCD3/aCD28 in the presence of human 10 μg/ml of PD‐L1 protein and 100 nM of MB for 6 h. Luciferase activity was measured as readout for CD28‐P85 interaction. Data information: Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. Error bars denote SEM. * P
    Figure Legend Snippet: MB suppress PD‐1 signaling through blocking SHP2 recruitment by PD‐1 Confocal cell images of Jurkat cells co‐expressing PD‐1‐EGFP and SHP2‐mCherry. Jurkat cells co‐expressing PD‐1‐EGFP and SHP2‐mCherry were incubated with 10 μg/ml of human PD‐L1 protein in the presence of 100 nM and 1 μM of MB for 2 min, fixed with 4% paraformaldehyde, and stained with DAPI. Colocalization signal was examined with confocal microscope (scale bar = 2 μm). Fluorescence complementation analysis of the effect of MB on the interaction between PD‐1 and SHP2. Jurkat cells co‐expressing PD1‐C‐Luc or PD1 Y248F ‐C‐Luc and SHP2‐N‐luc were seeded in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 with media supplemented with 10 μg/ml human PD‐L1 protein in the presence of MB at indicated concentrations for 6 h followed by analysis of luciferase activity. Co‐IP analysis of impact of MB on interaction between PD‐1 and SHP2. 293T cells co‐expressing PD‐1 and SHP2‐FLAG were treated with MB at indicated concentration for 6 h. Ability of MB to block interaction between PD‐1 with SHP2 revealed through ELISA. Y248‐phosphorylated human PD‐1 ITSM peptide was coated at 4 μg/ml in 96‐well plate overnight. Recombinant human SHP2 protein incubated with the coated peptide in the presence of MB for 2 h. Bound SHP2 was measured by monitoring the activity of horse peroxidase conjugated on secondary antibody targeting SHP2. Representative Western blot showing the levels of total and phosphorylated ZAP70, PKCθ, and PLCγ1 in the lysates of stimulated CTL, inhibited by PD‐L1 in the presence of 1 μM of MB. Representative Western blot showing the levels CD28 phosphorylation. CTL was stimulated with EG7 cells (parental), EG7‐L1, and EG7‐L1 in the presence of 100 nM of MB for 2 h. Protein immunoprecipitated (IP) with anti‐pY from the lysates of the indicated CTL‐EG7 co‐culture was subjected to SDS–PAGE separation and blotted with aCD28. β‐Actin served as loading control. EG7‐L1: EG7 overexpressing PD‐L1. Phos‐tag analysis of CD28 phosphorylation in CTL. CTLs were stimulated with EG7 or EG7‐L1 in the presence of 1 μM MB. Cell lysate was then separated by SDS–PAGE containing 50 μM of Phos‐tag and blotted with aCD28 antibody. Phosphorylated CD28 (slow‐moving) species were visualized through exposure. Fluorescence complementation analysis of the effect of MB on the interaction between CD28 with P85. Jurkat or Jurkat‐PD‐1 cells were co‐transfected with CD28‐C‐Luc or CD28 Y191F ‐C‐Luc and P85‐N‐Luc. Cells were seeded in a 96‐well plate coated with 10 μg/ml of aCD3/aCD28 in the presence of human 10 μg/ml of PD‐L1 protein and 100 nM of MB for 6 h. Luciferase activity was measured as readout for CD28‐P85 interaction. Data information: Data are representative of three independent experiments and were analyzed by unpaired t ‐test. Error bars denote SEM. * P

    Techniques Used: Blocking Assay, Expressing, Incubation, Staining, Microscopy, Fluorescence, Luciferase, Activity Assay, Co-Immunoprecipitation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Western Blot, Immunoprecipitation, Co-Culture Assay, SDS Page, Transfection

    MB enhanced activation and effector function of CTL Effect of PD‐1 antibody on the proliferation of OT‐I CTLs. Splenocytes from OT‐I mice were labeled with CFSE and seeded in a 96‐well plate. Media were supplemented with 10 nM of SINFEEKL peptide, 10 ng/ml of human IL‐2 and 10 μg/ml of mouse PD‐L1 protein in the presence of 10 μg/ml of PD‐1 antibody. Cell proliferation was measured by FACS. Bar graph of (Fig   EV2 A). MB enhancing production of cytokine and cytolytic granule by OT‐1 CTLs. CTLs were co‐incubated with CFSE‐labeled EG7‐L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule was determined by flow cytometry. aPD‐1 antibody served as positive control. EG7‐L1: EG7 overexpressing PD‐L1. Bar graph of IFNγ production of OT‐1 CTLs in (Fig   EV2 C). Bar graph of GZMB production of OT‐1 CTLs in (Fig   EV2 C). Data information: Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. Error bars denote SEM. * P
    Figure Legend Snippet: MB enhanced activation and effector function of CTL Effect of PD‐1 antibody on the proliferation of OT‐I CTLs. Splenocytes from OT‐I mice were labeled with CFSE and seeded in a 96‐well plate. Media were supplemented with 10 nM of SINFEEKL peptide, 10 ng/ml of human IL‐2 and 10 μg/ml of mouse PD‐L1 protein in the presence of 10 μg/ml of PD‐1 antibody. Cell proliferation was measured by FACS. Bar graph of (Fig  EV2 A). MB enhancing production of cytokine and cytolytic granule by OT‐1 CTLs. CTLs were co‐incubated with CFSE‐labeled EG7‐L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule was determined by flow cytometry. aPD‐1 antibody served as positive control. EG7‐L1: EG7 overexpressing PD‐L1. Bar graph of IFNγ production of OT‐1 CTLs in (Fig  EV2 C). Bar graph of GZMB production of OT‐1 CTLs in (Fig  EV2 C). Data information: Data are representative of three independent experiments and were analyzed by unpaired t ‐test. Error bars denote SEM. * P

    Techniques Used: Activation Assay, Mouse Assay, Labeling, FACS, Incubation, Expressing, Flow Cytometry, Positive Control

    MB enhanced activation and effector function of CTL Effect of MB on the proliferation of WT CTLs and PD‐1KO CTLs. Splenic cells were stained with 5 μM of CFSE and seeded into aCD3/aCD28‐coated 96‐well plates. 10 μg/ml of PD‐L1 was administered in media. Cell proliferation was checked by monitoring dilution of CFSE 48 h after CD3/CD28 stimulation by gating on CD8‐positive population through FACS analysis. WT: splenic cell from wild‐type C57BL/J mice. PD‐1KO: splenic cell from PD‐1 knockout mice. Statistical results of (A). FACS analysis of the effect of MB on the activation of OT‐I CTLs. OT‐I CTLs were stimulated with precoated aCD3/aCD28 and 10 μg/ml of mouse PD‐L1 protein in the presence of 100 nM MB or 10 μg/ml aPD1 (served as positive control). After 24 h, surface expression of CD25 and CD69 on OT‐1 CTLs was determined through FACS analysis. Statistical results of (C). Effect of MB on the activation status of Jurkat T cells. Luciferase activity is suppressed in JP‐luc by co‐culture with Raji‐L1 preloaded with 1 μg/ml superantigen (SEE). Treatment with MB enhanced luciferase expression (SEE‐loaded Raji (PD‐L1 negative) served as positive control). IC 50  is calculated to be 117 nM. JP‐luc: Jurkat cell harboring NFAT‐luciferase transgene and overexpressing PD‐1; Raji‐L1: Raji overexpressing PD‐L1. Impact of MB on luciferase activity of various engineered Jurkat T cells. J‐luc: Jurkat cell harboring NFAT‐luciferase transgene; J‐luc‐sgPD‐1: J‐luc cells treated with lentivirus expressing sgPD‐1/CAS9 simultaneously. qRT–PCR analysis of IL‐2 mRNA level in JP‐luc cells stimulated with precoated aCD3/aCD28 (10 μg/ml) in the presence of 10 μg/ml of PD‐L1 and MB at indicated concentrations. MB enhancing IL‐2 expression by JP‐luc stimulated with Raji‐L1 for 24 h quantified through ELISA analysis (aPD1 served as positive control). MB enhancing production of cytokine and cytolytic granule by OT‐I CTLs. CTLs were incubated with EG7‐L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule was determined by flow cytometry. aPD1 served as positive control. EG7‐L1: EG7 overexpressing PD‐L1. Statistical results of (I). Data information: Data are representative of three independent experiments. Unpaired  t ‐test; error bars denote SEM. * P
    Figure Legend Snippet: MB enhanced activation and effector function of CTL Effect of MB on the proliferation of WT CTLs and PD‐1KO CTLs. Splenic cells were stained with 5 μM of CFSE and seeded into aCD3/aCD28‐coated 96‐well plates. 10 μg/ml of PD‐L1 was administered in media. Cell proliferation was checked by monitoring dilution of CFSE 48 h after CD3/CD28 stimulation by gating on CD8‐positive population through FACS analysis. WT: splenic cell from wild‐type C57BL/J mice. PD‐1KO: splenic cell from PD‐1 knockout mice. Statistical results of (A). FACS analysis of the effect of MB on the activation of OT‐I CTLs. OT‐I CTLs were stimulated with precoated aCD3/aCD28 and 10 μg/ml of mouse PD‐L1 protein in the presence of 100 nM MB or 10 μg/ml aPD1 (served as positive control). After 24 h, surface expression of CD25 and CD69 on OT‐1 CTLs was determined through FACS analysis. Statistical results of (C). Effect of MB on the activation status of Jurkat T cells. Luciferase activity is suppressed in JP‐luc by co‐culture with Raji‐L1 preloaded with 1 μg/ml superantigen (SEE). Treatment with MB enhanced luciferase expression (SEE‐loaded Raji (PD‐L1 negative) served as positive control). IC 50 is calculated to be 117 nM. JP‐luc: Jurkat cell harboring NFAT‐luciferase transgene and overexpressing PD‐1; Raji‐L1: Raji overexpressing PD‐L1. Impact of MB on luciferase activity of various engineered Jurkat T cells. J‐luc: Jurkat cell harboring NFAT‐luciferase transgene; J‐luc‐sgPD‐1: J‐luc cells treated with lentivirus expressing sgPD‐1/CAS9 simultaneously. qRT–PCR analysis of IL‐2 mRNA level in JP‐luc cells stimulated with precoated aCD3/aCD28 (10 μg/ml) in the presence of 10 μg/ml of PD‐L1 and MB at indicated concentrations. MB enhancing IL‐2 expression by JP‐luc stimulated with Raji‐L1 for 24 h quantified through ELISA analysis (aPD1 served as positive control). MB enhancing production of cytokine and cytolytic granule by OT‐I CTLs. CTLs were incubated with EG7‐L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule was determined by flow cytometry. aPD1 served as positive control. EG7‐L1: EG7 overexpressing PD‐L1. Statistical results of (I). Data information: Data are representative of three independent experiments. Unpaired t ‐test; error bars denote SEM. * P

    Techniques Used: Activation Assay, Staining, FACS, Mouse Assay, Knock-Out, Positive Control, Expressing, Luciferase, Activity Assay, Co-Culture Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry

    3) Product Images from "Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors"

    Article Title: Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201911571

    MB is effective to activate human CD8 +  T cells Effect of MB on proliferation of human CD8 +  T cells. CFSE‐labeled human peripheral blood mononuclear cells (PBMC) were preincubated with DMSO, MB, 25 μg/ml of pembrolizumab, or 20 μg/ml of nivolumab for 1 h and then seeded in the 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 in the presence of 10 μg/ml human PD‐L1 protein. Cell proliferation was analyzed through FACS analysis of dilution of CFSE. Cytokine and cytolytic granule production in CD8 +  T cells. Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg/ml of phytohaemagglutinin for 2 days and then rested for 1 day. Cells were pretreated with DMSO, MB, 25 μg/ml of pembrolizumab (Pem), or 20 μg/ml of nivolumab (Niv) for 1 h and then seeded in a 96‐well plate precoated with 10 μg/ml aCD3/aCD28, with media supplemented with 10 μg/ml of human PD‐L1 protein. Cytokine and cytolytic granule production was determined by intracellular flow cytometry by gating on CD8 +  populations. Comparison of the efficacy of MB with PD1 inhibitors currently used in clinic to activate T cell. JP‐luc cells were stimulated with Raji‐L1 or parental Raji cells preloaded with 1 μg/ml of superantigen (SEE) in the presence of MB at indicated concentration or 25 μg/ml of pembrolizumab (Pem) or 20 μg/ml of nivolumab (Niv) for 6 h. Luciferase activity was measured by luminometer. Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. JP‐luc: Jurkat cell harboring NFAT‐luciferase transgene and overexpressing PD‐1; Raji‐L1: Raji overexpressing PD‐L1. Data information: Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. Error bars denote SEM. ** P
    Figure Legend Snippet: MB is effective to activate human CD8 + T cells Effect of MB on proliferation of human CD8 + T cells. CFSE‐labeled human peripheral blood mononuclear cells (PBMC) were preincubated with DMSO, MB, 25 μg/ml of pembrolizumab, or 20 μg/ml of nivolumab for 1 h and then seeded in the 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 in the presence of 10 μg/ml human PD‐L1 protein. Cell proliferation was analyzed through FACS analysis of dilution of CFSE. Cytokine and cytolytic granule production in CD8 + T cells. Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg/ml of phytohaemagglutinin for 2 days and then rested for 1 day. Cells were pretreated with DMSO, MB, 25 μg/ml of pembrolizumab (Pem), or 20 μg/ml of nivolumab (Niv) for 1 h and then seeded in a 96‐well plate precoated with 10 μg/ml aCD3/aCD28, with media supplemented with 10 μg/ml of human PD‐L1 protein. Cytokine and cytolytic granule production was determined by intracellular flow cytometry by gating on CD8 + populations. Comparison of the efficacy of MB with PD1 inhibitors currently used in clinic to activate T cell. JP‐luc cells were stimulated with Raji‐L1 or parental Raji cells preloaded with 1 μg/ml of superantigen (SEE) in the presence of MB at indicated concentration or 25 μg/ml of pembrolizumab (Pem) or 20 μg/ml of nivolumab (Niv) for 6 h. Luciferase activity was measured by luminometer. Data are representative of three independent experiments and were analyzed by unpaired t ‐test. JP‐luc: Jurkat cell harboring NFAT‐luciferase transgene and overexpressing PD‐1; Raji‐L1: Raji overexpressing PD‐L1. Data information: Data are representative of three independent experiments and were analyzed by unpaired t ‐test. Error bars denote SEM. ** P

    Techniques Used: Labeling, FACS, Flow Cytometry, Concentration Assay, Luciferase, Activity Assay

    MB suppress PD‐1 signaling through blocking SHP2 recruitment by PD‐1 Confocal cell images of Jurkat cells co‐expressing PD‐1‐EGFP and SHP2‐mCherry. Jurkat cells co‐expressing PD‐1‐EGFP and SHP2‐mCherry were incubated with 10 μg/ml of human PD‐L1 protein in the presence of 100 nM and 1 μM of MB for 2 min, fixed with 4% paraformaldehyde, and stained with DAPI. Colocalization signal was examined with confocal microscope (scale bar = 2 μm). Fluorescence complementation analysis of the effect of MB on the interaction between PD‐1 and SHP2. Jurkat cells co‐expressing PD1‐C‐Luc or PD1 Y248F ‐C‐Luc and SHP2‐N‐luc were seeded in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 with media supplemented with 10 μg/ml human PD‐L1 protein in the presence of MB at indicated concentrations for 6 h followed by analysis of luciferase activity. Co‐IP analysis of impact of MB on interaction between PD‐1 and SHP2. 293T cells co‐expressing PD‐1 and SHP2‐FLAG were treated with MB at indicated concentration for 6 h. Ability of MB to block interaction between PD‐1 with SHP2 revealed through ELISA. Y248‐phosphorylated human PD‐1 ITSM peptide was coated at 4 μg/ml in 96‐well plate overnight. Recombinant human SHP2 protein incubated with the coated peptide in the presence of MB for 2 h. Bound SHP2 was measured by monitoring the activity of horse peroxidase conjugated on secondary antibody targeting SHP2. Representative Western blot showing the levels of total and phosphorylated ZAP70, PKCθ, and PLCγ1 in the lysates of stimulated CTL, inhibited by PD‐L1 in the presence of 1 μM of MB. Representative Western blot showing the levels CD28 phosphorylation. CTL was stimulated with EG7 cells (parental), EG7‐L1, and EG7‐L1 in the presence of 100 nM of MB for 2 h. Protein immunoprecipitated (IP) with anti‐pY from the lysates of the indicated CTL‐EG7 co‐culture was subjected to SDS–PAGE separation and blotted with aCD28. β‐Actin served as loading control. EG7‐L1: EG7 overexpressing PD‐L1. Phos‐tag analysis of CD28 phosphorylation in CTL. CTLs were stimulated with EG7 or EG7‐L1 in the presence of 1 μM MB. Cell lysate was then separated by SDS–PAGE containing 50 μM of Phos‐tag and blotted with aCD28 antibody. Phosphorylated CD28 (slow‐moving) species were visualized through exposure. Fluorescence complementation analysis of the effect of MB on the interaction between CD28 with P85. Jurkat or Jurkat‐PD‐1 cells were co‐transfected with CD28‐C‐Luc or CD28 Y191F ‐C‐Luc and P85‐N‐Luc. Cells were seeded in a 96‐well plate coated with 10 μg/ml of aCD3/aCD28 in the presence of human 10 μg/ml of PD‐L1 protein and 100 nM of MB for 6 h. Luciferase activity was measured as readout for CD28‐P85 interaction. Data information: Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. Error bars denote SEM. * P
    Figure Legend Snippet: MB suppress PD‐1 signaling through blocking SHP2 recruitment by PD‐1 Confocal cell images of Jurkat cells co‐expressing PD‐1‐EGFP and SHP2‐mCherry. Jurkat cells co‐expressing PD‐1‐EGFP and SHP2‐mCherry were incubated with 10 μg/ml of human PD‐L1 protein in the presence of 100 nM and 1 μM of MB for 2 min, fixed with 4% paraformaldehyde, and stained with DAPI. Colocalization signal was examined with confocal microscope (scale bar = 2 μm). Fluorescence complementation analysis of the effect of MB on the interaction between PD‐1 and SHP2. Jurkat cells co‐expressing PD1‐C‐Luc or PD1 Y248F ‐C‐Luc and SHP2‐N‐luc were seeded in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 with media supplemented with 10 μg/ml human PD‐L1 protein in the presence of MB at indicated concentrations for 6 h followed by analysis of luciferase activity. Co‐IP analysis of impact of MB on interaction between PD‐1 and SHP2. 293T cells co‐expressing PD‐1 and SHP2‐FLAG were treated with MB at indicated concentration for 6 h. Ability of MB to block interaction between PD‐1 with SHP2 revealed through ELISA. Y248‐phosphorylated human PD‐1 ITSM peptide was coated at 4 μg/ml in 96‐well plate overnight. Recombinant human SHP2 protein incubated with the coated peptide in the presence of MB for 2 h. Bound SHP2 was measured by monitoring the activity of horse peroxidase conjugated on secondary antibody targeting SHP2. Representative Western blot showing the levels of total and phosphorylated ZAP70, PKCθ, and PLCγ1 in the lysates of stimulated CTL, inhibited by PD‐L1 in the presence of 1 μM of MB. Representative Western blot showing the levels CD28 phosphorylation. CTL was stimulated with EG7 cells (parental), EG7‐L1, and EG7‐L1 in the presence of 100 nM of MB for 2 h. Protein immunoprecipitated (IP) with anti‐pY from the lysates of the indicated CTL‐EG7 co‐culture was subjected to SDS–PAGE separation and blotted with aCD28. β‐Actin served as loading control. EG7‐L1: EG7 overexpressing PD‐L1. Phos‐tag analysis of CD28 phosphorylation in CTL. CTLs were stimulated with EG7 or EG7‐L1 in the presence of 1 μM MB. Cell lysate was then separated by SDS–PAGE containing 50 μM of Phos‐tag and blotted with aCD28 antibody. Phosphorylated CD28 (slow‐moving) species were visualized through exposure. Fluorescence complementation analysis of the effect of MB on the interaction between CD28 with P85. Jurkat or Jurkat‐PD‐1 cells were co‐transfected with CD28‐C‐Luc or CD28 Y191F ‐C‐Luc and P85‐N‐Luc. Cells were seeded in a 96‐well plate coated with 10 μg/ml of aCD3/aCD28 in the presence of human 10 μg/ml of PD‐L1 protein and 100 nM of MB for 6 h. Luciferase activity was measured as readout for CD28‐P85 interaction. Data information: Data are representative of three independent experiments and were analyzed by unpaired t ‐test. Error bars denote SEM. * P

    Techniques Used: Blocking Assay, Expressing, Incubation, Staining, Microscopy, Fluorescence, Luciferase, Activity Assay, Co-Immunoprecipitation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Western Blot, Immunoprecipitation, Co-Culture Assay, SDS Page, Transfection

    MB enhanced activation and effector function of CTL Effect of PD‐1 antibody on the proliferation of OT‐I CTLs. Splenocytes from OT‐I mice were labeled with CFSE and seeded in a 96‐well plate. Media were supplemented with 10 nM of SINFEEKL peptide, 10 ng/ml of human IL‐2 and 10 μg/ml of mouse PD‐L1 protein in the presence of 10 μg/ml of PD‐1 antibody. Cell proliferation was measured by FACS. Bar graph of (Fig   EV2 A). MB enhancing production of cytokine and cytolytic granule by OT‐1 CTLs. CTLs were co‐incubated with CFSE‐labeled EG7‐L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule was determined by flow cytometry. aPD‐1 antibody served as positive control. EG7‐L1: EG7 overexpressing PD‐L1. Bar graph of IFNγ production of OT‐1 CTLs in (Fig   EV2 C). Bar graph of GZMB production of OT‐1 CTLs in (Fig   EV2 C). Data information: Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. Error bars denote SEM. * P
    Figure Legend Snippet: MB enhanced activation and effector function of CTL Effect of PD‐1 antibody on the proliferation of OT‐I CTLs. Splenocytes from OT‐I mice were labeled with CFSE and seeded in a 96‐well plate. Media were supplemented with 10 nM of SINFEEKL peptide, 10 ng/ml of human IL‐2 and 10 μg/ml of mouse PD‐L1 protein in the presence of 10 μg/ml of PD‐1 antibody. Cell proliferation was measured by FACS. Bar graph of (Fig  EV2 A). MB enhancing production of cytokine and cytolytic granule by OT‐1 CTLs. CTLs were co‐incubated with CFSE‐labeled EG7‐L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule was determined by flow cytometry. aPD‐1 antibody served as positive control. EG7‐L1: EG7 overexpressing PD‐L1. Bar graph of IFNγ production of OT‐1 CTLs in (Fig  EV2 C). Bar graph of GZMB production of OT‐1 CTLs in (Fig  EV2 C). Data information: Data are representative of three independent experiments and were analyzed by unpaired t ‐test. Error bars denote SEM. * P

    Techniques Used: Activation Assay, Mouse Assay, Labeling, FACS, Incubation, Expressing, Flow Cytometry, Positive Control

    MB enhanced activation and effector function of CTL Effect of MB on the proliferation of WT CTLs and PD‐1KO CTLs. Splenic cells were stained with 5 μM of CFSE and seeded into aCD3/aCD28‐coated 96‐well plates. 10 μg/ml of PD‐L1 was administered in media. Cell proliferation was checked by monitoring dilution of CFSE 48 h after CD3/CD28 stimulation by gating on CD8‐positive population through FACS analysis. WT: splenic cell from wild‐type C57BL/J mice. PD‐1KO: splenic cell from PD‐1 knockout mice. Statistical results of (A). FACS analysis of the effect of MB on the activation of OT‐I CTLs. OT‐I CTLs were stimulated with precoated aCD3/aCD28 and 10 μg/ml of mouse PD‐L1 protein in the presence of 100 nM MB or 10 μg/ml aPD1 (served as positive control). After 24 h, surface expression of CD25 and CD69 on OT‐1 CTLs was determined through FACS analysis. Statistical results of (C). Effect of MB on the activation status of Jurkat T cells. Luciferase activity is suppressed in JP‐luc by co‐culture with Raji‐L1 preloaded with 1 μg/ml superantigen (SEE). Treatment with MB enhanced luciferase expression (SEE‐loaded Raji (PD‐L1 negative) served as positive control). IC 50  is calculated to be 117 nM. JP‐luc: Jurkat cell harboring NFAT‐luciferase transgene and overexpressing PD‐1; Raji‐L1: Raji overexpressing PD‐L1. Impact of MB on luciferase activity of various engineered Jurkat T cells. J‐luc: Jurkat cell harboring NFAT‐luciferase transgene; J‐luc‐sgPD‐1: J‐luc cells treated with lentivirus expressing sgPD‐1/CAS9 simultaneously. qRT–PCR analysis of IL‐2 mRNA level in JP‐luc cells stimulated with precoated aCD3/aCD28 (10 μg/ml) in the presence of 10 μg/ml of PD‐L1 and MB at indicated concentrations. MB enhancing IL‐2 expression by JP‐luc stimulated with Raji‐L1 for 24 h quantified through ELISA analysis (aPD1 served as positive control). MB enhancing production of cytokine and cytolytic granule by OT‐I CTLs. CTLs were incubated with EG7‐L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule was determined by flow cytometry. aPD1 served as positive control. EG7‐L1: EG7 overexpressing PD‐L1. Statistical results of (I). Data information: Data are representative of three independent experiments. Unpaired  t ‐test; error bars denote SEM. * P
    Figure Legend Snippet: MB enhanced activation and effector function of CTL Effect of MB on the proliferation of WT CTLs and PD‐1KO CTLs. Splenic cells were stained with 5 μM of CFSE and seeded into aCD3/aCD28‐coated 96‐well plates. 10 μg/ml of PD‐L1 was administered in media. Cell proliferation was checked by monitoring dilution of CFSE 48 h after CD3/CD28 stimulation by gating on CD8‐positive population through FACS analysis. WT: splenic cell from wild‐type C57BL/J mice. PD‐1KO: splenic cell from PD‐1 knockout mice. Statistical results of (A). FACS analysis of the effect of MB on the activation of OT‐I CTLs. OT‐I CTLs were stimulated with precoated aCD3/aCD28 and 10 μg/ml of mouse PD‐L1 protein in the presence of 100 nM MB or 10 μg/ml aPD1 (served as positive control). After 24 h, surface expression of CD25 and CD69 on OT‐1 CTLs was determined through FACS analysis. Statistical results of (C). Effect of MB on the activation status of Jurkat T cells. Luciferase activity is suppressed in JP‐luc by co‐culture with Raji‐L1 preloaded with 1 μg/ml superantigen (SEE). Treatment with MB enhanced luciferase expression (SEE‐loaded Raji (PD‐L1 negative) served as positive control). IC 50 is calculated to be 117 nM. JP‐luc: Jurkat cell harboring NFAT‐luciferase transgene and overexpressing PD‐1; Raji‐L1: Raji overexpressing PD‐L1. Impact of MB on luciferase activity of various engineered Jurkat T cells. J‐luc: Jurkat cell harboring NFAT‐luciferase transgene; J‐luc‐sgPD‐1: J‐luc cells treated with lentivirus expressing sgPD‐1/CAS9 simultaneously. qRT–PCR analysis of IL‐2 mRNA level in JP‐luc cells stimulated with precoated aCD3/aCD28 (10 μg/ml) in the presence of 10 μg/ml of PD‐L1 and MB at indicated concentrations. MB enhancing IL‐2 expression by JP‐luc stimulated with Raji‐L1 for 24 h quantified through ELISA analysis (aPD1 served as positive control). MB enhancing production of cytokine and cytolytic granule by OT‐I CTLs. CTLs were incubated with EG7‐L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule was determined by flow cytometry. aPD1 served as positive control. EG7‐L1: EG7 overexpressing PD‐L1. Statistical results of (I). Data information: Data are representative of three independent experiments. Unpaired t ‐test; error bars denote SEM. * P

    Techniques Used: Activation Assay, Staining, FACS, Mouse Assay, Knock-Out, Positive Control, Expressing, Luciferase, Activity Assay, Co-Culture Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry

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    Article Snippet: Bimolecular fluorescence complementation assay Jurkat cells were co‐transfected with hPD1‐C‐Luc or PD1Y248F ‐C‐Luc and hSHP2‐N‐luc or hCD28‐C‐Luc or hCD28Y191F‐C‐Luc and hP85‐N‐luc. .. After 24 h of rest, transfected cells were seeded in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28, with media with 10 μg/ml of hPD‐L1 protein in the presence of MB at indicated concentrations for 6 h followed by analysis of luciferase activity. ..

    Transfection:

    Article Title: Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors
    Article Snippet: Bimolecular fluorescence complementation assay Jurkat cells were co‐transfected with hPD1‐C‐Luc or PD1Y248F ‐C‐Luc and hSHP2‐N‐luc or hCD28‐C‐Luc or hCD28Y191F‐C‐Luc and hP85‐N‐luc. .. After 24 h of rest, transfected cells were seeded in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28, with media with 10 μg/ml of hPD‐L1 protein in the presence of MB at indicated concentrations for 6 h followed by analysis of luciferase activity. ..

    Luciferase:

    Article Title: Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors
    Article Snippet: Bimolecular fluorescence complementation assay Jurkat cells were co‐transfected with hPD1‐C‐Luc or PD1Y248F ‐C‐Luc and hSHP2‐N‐luc or hCD28‐C‐Luc or hCD28Y191F‐C‐Luc and hP85‐N‐luc. .. After 24 h of rest, transfected cells were seeded in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28, with media with 10 μg/ml of hPD‐L1 protein in the presence of MB at indicated concentrations for 6 h followed by analysis of luciferase activity. ..

    other:

    Article Title: Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors
    Article Snippet: Cells were pretreated with DMSO or MB or 25 μg/ml pembrolizumab or 20 μg/ml nivolumab for 1 h and then seeded in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28, with media supplemented with 10 μg/ml of hPD‐L1 protein.

    Incubation:

    Article Title: Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors
    Article Snippet: After resting for 24 h, transfected cells were seeded on Poly‐L‐lysine‐treated coverslips in 24‐well plate. .. Cells were then incubated with 10 μg/ml of hPD‐L1 protein or 2 μM of pervanadate (PVD) for 5 min in the presence of 100 nM or 1 μM of MB for 2 min before fixing with 4% paraformaldehyde and staining with DAPI. ..

    Staining:

    Article Title: Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors
    Article Snippet: After resting for 24 h, transfected cells were seeded on Poly‐L‐lysine‐treated coverslips in 24‐well plate. .. Cells were then incubated with 10 μg/ml of hPD‐L1 protein or 2 μM of pervanadate (PVD) for 5 min in the presence of 100 nM or 1 μM of MB for 2 min before fixing with 4% paraformaldehyde and staining with DAPI. ..

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    Sino Biological hpd l1 protein
    MB is effective to activate human CD8 +  T cells Effect of MB on proliferation of human CD8 +  T cells. CFSE‐labeled human peripheral blood mononuclear cells (PBMC) were preincubated with DMSO, MB, 25 μg/ml of pembrolizumab, or 20 μg/ml of nivolumab for 1 h and then seeded in the 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 in the presence of 10 μg/ml human PD‐L1 protein. Cell proliferation was analyzed through FACS analysis of dilution of CFSE. Cytokine and cytolytic granule production in CD8 +  T cells. Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg/ml of phytohaemagglutinin for 2 days and then rested for 1 day. Cells were pretreated with DMSO, MB, 25 μg/ml of pembrolizumab (Pem), or 20 μg/ml of nivolumab (Niv) for 1 h and then seeded in a 96‐well plate precoated with 10 μg/ml aCD3/aCD28, with media supplemented with 10 μg/ml of human PD‐L1 protein. Cytokine and cytolytic granule production was determined by intracellular flow cytometry by gating on CD8 +  populations. Comparison of the efficacy of MB with PD1 inhibitors currently used in clinic to activate T cell. JP‐luc cells were stimulated with Raji‐L1 or parental Raji cells preloaded with 1 μg/ml of superantigen (SEE) in the presence of MB at indicated concentration or 25 μg/ml of pembrolizumab (Pem) or 20 μg/ml of nivolumab (Niv) for 6 h. Luciferase activity was measured by luminometer. Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. JP‐luc: Jurkat cell harboring NFAT‐luciferase transgene and overexpressing PD‐1; Raji‐L1: Raji overexpressing PD‐L1. Data information: Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. Error bars denote SEM. ** P
    Hpd L1 Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MB is effective to activate human CD8 +  T cells Effect of MB on proliferation of human CD8 +  T cells. CFSE‐labeled human peripheral blood mononuclear cells (PBMC) were preincubated with DMSO, MB, 25 μg/ml of pembrolizumab, or 20 μg/ml of nivolumab for 1 h and then seeded in the 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 in the presence of 10 μg/ml human PD‐L1 protein. Cell proliferation was analyzed through FACS analysis of dilution of CFSE. Cytokine and cytolytic granule production in CD8 +  T cells. Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg/ml of phytohaemagglutinin for 2 days and then rested for 1 day. Cells were pretreated with DMSO, MB, 25 μg/ml of pembrolizumab (Pem), or 20 μg/ml of nivolumab (Niv) for 1 h and then seeded in a 96‐well plate precoated with 10 μg/ml aCD3/aCD28, with media supplemented with 10 μg/ml of human PD‐L1 protein. Cytokine and cytolytic granule production was determined by intracellular flow cytometry by gating on CD8 +  populations. Comparison of the efficacy of MB with PD1 inhibitors currently used in clinic to activate T cell. JP‐luc cells were stimulated with Raji‐L1 or parental Raji cells preloaded with 1 μg/ml of superantigen (SEE) in the presence of MB at indicated concentration or 25 μg/ml of pembrolizumab (Pem) or 20 μg/ml of nivolumab (Niv) for 6 h. Luciferase activity was measured by luminometer. Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. JP‐luc: Jurkat cell harboring NFAT‐luciferase transgene and overexpressing PD‐1; Raji‐L1: Raji overexpressing PD‐L1. Data information: Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. Error bars denote SEM. ** P

    Journal: EMBO Molecular Medicine

    Article Title: Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors

    doi: 10.15252/emmm.201911571

    Figure Lengend Snippet: MB is effective to activate human CD8 + T cells Effect of MB on proliferation of human CD8 + T cells. CFSE‐labeled human peripheral blood mononuclear cells (PBMC) were preincubated with DMSO, MB, 25 μg/ml of pembrolizumab, or 20 μg/ml of nivolumab for 1 h and then seeded in the 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 in the presence of 10 μg/ml human PD‐L1 protein. Cell proliferation was analyzed through FACS analysis of dilution of CFSE. Cytokine and cytolytic granule production in CD8 + T cells. Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg/ml of phytohaemagglutinin for 2 days and then rested for 1 day. Cells were pretreated with DMSO, MB, 25 μg/ml of pembrolizumab (Pem), or 20 μg/ml of nivolumab (Niv) for 1 h and then seeded in a 96‐well plate precoated with 10 μg/ml aCD3/aCD28, with media supplemented with 10 μg/ml of human PD‐L1 protein. Cytokine and cytolytic granule production was determined by intracellular flow cytometry by gating on CD8 + populations. Comparison of the efficacy of MB with PD1 inhibitors currently used in clinic to activate T cell. JP‐luc cells were stimulated with Raji‐L1 or parental Raji cells preloaded with 1 μg/ml of superantigen (SEE) in the presence of MB at indicated concentration or 25 μg/ml of pembrolizumab (Pem) or 20 μg/ml of nivolumab (Niv) for 6 h. Luciferase activity was measured by luminometer. Data are representative of three independent experiments and were analyzed by unpaired t ‐test. JP‐luc: Jurkat cell harboring NFAT‐luciferase transgene and overexpressing PD‐1; Raji‐L1: Raji overexpressing PD‐L1. Data information: Data are representative of three independent experiments and were analyzed by unpaired t ‐test. Error bars denote SEM. ** P

    Article Snippet: After 24 h of rest, transfected cells were seeded in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28, with media with 10 μg/ml of hPD‐L1 protein in the presence of MB at indicated concentrations for 6 h followed by analysis of luciferase activity.

    Techniques: Labeling, FACS, Flow Cytometry, Concentration Assay, Luciferase, Activity Assay

    MB suppress PD‐1 signaling through blocking SHP2 recruitment by PD‐1 Confocal cell images of Jurkat cells co‐expressing PD‐1‐EGFP and SHP2‐mCherry. Jurkat cells co‐expressing PD‐1‐EGFP and SHP2‐mCherry were incubated with 10 μg/ml of human PD‐L1 protein in the presence of 100 nM and 1 μM of MB for 2 min, fixed with 4% paraformaldehyde, and stained with DAPI. Colocalization signal was examined with confocal microscope (scale bar = 2 μm). Fluorescence complementation analysis of the effect of MB on the interaction between PD‐1 and SHP2. Jurkat cells co‐expressing PD1‐C‐Luc or PD1 Y248F ‐C‐Luc and SHP2‐N‐luc were seeded in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 with media supplemented with 10 μg/ml human PD‐L1 protein in the presence of MB at indicated concentrations for 6 h followed by analysis of luciferase activity. Co‐IP analysis of impact of MB on interaction between PD‐1 and SHP2. 293T cells co‐expressing PD‐1 and SHP2‐FLAG were treated with MB at indicated concentration for 6 h. Ability of MB to block interaction between PD‐1 with SHP2 revealed through ELISA. Y248‐phosphorylated human PD‐1 ITSM peptide was coated at 4 μg/ml in 96‐well plate overnight. Recombinant human SHP2 protein incubated with the coated peptide in the presence of MB for 2 h. Bound SHP2 was measured by monitoring the activity of horse peroxidase conjugated on secondary antibody targeting SHP2. Representative Western blot showing the levels of total and phosphorylated ZAP70, PKCθ, and PLCγ1 in the lysates of stimulated CTL, inhibited by PD‐L1 in the presence of 1 μM of MB. Representative Western blot showing the levels CD28 phosphorylation. CTL was stimulated with EG7 cells (parental), EG7‐L1, and EG7‐L1 in the presence of 100 nM of MB for 2 h. Protein immunoprecipitated (IP) with anti‐pY from the lysates of the indicated CTL‐EG7 co‐culture was subjected to SDS–PAGE separation and blotted with aCD28. β‐Actin served as loading control. EG7‐L1: EG7 overexpressing PD‐L1. Phos‐tag analysis of CD28 phosphorylation in CTL. CTLs were stimulated with EG7 or EG7‐L1 in the presence of 1 μM MB. Cell lysate was then separated by SDS–PAGE containing 50 μM of Phos‐tag and blotted with aCD28 antibody. Phosphorylated CD28 (slow‐moving) species were visualized through exposure. Fluorescence complementation analysis of the effect of MB on the interaction between CD28 with P85. Jurkat or Jurkat‐PD‐1 cells were co‐transfected with CD28‐C‐Luc or CD28 Y191F ‐C‐Luc and P85‐N‐Luc. Cells were seeded in a 96‐well plate coated with 10 μg/ml of aCD3/aCD28 in the presence of human 10 μg/ml of PD‐L1 protein and 100 nM of MB for 6 h. Luciferase activity was measured as readout for CD28‐P85 interaction. Data information: Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. Error bars denote SEM. * P

    Journal: EMBO Molecular Medicine

    Article Title: Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors

    doi: 10.15252/emmm.201911571

    Figure Lengend Snippet: MB suppress PD‐1 signaling through blocking SHP2 recruitment by PD‐1 Confocal cell images of Jurkat cells co‐expressing PD‐1‐EGFP and SHP2‐mCherry. Jurkat cells co‐expressing PD‐1‐EGFP and SHP2‐mCherry were incubated with 10 μg/ml of human PD‐L1 protein in the presence of 100 nM and 1 μM of MB for 2 min, fixed with 4% paraformaldehyde, and stained with DAPI. Colocalization signal was examined with confocal microscope (scale bar = 2 μm). Fluorescence complementation analysis of the effect of MB on the interaction between PD‐1 and SHP2. Jurkat cells co‐expressing PD1‐C‐Luc or PD1 Y248F ‐C‐Luc and SHP2‐N‐luc were seeded in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 with media supplemented with 10 μg/ml human PD‐L1 protein in the presence of MB at indicated concentrations for 6 h followed by analysis of luciferase activity. Co‐IP analysis of impact of MB on interaction between PD‐1 and SHP2. 293T cells co‐expressing PD‐1 and SHP2‐FLAG were treated with MB at indicated concentration for 6 h. Ability of MB to block interaction between PD‐1 with SHP2 revealed through ELISA. Y248‐phosphorylated human PD‐1 ITSM peptide was coated at 4 μg/ml in 96‐well plate overnight. Recombinant human SHP2 protein incubated with the coated peptide in the presence of MB for 2 h. Bound SHP2 was measured by monitoring the activity of horse peroxidase conjugated on secondary antibody targeting SHP2. Representative Western blot showing the levels of total and phosphorylated ZAP70, PKCθ, and PLCγ1 in the lysates of stimulated CTL, inhibited by PD‐L1 in the presence of 1 μM of MB. Representative Western blot showing the levels CD28 phosphorylation. CTL was stimulated with EG7 cells (parental), EG7‐L1, and EG7‐L1 in the presence of 100 nM of MB for 2 h. Protein immunoprecipitated (IP) with anti‐pY from the lysates of the indicated CTL‐EG7 co‐culture was subjected to SDS–PAGE separation and blotted with aCD28. β‐Actin served as loading control. EG7‐L1: EG7 overexpressing PD‐L1. Phos‐tag analysis of CD28 phosphorylation in CTL. CTLs were stimulated with EG7 or EG7‐L1 in the presence of 1 μM MB. Cell lysate was then separated by SDS–PAGE containing 50 μM of Phos‐tag and blotted with aCD28 antibody. Phosphorylated CD28 (slow‐moving) species were visualized through exposure. Fluorescence complementation analysis of the effect of MB on the interaction between CD28 with P85. Jurkat or Jurkat‐PD‐1 cells were co‐transfected with CD28‐C‐Luc or CD28 Y191F ‐C‐Luc and P85‐N‐Luc. Cells were seeded in a 96‐well plate coated with 10 μg/ml of aCD3/aCD28 in the presence of human 10 μg/ml of PD‐L1 protein and 100 nM of MB for 6 h. Luciferase activity was measured as readout for CD28‐P85 interaction. Data information: Data are representative of three independent experiments and were analyzed by unpaired t ‐test. Error bars denote SEM. * P

    Article Snippet: After 24 h of rest, transfected cells were seeded in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28, with media with 10 μg/ml of hPD‐L1 protein in the presence of MB at indicated concentrations for 6 h followed by analysis of luciferase activity.

    Techniques: Blocking Assay, Expressing, Incubation, Staining, Microscopy, Fluorescence, Luciferase, Activity Assay, Co-Immunoprecipitation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Western Blot, Immunoprecipitation, Co-Culture Assay, SDS Page, Transfection

    MB enhanced activation and effector function of CTL Effect of PD‐1 antibody on the proliferation of OT‐I CTLs. Splenocytes from OT‐I mice were labeled with CFSE and seeded in a 96‐well plate. Media were supplemented with 10 nM of SINFEEKL peptide, 10 ng/ml of human IL‐2 and 10 μg/ml of mouse PD‐L1 protein in the presence of 10 μg/ml of PD‐1 antibody. Cell proliferation was measured by FACS. Bar graph of (Fig   EV2 A). MB enhancing production of cytokine and cytolytic granule by OT‐1 CTLs. CTLs were co‐incubated with CFSE‐labeled EG7‐L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule was determined by flow cytometry. aPD‐1 antibody served as positive control. EG7‐L1: EG7 overexpressing PD‐L1. Bar graph of IFNγ production of OT‐1 CTLs in (Fig   EV2 C). Bar graph of GZMB production of OT‐1 CTLs in (Fig   EV2 C). Data information: Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. Error bars denote SEM. * P

    Journal: EMBO Molecular Medicine

    Article Title: Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors

    doi: 10.15252/emmm.201911571

    Figure Lengend Snippet: MB enhanced activation and effector function of CTL Effect of PD‐1 antibody on the proliferation of OT‐I CTLs. Splenocytes from OT‐I mice were labeled with CFSE and seeded in a 96‐well plate. Media were supplemented with 10 nM of SINFEEKL peptide, 10 ng/ml of human IL‐2 and 10 μg/ml of mouse PD‐L1 protein in the presence of 10 μg/ml of PD‐1 antibody. Cell proliferation was measured by FACS. Bar graph of (Fig  EV2 A). MB enhancing production of cytokine and cytolytic granule by OT‐1 CTLs. CTLs were co‐incubated with CFSE‐labeled EG7‐L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule was determined by flow cytometry. aPD‐1 antibody served as positive control. EG7‐L1: EG7 overexpressing PD‐L1. Bar graph of IFNγ production of OT‐1 CTLs in (Fig  EV2 C). Bar graph of GZMB production of OT‐1 CTLs in (Fig  EV2 C). Data information: Data are representative of three independent experiments and were analyzed by unpaired t ‐test. Error bars denote SEM. * P

    Article Snippet: After 24 h of rest, transfected cells were seeded in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28, with media with 10 μg/ml of hPD‐L1 protein in the presence of MB at indicated concentrations for 6 h followed by analysis of luciferase activity.

    Techniques: Activation Assay, Mouse Assay, Labeling, FACS, Incubation, Expressing, Flow Cytometry, Positive Control

    MB enhanced activation and effector function of CTL Effect of MB on the proliferation of WT CTLs and PD‐1KO CTLs. Splenic cells were stained with 5 μM of CFSE and seeded into aCD3/aCD28‐coated 96‐well plates. 10 μg/ml of PD‐L1 was administered in media. Cell proliferation was checked by monitoring dilution of CFSE 48 h after CD3/CD28 stimulation by gating on CD8‐positive population through FACS analysis. WT: splenic cell from wild‐type C57BL/J mice. PD‐1KO: splenic cell from PD‐1 knockout mice. Statistical results of (A). FACS analysis of the effect of MB on the activation of OT‐I CTLs. OT‐I CTLs were stimulated with precoated aCD3/aCD28 and 10 μg/ml of mouse PD‐L1 protein in the presence of 100 nM MB or 10 μg/ml aPD1 (served as positive control). After 24 h, surface expression of CD25 and CD69 on OT‐1 CTLs was determined through FACS analysis. Statistical results of (C). Effect of MB on the activation status of Jurkat T cells. Luciferase activity is suppressed in JP‐luc by co‐culture with Raji‐L1 preloaded with 1 μg/ml superantigen (SEE). Treatment with MB enhanced luciferase expression (SEE‐loaded Raji (PD‐L1 negative) served as positive control). IC 50  is calculated to be 117 nM. JP‐luc: Jurkat cell harboring NFAT‐luciferase transgene and overexpressing PD‐1; Raji‐L1: Raji overexpressing PD‐L1. Impact of MB on luciferase activity of various engineered Jurkat T cells. J‐luc: Jurkat cell harboring NFAT‐luciferase transgene; J‐luc‐sgPD‐1: J‐luc cells treated with lentivirus expressing sgPD‐1/CAS9 simultaneously. qRT–PCR analysis of IL‐2 mRNA level in JP‐luc cells stimulated with precoated aCD3/aCD28 (10 μg/ml) in the presence of 10 μg/ml of PD‐L1 and MB at indicated concentrations. MB enhancing IL‐2 expression by JP‐luc stimulated with Raji‐L1 for 24 h quantified through ELISA analysis (aPD1 served as positive control). MB enhancing production of cytokine and cytolytic granule by OT‐I CTLs. CTLs were incubated with EG7‐L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule was determined by flow cytometry. aPD1 served as positive control. EG7‐L1: EG7 overexpressing PD‐L1. Statistical results of (I). Data information: Data are representative of three independent experiments. Unpaired  t ‐test; error bars denote SEM. * P

    Journal: EMBO Molecular Medicine

    Article Title: Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors

    doi: 10.15252/emmm.201911571

    Figure Lengend Snippet: MB enhanced activation and effector function of CTL Effect of MB on the proliferation of WT CTLs and PD‐1KO CTLs. Splenic cells were stained with 5 μM of CFSE and seeded into aCD3/aCD28‐coated 96‐well plates. 10 μg/ml of PD‐L1 was administered in media. Cell proliferation was checked by monitoring dilution of CFSE 48 h after CD3/CD28 stimulation by gating on CD8‐positive population through FACS analysis. WT: splenic cell from wild‐type C57BL/J mice. PD‐1KO: splenic cell from PD‐1 knockout mice. Statistical results of (A). FACS analysis of the effect of MB on the activation of OT‐I CTLs. OT‐I CTLs were stimulated with precoated aCD3/aCD28 and 10 μg/ml of mouse PD‐L1 protein in the presence of 100 nM MB or 10 μg/ml aPD1 (served as positive control). After 24 h, surface expression of CD25 and CD69 on OT‐1 CTLs was determined through FACS analysis. Statistical results of (C). Effect of MB on the activation status of Jurkat T cells. Luciferase activity is suppressed in JP‐luc by co‐culture with Raji‐L1 preloaded with 1 μg/ml superantigen (SEE). Treatment with MB enhanced luciferase expression (SEE‐loaded Raji (PD‐L1 negative) served as positive control). IC 50 is calculated to be 117 nM. JP‐luc: Jurkat cell harboring NFAT‐luciferase transgene and overexpressing PD‐1; Raji‐L1: Raji overexpressing PD‐L1. Impact of MB on luciferase activity of various engineered Jurkat T cells. J‐luc: Jurkat cell harboring NFAT‐luciferase transgene; J‐luc‐sgPD‐1: J‐luc cells treated with lentivirus expressing sgPD‐1/CAS9 simultaneously. qRT–PCR analysis of IL‐2 mRNA level in JP‐luc cells stimulated with precoated aCD3/aCD28 (10 μg/ml) in the presence of 10 μg/ml of PD‐L1 and MB at indicated concentrations. MB enhancing IL‐2 expression by JP‐luc stimulated with Raji‐L1 for 24 h quantified through ELISA analysis (aPD1 served as positive control). MB enhancing production of cytokine and cytolytic granule by OT‐I CTLs. CTLs were incubated with EG7‐L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule was determined by flow cytometry. aPD1 served as positive control. EG7‐L1: EG7 overexpressing PD‐L1. Statistical results of (I). Data information: Data are representative of three independent experiments. Unpaired t ‐test; error bars denote SEM. * P

    Article Snippet: After 24 h of rest, transfected cells were seeded in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28, with media with 10 μg/ml of hPD‐L1 protein in the presence of MB at indicated concentrations for 6 h followed by analysis of luciferase activity.

    Techniques: Activation Assay, Staining, FACS, Mouse Assay, Knock-Out, Positive Control, Expressing, Luciferase, Activity Assay, Co-Culture Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry