pd l1  (Sino Biological)


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    Name:
    Cynomolgus Rhesus PD L1 B7 H1 CD274 Protein Biotinylated
    Description:
    A DNA sequence encoding the cynomolgus rhesus CD274 XP 015292694 1 Met1 Thr239 was expressed with a polyhistidine tag at the C terminus Cynomolgus and Rhesus CD274 sequences are identical The purified protein was biotinylated in vitro
    Catalog Number:
    90251-C08H-B
    Price:
    None
    Category:
    recombinant protein
    Host:
    HEK293 Cells
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    Structured Review

    Sino Biological pd l1
    <t>PD-1/PD-L1</t> interaction blockade and T-cell activation enhancement by SPE components in vitro . (A) Chemical components of SPE. (B) The ability of seven components from SPE to enhance T-cell functional activity was assessed using a PD-1/PD-L1 blockade bioassay. These seven components were used at a concentration of 2 µM. (C, D) A PD-1/PD-L1 blockade bioassay was performed to measure the inhibitory activity of apigenin and cosmosiin. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of apigenin or cosmosiin. (E, F) Competitive ELISA was performed using a PD-1/PD-L1 inhibitor ELISA-binding assay after treatment with the indicated concentrations of (E) apigenin and (F) cosmosiin. Bar graph (mean ± standard error of the mean) statistics were determined with data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P
    A DNA sequence encoding the cynomolgus rhesus CD274 XP 015292694 1 Met1 Thr239 was expressed with a polyhistidine tag at the C terminus Cynomolgus and Rhesus CD274 sequences are identical The purified protein was biotinylated in vitro
    https://www.bioz.com/result/pd l1/product/Sino Biological
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pd l1 - by Bioz Stars, 2021-07
    93/100 stars

    Images

    1) Product Images from "Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model"

    Article Title: Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.598556

    PD-1/PD-L1 interaction blockade and T-cell activation enhancement by SPE components in vitro . (A) Chemical components of SPE. (B) The ability of seven components from SPE to enhance T-cell functional activity was assessed using a PD-1/PD-L1 blockade bioassay. These seven components were used at a concentration of 2 µM. (C, D) A PD-1/PD-L1 blockade bioassay was performed to measure the inhibitory activity of apigenin and cosmosiin. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of apigenin or cosmosiin. (E, F) Competitive ELISA was performed using a PD-1/PD-L1 inhibitor ELISA-binding assay after treatment with the indicated concentrations of (E) apigenin and (F) cosmosiin. Bar graph (mean ± standard error of the mean) statistics were determined with data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P
    Figure Legend Snippet: PD-1/PD-L1 interaction blockade and T-cell activation enhancement by SPE components in vitro . (A) Chemical components of SPE. (B) The ability of seven components from SPE to enhance T-cell functional activity was assessed using a PD-1/PD-L1 blockade bioassay. These seven components were used at a concentration of 2 µM. (C, D) A PD-1/PD-L1 blockade bioassay was performed to measure the inhibitory activity of apigenin and cosmosiin. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of apigenin or cosmosiin. (E, F) Competitive ELISA was performed using a PD-1/PD-L1 inhibitor ELISA-binding assay after treatment with the indicated concentrations of (E) apigenin and (F) cosmosiin. Bar graph (mean ± standard error of the mean) statistics were determined with data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P

    Techniques Used: Activation Assay, In Vitro, Functional Assay, Activity Assay, Concentration Assay, Incubation, Competitive ELISA, Enzyme-linked Immunosorbent Assay, Binding Assay

    Cytotoxic and T-cell-mediated killing effect of SPE on colorectal cancer cells. (A) PD-L1 expression on the surface of humanized PD-L1-expressing MC38 cells (hPDL1-MCs) was determined using flow cytometry. (B–D) To measure the immunological activity of SPE, hPDL1-MCs (target cells) were co-cultured with humanized PD-1 mouse splenocytes (B, C) and CD8+ T-cells from the tumor (D) (hPD1-MSs; effector cells) at an effector cell-to-target cell ratio of 5:1 and were treated with SPE or 50% DMSO (control). (B) After 72 h of incubation, surviving hPD1-MSs in a 96-well plate underwent the CCK assay to assess cell viability. (C) Co-cultured CellTrace™ Far Red-labeled hPDL1-MCs were treated with SPE (25 μg/mL) or 50% DMSO (control), and the findings were compared with those in the absence of effector cells. The images were obtained under a fluorescence microscope. (D) The cytotoxicity of CD8+ T-cells against hPD-L1-MCs at an effector cell-to-target cell ratio of 5:1 was analyzed using an LDH assay kit (percentages calculated according to kit instruction). ***P
    Figure Legend Snippet: Cytotoxic and T-cell-mediated killing effect of SPE on colorectal cancer cells. (A) PD-L1 expression on the surface of humanized PD-L1-expressing MC38 cells (hPDL1-MCs) was determined using flow cytometry. (B–D) To measure the immunological activity of SPE, hPDL1-MCs (target cells) were co-cultured with humanized PD-1 mouse splenocytes (B, C) and CD8+ T-cells from the tumor (D) (hPD1-MSs; effector cells) at an effector cell-to-target cell ratio of 5:1 and were treated with SPE or 50% DMSO (control). (B) After 72 h of incubation, surviving hPD1-MSs in a 96-well plate underwent the CCK assay to assess cell viability. (C) Co-cultured CellTrace™ Far Red-labeled hPDL1-MCs were treated with SPE (25 μg/mL) or 50% DMSO (control), and the findings were compared with those in the absence of effector cells. The images were obtained under a fluorescence microscope. (D) The cytotoxicity of CD8+ T-cells against hPD-L1-MCs at an effector cell-to-target cell ratio of 5:1 was analyzed using an LDH assay kit (percentages calculated according to kit instruction). ***P

    Techniques Used: Expressing, Flow Cytometry, Activity Assay, Cell Culture, Incubation, Labeling, Fluorescence, Microscopy, Lactate Dehydrogenase Assay

    Protein–ligand docking simulation between PD-L1 (A) /PD-1 (B) and the Salvia plebeia R. Br. ethanol extract (SPE) component cosmosiin. Binding models were obtained through docking simulation of cosmosiin on PD-L1 and PD-1 derived from the PD-1/PD-L1 complex (PDB code 4ZQK) using AutoDock Vina. The hydrogen bonds and hydrophobic interactions between PD-L1/PD-1 and cosmosiin were analyzed using LigPlot+.
    Figure Legend Snippet: Protein–ligand docking simulation between PD-L1 (A) /PD-1 (B) and the Salvia plebeia R. Br. ethanol extract (SPE) component cosmosiin. Binding models were obtained through docking simulation of cosmosiin on PD-L1 and PD-1 derived from the PD-1/PD-L1 complex (PDB code 4ZQK) using AutoDock Vina. The hydrogen bonds and hydrophobic interactions between PD-L1/PD-1 and cosmosiin were analyzed using LigPlot+.

    Techniques Used: Binding Assay, Derivative Assay

    Global kinetic analysis of cosmosiin binding to biotinylated (A) PD-1 and (B) PD-L1 immobilized on a streptavidin biolayer interferometry (BLI) sensor. The kinetics of cosmosiin for immobilized PD-1 and PD-L1 were monitored with increasing concentration of cosmosiin (0, 0.125, 0.25, and 0.375 mM), dissolved in PBS buffer (pH 7.3) containing 1% DMSO.
    Figure Legend Snippet: Global kinetic analysis of cosmosiin binding to biotinylated (A) PD-1 and (B) PD-L1 immobilized on a streptavidin biolayer interferometry (BLI) sensor. The kinetics of cosmosiin for immobilized PD-1 and PD-L1 were monitored with increasing concentration of cosmosiin (0, 0.125, 0.25, and 0.375 mM), dissolved in PBS buffer (pH 7.3) containing 1% DMSO.

    Techniques Used: Binding Assay, Concentration Assay

    PD-1/PD-L1 interaction blockade by Salvia plebeia R. Br. ethanol extract (SPE). Competitive ELISA was performed using PD-1/PD-L1 inhibitor ELISA-screening assay with the indicated concentrations of PD-L1-blocking antibody, SPE, and SPE fractions. (A) Inhibition of the PD-1/PD-L1 interaction by PD-L1-blocking antibody, (B) SPE, and (C) SPE fractions (methylene chloride [MC], ethyl acetate [EA], and n-butanol [BuOH]). Cytotoxicity and T-cell activation by SPE and its ethyl acetate (EA) fraction in vitro . (D) The viabilities of PD-L1 aAPC/CHO-K1 and PD-1 effector cells were assessed using the Cell Counting Kit-8 (CCK) assay after treatment with the indicated concentrations of SPE for 24 h. (E–G) A PD-1/PD-L1 blockade bioassay was performed to assess the inhibitory activities of PD-L1-blocking antibody, SPE, and SPE-EA. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of (E) PD-L1-blocking antibody, (F) SPE, or (G) SPE-EA. Bar graph (mean ± standard error of the mean) statistics were determined with the data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P
    Figure Legend Snippet: PD-1/PD-L1 interaction blockade by Salvia plebeia R. Br. ethanol extract (SPE). Competitive ELISA was performed using PD-1/PD-L1 inhibitor ELISA-screening assay with the indicated concentrations of PD-L1-blocking antibody, SPE, and SPE fractions. (A) Inhibition of the PD-1/PD-L1 interaction by PD-L1-blocking antibody, (B) SPE, and (C) SPE fractions (methylene chloride [MC], ethyl acetate [EA], and n-butanol [BuOH]). Cytotoxicity and T-cell activation by SPE and its ethyl acetate (EA) fraction in vitro . (D) The viabilities of PD-L1 aAPC/CHO-K1 and PD-1 effector cells were assessed using the Cell Counting Kit-8 (CCK) assay after treatment with the indicated concentrations of SPE for 24 h. (E–G) A PD-1/PD-L1 blockade bioassay was performed to assess the inhibitory activities of PD-L1-blocking antibody, SPE, and SPE-EA. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of (E) PD-L1-blocking antibody, (F) SPE, or (G) SPE-EA. Bar graph (mean ± standard error of the mean) statistics were determined with the data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P

    Techniques Used: Competitive ELISA, Enzyme-linked Immunosorbent Assay, Screening Assay, Blocking Assay, Inhibition, Activation Assay, In Vitro, Cell Counting, Incubation

    Anti-tumor efficacy of SPE on hPD-L1 MC38 tumor growth in hPD-1 knock-in mice. (A) Representative images show the tumor development at 16 days after treatment. Mice were inoculated s.c. with hPD-L1 MC38 cells and were administrated with aPD-1 (5 mg/kg, i.p.) or SPE (100, 300 mg/kg, i.g.) (n=3). Tumor mice received an equivalent volume of vehicle (200 µL). (B) Image of subcutaneous tumor mass on day 16 (scale bars, 1 cm) (n=3). (C) Mean tumor weight (n=3). (D) Mean tumor diameter (n=3). (E) Immunohistochemistry staining of CD3+ in humanized PD-1 mouse model of tumor. (F) FACS analysis of tumor CD8+ T-cell populations from human PD-L1 knock in MC 38 tumor-bearing model of humanized PD-1 mice. Percentages of polyclonal CD8+ T-cells of CD45+ cells in tumor. Bar graph (mean ± standard error of the mean) statistics were determined with one-way ANOVA with Tukey’s post-hoc test; ***P
    Figure Legend Snippet: Anti-tumor efficacy of SPE on hPD-L1 MC38 tumor growth in hPD-1 knock-in mice. (A) Representative images show the tumor development at 16 days after treatment. Mice were inoculated s.c. with hPD-L1 MC38 cells and were administrated with aPD-1 (5 mg/kg, i.p.) or SPE (100, 300 mg/kg, i.g.) (n=3). Tumor mice received an equivalent volume of vehicle (200 µL). (B) Image of subcutaneous tumor mass on day 16 (scale bars, 1 cm) (n=3). (C) Mean tumor weight (n=3). (D) Mean tumor diameter (n=3). (E) Immunohistochemistry staining of CD3+ in humanized PD-1 mouse model of tumor. (F) FACS analysis of tumor CD8+ T-cell populations from human PD-L1 knock in MC 38 tumor-bearing model of humanized PD-1 mice. Percentages of polyclonal CD8+ T-cells of CD45+ cells in tumor. Bar graph (mean ± standard error of the mean) statistics were determined with one-way ANOVA with Tukey’s post-hoc test; ***P

    Techniques Used: Knock-In, Mouse Assay, Immunohistochemistry, Staining, FACS

    2) Product Images from "Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model"

    Article Title: Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.598556

    PD-1/PD-L1 interaction blockade and T-cell activation enhancement by SPE components in vitro . (A) Chemical components of SPE. (B) The ability of seven components from SPE to enhance T-cell functional activity was assessed using a PD-1/PD-L1 blockade bioassay. These seven components were used at a concentration of 2 µM. (C, D) A PD-1/PD-L1 blockade bioassay was performed to measure the inhibitory activity of apigenin and cosmosiin. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of apigenin or cosmosiin. (E, F) Competitive ELISA was performed using a PD-1/PD-L1 inhibitor ELISA-binding assay after treatment with the indicated concentrations of (E) apigenin and (F) cosmosiin. Bar graph (mean ± standard error of the mean) statistics were determined with data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P
    Figure Legend Snippet: PD-1/PD-L1 interaction blockade and T-cell activation enhancement by SPE components in vitro . (A) Chemical components of SPE. (B) The ability of seven components from SPE to enhance T-cell functional activity was assessed using a PD-1/PD-L1 blockade bioassay. These seven components were used at a concentration of 2 µM. (C, D) A PD-1/PD-L1 blockade bioassay was performed to measure the inhibitory activity of apigenin and cosmosiin. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of apigenin or cosmosiin. (E, F) Competitive ELISA was performed using a PD-1/PD-L1 inhibitor ELISA-binding assay after treatment with the indicated concentrations of (E) apigenin and (F) cosmosiin. Bar graph (mean ± standard error of the mean) statistics were determined with data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P

    Techniques Used: Activation Assay, In Vitro, Functional Assay, Activity Assay, Concentration Assay, Incubation, Competitive ELISA, Enzyme-linked Immunosorbent Assay, Binding Assay

    Cytotoxic and T-cell-mediated killing effect of SPE on colorectal cancer cells. (A) PD-L1 expression on the surface of humanized PD-L1-expressing MC38 cells (hPDL1-MCs) was determined using flow cytometry. (B–D) To measure the immunological activity of SPE, hPDL1-MCs (target cells) were co-cultured with humanized PD-1 mouse splenocytes (B, C) and CD8+ T-cells from the tumor (D) (hPD1-MSs; effector cells) at an effector cell-to-target cell ratio of 5:1 and were treated with SPE or 50% DMSO (control). (B) After 72 h of incubation, surviving hPD1-MSs in a 96-well plate underwent the CCK assay to assess cell viability. (C) Co-cultured CellTrace™ Far Red-labeled hPDL1-MCs were treated with SPE (25 μg/mL) or 50% DMSO (control), and the findings were compared with those in the absence of effector cells. The images were obtained under a fluorescence microscope. (D) The cytotoxicity of CD8+ T-cells against hPD-L1-MCs at an effector cell-to-target cell ratio of 5:1 was analyzed using an LDH assay kit (percentages calculated according to kit instruction). ***P
    Figure Legend Snippet: Cytotoxic and T-cell-mediated killing effect of SPE on colorectal cancer cells. (A) PD-L1 expression on the surface of humanized PD-L1-expressing MC38 cells (hPDL1-MCs) was determined using flow cytometry. (B–D) To measure the immunological activity of SPE, hPDL1-MCs (target cells) were co-cultured with humanized PD-1 mouse splenocytes (B, C) and CD8+ T-cells from the tumor (D) (hPD1-MSs; effector cells) at an effector cell-to-target cell ratio of 5:1 and were treated with SPE or 50% DMSO (control). (B) After 72 h of incubation, surviving hPD1-MSs in a 96-well plate underwent the CCK assay to assess cell viability. (C) Co-cultured CellTrace™ Far Red-labeled hPDL1-MCs were treated with SPE (25 μg/mL) or 50% DMSO (control), and the findings were compared with those in the absence of effector cells. The images were obtained under a fluorescence microscope. (D) The cytotoxicity of CD8+ T-cells against hPD-L1-MCs at an effector cell-to-target cell ratio of 5:1 was analyzed using an LDH assay kit (percentages calculated according to kit instruction). ***P

    Techniques Used: Expressing, Flow Cytometry, Activity Assay, Cell Culture, Incubation, Labeling, Fluorescence, Microscopy, Lactate Dehydrogenase Assay

    Protein–ligand docking simulation between PD-L1 (A) /PD-1 (B) and the Salvia plebeia R. Br. ethanol extract (SPE) component cosmosiin. Binding models were obtained through docking simulation of cosmosiin on PD-L1 and PD-1 derived from the PD-1/PD-L1 complex (PDB code 4ZQK) using AutoDock Vina. The hydrogen bonds and hydrophobic interactions between PD-L1/PD-1 and cosmosiin were analyzed using LigPlot+.
    Figure Legend Snippet: Protein–ligand docking simulation between PD-L1 (A) /PD-1 (B) and the Salvia plebeia R. Br. ethanol extract (SPE) component cosmosiin. Binding models were obtained through docking simulation of cosmosiin on PD-L1 and PD-1 derived from the PD-1/PD-L1 complex (PDB code 4ZQK) using AutoDock Vina. The hydrogen bonds and hydrophobic interactions between PD-L1/PD-1 and cosmosiin were analyzed using LigPlot+.

    Techniques Used: Binding Assay, Derivative Assay

    Global kinetic analysis of cosmosiin binding to biotinylated (A) PD-1 and (B) PD-L1 immobilized on a streptavidin biolayer interferometry (BLI) sensor. The kinetics of cosmosiin for immobilized PD-1 and PD-L1 were monitored with increasing concentration of cosmosiin (0, 0.125, 0.25, and 0.375 mM), dissolved in PBS buffer (pH 7.3) containing 1% DMSO.
    Figure Legend Snippet: Global kinetic analysis of cosmosiin binding to biotinylated (A) PD-1 and (B) PD-L1 immobilized on a streptavidin biolayer interferometry (BLI) sensor. The kinetics of cosmosiin for immobilized PD-1 and PD-L1 were monitored with increasing concentration of cosmosiin (0, 0.125, 0.25, and 0.375 mM), dissolved in PBS buffer (pH 7.3) containing 1% DMSO.

    Techniques Used: Binding Assay, Concentration Assay

    PD-1/PD-L1 interaction blockade by Salvia plebeia R. Br. ethanol extract (SPE). Competitive ELISA was performed using PD-1/PD-L1 inhibitor ELISA-screening assay with the indicated concentrations of PD-L1-blocking antibody, SPE, and SPE fractions. (A) Inhibition of the PD-1/PD-L1 interaction by PD-L1-blocking antibody, (B) SPE, and (C) SPE fractions (methylene chloride [MC], ethyl acetate [EA], and n-butanol [BuOH]). Cytotoxicity and T-cell activation by SPE and its ethyl acetate (EA) fraction in vitro . (D) The viabilities of PD-L1 aAPC/CHO-K1 and PD-1 effector cells were assessed using the Cell Counting Kit-8 (CCK) assay after treatment with the indicated concentrations of SPE for 24 h. (E–G) A PD-1/PD-L1 blockade bioassay was performed to assess the inhibitory activities of PD-L1-blocking antibody, SPE, and SPE-EA. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of (E) PD-L1-blocking antibody, (F) SPE, or (G) SPE-EA. Bar graph (mean ± standard error of the mean) statistics were determined with the data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P
    Figure Legend Snippet: PD-1/PD-L1 interaction blockade by Salvia plebeia R. Br. ethanol extract (SPE). Competitive ELISA was performed using PD-1/PD-L1 inhibitor ELISA-screening assay with the indicated concentrations of PD-L1-blocking antibody, SPE, and SPE fractions. (A) Inhibition of the PD-1/PD-L1 interaction by PD-L1-blocking antibody, (B) SPE, and (C) SPE fractions (methylene chloride [MC], ethyl acetate [EA], and n-butanol [BuOH]). Cytotoxicity and T-cell activation by SPE and its ethyl acetate (EA) fraction in vitro . (D) The viabilities of PD-L1 aAPC/CHO-K1 and PD-1 effector cells were assessed using the Cell Counting Kit-8 (CCK) assay after treatment with the indicated concentrations of SPE for 24 h. (E–G) A PD-1/PD-L1 blockade bioassay was performed to assess the inhibitory activities of PD-L1-blocking antibody, SPE, and SPE-EA. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of (E) PD-L1-blocking antibody, (F) SPE, or (G) SPE-EA. Bar graph (mean ± standard error of the mean) statistics were determined with the data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P

    Techniques Used: Competitive ELISA, Enzyme-linked Immunosorbent Assay, Screening Assay, Blocking Assay, Inhibition, Activation Assay, In Vitro, Cell Counting, Incubation

    Anti-tumor efficacy of SPE on hPD-L1 MC38 tumor growth in hPD-1 knock-in mice. (A) Representative images show the tumor development at 16 days after treatment. Mice were inoculated s.c. with hPD-L1 MC38 cells and were administrated with aPD-1 (5 mg/kg, i.p.) or SPE (100, 300 mg/kg, i.g.) (n=3). Tumor mice received an equivalent volume of vehicle (200 µL). (B) Image of subcutaneous tumor mass on day 16 (scale bars, 1 cm) (n=3). (C) Mean tumor weight (n=3). (D) Mean tumor diameter (n=3). (E) Immunohistochemistry staining of CD3+ in humanized PD-1 mouse model of tumor. (F) FACS analysis of tumor CD8+ T-cell populations from human PD-L1 knock in MC 38 tumor-bearing model of humanized PD-1 mice. Percentages of polyclonal CD8+ T-cells of CD45+ cells in tumor. Bar graph (mean ± standard error of the mean) statistics were determined with one-way ANOVA with Tukey’s post-hoc test; ***P
    Figure Legend Snippet: Anti-tumor efficacy of SPE on hPD-L1 MC38 tumor growth in hPD-1 knock-in mice. (A) Representative images show the tumor development at 16 days after treatment. Mice were inoculated s.c. with hPD-L1 MC38 cells and were administrated with aPD-1 (5 mg/kg, i.p.) or SPE (100, 300 mg/kg, i.g.) (n=3). Tumor mice received an equivalent volume of vehicle (200 µL). (B) Image of subcutaneous tumor mass on day 16 (scale bars, 1 cm) (n=3). (C) Mean tumor weight (n=3). (D) Mean tumor diameter (n=3). (E) Immunohistochemistry staining of CD3+ in humanized PD-1 mouse model of tumor. (F) FACS analysis of tumor CD8+ T-cell populations from human PD-L1 knock in MC 38 tumor-bearing model of humanized PD-1 mice. Percentages of polyclonal CD8+ T-cells of CD45+ cells in tumor. Bar graph (mean ± standard error of the mean) statistics were determined with one-way ANOVA with Tukey’s post-hoc test; ***P

    Techniques Used: Knock-In, Mouse Assay, Immunohistochemistry, Staining, FACS

    3) Product Images from "Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model"

    Article Title: Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.598556

    PD-1/PD-L1 interaction blockade and T-cell activation enhancement by SPE components in vitro . (A) Chemical components of SPE. (B) The ability of seven components from SPE to enhance T-cell functional activity was assessed using a PD-1/PD-L1 blockade bioassay. These seven components were used at a concentration of 2 µM. (C, D) A PD-1/PD-L1 blockade bioassay was performed to measure the inhibitory activity of apigenin and cosmosiin. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of apigenin or cosmosiin. (E, F) Competitive ELISA was performed using a PD-1/PD-L1 inhibitor ELISA-binding assay after treatment with the indicated concentrations of (E) apigenin and (F) cosmosiin. Bar graph (mean ± standard error of the mean) statistics were determined with data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P
    Figure Legend Snippet: PD-1/PD-L1 interaction blockade and T-cell activation enhancement by SPE components in vitro . (A) Chemical components of SPE. (B) The ability of seven components from SPE to enhance T-cell functional activity was assessed using a PD-1/PD-L1 blockade bioassay. These seven components were used at a concentration of 2 µM. (C, D) A PD-1/PD-L1 blockade bioassay was performed to measure the inhibitory activity of apigenin and cosmosiin. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of apigenin or cosmosiin. (E, F) Competitive ELISA was performed using a PD-1/PD-L1 inhibitor ELISA-binding assay after treatment with the indicated concentrations of (E) apigenin and (F) cosmosiin. Bar graph (mean ± standard error of the mean) statistics were determined with data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P

    Techniques Used: Activation Assay, In Vitro, Functional Assay, Activity Assay, Concentration Assay, Incubation, Competitive ELISA, Enzyme-linked Immunosorbent Assay, Binding Assay

    Cytotoxic and T-cell-mediated killing effect of SPE on colorectal cancer cells. (A) PD-L1 expression on the surface of humanized PD-L1-expressing MC38 cells (hPDL1-MCs) was determined using flow cytometry. (B–D) To measure the immunological activity of SPE, hPDL1-MCs (target cells) were co-cultured with humanized PD-1 mouse splenocytes (B, C) and CD8+ T-cells from the tumor (D) (hPD1-MSs; effector cells) at an effector cell-to-target cell ratio of 5:1 and were treated with SPE or 50% DMSO (control). (B) After 72 h of incubation, surviving hPD1-MSs in a 96-well plate underwent the CCK assay to assess cell viability. (C) Co-cultured CellTrace™ Far Red-labeled hPDL1-MCs were treated with SPE (25 μg/mL) or 50% DMSO (control), and the findings were compared with those in the absence of effector cells. The images were obtained under a fluorescence microscope. (D) The cytotoxicity of CD8+ T-cells against hPD-L1-MCs at an effector cell-to-target cell ratio of 5:1 was analyzed using an LDH assay kit (percentages calculated according to kit instruction). ***P
    Figure Legend Snippet: Cytotoxic and T-cell-mediated killing effect of SPE on colorectal cancer cells. (A) PD-L1 expression on the surface of humanized PD-L1-expressing MC38 cells (hPDL1-MCs) was determined using flow cytometry. (B–D) To measure the immunological activity of SPE, hPDL1-MCs (target cells) were co-cultured with humanized PD-1 mouse splenocytes (B, C) and CD8+ T-cells from the tumor (D) (hPD1-MSs; effector cells) at an effector cell-to-target cell ratio of 5:1 and were treated with SPE or 50% DMSO (control). (B) After 72 h of incubation, surviving hPD1-MSs in a 96-well plate underwent the CCK assay to assess cell viability. (C) Co-cultured CellTrace™ Far Red-labeled hPDL1-MCs were treated with SPE (25 μg/mL) or 50% DMSO (control), and the findings were compared with those in the absence of effector cells. The images were obtained under a fluorescence microscope. (D) The cytotoxicity of CD8+ T-cells against hPD-L1-MCs at an effector cell-to-target cell ratio of 5:1 was analyzed using an LDH assay kit (percentages calculated according to kit instruction). ***P

    Techniques Used: Expressing, Flow Cytometry, Activity Assay, Cell Culture, Incubation, Labeling, Fluorescence, Microscopy, Lactate Dehydrogenase Assay

    Protein–ligand docking simulation between PD-L1 (A) /PD-1 (B) and the Salvia plebeia R. Br. ethanol extract (SPE) component cosmosiin. Binding models were obtained through docking simulation of cosmosiin on PD-L1 and PD-1 derived from the PD-1/PD-L1 complex (PDB code 4ZQK) using AutoDock Vina. The hydrogen bonds and hydrophobic interactions between PD-L1/PD-1 and cosmosiin were analyzed using LigPlot+.
    Figure Legend Snippet: Protein–ligand docking simulation between PD-L1 (A) /PD-1 (B) and the Salvia plebeia R. Br. ethanol extract (SPE) component cosmosiin. Binding models were obtained through docking simulation of cosmosiin on PD-L1 and PD-1 derived from the PD-1/PD-L1 complex (PDB code 4ZQK) using AutoDock Vina. The hydrogen bonds and hydrophobic interactions between PD-L1/PD-1 and cosmosiin were analyzed using LigPlot+.

    Techniques Used: Binding Assay, Derivative Assay

    Global kinetic analysis of cosmosiin binding to biotinylated (A) PD-1 and (B) PD-L1 immobilized on a streptavidin biolayer interferometry (BLI) sensor. The kinetics of cosmosiin for immobilized PD-1 and PD-L1 were monitored with increasing concentration of cosmosiin (0, 0.125, 0.25, and 0.375 mM), dissolved in PBS buffer (pH 7.3) containing 1% DMSO.
    Figure Legend Snippet: Global kinetic analysis of cosmosiin binding to biotinylated (A) PD-1 and (B) PD-L1 immobilized on a streptavidin biolayer interferometry (BLI) sensor. The kinetics of cosmosiin for immobilized PD-1 and PD-L1 were monitored with increasing concentration of cosmosiin (0, 0.125, 0.25, and 0.375 mM), dissolved in PBS buffer (pH 7.3) containing 1% DMSO.

    Techniques Used: Binding Assay, Concentration Assay

    PD-1/PD-L1 interaction blockade by Salvia plebeia R. Br. ethanol extract (SPE). Competitive ELISA was performed using PD-1/PD-L1 inhibitor ELISA-screening assay with the indicated concentrations of PD-L1-blocking antibody, SPE, and SPE fractions. (A) Inhibition of the PD-1/PD-L1 interaction by PD-L1-blocking antibody, (B) SPE, and (C) SPE fractions (methylene chloride [MC], ethyl acetate [EA], and n-butanol [BuOH]). Cytotoxicity and T-cell activation by SPE and its ethyl acetate (EA) fraction in vitro . (D) The viabilities of PD-L1 aAPC/CHO-K1 and PD-1 effector cells were assessed using the Cell Counting Kit-8 (CCK) assay after treatment with the indicated concentrations of SPE for 24 h. (E–G) A PD-1/PD-L1 blockade bioassay was performed to assess the inhibitory activities of PD-L1-blocking antibody, SPE, and SPE-EA. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of (E) PD-L1-blocking antibody, (F) SPE, or (G) SPE-EA. Bar graph (mean ± standard error of the mean) statistics were determined with the data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P
    Figure Legend Snippet: PD-1/PD-L1 interaction blockade by Salvia plebeia R. Br. ethanol extract (SPE). Competitive ELISA was performed using PD-1/PD-L1 inhibitor ELISA-screening assay with the indicated concentrations of PD-L1-blocking antibody, SPE, and SPE fractions. (A) Inhibition of the PD-1/PD-L1 interaction by PD-L1-blocking antibody, (B) SPE, and (C) SPE fractions (methylene chloride [MC], ethyl acetate [EA], and n-butanol [BuOH]). Cytotoxicity and T-cell activation by SPE and its ethyl acetate (EA) fraction in vitro . (D) The viabilities of PD-L1 aAPC/CHO-K1 and PD-1 effector cells were assessed using the Cell Counting Kit-8 (CCK) assay after treatment with the indicated concentrations of SPE for 24 h. (E–G) A PD-1/PD-L1 blockade bioassay was performed to assess the inhibitory activities of PD-L1-blocking antibody, SPE, and SPE-EA. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of (E) PD-L1-blocking antibody, (F) SPE, or (G) SPE-EA. Bar graph (mean ± standard error of the mean) statistics were determined with the data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P

    Techniques Used: Competitive ELISA, Enzyme-linked Immunosorbent Assay, Screening Assay, Blocking Assay, Inhibition, Activation Assay, In Vitro, Cell Counting, Incubation

    Anti-tumor efficacy of SPE on hPD-L1 MC38 tumor growth in hPD-1 knock-in mice. (A) Representative images show the tumor development at 16 days after treatment. Mice were inoculated s.c. with hPD-L1 MC38 cells and were administrated with aPD-1 (5 mg/kg, i.p.) or SPE (100, 300 mg/kg, i.g.) (n=3). Tumor mice received an equivalent volume of vehicle (200 µL). (B) Image of subcutaneous tumor mass on day 16 (scale bars, 1 cm) (n=3). (C) Mean tumor weight (n=3). (D) Mean tumor diameter (n=3). (E) Immunohistochemistry staining of CD3+ in humanized PD-1 mouse model of tumor. (F) FACS analysis of tumor CD8+ T-cell populations from human PD-L1 knock in MC 38 tumor-bearing model of humanized PD-1 mice. Percentages of polyclonal CD8+ T-cells of CD45+ cells in tumor. Bar graph (mean ± standard error of the mean) statistics were determined with one-way ANOVA with Tukey’s post-hoc test; ***P
    Figure Legend Snippet: Anti-tumor efficacy of SPE on hPD-L1 MC38 tumor growth in hPD-1 knock-in mice. (A) Representative images show the tumor development at 16 days after treatment. Mice were inoculated s.c. with hPD-L1 MC38 cells and were administrated with aPD-1 (5 mg/kg, i.p.) or SPE (100, 300 mg/kg, i.g.) (n=3). Tumor mice received an equivalent volume of vehicle (200 µL). (B) Image of subcutaneous tumor mass on day 16 (scale bars, 1 cm) (n=3). (C) Mean tumor weight (n=3). (D) Mean tumor diameter (n=3). (E) Immunohistochemistry staining of CD3+ in humanized PD-1 mouse model of tumor. (F) FACS analysis of tumor CD8+ T-cell populations from human PD-L1 knock in MC 38 tumor-bearing model of humanized PD-1 mice. Percentages of polyclonal CD8+ T-cells of CD45+ cells in tumor. Bar graph (mean ± standard error of the mean) statistics were determined with one-way ANOVA with Tukey’s post-hoc test; ***P

    Techniques Used: Knock-In, Mouse Assay, Immunohistochemistry, Staining, FACS

    4) Product Images from "Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model"

    Article Title: Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.598556

    PD-1/PD-L1 interaction blockade and T-cell activation enhancement by SPE components in vitro . (A) Chemical components of SPE. (B) The ability of seven components from SPE to enhance T-cell functional activity was assessed using a PD-1/PD-L1 blockade bioassay. These seven components were used at a concentration of 2 µM. (C, D) A PD-1/PD-L1 blockade bioassay was performed to measure the inhibitory activity of apigenin and cosmosiin. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of apigenin or cosmosiin. (E, F) Competitive ELISA was performed using a PD-1/PD-L1 inhibitor ELISA-binding assay after treatment with the indicated concentrations of (E) apigenin and (F) cosmosiin. Bar graph (mean ± standard error of the mean) statistics were determined with data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P
    Figure Legend Snippet: PD-1/PD-L1 interaction blockade and T-cell activation enhancement by SPE components in vitro . (A) Chemical components of SPE. (B) The ability of seven components from SPE to enhance T-cell functional activity was assessed using a PD-1/PD-L1 blockade bioassay. These seven components were used at a concentration of 2 µM. (C, D) A PD-1/PD-L1 blockade bioassay was performed to measure the inhibitory activity of apigenin and cosmosiin. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of apigenin or cosmosiin. (E, F) Competitive ELISA was performed using a PD-1/PD-L1 inhibitor ELISA-binding assay after treatment with the indicated concentrations of (E) apigenin and (F) cosmosiin. Bar graph (mean ± standard error of the mean) statistics were determined with data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P

    Techniques Used: Activation Assay, In Vitro, Functional Assay, Activity Assay, Concentration Assay, Incubation, Competitive ELISA, Enzyme-linked Immunosorbent Assay, Binding Assay

    Cytotoxic and T-cell-mediated killing effect of SPE on colorectal cancer cells. (A) PD-L1 expression on the surface of humanized PD-L1-expressing MC38 cells (hPDL1-MCs) was determined using flow cytometry. (B–D) To measure the immunological activity of SPE, hPDL1-MCs (target cells) were co-cultured with humanized PD-1 mouse splenocytes (B, C) and CD8+ T-cells from the tumor (D) (hPD1-MSs; effector cells) at an effector cell-to-target cell ratio of 5:1 and were treated with SPE or 50% DMSO (control). (B) After 72 h of incubation, surviving hPD1-MSs in a 96-well plate underwent the CCK assay to assess cell viability. (C) Co-cultured CellTrace™ Far Red-labeled hPDL1-MCs were treated with SPE (25 μg/mL) or 50% DMSO (control), and the findings were compared with those in the absence of effector cells. The images were obtained under a fluorescence microscope. (D) The cytotoxicity of CD8+ T-cells against hPD-L1-MCs at an effector cell-to-target cell ratio of 5:1 was analyzed using an LDH assay kit (percentages calculated according to kit instruction). ***P
    Figure Legend Snippet: Cytotoxic and T-cell-mediated killing effect of SPE on colorectal cancer cells. (A) PD-L1 expression on the surface of humanized PD-L1-expressing MC38 cells (hPDL1-MCs) was determined using flow cytometry. (B–D) To measure the immunological activity of SPE, hPDL1-MCs (target cells) were co-cultured with humanized PD-1 mouse splenocytes (B, C) and CD8+ T-cells from the tumor (D) (hPD1-MSs; effector cells) at an effector cell-to-target cell ratio of 5:1 and were treated with SPE or 50% DMSO (control). (B) After 72 h of incubation, surviving hPD1-MSs in a 96-well plate underwent the CCK assay to assess cell viability. (C) Co-cultured CellTrace™ Far Red-labeled hPDL1-MCs were treated with SPE (25 μg/mL) or 50% DMSO (control), and the findings were compared with those in the absence of effector cells. The images were obtained under a fluorescence microscope. (D) The cytotoxicity of CD8+ T-cells against hPD-L1-MCs at an effector cell-to-target cell ratio of 5:1 was analyzed using an LDH assay kit (percentages calculated according to kit instruction). ***P

    Techniques Used: Expressing, Flow Cytometry, Activity Assay, Cell Culture, Incubation, Labeling, Fluorescence, Microscopy, Lactate Dehydrogenase Assay

    Protein–ligand docking simulation between PD-L1 (A) /PD-1 (B) and the Salvia plebeia R. Br. ethanol extract (SPE) component cosmosiin. Binding models were obtained through docking simulation of cosmosiin on PD-L1 and PD-1 derived from the PD-1/PD-L1 complex (PDB code 4ZQK) using AutoDock Vina. The hydrogen bonds and hydrophobic interactions between PD-L1/PD-1 and cosmosiin were analyzed using LigPlot+.
    Figure Legend Snippet: Protein–ligand docking simulation between PD-L1 (A) /PD-1 (B) and the Salvia plebeia R. Br. ethanol extract (SPE) component cosmosiin. Binding models were obtained through docking simulation of cosmosiin on PD-L1 and PD-1 derived from the PD-1/PD-L1 complex (PDB code 4ZQK) using AutoDock Vina. The hydrogen bonds and hydrophobic interactions between PD-L1/PD-1 and cosmosiin were analyzed using LigPlot+.

    Techniques Used: Binding Assay, Derivative Assay

    Global kinetic analysis of cosmosiin binding to biotinylated (A) PD-1 and (B) PD-L1 immobilized on a streptavidin biolayer interferometry (BLI) sensor. The kinetics of cosmosiin for immobilized PD-1 and PD-L1 were monitored with increasing concentration of cosmosiin (0, 0.125, 0.25, and 0.375 mM), dissolved in PBS buffer (pH 7.3) containing 1% DMSO.
    Figure Legend Snippet: Global kinetic analysis of cosmosiin binding to biotinylated (A) PD-1 and (B) PD-L1 immobilized on a streptavidin biolayer interferometry (BLI) sensor. The kinetics of cosmosiin for immobilized PD-1 and PD-L1 were monitored with increasing concentration of cosmosiin (0, 0.125, 0.25, and 0.375 mM), dissolved in PBS buffer (pH 7.3) containing 1% DMSO.

    Techniques Used: Binding Assay, Concentration Assay

    PD-1/PD-L1 interaction blockade by Salvia plebeia R. Br. ethanol extract (SPE). Competitive ELISA was performed using PD-1/PD-L1 inhibitor ELISA-screening assay with the indicated concentrations of PD-L1-blocking antibody, SPE, and SPE fractions. (A) Inhibition of the PD-1/PD-L1 interaction by PD-L1-blocking antibody, (B) SPE, and (C) SPE fractions (methylene chloride [MC], ethyl acetate [EA], and n-butanol [BuOH]). Cytotoxicity and T-cell activation by SPE and its ethyl acetate (EA) fraction in vitro . (D) The viabilities of PD-L1 aAPC/CHO-K1 and PD-1 effector cells were assessed using the Cell Counting Kit-8 (CCK) assay after treatment with the indicated concentrations of SPE for 24 h. (E–G) A PD-1/PD-L1 blockade bioassay was performed to assess the inhibitory activities of PD-L1-blocking antibody, SPE, and SPE-EA. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of (E) PD-L1-blocking antibody, (F) SPE, or (G) SPE-EA. Bar graph (mean ± standard error of the mean) statistics were determined with the data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P
    Figure Legend Snippet: PD-1/PD-L1 interaction blockade by Salvia plebeia R. Br. ethanol extract (SPE). Competitive ELISA was performed using PD-1/PD-L1 inhibitor ELISA-screening assay with the indicated concentrations of PD-L1-blocking antibody, SPE, and SPE fractions. (A) Inhibition of the PD-1/PD-L1 interaction by PD-L1-blocking antibody, (B) SPE, and (C) SPE fractions (methylene chloride [MC], ethyl acetate [EA], and n-butanol [BuOH]). Cytotoxicity and T-cell activation by SPE and its ethyl acetate (EA) fraction in vitro . (D) The viabilities of PD-L1 aAPC/CHO-K1 and PD-1 effector cells were assessed using the Cell Counting Kit-8 (CCK) assay after treatment with the indicated concentrations of SPE for 24 h. (E–G) A PD-1/PD-L1 blockade bioassay was performed to assess the inhibitory activities of PD-L1-blocking antibody, SPE, and SPE-EA. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of (E) PD-L1-blocking antibody, (F) SPE, or (G) SPE-EA. Bar graph (mean ± standard error of the mean) statistics were determined with the data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P

    Techniques Used: Competitive ELISA, Enzyme-linked Immunosorbent Assay, Screening Assay, Blocking Assay, Inhibition, Activation Assay, In Vitro, Cell Counting, Incubation

    Anti-tumor efficacy of SPE on hPD-L1 MC38 tumor growth in hPD-1 knock-in mice. (A) Representative images show the tumor development at 16 days after treatment. Mice were inoculated s.c. with hPD-L1 MC38 cells and were administrated with aPD-1 (5 mg/kg, i.p.) or SPE (100, 300 mg/kg, i.g.) (n=3). Tumor mice received an equivalent volume of vehicle (200 µL). (B) Image of subcutaneous tumor mass on day 16 (scale bars, 1 cm) (n=3). (C) Mean tumor weight (n=3). (D) Mean tumor diameter (n=3). (E) Immunohistochemistry staining of CD3+ in humanized PD-1 mouse model of tumor. (F) FACS analysis of tumor CD8+ T-cell populations from human PD-L1 knock in MC 38 tumor-bearing model of humanized PD-1 mice. Percentages of polyclonal CD8+ T-cells of CD45+ cells in tumor. Bar graph (mean ± standard error of the mean) statistics were determined with one-way ANOVA with Tukey’s post-hoc test; ***P
    Figure Legend Snippet: Anti-tumor efficacy of SPE on hPD-L1 MC38 tumor growth in hPD-1 knock-in mice. (A) Representative images show the tumor development at 16 days after treatment. Mice were inoculated s.c. with hPD-L1 MC38 cells and were administrated with aPD-1 (5 mg/kg, i.p.) or SPE (100, 300 mg/kg, i.g.) (n=3). Tumor mice received an equivalent volume of vehicle (200 µL). (B) Image of subcutaneous tumor mass on day 16 (scale bars, 1 cm) (n=3). (C) Mean tumor weight (n=3). (D) Mean tumor diameter (n=3). (E) Immunohistochemistry staining of CD3+ in humanized PD-1 mouse model of tumor. (F) FACS analysis of tumor CD8+ T-cell populations from human PD-L1 knock in MC 38 tumor-bearing model of humanized PD-1 mice. Percentages of polyclonal CD8+ T-cells of CD45+ cells in tumor. Bar graph (mean ± standard error of the mean) statistics were determined with one-way ANOVA with Tukey’s post-hoc test; ***P

    Techniques Used: Knock-In, Mouse Assay, Immunohistochemistry, Staining, FACS

    5) Product Images from "Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model"

    Article Title: Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.598556

    PD-1/PD-L1 interaction blockade and T-cell activation enhancement by SPE components in vitro . (A) Chemical components of SPE. (B) The ability of seven components from SPE to enhance T-cell functional activity was assessed using a PD-1/PD-L1 blockade bioassay. These seven components were used at a concentration of 2 µM. (C, D) A PD-1/PD-L1 blockade bioassay was performed to measure the inhibitory activity of apigenin and cosmosiin. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of apigenin or cosmosiin. (E, F) Competitive ELISA was performed using a PD-1/PD-L1 inhibitor ELISA-binding assay after treatment with the indicated concentrations of (E) apigenin and (F) cosmosiin. Bar graph (mean ± standard error of the mean) statistics were determined with data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P
    Figure Legend Snippet: PD-1/PD-L1 interaction blockade and T-cell activation enhancement by SPE components in vitro . (A) Chemical components of SPE. (B) The ability of seven components from SPE to enhance T-cell functional activity was assessed using a PD-1/PD-L1 blockade bioassay. These seven components were used at a concentration of 2 µM. (C, D) A PD-1/PD-L1 blockade bioassay was performed to measure the inhibitory activity of apigenin and cosmosiin. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of apigenin or cosmosiin. (E, F) Competitive ELISA was performed using a PD-1/PD-L1 inhibitor ELISA-binding assay after treatment with the indicated concentrations of (E) apigenin and (F) cosmosiin. Bar graph (mean ± standard error of the mean) statistics were determined with data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P

    Techniques Used: Activation Assay, In Vitro, Functional Assay, Activity Assay, Concentration Assay, Incubation, Competitive ELISA, Enzyme-linked Immunosorbent Assay, Binding Assay

    Cytotoxic and T-cell-mediated killing effect of SPE on colorectal cancer cells. (A) PD-L1 expression on the surface of humanized PD-L1-expressing MC38 cells (hPDL1-MCs) was determined using flow cytometry. (B–D) To measure the immunological activity of SPE, hPDL1-MCs (target cells) were co-cultured with humanized PD-1 mouse splenocytes (B, C) and CD8+ T-cells from the tumor (D) (hPD1-MSs; effector cells) at an effector cell-to-target cell ratio of 5:1 and were treated with SPE or 50% DMSO (control). (B) After 72 h of incubation, surviving hPD1-MSs in a 96-well plate underwent the CCK assay to assess cell viability. (C) Co-cultured CellTrace™ Far Red-labeled hPDL1-MCs were treated with SPE (25 μg/mL) or 50% DMSO (control), and the findings were compared with those in the absence of effector cells. The images were obtained under a fluorescence microscope. (D) The cytotoxicity of CD8+ T-cells against hPD-L1-MCs at an effector cell-to-target cell ratio of 5:1 was analyzed using an LDH assay kit (percentages calculated according to kit instruction). ***P
    Figure Legend Snippet: Cytotoxic and T-cell-mediated killing effect of SPE on colorectal cancer cells. (A) PD-L1 expression on the surface of humanized PD-L1-expressing MC38 cells (hPDL1-MCs) was determined using flow cytometry. (B–D) To measure the immunological activity of SPE, hPDL1-MCs (target cells) were co-cultured with humanized PD-1 mouse splenocytes (B, C) and CD8+ T-cells from the tumor (D) (hPD1-MSs; effector cells) at an effector cell-to-target cell ratio of 5:1 and were treated with SPE or 50% DMSO (control). (B) After 72 h of incubation, surviving hPD1-MSs in a 96-well plate underwent the CCK assay to assess cell viability. (C) Co-cultured CellTrace™ Far Red-labeled hPDL1-MCs were treated with SPE (25 μg/mL) or 50% DMSO (control), and the findings were compared with those in the absence of effector cells. The images were obtained under a fluorescence microscope. (D) The cytotoxicity of CD8+ T-cells against hPD-L1-MCs at an effector cell-to-target cell ratio of 5:1 was analyzed using an LDH assay kit (percentages calculated according to kit instruction). ***P

    Techniques Used: Expressing, Flow Cytometry, Activity Assay, Cell Culture, Incubation, Labeling, Fluorescence, Microscopy, Lactate Dehydrogenase Assay

    Protein–ligand docking simulation between PD-L1 (A) /PD-1 (B) and the Salvia plebeia R. Br. ethanol extract (SPE) component cosmosiin. Binding models were obtained through docking simulation of cosmosiin on PD-L1 and PD-1 derived from the PD-1/PD-L1 complex (PDB code 4ZQK) using AutoDock Vina. The hydrogen bonds and hydrophobic interactions between PD-L1/PD-1 and cosmosiin were analyzed using LigPlot+.
    Figure Legend Snippet: Protein–ligand docking simulation between PD-L1 (A) /PD-1 (B) and the Salvia plebeia R. Br. ethanol extract (SPE) component cosmosiin. Binding models were obtained through docking simulation of cosmosiin on PD-L1 and PD-1 derived from the PD-1/PD-L1 complex (PDB code 4ZQK) using AutoDock Vina. The hydrogen bonds and hydrophobic interactions between PD-L1/PD-1 and cosmosiin were analyzed using LigPlot+.

    Techniques Used: Binding Assay, Derivative Assay

    Global kinetic analysis of cosmosiin binding to biotinylated (A) PD-1 and (B) PD-L1 immobilized on a streptavidin biolayer interferometry (BLI) sensor. The kinetics of cosmosiin for immobilized PD-1 and PD-L1 were monitored with increasing concentration of cosmosiin (0, 0.125, 0.25, and 0.375 mM), dissolved in PBS buffer (pH 7.3) containing 1% DMSO.
    Figure Legend Snippet: Global kinetic analysis of cosmosiin binding to biotinylated (A) PD-1 and (B) PD-L1 immobilized on a streptavidin biolayer interferometry (BLI) sensor. The kinetics of cosmosiin for immobilized PD-1 and PD-L1 were monitored with increasing concentration of cosmosiin (0, 0.125, 0.25, and 0.375 mM), dissolved in PBS buffer (pH 7.3) containing 1% DMSO.

    Techniques Used: Binding Assay, Concentration Assay

    PD-1/PD-L1 interaction blockade by Salvia plebeia R. Br. ethanol extract (SPE). Competitive ELISA was performed using PD-1/PD-L1 inhibitor ELISA-screening assay with the indicated concentrations of PD-L1-blocking antibody, SPE, and SPE fractions. (A) Inhibition of the PD-1/PD-L1 interaction by PD-L1-blocking antibody, (B) SPE, and (C) SPE fractions (methylene chloride [MC], ethyl acetate [EA], and n-butanol [BuOH]). Cytotoxicity and T-cell activation by SPE and its ethyl acetate (EA) fraction in vitro . (D) The viabilities of PD-L1 aAPC/CHO-K1 and PD-1 effector cells were assessed using the Cell Counting Kit-8 (CCK) assay after treatment with the indicated concentrations of SPE for 24 h. (E–G) A PD-1/PD-L1 blockade bioassay was performed to assess the inhibitory activities of PD-L1-blocking antibody, SPE, and SPE-EA. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of (E) PD-L1-blocking antibody, (F) SPE, or (G) SPE-EA. Bar graph (mean ± standard error of the mean) statistics were determined with the data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P
    Figure Legend Snippet: PD-1/PD-L1 interaction blockade by Salvia plebeia R. Br. ethanol extract (SPE). Competitive ELISA was performed using PD-1/PD-L1 inhibitor ELISA-screening assay with the indicated concentrations of PD-L1-blocking antibody, SPE, and SPE fractions. (A) Inhibition of the PD-1/PD-L1 interaction by PD-L1-blocking antibody, (B) SPE, and (C) SPE fractions (methylene chloride [MC], ethyl acetate [EA], and n-butanol [BuOH]). Cytotoxicity and T-cell activation by SPE and its ethyl acetate (EA) fraction in vitro . (D) The viabilities of PD-L1 aAPC/CHO-K1 and PD-1 effector cells were assessed using the Cell Counting Kit-8 (CCK) assay after treatment with the indicated concentrations of SPE for 24 h. (E–G) A PD-1/PD-L1 blockade bioassay was performed to assess the inhibitory activities of PD-L1-blocking antibody, SPE, and SPE-EA. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of (E) PD-L1-blocking antibody, (F) SPE, or (G) SPE-EA. Bar graph (mean ± standard error of the mean) statistics were determined with the data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P

    Techniques Used: Competitive ELISA, Enzyme-linked Immunosorbent Assay, Screening Assay, Blocking Assay, Inhibition, Activation Assay, In Vitro, Cell Counting, Incubation

    Anti-tumor efficacy of SPE on hPD-L1 MC38 tumor growth in hPD-1 knock-in mice. (A) Representative images show the tumor development at 16 days after treatment. Mice were inoculated s.c. with hPD-L1 MC38 cells and were administrated with aPD-1 (5 mg/kg, i.p.) or SPE (100, 300 mg/kg, i.g.) (n=3). Tumor mice received an equivalent volume of vehicle (200 µL). (B) Image of subcutaneous tumor mass on day 16 (scale bars, 1 cm) (n=3). (C) Mean tumor weight (n=3). (D) Mean tumor diameter (n=3). (E) Immunohistochemistry staining of CD3+ in humanized PD-1 mouse model of tumor. (F) FACS analysis of tumor CD8+ T-cell populations from human PD-L1 knock in MC 38 tumor-bearing model of humanized PD-1 mice. Percentages of polyclonal CD8+ T-cells of CD45+ cells in tumor. Bar graph (mean ± standard error of the mean) statistics were determined with one-way ANOVA with Tukey’s post-hoc test; ***P
    Figure Legend Snippet: Anti-tumor efficacy of SPE on hPD-L1 MC38 tumor growth in hPD-1 knock-in mice. (A) Representative images show the tumor development at 16 days after treatment. Mice were inoculated s.c. with hPD-L1 MC38 cells and were administrated with aPD-1 (5 mg/kg, i.p.) or SPE (100, 300 mg/kg, i.g.) (n=3). Tumor mice received an equivalent volume of vehicle (200 µL). (B) Image of subcutaneous tumor mass on day 16 (scale bars, 1 cm) (n=3). (C) Mean tumor weight (n=3). (D) Mean tumor diameter (n=3). (E) Immunohistochemistry staining of CD3+ in humanized PD-1 mouse model of tumor. (F) FACS analysis of tumor CD8+ T-cell populations from human PD-L1 knock in MC 38 tumor-bearing model of humanized PD-1 mice. Percentages of polyclonal CD8+ T-cells of CD45+ cells in tumor. Bar graph (mean ± standard error of the mean) statistics were determined with one-way ANOVA with Tukey’s post-hoc test; ***P

    Techniques Used: Knock-In, Mouse Assay, Immunohistochemistry, Staining, FACS

    6) Product Images from "Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model"

    Article Title: Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.598556

    PD-1/PD-L1 interaction blockade and T-cell activation enhancement by SPE components in vitro . (A) Chemical components of SPE. (B) The ability of seven components from SPE to enhance T-cell functional activity was assessed using a PD-1/PD-L1 blockade bioassay. These seven components were used at a concentration of 2 µM. (C, D) A PD-1/PD-L1 blockade bioassay was performed to measure the inhibitory activity of apigenin and cosmosiin. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of apigenin or cosmosiin. (E, F) Competitive ELISA was performed using a PD-1/PD-L1 inhibitor ELISA-binding assay after treatment with the indicated concentrations of (E) apigenin and (F) cosmosiin. Bar graph (mean ± standard error of the mean) statistics were determined with data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P
    Figure Legend Snippet: PD-1/PD-L1 interaction blockade and T-cell activation enhancement by SPE components in vitro . (A) Chemical components of SPE. (B) The ability of seven components from SPE to enhance T-cell functional activity was assessed using a PD-1/PD-L1 blockade bioassay. These seven components were used at a concentration of 2 µM. (C, D) A PD-1/PD-L1 blockade bioassay was performed to measure the inhibitory activity of apigenin and cosmosiin. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of apigenin or cosmosiin. (E, F) Competitive ELISA was performed using a PD-1/PD-L1 inhibitor ELISA-binding assay after treatment with the indicated concentrations of (E) apigenin and (F) cosmosiin. Bar graph (mean ± standard error of the mean) statistics were determined with data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P

    Techniques Used: Activation Assay, In Vitro, Functional Assay, Activity Assay, Concentration Assay, Incubation, Competitive ELISA, Enzyme-linked Immunosorbent Assay, Binding Assay

    Cytotoxic and T-cell-mediated killing effect of SPE on colorectal cancer cells. (A) PD-L1 expression on the surface of humanized PD-L1-expressing MC38 cells (hPDL1-MCs) was determined using flow cytometry. (B–D) To measure the immunological activity of SPE, hPDL1-MCs (target cells) were co-cultured with humanized PD-1 mouse splenocytes (B, C) and CD8+ T-cells from the tumor (D) (hPD1-MSs; effector cells) at an effector cell-to-target cell ratio of 5:1 and were treated with SPE or 50% DMSO (control). (B) After 72 h of incubation, surviving hPD1-MSs in a 96-well plate underwent the CCK assay to assess cell viability. (C) Co-cultured CellTrace™ Far Red-labeled hPDL1-MCs were treated with SPE (25 μg/mL) or 50% DMSO (control), and the findings were compared with those in the absence of effector cells. The images were obtained under a fluorescence microscope. (D) The cytotoxicity of CD8+ T-cells against hPD-L1-MCs at an effector cell-to-target cell ratio of 5:1 was analyzed using an LDH assay kit (percentages calculated according to kit instruction). ***P
    Figure Legend Snippet: Cytotoxic and T-cell-mediated killing effect of SPE on colorectal cancer cells. (A) PD-L1 expression on the surface of humanized PD-L1-expressing MC38 cells (hPDL1-MCs) was determined using flow cytometry. (B–D) To measure the immunological activity of SPE, hPDL1-MCs (target cells) were co-cultured with humanized PD-1 mouse splenocytes (B, C) and CD8+ T-cells from the tumor (D) (hPD1-MSs; effector cells) at an effector cell-to-target cell ratio of 5:1 and were treated with SPE or 50% DMSO (control). (B) After 72 h of incubation, surviving hPD1-MSs in a 96-well plate underwent the CCK assay to assess cell viability. (C) Co-cultured CellTrace™ Far Red-labeled hPDL1-MCs were treated with SPE (25 μg/mL) or 50% DMSO (control), and the findings were compared with those in the absence of effector cells. The images were obtained under a fluorescence microscope. (D) The cytotoxicity of CD8+ T-cells against hPD-L1-MCs at an effector cell-to-target cell ratio of 5:1 was analyzed using an LDH assay kit (percentages calculated according to kit instruction). ***P

    Techniques Used: Expressing, Flow Cytometry, Activity Assay, Cell Culture, Incubation, Labeling, Fluorescence, Microscopy, Lactate Dehydrogenase Assay

    Protein–ligand docking simulation between PD-L1 (A) /PD-1 (B) and the Salvia plebeia R. Br. ethanol extract (SPE) component cosmosiin. Binding models were obtained through docking simulation of cosmosiin on PD-L1 and PD-1 derived from the PD-1/PD-L1 complex (PDB code 4ZQK) using AutoDock Vina. The hydrogen bonds and hydrophobic interactions between PD-L1/PD-1 and cosmosiin were analyzed using LigPlot+.
    Figure Legend Snippet: Protein–ligand docking simulation between PD-L1 (A) /PD-1 (B) and the Salvia plebeia R. Br. ethanol extract (SPE) component cosmosiin. Binding models were obtained through docking simulation of cosmosiin on PD-L1 and PD-1 derived from the PD-1/PD-L1 complex (PDB code 4ZQK) using AutoDock Vina. The hydrogen bonds and hydrophobic interactions between PD-L1/PD-1 and cosmosiin were analyzed using LigPlot+.

    Techniques Used: Binding Assay, Derivative Assay

    Global kinetic analysis of cosmosiin binding to biotinylated (A) PD-1 and (B) PD-L1 immobilized on a streptavidin biolayer interferometry (BLI) sensor. The kinetics of cosmosiin for immobilized PD-1 and PD-L1 were monitored with increasing concentration of cosmosiin (0, 0.125, 0.25, and 0.375 mM), dissolved in PBS buffer (pH 7.3) containing 1% DMSO.
    Figure Legend Snippet: Global kinetic analysis of cosmosiin binding to biotinylated (A) PD-1 and (B) PD-L1 immobilized on a streptavidin biolayer interferometry (BLI) sensor. The kinetics of cosmosiin for immobilized PD-1 and PD-L1 were monitored with increasing concentration of cosmosiin (0, 0.125, 0.25, and 0.375 mM), dissolved in PBS buffer (pH 7.3) containing 1% DMSO.

    Techniques Used: Binding Assay, Concentration Assay

    PD-1/PD-L1 interaction blockade by Salvia plebeia R. Br. ethanol extract (SPE). Competitive ELISA was performed using PD-1/PD-L1 inhibitor ELISA-screening assay with the indicated concentrations of PD-L1-blocking antibody, SPE, and SPE fractions. (A) Inhibition of the PD-1/PD-L1 interaction by PD-L1-blocking antibody, (B) SPE, and (C) SPE fractions (methylene chloride [MC], ethyl acetate [EA], and n-butanol [BuOH]). Cytotoxicity and T-cell activation by SPE and its ethyl acetate (EA) fraction in vitro . (D) The viabilities of PD-L1 aAPC/CHO-K1 and PD-1 effector cells were assessed using the Cell Counting Kit-8 (CCK) assay after treatment with the indicated concentrations of SPE for 24 h. (E–G) A PD-1/PD-L1 blockade bioassay was performed to assess the inhibitory activities of PD-L1-blocking antibody, SPE, and SPE-EA. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of (E) PD-L1-blocking antibody, (F) SPE, or (G) SPE-EA. Bar graph (mean ± standard error of the mean) statistics were determined with the data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P
    Figure Legend Snippet: PD-1/PD-L1 interaction blockade by Salvia plebeia R. Br. ethanol extract (SPE). Competitive ELISA was performed using PD-1/PD-L1 inhibitor ELISA-screening assay with the indicated concentrations of PD-L1-blocking antibody, SPE, and SPE fractions. (A) Inhibition of the PD-1/PD-L1 interaction by PD-L1-blocking antibody, (B) SPE, and (C) SPE fractions (methylene chloride [MC], ethyl acetate [EA], and n-butanol [BuOH]). Cytotoxicity and T-cell activation by SPE and its ethyl acetate (EA) fraction in vitro . (D) The viabilities of PD-L1 aAPC/CHO-K1 and PD-1 effector cells were assessed using the Cell Counting Kit-8 (CCK) assay after treatment with the indicated concentrations of SPE for 24 h. (E–G) A PD-1/PD-L1 blockade bioassay was performed to assess the inhibitory activities of PD-L1-blocking antibody, SPE, and SPE-EA. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of (E) PD-L1-blocking antibody, (F) SPE, or (G) SPE-EA. Bar graph (mean ± standard error of the mean) statistics were determined with the data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P

    Techniques Used: Competitive ELISA, Enzyme-linked Immunosorbent Assay, Screening Assay, Blocking Assay, Inhibition, Activation Assay, In Vitro, Cell Counting, Incubation

    Anti-tumor efficacy of SPE on hPD-L1 MC38 tumor growth in hPD-1 knock-in mice. (A) Representative images show the tumor development at 16 days after treatment. Mice were inoculated s.c. with hPD-L1 MC38 cells and were administrated with aPD-1 (5 mg/kg, i.p.) or SPE (100, 300 mg/kg, i.g.) (n=3). Tumor mice received an equivalent volume of vehicle (200 µL). (B) Image of subcutaneous tumor mass on day 16 (scale bars, 1 cm) (n=3). (C) Mean tumor weight (n=3). (D) Mean tumor diameter (n=3). (E) Immunohistochemistry staining of CD3+ in humanized PD-1 mouse model of tumor. (F) FACS analysis of tumor CD8+ T-cell populations from human PD-L1 knock in MC 38 tumor-bearing model of humanized PD-1 mice. Percentages of polyclonal CD8+ T-cells of CD45+ cells in tumor. Bar graph (mean ± standard error of the mean) statistics were determined with one-way ANOVA with Tukey’s post-hoc test; ***P
    Figure Legend Snippet: Anti-tumor efficacy of SPE on hPD-L1 MC38 tumor growth in hPD-1 knock-in mice. (A) Representative images show the tumor development at 16 days after treatment. Mice were inoculated s.c. with hPD-L1 MC38 cells and were administrated with aPD-1 (5 mg/kg, i.p.) or SPE (100, 300 mg/kg, i.g.) (n=3). Tumor mice received an equivalent volume of vehicle (200 µL). (B) Image of subcutaneous tumor mass on day 16 (scale bars, 1 cm) (n=3). (C) Mean tumor weight (n=3). (D) Mean tumor diameter (n=3). (E) Immunohistochemistry staining of CD3+ in humanized PD-1 mouse model of tumor. (F) FACS analysis of tumor CD8+ T-cell populations from human PD-L1 knock in MC 38 tumor-bearing model of humanized PD-1 mice. Percentages of polyclonal CD8+ T-cells of CD45+ cells in tumor. Bar graph (mean ± standard error of the mean) statistics were determined with one-way ANOVA with Tukey’s post-hoc test; ***P

    Techniques Used: Knock-In, Mouse Assay, Immunohistochemistry, Staining, FACS

    Related Articles

    Binding Assay:

    Article Title: Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model
    Article Snippet: As shown in , the increase of cosmosiin concentration led to a wavelength shift in the BLItz sensorgram, indicating an increase in the cosmosiin interacting with the biotinylated PD-1 and PD-L1, respectively, immobilized on the streptavidin sensor. .. The equilibrium dissociation constants (KD ) of cosmosiin to PD-1 and PD-L1 were 386 and 85 μM with co efficient of determinations, R2 , of 0.9804 and 0.9866, respectively, showing high goodness of fit to 1:1 binding model and that cosmosiin has 4.5-fold higher affinity to PD-L1 than PD-1.Furthermore, the ka and kd values of cosmosiin to PD-1 and PD-L1 in indicate that the higher affinity of cosmosiin to PD-L1 results from its higher binding rate to PD-L1 rather than insignificant difference in dissociation rates. .. Protein–Ligand Docking Simulation and Pharmacophore Analysis of SPE ComponentsPrevious studies have reported a crystal structure showing the interaction between hPD-1 and hPD-L1 (PDB code: 4ZQK) ( ) and the binding region of small molecules in PD-1 and PD-L1 ( ).

    Concentration Assay:

    Article Title: Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model
    Article Snippet: The Affinity of Small Molecule Cosmosiin With PD-1 and PD-L1 The affinity of cosmosiin with PD-1 and PD-L1 were evaluated by kinetic model using BLItz system from Pall ForteBio (Menlo Park, USA). .. As shown in , the increase of cosmosiin concentration led to a wavelength shift in the BLItz sensorgram, indicating an increase in the cosmosiin interacting with the biotinylated PD-1 and PD-L1, respectively, immobilized on the streptavidin sensor. .. The equilibrium dissociation constants (KD ) of cosmosiin to PD-1 and PD-L1 were 386 and 85 μM with co efficient of determinations, R2 , of 0.9804 and 0.9866, respectively, showing high goodness of fit to 1:1 binding model and that cosmosiin has 4.5-fold higher affinity to PD-L1 than PD-1.Furthermore, the ka and kd values of cosmosiin to PD-1 and PD-L1 in indicate that the higher affinity of cosmosiin to PD-L1 results from its higher binding rate to PD-L1 rather than insignificant difference in dissociation rates.

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    <t>PD-1/PD-L1</t> interaction blockade and T-cell activation enhancement by SPE components in vitro . (A) Chemical components of SPE. (B) The ability of seven components from SPE to enhance T-cell functional activity was assessed using a PD-1/PD-L1 blockade bioassay. These seven components were used at a concentration of 2 µM. (C, D) A PD-1/PD-L1 blockade bioassay was performed to measure the inhibitory activity of apigenin and cosmosiin. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of apigenin or cosmosiin. (E, F) Competitive ELISA was performed using a PD-1/PD-L1 inhibitor ELISA-binding assay after treatment with the indicated concentrations of (E) apigenin and (F) cosmosiin. Bar graph (mean ± standard error of the mean) statistics were determined with data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P
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    PD-1/PD-L1 interaction blockade and T-cell activation enhancement by SPE components in vitro . (A) Chemical components of SPE. (B) The ability of seven components from SPE to enhance T-cell functional activity was assessed using a PD-1/PD-L1 blockade bioassay. These seven components were used at a concentration of 2 µM. (C, D) A PD-1/PD-L1 blockade bioassay was performed to measure the inhibitory activity of apigenin and cosmosiin. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of apigenin or cosmosiin. (E, F) Competitive ELISA was performed using a PD-1/PD-L1 inhibitor ELISA-binding assay after treatment with the indicated concentrations of (E) apigenin and (F) cosmosiin. Bar graph (mean ± standard error of the mean) statistics were determined with data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P

    Journal: Frontiers in Immunology

    Article Title: Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model

    doi: 10.3389/fimmu.2020.598556

    Figure Lengend Snippet: PD-1/PD-L1 interaction blockade and T-cell activation enhancement by SPE components in vitro . (A) Chemical components of SPE. (B) The ability of seven components from SPE to enhance T-cell functional activity was assessed using a PD-1/PD-L1 blockade bioassay. These seven components were used at a concentration of 2 µM. (C, D) A PD-1/PD-L1 blockade bioassay was performed to measure the inhibitory activity of apigenin and cosmosiin. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of apigenin or cosmosiin. (E, F) Competitive ELISA was performed using a PD-1/PD-L1 inhibitor ELISA-binding assay after treatment with the indicated concentrations of (E) apigenin and (F) cosmosiin. Bar graph (mean ± standard error of the mean) statistics were determined with data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P

    Article Snippet: The equilibrium dissociation constants (KD ) of cosmosiin to PD-1 and PD-L1 were 386 and 85 μM with co efficient of determinations, R2 , of 0.9804 and 0.9866, respectively, showing high goodness of fit to 1:1 binding model and that cosmosiin has 4.5-fold higher affinity to PD-L1 than PD-1.Furthermore, the ka and kd values of cosmosiin to PD-1 and PD-L1 in indicate that the higher affinity of cosmosiin to PD-L1 results from its higher binding rate to PD-L1 rather than insignificant difference in dissociation rates.

    Techniques: Activation Assay, In Vitro, Functional Assay, Activity Assay, Concentration Assay, Incubation, Competitive ELISA, Enzyme-linked Immunosorbent Assay, Binding Assay

    Cytotoxic and T-cell-mediated killing effect of SPE on colorectal cancer cells. (A) PD-L1 expression on the surface of humanized PD-L1-expressing MC38 cells (hPDL1-MCs) was determined using flow cytometry. (B–D) To measure the immunological activity of SPE, hPDL1-MCs (target cells) were co-cultured with humanized PD-1 mouse splenocytes (B, C) and CD8+ T-cells from the tumor (D) (hPD1-MSs; effector cells) at an effector cell-to-target cell ratio of 5:1 and were treated with SPE or 50% DMSO (control). (B) After 72 h of incubation, surviving hPD1-MSs in a 96-well plate underwent the CCK assay to assess cell viability. (C) Co-cultured CellTrace™ Far Red-labeled hPDL1-MCs were treated with SPE (25 μg/mL) or 50% DMSO (control), and the findings were compared with those in the absence of effector cells. The images were obtained under a fluorescence microscope. (D) The cytotoxicity of CD8+ T-cells against hPD-L1-MCs at an effector cell-to-target cell ratio of 5:1 was analyzed using an LDH assay kit (percentages calculated according to kit instruction). ***P

    Journal: Frontiers in Immunology

    Article Title: Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model

    doi: 10.3389/fimmu.2020.598556

    Figure Lengend Snippet: Cytotoxic and T-cell-mediated killing effect of SPE on colorectal cancer cells. (A) PD-L1 expression on the surface of humanized PD-L1-expressing MC38 cells (hPDL1-MCs) was determined using flow cytometry. (B–D) To measure the immunological activity of SPE, hPDL1-MCs (target cells) were co-cultured with humanized PD-1 mouse splenocytes (B, C) and CD8+ T-cells from the tumor (D) (hPD1-MSs; effector cells) at an effector cell-to-target cell ratio of 5:1 and were treated with SPE or 50% DMSO (control). (B) After 72 h of incubation, surviving hPD1-MSs in a 96-well plate underwent the CCK assay to assess cell viability. (C) Co-cultured CellTrace™ Far Red-labeled hPDL1-MCs were treated with SPE (25 μg/mL) or 50% DMSO (control), and the findings were compared with those in the absence of effector cells. The images were obtained under a fluorescence microscope. (D) The cytotoxicity of CD8+ T-cells against hPD-L1-MCs at an effector cell-to-target cell ratio of 5:1 was analyzed using an LDH assay kit (percentages calculated according to kit instruction). ***P

    Article Snippet: The equilibrium dissociation constants (KD ) of cosmosiin to PD-1 and PD-L1 were 386 and 85 μM with co efficient of determinations, R2 , of 0.9804 and 0.9866, respectively, showing high goodness of fit to 1:1 binding model and that cosmosiin has 4.5-fold higher affinity to PD-L1 than PD-1.Furthermore, the ka and kd values of cosmosiin to PD-1 and PD-L1 in indicate that the higher affinity of cosmosiin to PD-L1 results from its higher binding rate to PD-L1 rather than insignificant difference in dissociation rates.

    Techniques: Expressing, Flow Cytometry, Activity Assay, Cell Culture, Incubation, Labeling, Fluorescence, Microscopy, Lactate Dehydrogenase Assay

    Protein–ligand docking simulation between PD-L1 (A) /PD-1 (B) and the Salvia plebeia R. Br. ethanol extract (SPE) component cosmosiin. Binding models were obtained through docking simulation of cosmosiin on PD-L1 and PD-1 derived from the PD-1/PD-L1 complex (PDB code 4ZQK) using AutoDock Vina. The hydrogen bonds and hydrophobic interactions between PD-L1/PD-1 and cosmosiin were analyzed using LigPlot+.

    Journal: Frontiers in Immunology

    Article Title: Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model

    doi: 10.3389/fimmu.2020.598556

    Figure Lengend Snippet: Protein–ligand docking simulation between PD-L1 (A) /PD-1 (B) and the Salvia plebeia R. Br. ethanol extract (SPE) component cosmosiin. Binding models were obtained through docking simulation of cosmosiin on PD-L1 and PD-1 derived from the PD-1/PD-L1 complex (PDB code 4ZQK) using AutoDock Vina. The hydrogen bonds and hydrophobic interactions between PD-L1/PD-1 and cosmosiin were analyzed using LigPlot+.

    Article Snippet: The equilibrium dissociation constants (KD ) of cosmosiin to PD-1 and PD-L1 were 386 and 85 μM with co efficient of determinations, R2 , of 0.9804 and 0.9866, respectively, showing high goodness of fit to 1:1 binding model and that cosmosiin has 4.5-fold higher affinity to PD-L1 than PD-1.Furthermore, the ka and kd values of cosmosiin to PD-1 and PD-L1 in indicate that the higher affinity of cosmosiin to PD-L1 results from its higher binding rate to PD-L1 rather than insignificant difference in dissociation rates.

    Techniques: Binding Assay, Derivative Assay

    Global kinetic analysis of cosmosiin binding to biotinylated (A) PD-1 and (B) PD-L1 immobilized on a streptavidin biolayer interferometry (BLI) sensor. The kinetics of cosmosiin for immobilized PD-1 and PD-L1 were monitored with increasing concentration of cosmosiin (0, 0.125, 0.25, and 0.375 mM), dissolved in PBS buffer (pH 7.3) containing 1% DMSO.

    Journal: Frontiers in Immunology

    Article Title: Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model

    doi: 10.3389/fimmu.2020.598556

    Figure Lengend Snippet: Global kinetic analysis of cosmosiin binding to biotinylated (A) PD-1 and (B) PD-L1 immobilized on a streptavidin biolayer interferometry (BLI) sensor. The kinetics of cosmosiin for immobilized PD-1 and PD-L1 were monitored with increasing concentration of cosmosiin (0, 0.125, 0.25, and 0.375 mM), dissolved in PBS buffer (pH 7.3) containing 1% DMSO.

    Article Snippet: The equilibrium dissociation constants (KD ) of cosmosiin to PD-1 and PD-L1 were 386 and 85 μM with co efficient of determinations, R2 , of 0.9804 and 0.9866, respectively, showing high goodness of fit to 1:1 binding model and that cosmosiin has 4.5-fold higher affinity to PD-L1 than PD-1.Furthermore, the ka and kd values of cosmosiin to PD-1 and PD-L1 in indicate that the higher affinity of cosmosiin to PD-L1 results from its higher binding rate to PD-L1 rather than insignificant difference in dissociation rates.

    Techniques: Binding Assay, Concentration Assay

    PD-1/PD-L1 interaction blockade by Salvia plebeia R. Br. ethanol extract (SPE). Competitive ELISA was performed using PD-1/PD-L1 inhibitor ELISA-screening assay with the indicated concentrations of PD-L1-blocking antibody, SPE, and SPE fractions. (A) Inhibition of the PD-1/PD-L1 interaction by PD-L1-blocking antibody, (B) SPE, and (C) SPE fractions (methylene chloride [MC], ethyl acetate [EA], and n-butanol [BuOH]). Cytotoxicity and T-cell activation by SPE and its ethyl acetate (EA) fraction in vitro . (D) The viabilities of PD-L1 aAPC/CHO-K1 and PD-1 effector cells were assessed using the Cell Counting Kit-8 (CCK) assay after treatment with the indicated concentrations of SPE for 24 h. (E–G) A PD-1/PD-L1 blockade bioassay was performed to assess the inhibitory activities of PD-L1-blocking antibody, SPE, and SPE-EA. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of (E) PD-L1-blocking antibody, (F) SPE, or (G) SPE-EA. Bar graph (mean ± standard error of the mean) statistics were determined with the data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P

    Journal: Frontiers in Immunology

    Article Title: Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model

    doi: 10.3389/fimmu.2020.598556

    Figure Lengend Snippet: PD-1/PD-L1 interaction blockade by Salvia plebeia R. Br. ethanol extract (SPE). Competitive ELISA was performed using PD-1/PD-L1 inhibitor ELISA-screening assay with the indicated concentrations of PD-L1-blocking antibody, SPE, and SPE fractions. (A) Inhibition of the PD-1/PD-L1 interaction by PD-L1-blocking antibody, (B) SPE, and (C) SPE fractions (methylene chloride [MC], ethyl acetate [EA], and n-butanol [BuOH]). Cytotoxicity and T-cell activation by SPE and its ethyl acetate (EA) fraction in vitro . (D) The viabilities of PD-L1 aAPC/CHO-K1 and PD-1 effector cells were assessed using the Cell Counting Kit-8 (CCK) assay after treatment with the indicated concentrations of SPE for 24 h. (E–G) A PD-1/PD-L1 blockade bioassay was performed to assess the inhibitory activities of PD-L1-blocking antibody, SPE, and SPE-EA. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of (E) PD-L1-blocking antibody, (F) SPE, or (G) SPE-EA. Bar graph (mean ± standard error of the mean) statistics were determined with the data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P

    Article Snippet: The equilibrium dissociation constants (KD ) of cosmosiin to PD-1 and PD-L1 were 386 and 85 μM with co efficient of determinations, R2 , of 0.9804 and 0.9866, respectively, showing high goodness of fit to 1:1 binding model and that cosmosiin has 4.5-fold higher affinity to PD-L1 than PD-1.Furthermore, the ka and kd values of cosmosiin to PD-1 and PD-L1 in indicate that the higher affinity of cosmosiin to PD-L1 results from its higher binding rate to PD-L1 rather than insignificant difference in dissociation rates.

    Techniques: Competitive ELISA, Enzyme-linked Immunosorbent Assay, Screening Assay, Blocking Assay, Inhibition, Activation Assay, In Vitro, Cell Counting, Incubation

    Anti-tumor efficacy of SPE on hPD-L1 MC38 tumor growth in hPD-1 knock-in mice. (A) Representative images show the tumor development at 16 days after treatment. Mice were inoculated s.c. with hPD-L1 MC38 cells and were administrated with aPD-1 (5 mg/kg, i.p.) or SPE (100, 300 mg/kg, i.g.) (n=3). Tumor mice received an equivalent volume of vehicle (200 µL). (B) Image of subcutaneous tumor mass on day 16 (scale bars, 1 cm) (n=3). (C) Mean tumor weight (n=3). (D) Mean tumor diameter (n=3). (E) Immunohistochemistry staining of CD3+ in humanized PD-1 mouse model of tumor. (F) FACS analysis of tumor CD8+ T-cell populations from human PD-L1 knock in MC 38 tumor-bearing model of humanized PD-1 mice. Percentages of polyclonal CD8+ T-cells of CD45+ cells in tumor. Bar graph (mean ± standard error of the mean) statistics were determined with one-way ANOVA with Tukey’s post-hoc test; ***P

    Journal: Frontiers in Immunology

    Article Title: Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model

    doi: 10.3389/fimmu.2020.598556

    Figure Lengend Snippet: Anti-tumor efficacy of SPE on hPD-L1 MC38 tumor growth in hPD-1 knock-in mice. (A) Representative images show the tumor development at 16 days after treatment. Mice were inoculated s.c. with hPD-L1 MC38 cells and were administrated with aPD-1 (5 mg/kg, i.p.) or SPE (100, 300 mg/kg, i.g.) (n=3). Tumor mice received an equivalent volume of vehicle (200 µL). (B) Image of subcutaneous tumor mass on day 16 (scale bars, 1 cm) (n=3). (C) Mean tumor weight (n=3). (D) Mean tumor diameter (n=3). (E) Immunohistochemistry staining of CD3+ in humanized PD-1 mouse model of tumor. (F) FACS analysis of tumor CD8+ T-cell populations from human PD-L1 knock in MC 38 tumor-bearing model of humanized PD-1 mice. Percentages of polyclonal CD8+ T-cells of CD45+ cells in tumor. Bar graph (mean ± standard error of the mean) statistics were determined with one-way ANOVA with Tukey’s post-hoc test; ***P

    Article Snippet: The equilibrium dissociation constants (KD ) of cosmosiin to PD-1 and PD-L1 were 386 and 85 μM with co efficient of determinations, R2 , of 0.9804 and 0.9866, respectively, showing high goodness of fit to 1:1 binding model and that cosmosiin has 4.5-fold higher affinity to PD-L1 than PD-1.Furthermore, the ka and kd values of cosmosiin to PD-1 and PD-L1 in indicate that the higher affinity of cosmosiin to PD-L1 results from its higher binding rate to PD-L1 rather than insignificant difference in dissociation rates.

    Techniques: Knock-In, Mouse Assay, Immunohistochemistry, Staining, FACS