il 21  (R&D Systems)

 
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    Name:
    Recombinant Human IL 21 Protein CF
    Description:
    The Recombinant Human IL 21 Protein from R D Systems is derived from E coli The Recombinant Human IL 21 Protein has been validated for the following applications Bioactivity
    Catalog Number:
    8879-IL-010/CF
    Price:
    229
    Category:
    Proteins and Enzymes
    Source:
    E. coli-derived Recombinant Human IL-21 Protein
    Applications:
    Bioactivity
    Purity:
    >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie« Blue Staining.
    Conjugate:
    Unconjugated
    Size:
    10 ug
    Buy from Supplier


    Structured Review

    R&D Systems il 21
    Recombinant Human IL 21 Protein CF
    The Recombinant Human IL 21 Protein from R D Systems is derived from E coli The Recombinant Human IL 21 Protein has been validated for the following applications Bioactivity
    https://www.bioz.com/result/il 21/product/R&D Systems
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    il 21 - by Bioz Stars, 2021-06
    86/100 stars

    Images

    1) Product Images from "T Follicular Helper Cells restricted by IRF8 contribute to T Cell-Mediated Inflammation"

    Article Title: T Follicular Helper Cells restricted by IRF8 contribute to T Cell-Mediated Inflammation

    Journal: Journal of autoimmunity

    doi: 10.1016/j.jaut.2018.09.001

    IRF8-deficient in T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from Irf8 wt/wt or Irf8 lck/lck mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between Irf8 wt/wt (n = 5) and Irf8 lck/lck (n = 5) CD4 + T cell cultures. Results shown are representative of three independent experiments. Data are given as means ± SEM.
    Figure Legend Snippet: IRF8-deficient in T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from Irf8 wt/wt or Irf8 lck/lck mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between Irf8 wt/wt (n = 5) and Irf8 lck/lck (n = 5) CD4 + T cell cultures. Results shown are representative of three independent experiments. Data are given as means ± SEM.

    Techniques Used: Ex Vivo, Purification, Mouse Assay, Expressing, Flow Cytometry, Cytometry

    IRF8-deficient CD4 + T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from WT or Irf8 −/− mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD-1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between WT (n = 5) and Irf8 −/− (n = 5) CD4 + T cell cultures. (D) Flow cytometry evaluation of IL-21 + CD4 + T cells in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). (E) ELISA of IL-21 levels in culture supernatant of WT and Irf8 ‒/‒ CD4 + T cells cultured under T FH condition (n = 4). (F) Quantitative real-time RT-PCR analysis of T FH -associated genes in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. Results shown are representative of three independent experiments. Data are given as means ± SEM.
    Figure Legend Snippet: IRF8-deficient CD4 + T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from WT or Irf8 −/− mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD-1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between WT (n = 5) and Irf8 −/− (n = 5) CD4 + T cell cultures. (D) Flow cytometry evaluation of IL-21 + CD4 + T cells in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). (E) ELISA of IL-21 levels in culture supernatant of WT and Irf8 ‒/‒ CD4 + T cells cultured under T FH condition (n = 4). (F) Quantitative real-time RT-PCR analysis of T FH -associated genes in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. Results shown are representative of three independent experiments. Data are given as means ± SEM.

    Techniques Used: Ex Vivo, Purification, Mouse Assay, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Quantitative RT-PCR

    Regulation of IRF4 expression by IRF8. (A) Purified CD4 + T cells from WT or Irf8 −/− mice were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Microarray analysis of the gene expression by WT and Irf8 ‒/‒ CD4 + T cells under T FH condition. (B) Quantitative real-time RT-PCR analysis of irf4 gene level in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition for time course (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. (C) IRF8 ChIP-seq signals at the IRF4 gene locus. Blue line denotes the range of the IRF4 gene locus, and the peak patterns in red represent the ChIP-seq signals of IRF8 binding. Signals represent DNA fragments that were captured by the IRF8 antibody through ChIP. (D) Purified CD4+ T cells from WT or Irf8 −/− mice were stimulated with anti-CD3 and anti-CD28 for 48h in the presence of IL-21, followed by ChIP assay. Three micrograms of an anti-IRF4 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the irf4-binding site of the IL-21 promoter region. Each bar represents mean ± S.D. from three independent experiments, unpaired Student’s t-test, *P
    Figure Legend Snippet: Regulation of IRF4 expression by IRF8. (A) Purified CD4 + T cells from WT or Irf8 −/− mice were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Microarray analysis of the gene expression by WT and Irf8 ‒/‒ CD4 + T cells under T FH condition. (B) Quantitative real-time RT-PCR analysis of irf4 gene level in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition for time course (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. (C) IRF8 ChIP-seq signals at the IRF4 gene locus. Blue line denotes the range of the IRF4 gene locus, and the peak patterns in red represent the ChIP-seq signals of IRF8 binding. Signals represent DNA fragments that were captured by the IRF8 antibody through ChIP. (D) Purified CD4+ T cells from WT or Irf8 −/− mice were stimulated with anti-CD3 and anti-CD28 for 48h in the presence of IL-21, followed by ChIP assay. Three micrograms of an anti-IRF4 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the irf4-binding site of the IL-21 promoter region. Each bar represents mean ± S.D. from three independent experiments, unpaired Student’s t-test, *P

    Techniques Used: Expressing, Purification, Mouse Assay, Microarray, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction

    2) Product Images from "Exploratory analysis of circulating cytokines in patients with metastatic breast cancer treated with eribulin: the TRANSERI-GONO (Gruppo Oncologico del Nord Ovest) study"

    Article Title: Exploratory analysis of circulating cytokines in patients with metastatic breast cancer treated with eribulin: the TRANSERI-GONO (Gruppo Oncologico del Nord Ovest) study

    Journal: ESMO Open

    doi: 10.1136/esmoopen-2020-000876

    Cox regression model for TGF-β, IL-8 and IL-21 at T 1 divided in two groups. Group 1: patients with cytokine levels above the median. Group 2: patients with cytokine levels below or equal to the median. IL, interleukin; OS, overall survival; PFS, progression free survival; TGF-β, transforming growth factor β.
    Figure Legend Snippet: Cox regression model for TGF-β, IL-8 and IL-21 at T 1 divided in two groups. Group 1: patients with cytokine levels above the median. Group 2: patients with cytokine levels below or equal to the median. IL, interleukin; OS, overall survival; PFS, progression free survival; TGF-β, transforming growth factor β.

    Techniques Used:

    Cox regression model for TGF-β, IL-21 and IL-8 comparing T 0 with T 1 . Group 1: patients with T 1 > T 0 . Group 2: patients with T 1 ≤ T 0 . IL, interleukin; OS, overall survival. PFS, progression free survival;TGF, transforming growth factor.
    Figure Legend Snippet: Cox regression model for TGF-β, IL-21 and IL-8 comparing T 0 with T 1 . Group 1: patients with T 1 > T 0 . Group 2: patients with T 1 ≤ T 0 . IL, interleukin; OS, overall survival. PFS, progression free survival;TGF, transforming growth factor.

    Techniques Used:

    3) Product Images from "T Follicular Helper Cells restricted by IRF8 contribute to T Cell-Mediated Inflammation"

    Article Title: T Follicular Helper Cells restricted by IRF8 contribute to T Cell-Mediated Inflammation

    Journal: Journal of autoimmunity

    doi: 10.1016/j.jaut.2018.09.001

    IRF8-deficient in T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from Irf8 wt/wt or Irf8 lck/lck mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between Irf8 wt/wt (n = 5) and Irf8 lck/lck (n = 5) CD4 + T cell cultures. Results shown are representative of three independent experiments. Data are given as means ± SEM.
    Figure Legend Snippet: IRF8-deficient in T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from Irf8 wt/wt or Irf8 lck/lck mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between Irf8 wt/wt (n = 5) and Irf8 lck/lck (n = 5) CD4 + T cell cultures. Results shown are representative of three independent experiments. Data are given as means ± SEM.

    Techniques Used: Ex Vivo, Purification, Mouse Assay, Expressing, Flow Cytometry, Cytometry

    IRF8-deficient CD4 + T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from WT or Irf8 −/− mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD-1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between WT (n = 5) and Irf8 −/− (n = 5) CD4 + T cell cultures. (D) Flow cytometry evaluation of IL-21 + CD4 + T cells in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). (E) ELISA of IL-21 levels in culture supernatant of WT and Irf8 ‒/‒ CD4 + T cells cultured under T FH condition (n = 4). (F) Quantitative real-time RT-PCR analysis of T FH -associated genes in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. Results shown are representative of three independent experiments. Data are given as means ± SEM.
    Figure Legend Snippet: IRF8-deficient CD4 + T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from WT or Irf8 −/− mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD-1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between WT (n = 5) and Irf8 −/− (n = 5) CD4 + T cell cultures. (D) Flow cytometry evaluation of IL-21 + CD4 + T cells in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). (E) ELISA of IL-21 levels in culture supernatant of WT and Irf8 ‒/‒ CD4 + T cells cultured under T FH condition (n = 4). (F) Quantitative real-time RT-PCR analysis of T FH -associated genes in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. Results shown are representative of three independent experiments. Data are given as means ± SEM.

    Techniques Used: Ex Vivo, Purification, Mouse Assay, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Quantitative RT-PCR

    Regulation of IRF4 expression by IRF8. (A) Purified CD4 + T cells from WT or Irf8 −/− mice were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Microarray analysis of the gene expression by WT and Irf8 ‒/‒ CD4 + T cells under T FH condition. (B) Quantitative real-time RT-PCR analysis of irf4 gene level in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition for time course (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. (C) IRF8 ChIP-seq signals at the IRF4 gene locus. Blue line denotes the range of the IRF4 gene locus, and the peak patterns in red represent the ChIP-seq signals of IRF8 binding. Signals represent DNA fragments that were captured by the IRF8 antibody through ChIP. (D) Purified CD4+ T cells from WT or Irf8 −/− mice were stimulated with anti-CD3 and anti-CD28 for 48h in the presence of IL-21, followed by ChIP assay. Three micrograms of an anti-IRF4 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the irf4-binding site of the IL-21 promoter region. Each bar represents mean ± S.D. from three independent experiments, unpaired Student’s t-test, *P
    Figure Legend Snippet: Regulation of IRF4 expression by IRF8. (A) Purified CD4 + T cells from WT or Irf8 −/− mice were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Microarray analysis of the gene expression by WT and Irf8 ‒/‒ CD4 + T cells under T FH condition. (B) Quantitative real-time RT-PCR analysis of irf4 gene level in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition for time course (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. (C) IRF8 ChIP-seq signals at the IRF4 gene locus. Blue line denotes the range of the IRF4 gene locus, and the peak patterns in red represent the ChIP-seq signals of IRF8 binding. Signals represent DNA fragments that were captured by the IRF8 antibody through ChIP. (D) Purified CD4+ T cells from WT or Irf8 −/− mice were stimulated with anti-CD3 and anti-CD28 for 48h in the presence of IL-21, followed by ChIP assay. Three micrograms of an anti-IRF4 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the irf4-binding site of the IL-21 promoter region. Each bar represents mean ± S.D. from three independent experiments, unpaired Student’s t-test, *P

    Techniques Used: Expressing, Purification, Mouse Assay, Microarray, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction

    4) Product Images from "Interleukin-21 differentially affects human natural killer cell subsets"

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets

    Journal:

    doi: 10.1111/j.1365-2567.2007.02675.x

    FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5
    Figure Legend Snippet: FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5

    Techniques Used: FACS

    IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)
    Figure Legend Snippet: IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)

    Techniques Used:

    Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.
    Figure Legend Snippet: Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.

    Techniques Used: Activation Assay

    Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open
    Figure Legend Snippet: Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open

    Techniques Used: Activation Assay, Flow Cytometry

    5) Product Images from "Dysregulated TFR and TFH cells correlate with B‐cell differentiation and antibody production in autoimmune hepatitis. Dysregulated TFR and TFH cells correlate with B‐cell differentiation and antibody production in autoimmune hepatitis"

    Article Title: Dysregulated TFR and TFH cells correlate with B‐cell differentiation and antibody production in autoimmune hepatitis. Dysregulated TFR and TFH cells correlate with B‐cell differentiation and antibody production in autoimmune hepatitis

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14997

    Correlation among the numbers of different subsets of TFR and TFH cells and the levels of serum IL‐21, IL‐10 and TGF‐β1 in AIH patients. A, Correlation between the numbers of TFR cells and the numbers of TFH cells in AIH patients; B, correlation between the numbers of TFH cells and serum levels of IL‐21 in AIH patients; C, correlation between the numbers of TFR cells and serum levels of IL‐21 in AIH patients; D, correlation between the numbers of TFR cells and serum levels of IL‐10 in AIH patients; E, correlation between the numbers of TFR cells and serum levels of TGF‐β1 in AIH patients
    Figure Legend Snippet: Correlation among the numbers of different subsets of TFR and TFH cells and the levels of serum IL‐21, IL‐10 and TGF‐β1 in AIH patients. A, Correlation between the numbers of TFR cells and the numbers of TFH cells in AIH patients; B, correlation between the numbers of TFH cells and serum levels of IL‐21 in AIH patients; C, correlation between the numbers of TFR cells and serum levels of IL‐21 in AIH patients; D, correlation between the numbers of TFR cells and serum levels of IL‐10 in AIH patients; E, correlation between the numbers of TFR cells and serum levels of TGF‐β1 in AIH patients

    Techniques Used:

    Correlation between serum levels of TFR/TFH‐type cytokines and the values of clinical parameters in AIH patients. A‐B, Correlation between serum levels of IL‐21 and titre IgG/IgM in AIH patients; C‐D, correlation between serum levels of IL‐10 and titre IgG/IgM in AIH patients
    Figure Legend Snippet: Correlation between serum levels of TFR/TFH‐type cytokines and the values of clinical parameters in AIH patients. A‐B, Correlation between serum levels of IL‐21 and titre IgG/IgM in AIH patients; C‐D, correlation between serum levels of IL‐10 and titre IgG/IgM in AIH patients

    Techniques Used:

    Serum levels of TFR/TFH‐type cytokines in AIH patients and HCs. Serum levels of A, IL‐21; B, IL‐10; C, TGF‐β1in AIH patients and HCs. Data shown are the mean levels of each serum cytokine in individual subjects from two separate experiments. The horizontal lines indicate the mean values for the different groups
    Figure Legend Snippet: Serum levels of TFR/TFH‐type cytokines in AIH patients and HCs. Serum levels of A, IL‐21; B, IL‐10; C, TGF‐β1in AIH patients and HCs. Data shown are the mean levels of each serum cytokine in individual subjects from two separate experiments. The horizontal lines indicate the mean values for the different groups

    Techniques Used:

    Flow cytometry analysis of the numbers of circulating TFR and TFH cells in AIH patients and HCs. PBMCs 5*10 5 /tube were isolated from individual subjects and were stained in duplicate with anti‐CD4, anti‐CXCR5, anti‐ICOS, anti‐PD‐1, anti‐CTLA‐4, anti‐CD25 and intracellular anti‐Foxp3, anti‐IL‐21 or IL‐10, respectively. The cells were characterized by flow cytometry analysis by gating initially on living lymphocytes, and then on CD4 + CXCR5 + Foxp3 + TFR and CD4 + CXCR5 + Foxp3 ‐ TFH cells. Subsequently, the numbers of different subsets of TFR and TFH cells were calculated, according to the total numbers of PBMCs, the frequency of TFR and TFH cells. A, Flow cytometry analysis of TFR and TFH cells; B, the numbers of CD4 + CXCR5 + Foxp3 + TFR, CD4 + CXCR5 + Foxp3 ‐ TFH cells; and TFR/TFH ratio; C, flow cytometry analysis of different subsets of TFR and TFH cell; D, the numbers of ICOS + , PD‐1 + , CTLA‐4 + , CD25 + and IL‐10 + TFR and TFH cells. Data shown are representative FACS charts or the mean numbers of each type of cells per mL of peripheral blood in individual subjects from two separate experiments. The horizontal lines indicate the median values for each group
    Figure Legend Snippet: Flow cytometry analysis of the numbers of circulating TFR and TFH cells in AIH patients and HCs. PBMCs 5*10 5 /tube were isolated from individual subjects and were stained in duplicate with anti‐CD4, anti‐CXCR5, anti‐ICOS, anti‐PD‐1, anti‐CTLA‐4, anti‐CD25 and intracellular anti‐Foxp3, anti‐IL‐21 or IL‐10, respectively. The cells were characterized by flow cytometry analysis by gating initially on living lymphocytes, and then on CD4 + CXCR5 + Foxp3 + TFR and CD4 + CXCR5 + Foxp3 ‐ TFH cells. Subsequently, the numbers of different subsets of TFR and TFH cells were calculated, according to the total numbers of PBMCs, the frequency of TFR and TFH cells. A, Flow cytometry analysis of TFR and TFH cells; B, the numbers of CD4 + CXCR5 + Foxp3 + TFR, CD4 + CXCR5 + Foxp3 ‐ TFH cells; and TFR/TFH ratio; C, flow cytometry analysis of different subsets of TFR and TFH cell; D, the numbers of ICOS + , PD‐1 + , CTLA‐4 + , CD25 + and IL‐10 + TFR and TFH cells. Data shown are representative FACS charts or the mean numbers of each type of cells per mL of peripheral blood in individual subjects from two separate experiments. The horizontal lines indicate the median values for each group

    Techniques Used: Flow Cytometry, Isolation, Staining, FACS

    6) Product Images from "Interleukin-21 differentially affects human natural killer cell subsets"

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets

    Journal:

    doi: 10.1111/j.1365-2567.2007.02675.x

    FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5
    Figure Legend Snippet: FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5

    Techniques Used: FACS

    IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)
    Figure Legend Snippet: IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)

    Techniques Used:

    Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.
    Figure Legend Snippet: Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.

    Techniques Used: Activation Assay

    Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open
    Figure Legend Snippet: Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open

    Techniques Used: Activation Assay, Flow Cytometry

    7) Product Images from "Interleukin-21 differentially affects human natural killer cell subsets"

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets

    Journal:

    doi: 10.1111/j.1365-2567.2007.02675.x

    FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5
    Figure Legend Snippet: FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5

    Techniques Used: FACS

    IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)
    Figure Legend Snippet: IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)

    Techniques Used:

    Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.
    Figure Legend Snippet: Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.

    Techniques Used: Activation Assay

    Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open
    Figure Legend Snippet: Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open

    Techniques Used: Activation Assay, Flow Cytometry

    8) Product Images from "HLA-DR-Positive NK Cells Expand in Response to Mycobacterium Tuberculosis Antigens and Mediate Mycobacteria-Induced T Cell Activation"

    Article Title: HLA-DR-Positive NK Cells Expand in Response to Mycobacterium Tuberculosis Antigens and Mediate Mycobacteria-Induced T Cell Activation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.662128

    | Production of IFNγ by HLA-DR + and HLA-DR – NK cells after 6 days incubation with IL-2+IL-21 and re-stimulation with IL-12+IL-15 and 2 μg/ml sonicate. (A) Representative staining and (B) summarized data of four independent experiments are shown: median, 10-90% percentile and scatter. Statistical difference was evaluated by paired t-test, *p
    Figure Legend Snippet: | Production of IFNγ by HLA-DR + and HLA-DR – NK cells after 6 days incubation with IL-2+IL-21 and re-stimulation with IL-12+IL-15 and 2 μg/ml sonicate. (A) Representative staining and (B) summarized data of four independent experiments are shown: median, 10-90% percentile and scatter. Statistical difference was evaluated by paired t-test, *p

    Techniques Used: Incubation, Staining

    | Degranulation activity of CD56 + CD16 low HLA-DR + , CD56 + CD16 low HLA-DR – , CD56 + CD16 hi HLA-DR + and CD56 + CD16 hi HLA-DR − NK cells after 6 days incubation with IL-2+IL-21 and re-stimulation with IL-2 and 2 μg/ml sonicate. (A) Summarized data of three independent experiments (Mean ± SEM) and (B) representative staining are shown. Statistical difference was evaluated by two-way ANOVA., *p
    Figure Legend Snippet: | Degranulation activity of CD56 + CD16 low HLA-DR + , CD56 + CD16 low HLA-DR – , CD56 + CD16 hi HLA-DR + and CD56 + CD16 hi HLA-DR − NK cells after 6 days incubation with IL-2+IL-21 and re-stimulation with IL-2 and 2 μg/ml sonicate. (A) Summarized data of three independent experiments (Mean ± SEM) and (B) representative staining are shown. Statistical difference was evaluated by two-way ANOVA., *p

    Techniques Used: Activity Assay, Incubation, Staining

    9) Product Images from "Interleukin-21 differentially affects human natural killer cell subsets"

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets

    Journal:

    doi: 10.1111/j.1365-2567.2007.02675.x

    FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5
    Figure Legend Snippet: FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5

    Techniques Used: FACS

    IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)
    Figure Legend Snippet: IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)

    Techniques Used:

    Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.
    Figure Legend Snippet: Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.

    Techniques Used: Activation Assay

    Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open
    Figure Legend Snippet: Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open

    Techniques Used: Activation Assay, Flow Cytometry

    10) Product Images from "Interleukin-21 differentially affects human natural killer cell subsets"

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets

    Journal:

    doi: 10.1111/j.1365-2567.2007.02675.x

    FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5
    Figure Legend Snippet: FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5

    Techniques Used: FACS

    IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)
    Figure Legend Snippet: IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)

    Techniques Used:

    Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.
    Figure Legend Snippet: Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.

    Techniques Used: Activation Assay

    Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open
    Figure Legend Snippet: Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open

    Techniques Used: Activation Assay, Flow Cytometry

    11) Product Images from "Interleukin-21 differentially affects human natural killer cell subsets"

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets

    Journal:

    doi: 10.1111/j.1365-2567.2007.02675.x

    FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5
    Figure Legend Snippet: FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5

    Techniques Used: FACS

    IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)
    Figure Legend Snippet: IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)

    Techniques Used:

    Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.
    Figure Legend Snippet: Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.

    Techniques Used: Activation Assay

    Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open
    Figure Legend Snippet: Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open

    Techniques Used: Activation Assay, Flow Cytometry

    12) Product Images from "Exploratory analysis of circulating cytokines in patients with metastatic breast cancer treated with eribulin: the TRANSERI-GONO (Gruppo Oncologico del Nord Ovest) study"

    Article Title: Exploratory analysis of circulating cytokines in patients with metastatic breast cancer treated with eribulin: the TRANSERI-GONO (Gruppo Oncologico del Nord Ovest) study

    Journal: ESMO Open

    doi: 10.1136/esmoopen-2020-000876

    Cox regression model for TGF-β, IL-8 and IL-21 at T 1 divided in two groups. Group 1: patients with cytokine levels above the median. Group 2: patients with cytokine levels below or equal to the median. IL, interleukin; OS, overall survival; PFS, progression free survival; TGF-β, transforming growth factor β.
    Figure Legend Snippet: Cox regression model for TGF-β, IL-8 and IL-21 at T 1 divided in two groups. Group 1: patients with cytokine levels above the median. Group 2: patients with cytokine levels below or equal to the median. IL, interleukin; OS, overall survival; PFS, progression free survival; TGF-β, transforming growth factor β.

    Techniques Used:

    Cox regression model for TGF-β, IL-21 and IL-8 comparing T 0 with T 1 . Group 1: patients with T 1 > T 0 . Group 2: patients with T 1 ≤ T 0 . IL, interleukin; OS, overall survival. PFS, progression free survival;TGF, transforming growth factor.
    Figure Legend Snippet: Cox regression model for TGF-β, IL-21 and IL-8 comparing T 0 with T 1 . Group 1: patients with T 1 > T 0 . Group 2: patients with T 1 ≤ T 0 . IL, interleukin; OS, overall survival. PFS, progression free survival;TGF, transforming growth factor.

    Techniques Used:

    13) Product Images from "T Follicular Helper Cells restricted by IRF8 contribute to T Cell-Mediated Inflammation"

    Article Title: T Follicular Helper Cells restricted by IRF8 contribute to T Cell-Mediated Inflammation

    Journal: Journal of autoimmunity

    doi: 10.1016/j.jaut.2018.09.001

    IRF8-deficient in T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from Irf8 wt/wt or Irf8 lck/lck mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between Irf8 wt/wt (n = 5) and Irf8 lck/lck (n = 5) CD4 + T cell cultures. Results shown are representative of three independent experiments. Data are given as means ± SEM.
    Figure Legend Snippet: IRF8-deficient in T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from Irf8 wt/wt or Irf8 lck/lck mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between Irf8 wt/wt (n = 5) and Irf8 lck/lck (n = 5) CD4 + T cell cultures. Results shown are representative of three independent experiments. Data are given as means ± SEM.

    Techniques Used: Ex Vivo, Purification, Mouse Assay, Expressing, Flow Cytometry, Cytometry

    IRF8-deficient CD4 + T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from WT or Irf8 −/− mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD-1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between WT (n = 5) and Irf8 −/− (n = 5) CD4 + T cell cultures. (D) Flow cytometry evaluation of IL-21 + CD4 + T cells in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). (E) ELISA of IL-21 levels in culture supernatant of WT and Irf8 ‒/‒ CD4 + T cells cultured under T FH condition (n = 4). (F) Quantitative real-time RT-PCR analysis of T FH -associated genes in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. Results shown are representative of three independent experiments. Data are given as means ± SEM.
    Figure Legend Snippet: IRF8-deficient CD4 + T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from WT or Irf8 −/− mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD-1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between WT (n = 5) and Irf8 −/− (n = 5) CD4 + T cell cultures. (D) Flow cytometry evaluation of IL-21 + CD4 + T cells in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). (E) ELISA of IL-21 levels in culture supernatant of WT and Irf8 ‒/‒ CD4 + T cells cultured under T FH condition (n = 4). (F) Quantitative real-time RT-PCR analysis of T FH -associated genes in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. Results shown are representative of three independent experiments. Data are given as means ± SEM.

    Techniques Used: Ex Vivo, Purification, Mouse Assay, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Quantitative RT-PCR

    Regulation of IRF4 expression by IRF8. (A) Purified CD4 + T cells from WT or Irf8 −/− mice were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Microarray analysis of the gene expression by WT and Irf8 ‒/‒ CD4 + T cells under T FH condition. (B) Quantitative real-time RT-PCR analysis of irf4 gene level in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition for time course (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. (C) IRF8 ChIP-seq signals at the IRF4 gene locus. Blue line denotes the range of the IRF4 gene locus, and the peak patterns in red represent the ChIP-seq signals of IRF8 binding. Signals represent DNA fragments that were captured by the IRF8 antibody through ChIP. (D) Purified CD4+ T cells from WT or Irf8 −/− mice were stimulated with anti-CD3 and anti-CD28 for 48h in the presence of IL-21, followed by ChIP assay. Three micrograms of an anti-IRF4 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the irf4-binding site of the IL-21 promoter region. Each bar represents mean ± S.D. from three independent experiments, unpaired Student’s t-test, *P
    Figure Legend Snippet: Regulation of IRF4 expression by IRF8. (A) Purified CD4 + T cells from WT or Irf8 −/− mice were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Microarray analysis of the gene expression by WT and Irf8 ‒/‒ CD4 + T cells under T FH condition. (B) Quantitative real-time RT-PCR analysis of irf4 gene level in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition for time course (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. (C) IRF8 ChIP-seq signals at the IRF4 gene locus. Blue line denotes the range of the IRF4 gene locus, and the peak patterns in red represent the ChIP-seq signals of IRF8 binding. Signals represent DNA fragments that were captured by the IRF8 antibody through ChIP. (D) Purified CD4+ T cells from WT or Irf8 −/− mice were stimulated with anti-CD3 and anti-CD28 for 48h in the presence of IL-21, followed by ChIP assay. Three micrograms of an anti-IRF4 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the irf4-binding site of the IL-21 promoter region. Each bar represents mean ± S.D. from three independent experiments, unpaired Student’s t-test, *P

    Techniques Used: Expressing, Purification, Mouse Assay, Microarray, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction

    14) Product Images from "T Follicular Helper Cells restricted by IRF8 contribute to T Cell-Mediated Inflammation"

    Article Title: T Follicular Helper Cells restricted by IRF8 contribute to T Cell-Mediated Inflammation

    Journal: Journal of autoimmunity

    doi: 10.1016/j.jaut.2018.09.001

    IRF8-deficient in T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from Irf8 wt/wt or Irf8 lck/lck mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between Irf8 wt/wt (n = 5) and Irf8 lck/lck (n = 5) CD4 + T cell cultures. Results shown are representative of three independent experiments. Data are given as means ± SEM.
    Figure Legend Snippet: IRF8-deficient in T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from Irf8 wt/wt or Irf8 lck/lck mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between Irf8 wt/wt (n = 5) and Irf8 lck/lck (n = 5) CD4 + T cell cultures. Results shown are representative of three independent experiments. Data are given as means ± SEM.

    Techniques Used: Ex Vivo, Purification, Mouse Assay, Expressing, Flow Cytometry, Cytometry

    IRF8-deficient CD4 + T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from WT or Irf8 −/− mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD-1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between WT (n = 5) and Irf8 −/− (n = 5) CD4 + T cell cultures. (D) Flow cytometry evaluation of IL-21 + CD4 + T cells in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). (E) ELISA of IL-21 levels in culture supernatant of WT and Irf8 ‒/‒ CD4 + T cells cultured under T FH condition (n = 4). (F) Quantitative real-time RT-PCR analysis of T FH -associated genes in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. Results shown are representative of three independent experiments. Data are given as means ± SEM.
    Figure Legend Snippet: IRF8-deficient CD4 + T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from WT or Irf8 −/− mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD-1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between WT (n = 5) and Irf8 −/− (n = 5) CD4 + T cell cultures. (D) Flow cytometry evaluation of IL-21 + CD4 + T cells in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). (E) ELISA of IL-21 levels in culture supernatant of WT and Irf8 ‒/‒ CD4 + T cells cultured under T FH condition (n = 4). (F) Quantitative real-time RT-PCR analysis of T FH -associated genes in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. Results shown are representative of three independent experiments. Data are given as means ± SEM.

    Techniques Used: Ex Vivo, Purification, Mouse Assay, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Quantitative RT-PCR

    Regulation of IRF4 expression by IRF8. (A) Purified CD4 + T cells from WT or Irf8 −/− mice were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Microarray analysis of the gene expression by WT and Irf8 ‒/‒ CD4 + T cells under T FH condition. (B) Quantitative real-time RT-PCR analysis of irf4 gene level in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition for time course (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. (C) IRF8 ChIP-seq signals at the IRF4 gene locus. Blue line denotes the range of the IRF4 gene locus, and the peak patterns in red represent the ChIP-seq signals of IRF8 binding. Signals represent DNA fragments that were captured by the IRF8 antibody through ChIP. (D) Purified CD4+ T cells from WT or Irf8 −/− mice were stimulated with anti-CD3 and anti-CD28 for 48h in the presence of IL-21, followed by ChIP assay. Three micrograms of an anti-IRF4 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the irf4-binding site of the IL-21 promoter region. Each bar represents mean ± S.D. from three independent experiments, unpaired Student’s t-test, *P
    Figure Legend Snippet: Regulation of IRF4 expression by IRF8. (A) Purified CD4 + T cells from WT or Irf8 −/− mice were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Microarray analysis of the gene expression by WT and Irf8 ‒/‒ CD4 + T cells under T FH condition. (B) Quantitative real-time RT-PCR analysis of irf4 gene level in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition for time course (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. (C) IRF8 ChIP-seq signals at the IRF4 gene locus. Blue line denotes the range of the IRF4 gene locus, and the peak patterns in red represent the ChIP-seq signals of IRF8 binding. Signals represent DNA fragments that were captured by the IRF8 antibody through ChIP. (D) Purified CD4+ T cells from WT or Irf8 −/− mice were stimulated with anti-CD3 and anti-CD28 for 48h in the presence of IL-21, followed by ChIP assay. Three micrograms of an anti-IRF4 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the irf4-binding site of the IL-21 promoter region. Each bar represents mean ± S.D. from three independent experiments, unpaired Student’s t-test, *P

    Techniques Used: Expressing, Purification, Mouse Assay, Microarray, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction

    15) Product Images from "Ablation of RhoA impairs Th17 cell differentiation and alleviates house dust mite-triggered allergic airway inflammation"

    Article Title: Ablation of RhoA impairs Th17 cell differentiation and alleviates house dust mite-triggered allergic airway inflammation

    Journal: Journal of leukocyte biology

    doi: 10.1002/JLB.3A0119-025RRR

    RhoA specific inhibitor Y16 blocks Th17 differentiation. ( A ) Purified naïve CD4 + T cells (1 × 10 6 /mL) pooled from 8 WT mice were stimulated with plate-bound anti-CD3 plus free anti-CD28 for 2 days without or with Y16 (0~50 μM). IL-17 and IL-21 in the culture supernatants were determined by ELISA. ( B - D ) Naïve CD4 + T cells were differentiated under Th17 conditions for 4 days and restimulated with PMA plus ionomycin for 5 h, in the presence of vehicle (Veh) or Y16 (30 μM) throughout the culture. Cells were collected for IL-17/IFN-γ intracellular staining. Percentages of IL-17 + or IFN-γ + CD4 + T cells are shown in representative dot plots ( B ) and summarized in a bar graph ( C ). IL-17 and IL-21 in the culture supernatants were determined by ELISA ( D ). Data are representative of 2 independent experiments. ** P
    Figure Legend Snippet: RhoA specific inhibitor Y16 blocks Th17 differentiation. ( A ) Purified naïve CD4 + T cells (1 × 10 6 /mL) pooled from 8 WT mice were stimulated with plate-bound anti-CD3 plus free anti-CD28 for 2 days without or with Y16 (0~50 μM). IL-17 and IL-21 in the culture supernatants were determined by ELISA. ( B - D ) Naïve CD4 + T cells were differentiated under Th17 conditions for 4 days and restimulated with PMA plus ionomycin for 5 h, in the presence of vehicle (Veh) or Y16 (30 μM) throughout the culture. Cells were collected for IL-17/IFN-γ intracellular staining. Percentages of IL-17 + or IFN-γ + CD4 + T cells are shown in representative dot plots ( B ) and summarized in a bar graph ( C ). IL-17 and IL-21 in the culture supernatants were determined by ELISA ( D ). Data are representative of 2 independent experiments. ** P

    Techniques Used: Purification, Mouse Assay, Enzyme-linked Immunosorbent Assay, Staining

    16) Product Images from "T Follicular Helper Cells restricted by IRF8 contribute to T Cell-Mediated Inflammation"

    Article Title: T Follicular Helper Cells restricted by IRF8 contribute to T Cell-Mediated Inflammation

    Journal: Journal of autoimmunity

    doi: 10.1016/j.jaut.2018.09.001

    IRF8-deficient in T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from Irf8 wt/wt or Irf8 lck/lck mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between Irf8 wt/wt (n = 5) and Irf8 lck/lck (n = 5) CD4 + T cell cultures. Results shown are representative of three independent experiments. Data are given as means ± SEM.
    Figure Legend Snippet: IRF8-deficient in T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from Irf8 wt/wt or Irf8 lck/lck mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between Irf8 wt/wt (n = 5) and Irf8 lck/lck (n = 5) CD4 + T cell cultures. Results shown are representative of three independent experiments. Data are given as means ± SEM.

    Techniques Used: Ex Vivo, Purification, Mouse Assay, Expressing, Flow Cytometry, Cytometry

    IRF8-deficient CD4 + T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from WT or Irf8 −/− mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD-1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between WT (n = 5) and Irf8 −/− (n = 5) CD4 + T cell cultures. (D) Flow cytometry evaluation of IL-21 + CD4 + T cells in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). (E) ELISA of IL-21 levels in culture supernatant of WT and Irf8 ‒/‒ CD4 + T cells cultured under T FH condition (n = 4). (F) Quantitative real-time RT-PCR analysis of T FH -associated genes in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. Results shown are representative of three independent experiments. Data are given as means ± SEM.
    Figure Legend Snippet: IRF8-deficient CD4 + T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from WT or Irf8 −/− mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD-1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between WT (n = 5) and Irf8 −/− (n = 5) CD4 + T cell cultures. (D) Flow cytometry evaluation of IL-21 + CD4 + T cells in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). (E) ELISA of IL-21 levels in culture supernatant of WT and Irf8 ‒/‒ CD4 + T cells cultured under T FH condition (n = 4). (F) Quantitative real-time RT-PCR analysis of T FH -associated genes in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. Results shown are representative of three independent experiments. Data are given as means ± SEM.

    Techniques Used: Ex Vivo, Purification, Mouse Assay, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Quantitative RT-PCR

    Regulation of IRF4 expression by IRF8. (A) Purified CD4 + T cells from WT or Irf8 −/− mice were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Microarray analysis of the gene expression by WT and Irf8 ‒/‒ CD4 + T cells under T FH condition. (B) Quantitative real-time RT-PCR analysis of irf4 gene level in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition for time course (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. (C) IRF8 ChIP-seq signals at the IRF4 gene locus. Blue line denotes the range of the IRF4 gene locus, and the peak patterns in red represent the ChIP-seq signals of IRF8 binding. Signals represent DNA fragments that were captured by the IRF8 antibody through ChIP. (D) Purified CD4+ T cells from WT or Irf8 −/− mice were stimulated with anti-CD3 and anti-CD28 for 48h in the presence of IL-21, followed by ChIP assay. Three micrograms of an anti-IRF4 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the irf4-binding site of the IL-21 promoter region. Each bar represents mean ± S.D. from three independent experiments, unpaired Student’s t-test, *P
    Figure Legend Snippet: Regulation of IRF4 expression by IRF8. (A) Purified CD4 + T cells from WT or Irf8 −/− mice were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Microarray analysis of the gene expression by WT and Irf8 ‒/‒ CD4 + T cells under T FH condition. (B) Quantitative real-time RT-PCR analysis of irf4 gene level in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition for time course (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. (C) IRF8 ChIP-seq signals at the IRF4 gene locus. Blue line denotes the range of the IRF4 gene locus, and the peak patterns in red represent the ChIP-seq signals of IRF8 binding. Signals represent DNA fragments that were captured by the IRF8 antibody through ChIP. (D) Purified CD4+ T cells from WT or Irf8 −/− mice were stimulated with anti-CD3 and anti-CD28 for 48h in the presence of IL-21, followed by ChIP assay. Three micrograms of an anti-IRF4 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the irf4-binding site of the IL-21 promoter region. Each bar represents mean ± S.D. from three independent experiments, unpaired Student’s t-test, *P

    Techniques Used: Expressing, Purification, Mouse Assay, Microarray, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction

    17) Product Images from "T Follicular Helper Cells restricted by IRF8 contribute to T Cell-Mediated Inflammation"

    Article Title: T Follicular Helper Cells restricted by IRF8 contribute to T Cell-Mediated Inflammation

    Journal: Journal of autoimmunity

    doi: 10.1016/j.jaut.2018.09.001

    IRF8-deficient in T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from Irf8 wt/wt or Irf8 lck/lck mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between Irf8 wt/wt (n = 5) and Irf8 lck/lck (n = 5) CD4 + T cell cultures. Results shown are representative of three independent experiments. Data are given as means ± SEM.
    Figure Legend Snippet: IRF8-deficient in T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from Irf8 wt/wt or Irf8 lck/lck mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between Irf8 wt/wt (n = 5) and Irf8 lck/lck (n = 5) CD4 + T cell cultures. Results shown are representative of three independent experiments. Data are given as means ± SEM.

    Techniques Used: Ex Vivo, Purification, Mouse Assay, Expressing, Flow Cytometry, Cytometry

    IRF8-deficient CD4 + T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from WT or Irf8 −/− mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD-1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between WT (n = 5) and Irf8 −/− (n = 5) CD4 + T cell cultures. (D) Flow cytometry evaluation of IL-21 + CD4 + T cells in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). (E) ELISA of IL-21 levels in culture supernatant of WT and Irf8 ‒/‒ CD4 + T cells cultured under T FH condition (n = 4). (F) Quantitative real-time RT-PCR analysis of T FH -associated genes in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. Results shown are representative of three independent experiments. Data are given as means ± SEM.
    Figure Legend Snippet: IRF8-deficient CD4 + T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from WT or Irf8 −/− mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD-1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between WT (n = 5) and Irf8 −/− (n = 5) CD4 + T cell cultures. (D) Flow cytometry evaluation of IL-21 + CD4 + T cells in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). (E) ELISA of IL-21 levels in culture supernatant of WT and Irf8 ‒/‒ CD4 + T cells cultured under T FH condition (n = 4). (F) Quantitative real-time RT-PCR analysis of T FH -associated genes in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. Results shown are representative of three independent experiments. Data are given as means ± SEM.

    Techniques Used: Ex Vivo, Purification, Mouse Assay, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Quantitative RT-PCR

    Regulation of IRF4 expression by IRF8. (A) Purified CD4 + T cells from WT or Irf8 −/− mice were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Microarray analysis of the gene expression by WT and Irf8 ‒/‒ CD4 + T cells under T FH condition. (B) Quantitative real-time RT-PCR analysis of irf4 gene level in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition for time course (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. (C) IRF8 ChIP-seq signals at the IRF4 gene locus. Blue line denotes the range of the IRF4 gene locus, and the peak patterns in red represent the ChIP-seq signals of IRF8 binding. Signals represent DNA fragments that were captured by the IRF8 antibody through ChIP. (D) Purified CD4+ T cells from WT or Irf8 −/− mice were stimulated with anti-CD3 and anti-CD28 for 48h in the presence of IL-21, followed by ChIP assay. Three micrograms of an anti-IRF4 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the irf4-binding site of the IL-21 promoter region. Each bar represents mean ± S.D. from three independent experiments, unpaired Student’s t-test, *P
    Figure Legend Snippet: Regulation of IRF4 expression by IRF8. (A) Purified CD4 + T cells from WT or Irf8 −/− mice were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Microarray analysis of the gene expression by WT and Irf8 ‒/‒ CD4 + T cells under T FH condition. (B) Quantitative real-time RT-PCR analysis of irf4 gene level in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition for time course (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. (C) IRF8 ChIP-seq signals at the IRF4 gene locus. Blue line denotes the range of the IRF4 gene locus, and the peak patterns in red represent the ChIP-seq signals of IRF8 binding. Signals represent DNA fragments that were captured by the IRF8 antibody through ChIP. (D) Purified CD4+ T cells from WT or Irf8 −/− mice were stimulated with anti-CD3 and anti-CD28 for 48h in the presence of IL-21, followed by ChIP assay. Three micrograms of an anti-IRF4 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the irf4-binding site of the IL-21 promoter region. Each bar represents mean ± S.D. from three independent experiments, unpaired Student’s t-test, *P

    Techniques Used: Expressing, Purification, Mouse Assay, Microarray, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction

    18) Product Images from "CD8+ T cell priming in established chronic viral infection preferentially directs differentiation of memory-like cells for sustained immunity"

    Article Title: CD8+ T cell priming in established chronic viral infection preferentially directs differentiation of memory-like cells for sustained immunity

    Journal: Immunity

    doi: 10.1016/j.immuni.2018.08.002

    CD4 + T cell help and differential IL-2 and IL-21 usage drive differentiation of TCF1 + into GzmB + effector antiviral CD8 + T cell subsets at distinct times after infection. (A) STAT5a and STAT3 phosphorylation by P14 Tn, (gray), Tep (black) or Tlp (red) cells isolated from mice 60hrs after priming and then cultured without stimulation (media) or following IL-2 or IL-21 stimulation for 30 minutes. (B C) Naïve mice immediately infected with Cl13 or mice infected 21 days prior with Cl13 received P14 T cells and were treated with (B) isotype (gray) or anti-IL-2 (blue); (C) isotype (gray) or anti-IL-21R (red) antibodies. Flow plots depict the proportion and bar graphs the number of total, GzmB + and TCF1 + virus-specific CD8 + Tep and Tlp P14 cells 8 days after priming. (D) Mice were CD4 + depleted or isotype treated (undepleted) prior to Cl13 infection. (Top) Flow plots depict the proportion and bar graphs show the numbers of total, GzmB + and TCF1 + subsets of Tep or Tlp cells 8 days after priming. (Bottom) Proportion of IFNγ and TNFα producing Tep or Tlp cells upon peptide restimulation 8 days after priming. Data represent 2 independent experiments with 4-5 mice per group. Error bars indicate SD. Significance was determined by t-test. *, p
    Figure Legend Snippet: CD4 + T cell help and differential IL-2 and IL-21 usage drive differentiation of TCF1 + into GzmB + effector antiviral CD8 + T cell subsets at distinct times after infection. (A) STAT5a and STAT3 phosphorylation by P14 Tn, (gray), Tep (black) or Tlp (red) cells isolated from mice 60hrs after priming and then cultured without stimulation (media) or following IL-2 or IL-21 stimulation for 30 minutes. (B C) Naïve mice immediately infected with Cl13 or mice infected 21 days prior with Cl13 received P14 T cells and were treated with (B) isotype (gray) or anti-IL-2 (blue); (C) isotype (gray) or anti-IL-21R (red) antibodies. Flow plots depict the proportion and bar graphs the number of total, GzmB + and TCF1 + virus-specific CD8 + Tep and Tlp P14 cells 8 days after priming. (D) Mice were CD4 + depleted or isotype treated (undepleted) prior to Cl13 infection. (Top) Flow plots depict the proportion and bar graphs show the numbers of total, GzmB + and TCF1 + subsets of Tep or Tlp cells 8 days after priming. (Bottom) Proportion of IFNγ and TNFα producing Tep or Tlp cells upon peptide restimulation 8 days after priming. Data represent 2 independent experiments with 4-5 mice per group. Error bars indicate SD. Significance was determined by t-test. *, p

    Techniques Used: Infection, Isolation, Mouse Assay, Cell Culture

    19) Product Images from "T Follicular Helper Cells restricted by IRF8 contribute to T Cell-Mediated Inflammation"

    Article Title: T Follicular Helper Cells restricted by IRF8 contribute to T Cell-Mediated Inflammation

    Journal: Journal of autoimmunity

    doi: 10.1016/j.jaut.2018.09.001

    IRF8-deficient in T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from Irf8 wt/wt or Irf8 lck/lck mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between Irf8 wt/wt (n = 5) and Irf8 lck/lck (n = 5) CD4 + T cell cultures. Results shown are representative of three independent experiments. Data are given as means ± SEM.
    Figure Legend Snippet: IRF8-deficient in T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from Irf8 wt/wt or Irf8 lck/lck mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between Irf8 wt/wt (n = 5) and Irf8 lck/lck (n = 5) CD4 + T cell cultures. Results shown are representative of three independent experiments. Data are given as means ± SEM.

    Techniques Used: Ex Vivo, Purification, Mouse Assay, Expressing, Flow Cytometry, Cytometry

    IRF8-deficient CD4 + T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from WT or Irf8 −/− mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD-1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between WT (n = 5) and Irf8 −/− (n = 5) CD4 + T cell cultures. (D) Flow cytometry evaluation of IL-21 + CD4 + T cells in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). (E) ELISA of IL-21 levels in culture supernatant of WT and Irf8 ‒/‒ CD4 + T cells cultured under T FH condition (n = 4). (F) Quantitative real-time RT-PCR analysis of T FH -associated genes in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. Results shown are representative of three independent experiments. Data are given as means ± SEM.
    Figure Legend Snippet: IRF8-deficient CD4 + T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from WT or Irf8 −/− mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD-1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between WT (n = 5) and Irf8 −/− (n = 5) CD4 + T cell cultures. (D) Flow cytometry evaluation of IL-21 + CD4 + T cells in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). (E) ELISA of IL-21 levels in culture supernatant of WT and Irf8 ‒/‒ CD4 + T cells cultured under T FH condition (n = 4). (F) Quantitative real-time RT-PCR analysis of T FH -associated genes in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. Results shown are representative of three independent experiments. Data are given as means ± SEM.

    Techniques Used: Ex Vivo, Purification, Mouse Assay, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Quantitative RT-PCR

    Regulation of IRF4 expression by IRF8. (A) Purified CD4 + T cells from WT or Irf8 −/− mice were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Microarray analysis of the gene expression by WT and Irf8 ‒/‒ CD4 + T cells under T FH condition. (B) Quantitative real-time RT-PCR analysis of irf4 gene level in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition for time course (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. (C) IRF8 ChIP-seq signals at the IRF4 gene locus. Blue line denotes the range of the IRF4 gene locus, and the peak patterns in red represent the ChIP-seq signals of IRF8 binding. Signals represent DNA fragments that were captured by the IRF8 antibody through ChIP. (D) Purified CD4+ T cells from WT or Irf8 −/− mice were stimulated with anti-CD3 and anti-CD28 for 48h in the presence of IL-21, followed by ChIP assay. Three micrograms of an anti-IRF4 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the irf4-binding site of the IL-21 promoter region. Each bar represents mean ± S.D. from three independent experiments, unpaired Student’s t-test, *P
    Figure Legend Snippet: Regulation of IRF4 expression by IRF8. (A) Purified CD4 + T cells from WT or Irf8 −/− mice were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Microarray analysis of the gene expression by WT and Irf8 ‒/‒ CD4 + T cells under T FH condition. (B) Quantitative real-time RT-PCR analysis of irf4 gene level in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition for time course (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. (C) IRF8 ChIP-seq signals at the IRF4 gene locus. Blue line denotes the range of the IRF4 gene locus, and the peak patterns in red represent the ChIP-seq signals of IRF8 binding. Signals represent DNA fragments that were captured by the IRF8 antibody through ChIP. (D) Purified CD4+ T cells from WT or Irf8 −/− mice were stimulated with anti-CD3 and anti-CD28 for 48h in the presence of IL-21, followed by ChIP assay. Three micrograms of an anti-IRF4 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the irf4-binding site of the IL-21 promoter region. Each bar represents mean ± S.D. from three independent experiments, unpaired Student’s t-test, *P

    Techniques Used: Expressing, Purification, Mouse Assay, Microarray, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction

    20) Product Images from "Interleukin-21 differentially affects human natural killer cell subsets"

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets

    Journal:

    doi: 10.1111/j.1365-2567.2007.02675.x

    FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5
    Figure Legend Snippet: FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5

    Techniques Used: FACS

    IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)
    Figure Legend Snippet: IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)

    Techniques Used:

    Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.
    Figure Legend Snippet: Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.

    Techniques Used: Activation Assay

    Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open
    Figure Legend Snippet: Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open

    Techniques Used: Activation Assay, Flow Cytometry

    21) Product Images from "Interleukin-21 differentially affects human natural killer cell subsets"

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets

    Journal:

    doi: 10.1111/j.1365-2567.2007.02675.x

    FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5
    Figure Legend Snippet: FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5

    Techniques Used: FACS

    IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)
    Figure Legend Snippet: IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)

    Techniques Used:

    Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.
    Figure Legend Snippet: Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.

    Techniques Used: Activation Assay

    Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open
    Figure Legend Snippet: Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open

    Techniques Used: Activation Assay, Flow Cytometry

    22) Product Images from "Interleukin-21 differentially affects human natural killer cell subsets"

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets

    Journal:

    doi: 10.1111/j.1365-2567.2007.02675.x

    FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5
    Figure Legend Snippet: FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5

    Techniques Used: FACS

    IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)
    Figure Legend Snippet: IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)

    Techniques Used:

    Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.
    Figure Legend Snippet: Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.

    Techniques Used: Activation Assay

    Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open
    Figure Legend Snippet: Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open

    Techniques Used: Activation Assay, Flow Cytometry

    23) Product Images from "Interleukin-21 differentially affects human natural killer cell subsets"

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets

    Journal:

    doi: 10.1111/j.1365-2567.2007.02675.x

    FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5
    Figure Legend Snippet: FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5

    Techniques Used: FACS

    IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)
    Figure Legend Snippet: IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)

    Techniques Used:

    Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.
    Figure Legend Snippet: Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.

    Techniques Used: Activation Assay

    Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open
    Figure Legend Snippet: Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open

    Techniques Used: Activation Assay, Flow Cytometry

    24) Product Images from "Interleukin-21 differentially affects human natural killer cell subsets"

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets

    Journal:

    doi: 10.1111/j.1365-2567.2007.02675.x

    FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5
    Figure Legend Snippet: FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5

    Techniques Used: FACS

    IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)
    Figure Legend Snippet: IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)

    Techniques Used:

    Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.
    Figure Legend Snippet: Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.

    Techniques Used: Activation Assay

    Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open
    Figure Legend Snippet: Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open

    Techniques Used: Activation Assay, Flow Cytometry

    25) Product Images from "Overexpression of T-bet in HIV infection is associated with accumulation of B cells outside germinal centers and poor affinity maturation"

    Article Title: Overexpression of T-bet in HIV infection is associated with accumulation of B cells outside germinal centers and poor affinity maturation

    Journal: Science translational medicine

    doi: 10.1126/scitranslmed.aax0904

    T H 1 and T FH cytokines modulate expression of T-bet and migration receptors. ( A ) Conventional flow cytometry performed on LN GCBC and CD19 hi MBC depicting the expression of Bcl6, T-bet, and CXCR5 expression of an HIV-infected individual. Red pattern and numbers identify Bcl6 frequencies within each quadrant. ( B ) Conventional flow cytometry performed on tonsillar B cells isolated from HIV-negative donors depicting Bcl6 and T-bet expression before and after 48 hours of stimulation of IgD + cells with anti-human BCR and CD40L with or without IFN-γ or IL-21. ( C ) Histograms and comparison ( n = 10) of T-bet and CXCR5 protein expression under conditions in (B). ( D ) Comparison ( n = 6) of gene expression for TBX21 , S1PR2 , and BACH2 under conditions in (B). * P
    Figure Legend Snippet: T H 1 and T FH cytokines modulate expression of T-bet and migration receptors. ( A ) Conventional flow cytometry performed on LN GCBC and CD19 hi MBC depicting the expression of Bcl6, T-bet, and CXCR5 expression of an HIV-infected individual. Red pattern and numbers identify Bcl6 frequencies within each quadrant. ( B ) Conventional flow cytometry performed on tonsillar B cells isolated from HIV-negative donors depicting Bcl6 and T-bet expression before and after 48 hours of stimulation of IgD + cells with anti-human BCR and CD40L with or without IFN-γ or IL-21. ( C ) Histograms and comparison ( n = 10) of T-bet and CXCR5 protein expression under conditions in (B). ( D ) Comparison ( n = 6) of gene expression for TBX21 , S1PR2 , and BACH2 under conditions in (B). * P

    Techniques Used: Expressing, Migration, Flow Cytometry, Infection, Isolation

    26) Product Images from "T Follicular Helper Cells restricted by IRF8 contribute to T Cell-Mediated Inflammation"

    Article Title: T Follicular Helper Cells restricted by IRF8 contribute to T Cell-Mediated Inflammation

    Journal: Journal of autoimmunity

    doi: 10.1016/j.jaut.2018.09.001

    IRF8-deficient in T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from Irf8 wt/wt or Irf8 lck/lck mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between Irf8 wt/wt (n = 5) and Irf8 lck/lck (n = 5) CD4 + T cell cultures. Results shown are representative of three independent experiments. Data are given as means ± SEM.
    Figure Legend Snippet: IRF8-deficient in T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from Irf8 wt/wt or Irf8 lck/lck mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between Irf8 wt/wt (n = 5) and Irf8 lck/lck (n = 5) CD4 + T cell cultures. Results shown are representative of three independent experiments. Data are given as means ± SEM.

    Techniques Used: Ex Vivo, Purification, Mouse Assay, Expressing, Flow Cytometry, Cytometry

    IRF8-deficient CD4 + T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from WT or Irf8 −/− mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD-1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between WT (n = 5) and Irf8 −/− (n = 5) CD4 + T cell cultures. (D) Flow cytometry evaluation of IL-21 + CD4 + T cells in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). (E) ELISA of IL-21 levels in culture supernatant of WT and Irf8 ‒/‒ CD4 + T cells cultured under T FH condition (n = 4). (F) Quantitative real-time RT-PCR analysis of T FH -associated genes in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. Results shown are representative of three independent experiments. Data are given as means ± SEM.
    Figure Legend Snippet: IRF8-deficient CD4 + T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from WT or Irf8 −/− mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD-1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between WT (n = 5) and Irf8 −/− (n = 5) CD4 + T cell cultures. (D) Flow cytometry evaluation of IL-21 + CD4 + T cells in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). (E) ELISA of IL-21 levels in culture supernatant of WT and Irf8 ‒/‒ CD4 + T cells cultured under T FH condition (n = 4). (F) Quantitative real-time RT-PCR analysis of T FH -associated genes in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. Results shown are representative of three independent experiments. Data are given as means ± SEM.

    Techniques Used: Ex Vivo, Purification, Mouse Assay, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Quantitative RT-PCR

    Regulation of IRF4 expression by IRF8. (A) Purified CD4 + T cells from WT or Irf8 −/− mice were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Microarray analysis of the gene expression by WT and Irf8 ‒/‒ CD4 + T cells under T FH condition. (B) Quantitative real-time RT-PCR analysis of irf4 gene level in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition for time course (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. (C) IRF8 ChIP-seq signals at the IRF4 gene locus. Blue line denotes the range of the IRF4 gene locus, and the peak patterns in red represent the ChIP-seq signals of IRF8 binding. Signals represent DNA fragments that were captured by the IRF8 antibody through ChIP. (D) Purified CD4+ T cells from WT or Irf8 −/− mice were stimulated with anti-CD3 and anti-CD28 for 48h in the presence of IL-21, followed by ChIP assay. Three micrograms of an anti-IRF4 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the irf4-binding site of the IL-21 promoter region. Each bar represents mean ± S.D. from three independent experiments, unpaired Student’s t-test, *P
    Figure Legend Snippet: Regulation of IRF4 expression by IRF8. (A) Purified CD4 + T cells from WT or Irf8 −/− mice were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Microarray analysis of the gene expression by WT and Irf8 ‒/‒ CD4 + T cells under T FH condition. (B) Quantitative real-time RT-PCR analysis of irf4 gene level in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition for time course (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. (C) IRF8 ChIP-seq signals at the IRF4 gene locus. Blue line denotes the range of the IRF4 gene locus, and the peak patterns in red represent the ChIP-seq signals of IRF8 binding. Signals represent DNA fragments that were captured by the IRF8 antibody through ChIP. (D) Purified CD4+ T cells from WT or Irf8 −/− mice were stimulated with anti-CD3 and anti-CD28 for 48h in the presence of IL-21, followed by ChIP assay. Three micrograms of an anti-IRF4 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the irf4-binding site of the IL-21 promoter region. Each bar represents mean ± S.D. from three independent experiments, unpaired Student’s t-test, *P

    Techniques Used: Expressing, Purification, Mouse Assay, Microarray, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction

    27) Product Images from "T Follicular Helper Cells restricted by IRF8 contribute to T Cell-Mediated Inflammation"

    Article Title: T Follicular Helper Cells restricted by IRF8 contribute to T Cell-Mediated Inflammation

    Journal: Journal of autoimmunity

    doi: 10.1016/j.jaut.2018.09.001

    IRF8-deficient in T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from Irf8 wt/wt or Irf8 lck/lck mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between Irf8 wt/wt (n = 5) and Irf8 lck/lck (n = 5) CD4 + T cell cultures. Results shown are representative of three independent experiments. Data are given as means ± SEM.
    Figure Legend Snippet: IRF8-deficient in T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from Irf8 wt/wt or Irf8 lck/lck mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between Irf8 wt/wt (n = 5) and Irf8 lck/lck (n = 5) CD4 + T cell cultures. Results shown are representative of three independent experiments. Data are given as means ± SEM.

    Techniques Used: Ex Vivo, Purification, Mouse Assay, Expressing, Flow Cytometry, Cytometry

    IRF8-deficient CD4 + T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from WT or Irf8 −/− mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD-1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between WT (n = 5) and Irf8 −/− (n = 5) CD4 + T cell cultures. (D) Flow cytometry evaluation of IL-21 + CD4 + T cells in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). (E) ELISA of IL-21 levels in culture supernatant of WT and Irf8 ‒/‒ CD4 + T cells cultured under T FH condition (n = 4). (F) Quantitative real-time RT-PCR analysis of T FH -associated genes in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. Results shown are representative of three independent experiments. Data are given as means ± SEM.
    Figure Legend Snippet: IRF8-deficient CD4 + T cells show enhanced T FH polarization after ex vivo stimulation. Purified CD4 + T cells from WT or Irf8 −/− mice (A, B, and C) were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Cell surface expression of PD-1, ICOS, CXCR5 and intracellular expression of Bcl-6 were analyzed by flow cytometry. The percentages of CXCR5 + PD1 + , CXCR5 + ICOS + and CXCR5 + Bcl6 + cells were compared between WT (n = 5) and Irf8 −/− (n = 5) CD4 + T cell cultures. (D) Flow cytometry evaluation of IL-21 + CD4 + T cells in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). (E) ELISA of IL-21 levels in culture supernatant of WT and Irf8 ‒/‒ CD4 + T cells cultured under T FH condition (n = 4). (F) Quantitative real-time RT-PCR analysis of T FH -associated genes in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. Results shown are representative of three independent experiments. Data are given as means ± SEM.

    Techniques Used: Ex Vivo, Purification, Mouse Assay, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Quantitative RT-PCR

    Regulation of IRF4 expression by IRF8. (A) Purified CD4 + T cells from WT or Irf8 −/− mice were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Microarray analysis of the gene expression by WT and Irf8 ‒/‒ CD4 + T cells under T FH condition. (B) Quantitative real-time RT-PCR analysis of irf4 gene level in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition for time course (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. (C) IRF8 ChIP-seq signals at the IRF4 gene locus. Blue line denotes the range of the IRF4 gene locus, and the peak patterns in red represent the ChIP-seq signals of IRF8 binding. Signals represent DNA fragments that were captured by the IRF8 antibody through ChIP. (D) Purified CD4+ T cells from WT or Irf8 −/− mice were stimulated with anti-CD3 and anti-CD28 for 48h in the presence of IL-21, followed by ChIP assay. Three micrograms of an anti-IRF4 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the irf4-binding site of the IL-21 promoter region. Each bar represents mean ± S.D. from three independent experiments, unpaired Student’s t-test, *P
    Figure Legend Snippet: Regulation of IRF4 expression by IRF8. (A) Purified CD4 + T cells from WT or Irf8 −/− mice were stimulated with or without anti-CD3 and anti-CD28 for 48h in the presence of IL-21. Microarray analysis of the gene expression by WT and Irf8 ‒/‒ CD4 + T cells under T FH condition. (B) Quantitative real-time RT-PCR analysis of irf4 gene level in WT and Irf8 ‒/‒ CD4 + T cells under T FH condition for time course (n = 5). The ubiquitin gene ( Ubi ) was used as an internal control. (C) IRF8 ChIP-seq signals at the IRF4 gene locus. Blue line denotes the range of the IRF4 gene locus, and the peak patterns in red represent the ChIP-seq signals of IRF8 binding. Signals represent DNA fragments that were captured by the IRF8 antibody through ChIP. (D) Purified CD4+ T cells from WT or Irf8 −/− mice were stimulated with anti-CD3 and anti-CD28 for 48h in the presence of IL-21, followed by ChIP assay. Three micrograms of an anti-IRF4 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the irf4-binding site of the IL-21 promoter region. Each bar represents mean ± S.D. from three independent experiments, unpaired Student’s t-test, *P

    Techniques Used: Expressing, Purification, Mouse Assay, Microarray, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction

    28) Product Images from "Interleukin-21 differentially affects human natural killer cell subsets"

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets

    Journal:

    doi: 10.1111/j.1365-2567.2007.02675.x

    FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5
    Figure Legend Snippet: FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5

    Techniques Used: FACS

    IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)
    Figure Legend Snippet: IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)

    Techniques Used:

    Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.
    Figure Legend Snippet: Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.

    Techniques Used: Activation Assay

    Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open
    Figure Legend Snippet: Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open

    Techniques Used: Activation Assay, Flow Cytometry

    29) Product Images from "Interleukin-21 differentially affects human natural killer cell subsets"

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets

    Journal:

    doi: 10.1111/j.1365-2567.2007.02675.x

    FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5
    Figure Legend Snippet: FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5

    Techniques Used: FACS

    IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)
    Figure Legend Snippet: IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)

    Techniques Used:

    Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.
    Figure Legend Snippet: Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.

    Techniques Used: Activation Assay

    Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open
    Figure Legend Snippet: Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open

    Techniques Used: Activation Assay, Flow Cytometry

    30) Product Images from "Interleukin-21 differentially affects human natural killer cell subsets"

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets

    Journal:

    doi: 10.1111/j.1365-2567.2007.02675.x

    FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5
    Figure Legend Snippet: FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5

    Techniques Used: FACS

    IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)
    Figure Legend Snippet: IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)

    Techniques Used:

    Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.
    Figure Legend Snippet: Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.

    Techniques Used: Activation Assay

    Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open
    Figure Legend Snippet: Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open

    Techniques Used: Activation Assay, Flow Cytometry

    31) Product Images from "Dysregulated TFR and TFH cells correlate with B‐cell differentiation and antibody production in autoimmune hepatitis. Dysregulated TFR and TFH cells correlate with B‐cell differentiation and antibody production in autoimmune hepatitis"

    Article Title: Dysregulated TFR and TFH cells correlate with B‐cell differentiation and antibody production in autoimmune hepatitis. Dysregulated TFR and TFH cells correlate with B‐cell differentiation and antibody production in autoimmune hepatitis

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14997

    Correlation among the numbers of different subsets of TFR and TFH cells and the levels of serum IL‐21, IL‐10 and TGF‐β1 in AIH patients. A, Correlation between the numbers of TFR cells and the numbers of TFH cells in AIH patients; B, correlation between the numbers of TFH cells and serum levels of IL‐21 in AIH patients; C, correlation between the numbers of TFR cells and serum levels of IL‐21 in AIH patients; D, correlation between the numbers of TFR cells and serum levels of IL‐10 in AIH patients; E, correlation between the numbers of TFR cells and serum levels of TGF‐β1 in AIH patients
    Figure Legend Snippet: Correlation among the numbers of different subsets of TFR and TFH cells and the levels of serum IL‐21, IL‐10 and TGF‐β1 in AIH patients. A, Correlation between the numbers of TFR cells and the numbers of TFH cells in AIH patients; B, correlation between the numbers of TFH cells and serum levels of IL‐21 in AIH patients; C, correlation between the numbers of TFR cells and serum levels of IL‐21 in AIH patients; D, correlation between the numbers of TFR cells and serum levels of IL‐10 in AIH patients; E, correlation between the numbers of TFR cells and serum levels of TGF‐β1 in AIH patients

    Techniques Used:

    Correlation between serum levels of TFR/TFH‐type cytokines and the values of clinical parameters in AIH patients. A‐B, Correlation between serum levels of IL‐21 and titre IgG/IgM in AIH patients; C‐D, correlation between serum levels of IL‐10 and titre IgG/IgM in AIH patients
    Figure Legend Snippet: Correlation between serum levels of TFR/TFH‐type cytokines and the values of clinical parameters in AIH patients. A‐B, Correlation between serum levels of IL‐21 and titre IgG/IgM in AIH patients; C‐D, correlation between serum levels of IL‐10 and titre IgG/IgM in AIH patients

    Techniques Used:

    Serum levels of TFR/TFH‐type cytokines in AIH patients and HCs. Serum levels of A, IL‐21; B, IL‐10; C, TGF‐β1in AIH patients and HCs. Data shown are the mean levels of each serum cytokine in individual subjects from two separate experiments. The horizontal lines indicate the mean values for the different groups
    Figure Legend Snippet: Serum levels of TFR/TFH‐type cytokines in AIH patients and HCs. Serum levels of A, IL‐21; B, IL‐10; C, TGF‐β1in AIH patients and HCs. Data shown are the mean levels of each serum cytokine in individual subjects from two separate experiments. The horizontal lines indicate the mean values for the different groups

    Techniques Used:

    Flow cytometry analysis of the numbers of circulating TFR and TFH cells in AIH patients and HCs. PBMCs 5*10 5 /tube were isolated from individual subjects and were stained in duplicate with anti‐CD4, anti‐CXCR5, anti‐ICOS, anti‐PD‐1, anti‐CTLA‐4, anti‐CD25 and intracellular anti‐Foxp3, anti‐IL‐21 or IL‐10, respectively. The cells were characterized by flow cytometry analysis by gating initially on living lymphocytes, and then on CD4 + CXCR5 + Foxp3 + TFR and CD4 + CXCR5 + Foxp3 ‐ TFH cells. Subsequently, the numbers of different subsets of TFR and TFH cells were calculated, according to the total numbers of PBMCs, the frequency of TFR and TFH cells. A, Flow cytometry analysis of TFR and TFH cells; B, the numbers of CD4 + CXCR5 + Foxp3 + TFR, CD4 + CXCR5 + Foxp3 ‐ TFH cells; and TFR/TFH ratio; C, flow cytometry analysis of different subsets of TFR and TFH cell; D, the numbers of ICOS + , PD‐1 + , CTLA‐4 + , CD25 + and IL‐10 + TFR and TFH cells. Data shown are representative FACS charts or the mean numbers of each type of cells per mL of peripheral blood in individual subjects from two separate experiments. The horizontal lines indicate the median values for each group
    Figure Legend Snippet: Flow cytometry analysis of the numbers of circulating TFR and TFH cells in AIH patients and HCs. PBMCs 5*10 5 /tube were isolated from individual subjects and were stained in duplicate with anti‐CD4, anti‐CXCR5, anti‐ICOS, anti‐PD‐1, anti‐CTLA‐4, anti‐CD25 and intracellular anti‐Foxp3, anti‐IL‐21 or IL‐10, respectively. The cells were characterized by flow cytometry analysis by gating initially on living lymphocytes, and then on CD4 + CXCR5 + Foxp3 + TFR and CD4 + CXCR5 + Foxp3 ‐ TFH cells. Subsequently, the numbers of different subsets of TFR and TFH cells were calculated, according to the total numbers of PBMCs, the frequency of TFR and TFH cells. A, Flow cytometry analysis of TFR and TFH cells; B, the numbers of CD4 + CXCR5 + Foxp3 + TFR, CD4 + CXCR5 + Foxp3 ‐ TFH cells; and TFR/TFH ratio; C, flow cytometry analysis of different subsets of TFR and TFH cell; D, the numbers of ICOS + , PD‐1 + , CTLA‐4 + , CD25 + and IL‐10 + TFR and TFH cells. Data shown are representative FACS charts or the mean numbers of each type of cells per mL of peripheral blood in individual subjects from two separate experiments. The horizontal lines indicate the median values for each group

    Techniques Used: Flow Cytometry, Isolation, Staining, FACS

    32) Product Images from "Exploratory analysis of circulating cytokines in patients with metastatic breast cancer treated with eribulin: the TRANSERI-GONO (Gruppo Oncologico del Nord Ovest) study"

    Article Title: Exploratory analysis of circulating cytokines in patients with metastatic breast cancer treated with eribulin: the TRANSERI-GONO (Gruppo Oncologico del Nord Ovest) study

    Journal: ESMO Open

    doi: 10.1136/esmoopen-2020-000876

    Cox regression model for TGF-β, IL-8 and IL-21 at T 1 divided in two groups. Group 1: patients with cytokine levels above the median. Group 2: patients with cytokine levels below or equal to the median. IL, interleukin; OS, overall survival; PFS, progression free survival; TGF-β, transforming growth factor β.
    Figure Legend Snippet: Cox regression model for TGF-β, IL-8 and IL-21 at T 1 divided in two groups. Group 1: patients with cytokine levels above the median. Group 2: patients with cytokine levels below or equal to the median. IL, interleukin; OS, overall survival; PFS, progression free survival; TGF-β, transforming growth factor β.

    Techniques Used:

    Cox regression model for TGF-β, IL-21 and IL-8 comparing T 0 with T 1 . Group 1: patients with T 1 > T 0 . Group 2: patients with T 1 ≤ T 0 . IL, interleukin; OS, overall survival. PFS, progression free survival;TGF, transforming growth factor.
    Figure Legend Snippet: Cox regression model for TGF-β, IL-21 and IL-8 comparing T 0 with T 1 . Group 1: patients with T 1 > T 0 . Group 2: patients with T 1 ≤ T 0 . IL, interleukin; OS, overall survival. PFS, progression free survival;TGF, transforming growth factor.

    Techniques Used:

    33) Product Images from "Human PD-1hiCD8+ T Cells Are a Cellular Source of IL-21 in Rheumatoid Arthritis"

    Article Title: Human PD-1hiCD8+ T Cells Are a Cellular Source of IL-21 in Rheumatoid Arthritis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.654623

    IL-21-producing CD8 + T cells are most efficiently induced by IL-12 and IL-21 in combination with CD3/28. (A) Naïve CD8 + T cells (CD45RA + CCR7 + CD8 + T cells) in HCs were purified by flow cytometry and then stimulated with the indicated cytokines in combination with CD3/28 beads for 72 h. Cells were harvested, and transcriptions of IL21 were evaluated by qPCR. (B) Purified naïve CD8 + T cells in HCs were stimulated with the indicated cytokines for 5 days, and IL-21 production was analyzed by intracellular staining. Representative data are shown and summarized results are shown in (C) (N=4). (D) Naïve CD8 + T cells in HCs were stimulated with IL-12 and IL-21 in conjunction with CD3/28 beads. The panels show expression of CD45RA, PD-1, CD28 and IFN-γ on IL-21 + and IL-21 - CD8 + T cells. Representative data are depicted, and summarized graphs of the results are shown (N=4). ** P
    Figure Legend Snippet: IL-21-producing CD8 + T cells are most efficiently induced by IL-12 and IL-21 in combination with CD3/28. (A) Naïve CD8 + T cells (CD45RA + CCR7 + CD8 + T cells) in HCs were purified by flow cytometry and then stimulated with the indicated cytokines in combination with CD3/28 beads for 72 h. Cells were harvested, and transcriptions of IL21 were evaluated by qPCR. (B) Purified naïve CD8 + T cells in HCs were stimulated with the indicated cytokines for 5 days, and IL-21 production was analyzed by intracellular staining. Representative data are shown and summarized results are shown in (C) (N=4). (D) Naïve CD8 + T cells in HCs were stimulated with IL-12 and IL-21 in conjunction with CD3/28 beads. The panels show expression of CD45RA, PD-1, CD28 and IFN-γ on IL-21 + and IL-21 - CD8 + T cells. Representative data are depicted, and summarized graphs of the results are shown (N=4). ** P

    Techniques Used: Purification, Flow Cytometry, Real-time Polymerase Chain Reaction, Staining, Expressing

    The subsets of IL-21-producing CD8 + T cells. (A) PBMCs from HCs were stimulated with CD3/28 beads for 72 h. Production of IL-21, IFN-γ and granzyme B in naïve (CD45RA + CCR7 + ), memory (CD45RA - ) and terminal effector (CD45RA + CCR7 - ) cells was analyzed by intracellular staining (added PMA and Ionomycin for the last 6 h). Left panels are representative data and right graphs summarize the results (N=3). (B) PBMCs from HCs were stimulated under the same condition as (A) , and then analyzed by flow cytometry. The panel shows four fractions of CD8 + T cells (CD45RA + , CD45RA - PD-1 -/low , CD45RA - PD-1 int and CD45RA - PD-1 hi ). The panels show IL-21 production from four populations including CD45RA + , CD45RA - PD-1 -/low , CD45RA - PD-1 int and CD45RA - PD-1 hi CD8 + T cells by intracellular staining. * P
    Figure Legend Snippet: The subsets of IL-21-producing CD8 + T cells. (A) PBMCs from HCs were stimulated with CD3/28 beads for 72 h. Production of IL-21, IFN-γ and granzyme B in naïve (CD45RA + CCR7 + ), memory (CD45RA - ) and terminal effector (CD45RA + CCR7 - ) cells was analyzed by intracellular staining (added PMA and Ionomycin for the last 6 h). Left panels are representative data and right graphs summarize the results (N=3). (B) PBMCs from HCs were stimulated under the same condition as (A) , and then analyzed by flow cytometry. The panel shows four fractions of CD8 + T cells (CD45RA + , CD45RA - PD-1 -/low , CD45RA - PD-1 int and CD45RA - PD-1 hi ). The panels show IL-21 production from four populations including CD45RA + , CD45RA - PD-1 -/low , CD45RA - PD-1 int and CD45RA - PD-1 hi CD8 + T cells by intracellular staining. * P

    Techniques Used: Staining, Flow Cytometry

    IL-21-producing CD8 + T cells are enriched in memory PD-1 hi fraction in RAPB and RASF. (A) The upper panels show the representative data regarding the percentage of IL-21-producing CD4 + and CD8 + T cells in PB and SF of patients with RA and the lower panel summarizes the results (N=18). (B) Correlation of the percentage of IL-21 + CD4 + T cells with that of IL-21 + CD8 + T cells in RASF (N=18). (C) The panel summarizes the frequency of synovial IL-21-producing CD8 + T cells in RF-positive and -negative patients with RA. (D) The left panels are representative data of the percentage of CD45RA - PD-1 hi fraction among whole CD8 + T cells in OASF (N=3), RAPB (N=12) and RASF (N=18) and right panel summarizes the results. (E) The left panels are representative data of the percentage of IL-21-producing CD8 + T cells among CD45RA + , CD45RA - PD-1 -/low , CD45RA - PD-1 int and CD45RA - PD-1 hi fractions in RASF and the right panel summarizes the results (N=12). * P
    Figure Legend Snippet: IL-21-producing CD8 + T cells are enriched in memory PD-1 hi fraction in RAPB and RASF. (A) The upper panels show the representative data regarding the percentage of IL-21-producing CD4 + and CD8 + T cells in PB and SF of patients with RA and the lower panel summarizes the results (N=18). (B) Correlation of the percentage of IL-21 + CD4 + T cells with that of IL-21 + CD8 + T cells in RASF (N=18). (C) The panel summarizes the frequency of synovial IL-21-producing CD8 + T cells in RF-positive and -negative patients with RA. (D) The left panels are representative data of the percentage of CD45RA - PD-1 hi fraction among whole CD8 + T cells in OASF (N=3), RAPB (N=12) and RASF (N=18) and right panel summarizes the results. (E) The left panels are representative data of the percentage of IL-21-producing CD8 + T cells among CD45RA + , CD45RA - PD-1 -/low , CD45RA - PD-1 int and CD45RA - PD-1 hi fractions in RASF and the right panel summarizes the results (N=12). * P

    Techniques Used:

    Memory PD-1 hi CD8 + T cells promote the differentiation of B cells to plasmablasts and IgG production in an IL-21-dependent manner. (A) Upon stimulation of PBMCs from HCs with CD3/28 beads for 72 h, CD45RA + CD8 + T cells, CD45RA - PD-1 hi CD8 + T cells and CD45RA - PD-1 hi CD4 + T cells were purified by flow cytometry. Memory B (CD19 + CD20 + CD27 + ) cells were co-cultured with CD45RA + CD8 + T cells, CD45RA - PD-1 hi CD8 + T cells and CD45RA - PD-1 hi CD4 + T cells in the presence of anti-BCR, CD3/28 beads and CpG for 6 days. Representative data on the expression of CD27 and CD38 are depicted in (A) and the results are summarized in (B) (N=4). (C) Comparison of IgG production from plasmablasts induced by co-cultured CD45RA - PD-1 hi CD4 + T cells and CD45RA - PD-1 hi CD8 + T cells. (D) Effect of IL-21 blockade on the frequency of plasmablasts and IgG production induced by co-cultured CD45RA - PD-1 hi CD8 + T cells. * P
    Figure Legend Snippet: Memory PD-1 hi CD8 + T cells promote the differentiation of B cells to plasmablasts and IgG production in an IL-21-dependent manner. (A) Upon stimulation of PBMCs from HCs with CD3/28 beads for 72 h, CD45RA + CD8 + T cells, CD45RA - PD-1 hi CD8 + T cells and CD45RA - PD-1 hi CD4 + T cells were purified by flow cytometry. Memory B (CD19 + CD20 + CD27 + ) cells were co-cultured with CD45RA + CD8 + T cells, CD45RA - PD-1 hi CD8 + T cells and CD45RA - PD-1 hi CD4 + T cells in the presence of anti-BCR, CD3/28 beads and CpG for 6 days. Representative data on the expression of CD27 and CD38 are depicted in (A) and the results are summarized in (B) (N=4). (C) Comparison of IgG production from plasmablasts induced by co-cultured CD45RA - PD-1 hi CD4 + T cells and CD45RA - PD-1 hi CD8 + T cells. (D) Effect of IL-21 blockade on the frequency of plasmablasts and IgG production induced by co-cultured CD45RA - PD-1 hi CD8 + T cells. * P

    Techniques Used: Purification, Flow Cytometry, Cell Culture, Expressing

    The phenotype of IL-21-producing CD8 + T cells. (A) PBMCs from HCs were stimulated in the absence or presence of CD3/28 beads for 72 h and then IL-21 production from CD4 + and CD8 + T cells was analyzed by intracellular staining (added PMA and Ionomycin for the last 6 h). Left panels are representative data on IL-21 production from CD4 + and CD8 + T cells stimulated with CD3/28 and the right graph summarizes the results (N=4). (B) PBMCs from HCs were stimulated under the same condition as (A) , and IFN-γ and IL-17 production from IL-21 + and IL-21 - CD8 + T cells was analyzed by intracellular staining. (C) PBMCs from HCs were stimulated under the same condition as (A) , and expression levels of PD-1, ICOS, CD95, CD69, CD28 and HLA-DR in IL-21 + and IL-21 - CD8 + T cells were analyzed by flow cytometry. The graph summarizes the results (N=4). * P
    Figure Legend Snippet: The phenotype of IL-21-producing CD8 + T cells. (A) PBMCs from HCs were stimulated in the absence or presence of CD3/28 beads for 72 h and then IL-21 production from CD4 + and CD8 + T cells was analyzed by intracellular staining (added PMA and Ionomycin for the last 6 h). Left panels are representative data on IL-21 production from CD4 + and CD8 + T cells stimulated with CD3/28 and the right graph summarizes the results (N=4). (B) PBMCs from HCs were stimulated under the same condition as (A) , and IFN-γ and IL-17 production from IL-21 + and IL-21 - CD8 + T cells was analyzed by intracellular staining. (C) PBMCs from HCs were stimulated under the same condition as (A) , and expression levels of PD-1, ICOS, CD95, CD69, CD28 and HLA-DR in IL-21 + and IL-21 - CD8 + T cells were analyzed by flow cytometry. The graph summarizes the results (N=4). * P

    Techniques Used: Staining, Expressing, Flow Cytometry

    Our hypothetical model in this study. Both IL-12 and IL-21 play a pivotal role in the generation of IL-21-producing PD-1 + CD8 + T cells. The provision of IL-12 and IL-21 to CD8 + T cells in this case could be from DCs and Tph cells, respectively. Memory PD-1 hi CD8 + T cells, in concert with Tph cells, play a role in promoting plasmablast differentiation and antibody production at the inflammatory sites such as RA synovium.
    Figure Legend Snippet: Our hypothetical model in this study. Both IL-12 and IL-21 play a pivotal role in the generation of IL-21-producing PD-1 + CD8 + T cells. The provision of IL-12 and IL-21 to CD8 + T cells in this case could be from DCs and Tph cells, respectively. Memory PD-1 hi CD8 + T cells, in concert with Tph cells, play a role in promoting plasmablast differentiation and antibody production at the inflammatory sites such as RA synovium.

    Techniques Used:

    34) Product Images from "P2X7 receptor restrains pathogenic Tfh cell generation in systemic lupus erythematosus"

    Article Title: P2X7 receptor restrains pathogenic Tfh cell generation in systemic lupus erythematosus

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20171976

    Increased PIL severity and IFN-γ secretion by ICOS + PSGL-1 lo/− CD4 T cells in mice with conditional deletion of P2rx7 in T cells. (A) Spleen weight of untreated CD4-Cre P2rx7 WT/WT ( n = 5), CD4-Cre P2rx7 fl/fl ( n = 12), pristane-treated CD4-Cre P2rx7 WT/WT ( n = 21), and CD4-Cre P2rx7 fl/fl ( n = 15) mice. (B) Proteinuria score. (C) Serum IgG concentration. (D) Semiquantitative detection of self-reactive IgG by ELISA (QUANTA-Lite ANA) in the same mice. ( E–G ) Absolute numbers of IgG-secreting cells (E) and frequencies and absolute numbers of CD19 + B220 + PNA + Fas + (F) and TCRβ + CD4 + CXCR5 + ICOS + (G) cells in the spleen of the same mice. (H) Representative contour plots for ICOS and PSGL-1 staining among CD4 + T cells from spleen and frequency of IFN-γ secreting cells within the ICOS + PSGL-1 lo/− CD4 + T cells of the same mice. Each dot represents an individual mouse; median or mean ± SEM is shown. Two-tailed Mann–Whitney U test. (I) Representative contour plots for ICOS and PSGL-1 staining among CD4 + T cells from the spleen of pristane-treated WT and P2rx7 −/− mice, and frequency of IFN-γ–secreting cells within the indicated subsets of untreated and treated mice. Statistics from three independent experiments are shown. Mean ± SEM (untreated mice, n ≥ 4; treated mice, n ≥ 8). (J) Representative contour plots for intracellular staining of IL-21, IL-17, and IFN-γ in CD4 + T cells from spleen of pristane-treated WT, P2rx7 −/− , and Icos −/− P2rx7 −/− mice and statistics of frequencies. Mean ± SEM ( n = 2 independent experiments with at least five mice). Student’s t test. *, P
    Figure Legend Snippet: Increased PIL severity and IFN-γ secretion by ICOS + PSGL-1 lo/− CD4 T cells in mice with conditional deletion of P2rx7 in T cells. (A) Spleen weight of untreated CD4-Cre P2rx7 WT/WT ( n = 5), CD4-Cre P2rx7 fl/fl ( n = 12), pristane-treated CD4-Cre P2rx7 WT/WT ( n = 21), and CD4-Cre P2rx7 fl/fl ( n = 15) mice. (B) Proteinuria score. (C) Serum IgG concentration. (D) Semiquantitative detection of self-reactive IgG by ELISA (QUANTA-Lite ANA) in the same mice. ( E–G ) Absolute numbers of IgG-secreting cells (E) and frequencies and absolute numbers of CD19 + B220 + PNA + Fas + (F) and TCRβ + CD4 + CXCR5 + ICOS + (G) cells in the spleen of the same mice. (H) Representative contour plots for ICOS and PSGL-1 staining among CD4 + T cells from spleen and frequency of IFN-γ secreting cells within the ICOS + PSGL-1 lo/− CD4 + T cells of the same mice. Each dot represents an individual mouse; median or mean ± SEM is shown. Two-tailed Mann–Whitney U test. (I) Representative contour plots for ICOS and PSGL-1 staining among CD4 + T cells from the spleen of pristane-treated WT and P2rx7 −/− mice, and frequency of IFN-γ–secreting cells within the indicated subsets of untreated and treated mice. Statistics from three independent experiments are shown. Mean ± SEM (untreated mice, n ≥ 4; treated mice, n ≥ 8). (J) Representative contour plots for intracellular staining of IL-21, IL-17, and IFN-γ in CD4 + T cells from spleen of pristane-treated WT, P2rx7 −/− , and Icos −/− P2rx7 −/− mice and statistics of frequencies. Mean ± SEM ( n = 2 independent experiments with at least five mice). Student’s t test. *, P

    Techniques Used: Mouse Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Two Tailed Test, MANN-WHITNEY

    P2X7-mediated control of PSGL-1 down-regulation and IFN-γ secretion in pristane-treated mice . (A) Frequency of PNA + Fas + GC splenic B cells (untreated WT, n = 3; P2rx7 −/− , n = 3; and Icos −/− P2rx7 −/− , n = 3; pristane-treated WT, n = 9; P2rx7 −/− , n = 9; and Icos −/− P2rx7 −/− , n = 5). (B) Serum ANA IgG detection by ELISA (MRL/ lpr , n = 2; untreated WT, n = 4; P2rx7 −/− , n = 4; and Icos −/− P2rx7 −/− , n = 5; pristane-treated WT, n = 9; P2rx7 −/− , n = 10; and Icos −/− P2rx7 −/− , n = 10). (C) Proteinuria score (untreated WT, n = 3; P2rx7 −/− , n = 3; and Icos −/− P2rx7 −/− , n = 4; pristane-treated WT, n = 8; P2rx7 −/− , n = 8; and Icos −/− P2rx7 −/− , n = 8) in the indicated mice at 33 wk after pristane injection. Each dot represents an individual mouse, and horizontal lines represent median values. (D) Representative contour plots for PSGL-1 and CD62L on splenic CD4 + T cells, frequency and absolute number (mean ± SEM) of PSGL1 lo/− CD62L − cells from untreated WT ( n = 12), P2rx7 −/− ( n = 10), Icos −/− P2rx7 −/− ( n = 5), treated WT ( n = 38), P2rx7 −/− ( n = 37), and Icos −/− P2rx7 −/− ( n = 8) mice. (E) Contour plots show representative intracellular staining for IL-21, IL-17, and IFN-γ on gated CD4 + ICOS + PSGL1 lo/− cells from spleens of treated WT and P2rx7 −/− mice. Statistics from three independent experiments are shown (mean ± SEM, untreated mice, n ≥ 4; treated mice, n ≥ 8). Two-tailed Mann–Whitney U test. *, P
    Figure Legend Snippet: P2X7-mediated control of PSGL-1 down-regulation and IFN-γ secretion in pristane-treated mice . (A) Frequency of PNA + Fas + GC splenic B cells (untreated WT, n = 3; P2rx7 −/− , n = 3; and Icos −/− P2rx7 −/− , n = 3; pristane-treated WT, n = 9; P2rx7 −/− , n = 9; and Icos −/− P2rx7 −/− , n = 5). (B) Serum ANA IgG detection by ELISA (MRL/ lpr , n = 2; untreated WT, n = 4; P2rx7 −/− , n = 4; and Icos −/− P2rx7 −/− , n = 5; pristane-treated WT, n = 9; P2rx7 −/− , n = 10; and Icos −/− P2rx7 −/− , n = 10). (C) Proteinuria score (untreated WT, n = 3; P2rx7 −/− , n = 3; and Icos −/− P2rx7 −/− , n = 4; pristane-treated WT, n = 8; P2rx7 −/− , n = 8; and Icos −/− P2rx7 −/− , n = 8) in the indicated mice at 33 wk after pristane injection. Each dot represents an individual mouse, and horizontal lines represent median values. (D) Representative contour plots for PSGL-1 and CD62L on splenic CD4 + T cells, frequency and absolute number (mean ± SEM) of PSGL1 lo/− CD62L − cells from untreated WT ( n = 12), P2rx7 −/− ( n = 10), Icos −/− P2rx7 −/− ( n = 5), treated WT ( n = 38), P2rx7 −/− ( n = 37), and Icos −/− P2rx7 −/− ( n = 8) mice. (E) Contour plots show representative intracellular staining for IL-21, IL-17, and IFN-γ on gated CD4 + ICOS + PSGL1 lo/− cells from spleens of treated WT and P2rx7 −/− mice. Statistics from three independent experiments are shown (mean ± SEM, untreated mice, n ≥ 4; treated mice, n ≥ 8). Two-tailed Mann–Whitney U test. *, P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Injection, Staining, Two Tailed Test, MANN-WHITNEY

    35) Product Images from "Interleukin-21 differentially affects human natural killer cell subsets"

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets

    Journal:

    doi: 10.1111/j.1365-2567.2007.02675.x

    FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5
    Figure Legend Snippet: FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5

    Techniques Used: FACS

    IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)
    Figure Legend Snippet: IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)

    Techniques Used:

    Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.
    Figure Legend Snippet: Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.

    Techniques Used: Activation Assay

    Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open
    Figure Legend Snippet: Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open

    Techniques Used: Activation Assay, Flow Cytometry

    36) Product Images from "Preclinical Efficacy and Anti-Inflammatory Mechanisms of Action of the Bruton Tyrosine Kinase Inhibitor Rilzabrutinib for Immune-Mediated Disease"

    Article Title: Preclinical Efficacy and Anti-Inflammatory Mechanisms of Action of the Bruton Tyrosine Kinase Inhibitor Rilzabrutinib for Immune-Mediated Disease

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.2001130

    Cellular characterization of rilzabrutinib by evaluating inhibition of B cell activation and in vitro IgG and IgM Ab production. ( A ) Inhibition of B cell activation as measured by anti-IgM–induced CD69 expression on CD20 + B cells from human whole blood treated with increasing concentrations of rilzabrutinib, as measured by flow cytometry ( n = 4). ( B ) Inhibition of IgG and IgM production. Rilzabrutinib-treated enriched human B cells were stimulated with CpG ( n = 4), TNP-LPS ( n = 4–6) or anti-CD40 + IL-21 ( n = 2–7) for 7 d. Measurement of IgG and IgM showed that both T-dependent and T-independent Ab production were inhibited by rilzabrutinib. Curves are representative experiments. Table provides IC 50 profiles of IgG and IgM production in rilzabrutinib-treated B cells.
    Figure Legend Snippet: Cellular characterization of rilzabrutinib by evaluating inhibition of B cell activation and in vitro IgG and IgM Ab production. ( A ) Inhibition of B cell activation as measured by anti-IgM–induced CD69 expression on CD20 + B cells from human whole blood treated with increasing concentrations of rilzabrutinib, as measured by flow cytometry ( n = 4). ( B ) Inhibition of IgG and IgM production. Rilzabrutinib-treated enriched human B cells were stimulated with CpG ( n = 4), TNP-LPS ( n = 4–6) or anti-CD40 + IL-21 ( n = 2–7) for 7 d. Measurement of IgG and IgM showed that both T-dependent and T-independent Ab production were inhibited by rilzabrutinib. Curves are representative experiments. Table provides IC 50 profiles of IgG and IgM production in rilzabrutinib-treated B cells.

    Techniques Used: Inhibition, Activation Assay, In Vitro, Expressing, Flow Cytometry

    37) Product Images from "Exploratory analysis of circulating cytokines in patients with metastatic breast cancer treated with eribulin: the TRANSERI-GONO (Gruppo Oncologico del Nord Ovest) study"

    Article Title: Exploratory analysis of circulating cytokines in patients with metastatic breast cancer treated with eribulin: the TRANSERI-GONO (Gruppo Oncologico del Nord Ovest) study

    Journal: ESMO Open

    doi: 10.1136/esmoopen-2020-000876

    Cox regression model for TGF-β, IL-8 and IL-21 at T 1 divided in two groups. Group 1: patients with cytokine levels above the median. Group 2: patients with cytokine levels below or equal to the median. IL, interleukin; OS, overall survival; PFS, progression free survival; TGF-β, transforming growth factor β.
    Figure Legend Snippet: Cox regression model for TGF-β, IL-8 and IL-21 at T 1 divided in two groups. Group 1: patients with cytokine levels above the median. Group 2: patients with cytokine levels below or equal to the median. IL, interleukin; OS, overall survival; PFS, progression free survival; TGF-β, transforming growth factor β.

    Techniques Used:

    Cox regression model for TGF-β, IL-21 and IL-8 comparing T 0 with T 1 . Group 1: patients with T 1 > T 0 . Group 2: patients with T 1 ≤ T 0 . IL, interleukin; OS, overall survival. PFS, progression free survival;TGF, transforming growth factor.
    Figure Legend Snippet: Cox regression model for TGF-β, IL-21 and IL-8 comparing T 0 with T 1 . Group 1: patients with T 1 > T 0 . Group 2: patients with T 1 ≤ T 0 . IL, interleukin; OS, overall survival. PFS, progression free survival;TGF, transforming growth factor.

    Techniques Used:

    38) Product Images from "Interleukin-21 differentially affects human natural killer cell subsets"

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets

    Journal:

    doi: 10.1111/j.1365-2567.2007.02675.x

    FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5
    Figure Legend Snippet: FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5

    Techniques Used: FACS

    IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)
    Figure Legend Snippet: IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr 51 Cr-release assays; lytic units (LU 20 /10 7 cells)

    Techniques Used:

    Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.
    Figure Legend Snippet: Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [ 3 H]thymidine assay. Mean values of seven assays ±SEM are shown.

    Techniques Used: Activation Assay

    Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open
    Figure Legend Snippet: Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors ( n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56 dim (open

    Techniques Used: Activation Assay, Flow Cytometry

    39) Product Images from "Reduced immune responses to hepatitis B primary vaccination in obese individuals with nonalcoholic fatty liver disease (NAFLD)"

    Article Title: Reduced immune responses to hepatitis B primary vaccination in obese individuals with nonalcoholic fatty liver disease (NAFLD)

    Journal: NPJ Vaccines

    doi: 10.1038/s41541-020-00266-4

    Peripheral follicular helper T (pTFH) cell analysis. A Serum CXCL13 B serum IL-21 levels in pg/mL at baseline and post vaccination in 31 NAFLD patients. C Spearman’s rank correlation between baseline CXCL13 and anti-HBs levels in NAFLD, r = −0.5, p = 0.009. D Frequency of pTFH in PBMC among NAFLD patients. E HBsAg specific CXCL13 production by pTFH cocultured with B cells. Sorting experiments were carried out in N = 19/21 responders and N = 9 non/low responders. Data are presented as mean ± standard deviation. R normal and high responders, anti-HBs > 100 IU/L, NR/LR nonresponders and low responders, anti-HBs
    Figure Legend Snippet: Peripheral follicular helper T (pTFH) cell analysis. A Serum CXCL13 B serum IL-21 levels in pg/mL at baseline and post vaccination in 31 NAFLD patients. C Spearman’s rank correlation between baseline CXCL13 and anti-HBs levels in NAFLD, r = −0.5, p = 0.009. D Frequency of pTFH in PBMC among NAFLD patients. E HBsAg specific CXCL13 production by pTFH cocultured with B cells. Sorting experiments were carried out in N = 19/21 responders and N = 9 non/low responders. Data are presented as mean ± standard deviation. R normal and high responders, anti-HBs > 100 IU/L, NR/LR nonresponders and low responders, anti-HBs

    Techniques Used: Standard Deviation

    40) Product Images from "IBP (IRF-4 Binding Protein) inhibits IL-17 and IL-21 production by controlling IRF-4 function"

    Article Title: IBP (IRF-4 Binding Protein) inhibits IL-17 and IL-21 production by controlling IRF-4 function

    Journal:

    doi: 10.1016/j.immuni.2008.10.011

    Lack of IBP leads to aberrant production of IL-17 and IL-21 in vitro and in vivo . A. Naïve CD4 + T cells derived from 6 wks. old IBP +/+ DO11.10 (white bars) or IBP trap/trap DO11.10 (black bars) mice were cultured with IBP +/+ APCs pulsed with 1
    Figure Legend Snippet: Lack of IBP leads to aberrant production of IL-17 and IL-21 in vitro and in vivo . A. Naïve CD4 + T cells derived from 6 wks. old IBP +/+ DO11.10 (white bars) or IBP trap/trap DO11.10 (black bars) mice were cultured with IBP +/+ APCs pulsed with 1

    Techniques Used: In Vitro, In Vivo, Derivative Assay, Mouse Assay, Cell Culture

    The deregulated IL-17 and IL-21 production observed in the absence of IBP is dependent on IRF-4 . A. IL-17 production by wt, IBP trap/trap , IRF-4 −/− , and IRF-4 −/− IBP trap/trap CD4 + T cells. Purified naïve CD4 + T cells
    Figure Legend Snippet: The deregulated IL-17 and IL-21 production observed in the absence of IBP is dependent on IRF-4 . A. IL-17 production by wt, IBP trap/trap , IRF-4 −/− , and IRF-4 −/− IBP trap/trap CD4 + T cells. Purified naïve CD4 + T cells

    Techniques Used: Purification

    The absence of IBP leads to enhanced targeting of the IL-17 and IL-21 regulatory regions . A. IBP can be found within the nuclear compartment. Purified CD4 + T cells were either left unstimulated or stimulated with anti-CD3 and anti-CD28 Abs for 48 hrs.
    Figure Legend Snippet: The absence of IBP leads to enhanced targeting of the IL-17 and IL-21 regulatory regions . A. IBP can be found within the nuclear compartment. Purified CD4 + T cells were either left unstimulated or stimulated with anti-CD3 and anti-CD28 Abs for 48 hrs.

    Techniques Used: Purification

    IRF-4 is a critical regulator of both IL-17 and IL-21 production. A. IL-17 production by wt and IRF-4 −/− CD4 + T cells. Purified naïve CD4 + T cells were stimulated under either TH0 (white bars) or TH17 (black bars) conditions for
    Figure Legend Snippet: IRF-4 is a critical regulator of both IL-17 and IL-21 production. A. IL-17 production by wt and IRF-4 −/− CD4 + T cells. Purified naïve CD4 + T cells were stimulated under either TH0 (white bars) or TH17 (black bars) conditions for

    Techniques Used: Purification

    Related Articles

    other:

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets
    Article Snippet: PBMCs (3 × 106 /ml) of seven different donors were stimulated with IL-2 and/or IL-21 as indicated above.

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets
    Article Snippet: In combination, IL-21 and IL-2 caused a more than doubling of the lytic units of both subsets as compared to the respective medium controls.

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets
    Article Snippet: Thus, coadministration of IL-21 and low-dose IL-2 could be a potent cancer therapy by inducing both an immediate innate antitumour response by NK cells and a subsequent long lasting immunity mediated by tumour-specific CD8+ T cells.

    Article Title: Exploratory analysis of circulating cytokines in patients with metastatic breast cancer treated with eribulin: the TRANSERI-GONO (Gruppo Oncologico del Nord Ovest) study
    Article Snippet: Therefore, our findings suggest that the ability of the ‘context’ to switch the role of IL-21 from positive to negative and vice versa might be driven by TGF-β.

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets
    Article Snippet: The PBMCs activated with IL-2 and/or IL-21 were analysed for phosphorylation of STAT1, STAT3 and STAT5.

    Article Title: T Follicular Helper Cells restricted by IRF8 contribute to T Cell-Mediated Inflammation
    Article Snippet: Early studies revealed that IL-21 is markedly overproduced in inflamed guts of patients with IBD compared to non-inflamed controls .

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets
    Article Snippet: We argue that IL-21 and/or IL-2, respectively, mediate the formation of particular dimers, which are responsible for differential regulation of the functions determined in CD56dim and CD56bright NK cells.

    Incubation:

    Article Title: Interleukin-21 differentially affects human natural killer cell subsets
    Article Snippet: This could be caused by the observed positive effect of IL-21 on CD25 expression, thus increasing the number of high-affinity IL-2 receptor complexes. .. CD56dim NK cells exhibited a low proliferative response towards IL-21 and IL-2 but exerted increased cytotoxicity when incubated with IL-21 alone. .. This is in line with a study demonstrating that the cytokine augments the cytotoxicity of human NK cells in vitro .

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    R&D Systems il 21r
    IL-2/IL-21 and upregulation of <t>IL-21R</t> expression replace CD4 + T cell help of CD8 + T cell expansion in vitro. ( A ) IL-21R expression on CD8 + T cells stimulated with aAPC/mOKT3 in the presence or absence of CD4 + T cells was studied by flow cytometry. On the left, histogram plots for 1 donor is shown and, on the right, IL-21R expression on day 4 is displayed for 5 donors. ( B ) IL-21R expression on CD8 + T cells ectopically transduced with mock or IL-21R is shown (left). Expansion of transduced CD8 + T cells stimulated twice by aAPC/mOKT3 with or without IL-21 is compared (right). Percent expansion was calculated by dividing the number of expanded transduced CD8 + T cells by that of CD8 + T cells stimulated in the presence of CD4 + T cells. Values indicate mean of four independent experiments; error bars show s.d. * P
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    Cytokine synergy of TGF-β3 and interleukin-10 <t>(IL-10)</t> inhibits mammalian target of rapamycin complex 1 (mTORC1) activity in lipopolysaccharide (LPS)-stimulated B cells. (A) The seven most related cellular functions and canonical pathways in genes of cluster 6 in Figure 4 A were analyzed by in Ingenuity Pathway Analysis (IPA) software. (B) Heatmap visualization of activation z -score ratios of LPS-stimulated B cells to LPS-stimulated B cells with IL-10/TGF-β3 less than −3.1 calculated by the upstream regulator analysis in IPA software. All of the 1,895 differentially expressed genes depicted in Figure 4 A were utilized in this analysis. (C) Representative western blot analyses of total and phosphorylated protein levels in LPS-stimulated B cells treated either with TGF-β3 and/or IL-10 for 24–48 h. (D) Flow cytometric (FCM) quantification of phosphorylated S6RP at Ser235/236 and 4E-BP1 at Thr37/46 in LPS-stimulated B cells either with or without TGF-β3 and/or IL-10 for 72 h ( n = 3). (E) Total IgG antibody titers in the supernatants of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 2 µM MHY1485 for 7 days quantified by ELISA ( n = 3). Data are representative of more than two independent experiments in (C–E) . (F) FCM quantification of phosphorylated S6RP at Ser235/236 in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). P
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    IL-2/IL-21 and upregulation of IL-21R expression replace CD4 + T cell help of CD8 + T cell expansion in vitro. ( A ) IL-21R expression on CD8 + T cells stimulated with aAPC/mOKT3 in the presence or absence of CD4 + T cells was studied by flow cytometry. On the left, histogram plots for 1 donor is shown and, on the right, IL-21R expression on day 4 is displayed for 5 donors. ( B ) IL-21R expression on CD8 + T cells ectopically transduced with mock or IL-21R is shown (left). Expansion of transduced CD8 + T cells stimulated twice by aAPC/mOKT3 with or without IL-21 is compared (right). Percent expansion was calculated by dividing the number of expanded transduced CD8 + T cells by that of CD8 + T cells stimulated in the presence of CD4 + T cells. Values indicate mean of four independent experiments; error bars show s.d. * P

    Journal: PLoS ONE

    Article Title: Ex Vivo Expansion of Human CD8+ T Cells Using Autologous CD4+ T Cell Help

    doi: 10.1371/journal.pone.0030229

    Figure Lengend Snippet: IL-2/IL-21 and upregulation of IL-21R expression replace CD4 + T cell help of CD8 + T cell expansion in vitro. ( A ) IL-21R expression on CD8 + T cells stimulated with aAPC/mOKT3 in the presence or absence of CD4 + T cells was studied by flow cytometry. On the left, histogram plots for 1 donor is shown and, on the right, IL-21R expression on day 4 is displayed for 5 donors. ( B ) IL-21R expression on CD8 + T cells ectopically transduced with mock or IL-21R is shown (left). Expansion of transduced CD8 + T cells stimulated twice by aAPC/mOKT3 with or without IL-21 is compared (right). Percent expansion was calculated by dividing the number of expanded transduced CD8 + T cells by that of CD8 + T cells stimulated in the presence of CD4 + T cells. Values indicate mean of four independent experiments; error bars show s.d. * P

    Article Snippet: Analysis of cultured T cells Flow cytometry analysis was performed using mAbs for the following antigens: CD4, CD8, CD25, CD28, CD56, CD62L, and IL-2Rβ (Coulter, CA); CD40 ligand, CD80, IL-7Rα, OX40, OX40 ligand, and 4-1BB (BD Biosciences, CA); CD27, CD45RA, CD45RO and CD83 (Invitrogen, CA); CCR4 and CCR7 (R & D Systems, MN); ICOS, NKG2D, and PD-1 (eBioscience, CA); CD38, Foxp3, HLA-DR, and 4-1BB ligand (Biolegend, CA); CD40 and CD70 (Ancell, MN); IL-21R (R & D Systems, MN; or BD Biosciences, CA).

    Techniques: Expressing, In Vitro, Flow Cytometry, Cytometry, Transduction

    Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) inhibits mammalian target of rapamycin complex 1 (mTORC1) activity in lipopolysaccharide (LPS)-stimulated B cells. (A) The seven most related cellular functions and canonical pathways in genes of cluster 6 in Figure 4 A were analyzed by in Ingenuity Pathway Analysis (IPA) software. (B) Heatmap visualization of activation z -score ratios of LPS-stimulated B cells to LPS-stimulated B cells with IL-10/TGF-β3 less than −3.1 calculated by the upstream regulator analysis in IPA software. All of the 1,895 differentially expressed genes depicted in Figure 4 A were utilized in this analysis. (C) Representative western blot analyses of total and phosphorylated protein levels in LPS-stimulated B cells treated either with TGF-β3 and/or IL-10 for 24–48 h. (D) Flow cytometric (FCM) quantification of phosphorylated S6RP at Ser235/236 and 4E-BP1 at Thr37/46 in LPS-stimulated B cells either with or without TGF-β3 and/or IL-10 for 72 h ( n = 3). (E) Total IgG antibody titers in the supernatants of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 2 µM MHY1485 for 7 days quantified by ELISA ( n = 3). Data are representative of more than two independent experiments in (C–E) . (F) FCM quantification of phosphorylated S6RP at Ser235/236 in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). P

    Journal: Frontiers in Immunology

    Article Title: Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals

    doi: 10.3389/fimmu.2018.01364

    Figure Lengend Snippet: Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) inhibits mammalian target of rapamycin complex 1 (mTORC1) activity in lipopolysaccharide (LPS)-stimulated B cells. (A) The seven most related cellular functions and canonical pathways in genes of cluster 6 in Figure 4 A were analyzed by in Ingenuity Pathway Analysis (IPA) software. (B) Heatmap visualization of activation z -score ratios of LPS-stimulated B cells to LPS-stimulated B cells with IL-10/TGF-β3 less than −3.1 calculated by the upstream regulator analysis in IPA software. All of the 1,895 differentially expressed genes depicted in Figure 4 A were utilized in this analysis. (C) Representative western blot analyses of total and phosphorylated protein levels in LPS-stimulated B cells treated either with TGF-β3 and/or IL-10 for 24–48 h. (D) Flow cytometric (FCM) quantification of phosphorylated S6RP at Ser235/236 and 4E-BP1 at Thr37/46 in LPS-stimulated B cells either with or without TGF-β3 and/or IL-10 for 72 h ( n = 3). (E) Total IgG antibody titers in the supernatants of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 2 µM MHY1485 for 7 days quantified by ELISA ( n = 3). Data are representative of more than two independent experiments in (C–E) . (F) FCM quantification of phosphorylated S6RP at Ser235/236 in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). P

    Article Snippet: In some experiments, 1 µg/ml anti-IL-10 antibody (R & D Systems) was used.

    Techniques: Activity Assay, Indirect Immunoperoxidase Assay, Software, Activation Assay, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Mouse Assay

    TGF-β and interleukin-10 (IL-10) synergistically suppress toll-like receptor-mediated humoral immune responses. (A) Histogram plots and percentages of CFSE-labeled B220 + B cells stimulated by 10 µg/ml lipopolysaccharides (LPS) either with or without 2 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 3 days ( n = 3). (B) Total IgG antibody titers in the supernatants of 3 µg/ml LPS-stimulated B cells either with 10 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 7 days were quantified by ELISA ( n = 3). (C) B cells were cultured under 200 ng/ml R848 and anti-IgM stimulation either with or without TGF-β1 or TGF-β3 for 7 days and total IgG antibody production was assessed by ELISA ( n = 3). (D) Total IgG antibody titer in the supernatants of B cells cultured with LPS and 25 ng/ml IL-6 (left) or 1 ng/ml IL-17 (right) either with TGF-β1 or TGF-β3 for 7 days ( n = 3). Data are representative of more than two independent experiments in (A–D) . (E) Serum anti-NP-bovine serum albumin (BSA) antibody titers from NP-LPS-immunized B6 mice treated with 300 µg anti-IL-10 antibody and 300 µg anti-TGF-β antibody were quantified by ELISA ( n = 9–10). (F,G) Anti-NP-BSA antibody titers from NP-KLH/CFA-immunized ( n = 11–16) (F) or NP-LPS-immunized ( n = 9–10) (G) mice with indicated pCAGGS plasmid vectors were quantified. P

    Journal: Frontiers in Immunology

    Article Title: Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals

    doi: 10.3389/fimmu.2018.01364

    Figure Lengend Snippet: TGF-β and interleukin-10 (IL-10) synergistically suppress toll-like receptor-mediated humoral immune responses. (A) Histogram plots and percentages of CFSE-labeled B220 + B cells stimulated by 10 µg/ml lipopolysaccharides (LPS) either with or without 2 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 3 days ( n = 3). (B) Total IgG antibody titers in the supernatants of 3 µg/ml LPS-stimulated B cells either with 10 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 7 days were quantified by ELISA ( n = 3). (C) B cells were cultured under 200 ng/ml R848 and anti-IgM stimulation either with or without TGF-β1 or TGF-β3 for 7 days and total IgG antibody production was assessed by ELISA ( n = 3). (D) Total IgG antibody titer in the supernatants of B cells cultured with LPS and 25 ng/ml IL-6 (left) or 1 ng/ml IL-17 (right) either with TGF-β1 or TGF-β3 for 7 days ( n = 3). Data are representative of more than two independent experiments in (A–D) . (E) Serum anti-NP-bovine serum albumin (BSA) antibody titers from NP-LPS-immunized B6 mice treated with 300 µg anti-IL-10 antibody and 300 µg anti-TGF-β antibody were quantified by ELISA ( n = 9–10). (F,G) Anti-NP-BSA antibody titers from NP-KLH/CFA-immunized ( n = 11–16) (F) or NP-LPS-immunized ( n = 9–10) (G) mice with indicated pCAGGS plasmid vectors were quantified. P

    Article Snippet: In some experiments, 1 µg/ml anti-IL-10 antibody (R & D Systems) was used.

    Techniques: Labeling, Enzyme-linked Immunosorbent Assay, Cell Culture, Mouse Assay, Plasmid Preparation

    TGF-β3 exerts therapeutic effects in mouse models of lupus under the presence of interleukin-10 (IL-10). (A) Indicated pCAGGS vectors were i.v. administered every 2 weeks to BALB/c mice treated with 1.25 mg imiquimod epicutaneously three times weekly. Serum anti-dsDNA antibody titers were quantified by ELISA ( n = 12). (B) Serum IL-10 levels in 8- to 16-week-old MRL/+ and MRL/ lpr mice were analyzed by ELISA ( n = 3). (C–G) MRL/ lpr mice treated with indicated pCAGGS vectors every 4 weeks were analyzed at an age of 21 weeks. Serum anti-dsDNA antibody levels (C) , gross appearances and weights of spleens (D) ( n = 12–18), and chronological proteinuria progression (E) ( n = 12–25). Flow cytometric quantification of CXCR5 + PD-1 + T FH cells (F) and B220 + CD138 + plasmablasts (G) ( n = 12–18). P

    Journal: Frontiers in Immunology

    Article Title: Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals

    doi: 10.3389/fimmu.2018.01364

    Figure Lengend Snippet: TGF-β3 exerts therapeutic effects in mouse models of lupus under the presence of interleukin-10 (IL-10). (A) Indicated pCAGGS vectors were i.v. administered every 2 weeks to BALB/c mice treated with 1.25 mg imiquimod epicutaneously three times weekly. Serum anti-dsDNA antibody titers were quantified by ELISA ( n = 12). (B) Serum IL-10 levels in 8- to 16-week-old MRL/+ and MRL/ lpr mice were analyzed by ELISA ( n = 3). (C–G) MRL/ lpr mice treated with indicated pCAGGS vectors every 4 weeks were analyzed at an age of 21 weeks. Serum anti-dsDNA antibody levels (C) , gross appearances and weights of spleens (D) ( n = 12–18), and chronological proteinuria progression (E) ( n = 12–25). Flow cytometric quantification of CXCR5 + PD-1 + T FH cells (F) and B220 + CD138 + plasmablasts (G) ( n = 12–18). P

    Article Snippet: In some experiments, 1 µg/ml anti-IL-10 antibody (R & D Systems) was used.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

    Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) modulate transcriptional programs in lipopolysaccharide (LPS)-stimulated B cells. (A) Heatmap visualization and hierarchical clustering analysis of differentially expressed genes of whole genome RNA-sequencing from B220 + 7-AAD − B cells stimulated by LPS with either TGF-β3 and/or IL-10 for 3 days. The clustered genes were subdivided into 16 categories labeled by different colors. (B,C) qRT-PCR analysis of genes downregulated (B) or upregulated (C) with TGF-β3 and IL-10 more than those in LPS-stimulated B cells ( n = 3). P

    Journal: Frontiers in Immunology

    Article Title: Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals

    doi: 10.3389/fimmu.2018.01364

    Figure Lengend Snippet: Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) modulate transcriptional programs in lipopolysaccharide (LPS)-stimulated B cells. (A) Heatmap visualization and hierarchical clustering analysis of differentially expressed genes of whole genome RNA-sequencing from B220 + 7-AAD − B cells stimulated by LPS with either TGF-β3 and/or IL-10 for 3 days. The clustered genes were subdivided into 16 categories labeled by different colors. (B,C) qRT-PCR analysis of genes downregulated (B) or upregulated (C) with TGF-β3 and IL-10 more than those in LPS-stimulated B cells ( n = 3). P

    Article Snippet: In some experiments, 1 µg/ml anti-IL-10 antibody (R & D Systems) was used.

    Techniques: RNA Sequencing Assay, Labeling, Quantitative RT-PCR

    Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) regulates metabolism in toll-like receptor-stimulated B cells. (A) Splenic B cells from LC3-green fluorescent protein (GFP) mice were stimulated by lipopolysaccharides (LPS) either with TGF-β3 and/or IL-10 for 3 days. GFP − LC3 + spots captured by fluorescence microscopy were counted manually ( n = 20). (B) Relative Prdm1 expression in LPS-stimulated B cells either with 1 mM 2-DG, 10 mM sodium pyruvate, 0.5 µM rotenone (Rot)/antimycin A (AA), or 10 µM M1/Mdivi1 analyzed by qRT-PCR ( n = 3). (C) Flow cytometric analysis of MitoTracker-stained, LPS-stimulated B cells with each cytokine cultured for 3 days. (D,E) Oxygen consumption rate (OCR) (D) and ECAR (E) of LPS-stimulated B cells with each cytokine measured by extracellular flux analyzer ( n = 3). (F,G) IgG antibody titers quantified by ELISA (F) and OCR measured by extracellular analyzer (G) of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 10 µM M1/Mdivi1 ( n = 3). (H) Mean fluorescence intensity (MFI) of MitoTracker Green in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). (I) Human B cells were stimulated by 2.5 µg/ml CpG-ODN, 1,000 U/ml IL-2, 10 ng/ml IL-6, and 0.5 µg/ml anti-CD40 with 10 pg/ml TGF-β3, 10 ng/ml IL-10, and/or 1 µg/ml anti-IL-10 Ab for 7 days ( n = 3). IgG2 antibody titers of the supernatants were quantified by ELISA. (J) OCR of CpG-ODN-stimulated human B cells with each cytokine for 3 days measured by extracellular flux analyzer ( n = 3). P

    Journal: Frontiers in Immunology

    Article Title: Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals

    doi: 10.3389/fimmu.2018.01364

    Figure Lengend Snippet: Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) regulates metabolism in toll-like receptor-stimulated B cells. (A) Splenic B cells from LC3-green fluorescent protein (GFP) mice were stimulated by lipopolysaccharides (LPS) either with TGF-β3 and/or IL-10 for 3 days. GFP − LC3 + spots captured by fluorescence microscopy were counted manually ( n = 20). (B) Relative Prdm1 expression in LPS-stimulated B cells either with 1 mM 2-DG, 10 mM sodium pyruvate, 0.5 µM rotenone (Rot)/antimycin A (AA), or 10 µM M1/Mdivi1 analyzed by qRT-PCR ( n = 3). (C) Flow cytometric analysis of MitoTracker-stained, LPS-stimulated B cells with each cytokine cultured for 3 days. (D,E) Oxygen consumption rate (OCR) (D) and ECAR (E) of LPS-stimulated B cells with each cytokine measured by extracellular flux analyzer ( n = 3). (F,G) IgG antibody titers quantified by ELISA (F) and OCR measured by extracellular analyzer (G) of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 10 µM M1/Mdivi1 ( n = 3). (H) Mean fluorescence intensity (MFI) of MitoTracker Green in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). (I) Human B cells were stimulated by 2.5 µg/ml CpG-ODN, 1,000 U/ml IL-2, 10 ng/ml IL-6, and 0.5 µg/ml anti-CD40 with 10 pg/ml TGF-β3, 10 ng/ml IL-10, and/or 1 µg/ml anti-IL-10 Ab for 7 days ( n = 3). IgG2 antibody titers of the supernatants were quantified by ELISA. (J) OCR of CpG-ODN-stimulated human B cells with each cytokine for 3 days measured by extracellular flux analyzer ( n = 3). P

    Article Snippet: In some experiments, 1 µg/ml anti-IL-10 antibody (R & D Systems) was used.

    Techniques: Mouse Assay, Fluorescence, Microscopy, Expressing, Quantitative RT-PCR, Flow Cytometry, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay

    TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml anti-CD40 and 10 µg/ml anti-IgM either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P

    Journal: Frontiers in Immunology

    Article Title: Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals

    doi: 10.3389/fimmu.2018.01364

    Figure Lengend Snippet: TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml anti-CD40 and 10 µg/ml anti-IgM either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P

    Article Snippet: In some experiments, 1 µg/ml anti-IL-10 antibody (R & D Systems) was used.

    Techniques: Activation Assay, Labeling, Fluorescence, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Expressing, Mouse Assay, Plasmid Preparation, Injection

    Comparison of therapeutic efficacy using Saa3 versus IL1E/IL6P promoter for disease-regulated Il1rn expression. ( a ) Promoter activity in transduced synovium of naive and arthritic C57Bl/6 mice. Knee joints were injected with 300 ng p24 gag equivalent lentivirus encoding PGK , Saa3, or IL1E/IL6P -luciferase. Seven days after transduction, arthritis was induced by intra-articular injection of 180 µg zymosan A. After 24 hours, luciferase activity was assessed ex vivo . Data are represented as individual relative luciferase activities; horizontal bars indicate the means per group. ( b ) Efficacy of transcriptionally targeted adenoviral vectors expressing Il1rn . NIH-3T3-5xNF-κB-Luc were transduced at a multiplicity of infection (MOI) of 10 with control vector (del) or adenovirus encoding CMV, Saa3 , and IL1E/IL6P -driven Il1rn . After 24 hours, cells were transduced at an MOI of 10 with control vector (del) or Ad5.CMV- Il1b . The day thereafter, IL-1β-induced NF-κB activation was assessed by luciferase assay. Data are represented as relative luciferase activities ± SEM ( n = 4), and differences were determined using analysis of variance with Dunnett's post-test. ** P

    Journal: Molecular Therapy

    Article Title: Computational Design and Application of Endogenous Promoters for Transcriptionally Targeted Gene Therapy for Rheumatoid Arthritis

    doi: 10.1038/mt.2009.182

    Figure Lengend Snippet: Comparison of therapeutic efficacy using Saa3 versus IL1E/IL6P promoter for disease-regulated Il1rn expression. ( a ) Promoter activity in transduced synovium of naive and arthritic C57Bl/6 mice. Knee joints were injected with 300 ng p24 gag equivalent lentivirus encoding PGK , Saa3, or IL1E/IL6P -luciferase. Seven days after transduction, arthritis was induced by intra-articular injection of 180 µg zymosan A. After 24 hours, luciferase activity was assessed ex vivo . Data are represented as individual relative luciferase activities; horizontal bars indicate the means per group. ( b ) Efficacy of transcriptionally targeted adenoviral vectors expressing Il1rn . NIH-3T3-5xNF-κB-Luc were transduced at a multiplicity of infection (MOI) of 10 with control vector (del) or adenovirus encoding CMV, Saa3 , and IL1E/IL6P -driven Il1rn . After 24 hours, cells were transduced at an MOI of 10 with control vector (del) or Ad5.CMV- Il1b . The day thereafter, IL-1β-induced NF-κB activation was assessed by luciferase assay. Data are represented as relative luciferase activities ± SEM ( n = 4), and differences were determined using analysis of variance with Dunnett's post-test. ** P

    Article Snippet: Cells were serum-starved (1% FCS) for 2 days and subsequently stimulated with recombinant human IL-1β (R & D Systems Europe, Oxford, UK), TNFα (Abcam, Cambridge, UK), or E. Coli lipopolysaccharide (Sigma) for indicated hours and subsequently lysed in ice-cold lysis buffer (0.5% NP-40, 1 mmol/l DTT, 1 mmol/l EDTA, 5 mmol/l MgCl2 , 100 mmol/l KCl, and 10 mmol/l Tris-HCl pH 7.5).

    Techniques: Expressing, Activity Assay, Mouse Assay, Injection, Luciferase, Transduction, Ex Vivo, Infection, Plasmid Preparation, Activation Assay

    Experimental verification of transcriptionally targeted vectors. ( a ) Dual luciferase assay of basal promoter activity in NIH-3T3 fibroblasts. Cells were co-transfected with 500 ng of transcriptional targeted vector and 50 ng of pRL-TK. Promoterless (empty) and cytomegalovirus-promoter vectors (black bars) served as negative and positive controls, respectively. Promoter activity is expressed as relative (firefly/Renilla) luciferase activity ± SEM ( n = 3). Kinetics of promoter induction by toll-like receptor 4 triggering in ( b ) murine RAW 264.7 macrophages and ( c ) NIH-3T3 fibroblasts. Two days after transduction with lentiviral vectors, cells were stimulated for indicated time points with lipopolysaccharide (50 ng/ml). Luciferase activities are represented as fold induction over unstimulated conditions. ( d ) Induction of promoter activity in rheumatoid arthritis synovial fibroblasts ( n = 4 donors). Fibroblasts were transduced with lentiviral vectors and stimulated for 24 hours with either recombinant human IL-1β (0.25 ng/ml) or hTNFα (1 ng/ml) alone, or the combination. Luciferase activities are expressed as fold induction ± SEM. LPS, lipopolysaccharide.

    Journal: Molecular Therapy

    Article Title: Computational Design and Application of Endogenous Promoters for Transcriptionally Targeted Gene Therapy for Rheumatoid Arthritis

    doi: 10.1038/mt.2009.182

    Figure Lengend Snippet: Experimental verification of transcriptionally targeted vectors. ( a ) Dual luciferase assay of basal promoter activity in NIH-3T3 fibroblasts. Cells were co-transfected with 500 ng of transcriptional targeted vector and 50 ng of pRL-TK. Promoterless (empty) and cytomegalovirus-promoter vectors (black bars) served as negative and positive controls, respectively. Promoter activity is expressed as relative (firefly/Renilla) luciferase activity ± SEM ( n = 3). Kinetics of promoter induction by toll-like receptor 4 triggering in ( b ) murine RAW 264.7 macrophages and ( c ) NIH-3T3 fibroblasts. Two days after transduction with lentiviral vectors, cells were stimulated for indicated time points with lipopolysaccharide (50 ng/ml). Luciferase activities are represented as fold induction over unstimulated conditions. ( d ) Induction of promoter activity in rheumatoid arthritis synovial fibroblasts ( n = 4 donors). Fibroblasts were transduced with lentiviral vectors and stimulated for 24 hours with either recombinant human IL-1β (0.25 ng/ml) or hTNFα (1 ng/ml) alone, or the combination. Luciferase activities are expressed as fold induction ± SEM. LPS, lipopolysaccharide.

    Article Snippet: Cells were serum-starved (1% FCS) for 2 days and subsequently stimulated with recombinant human IL-1β (R & D Systems Europe, Oxford, UK), TNFα (Abcam, Cambridge, UK), or E. Coli lipopolysaccharide (Sigma) for indicated hours and subsequently lysed in ice-cold lysis buffer (0.5% NP-40, 1 mmol/l DTT, 1 mmol/l EDTA, 5 mmol/l MgCl2 , 100 mmol/l KCl, and 10 mmol/l Tris-HCl pH 7.5).

    Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Transduction, Recombinant

    Co-culture with recombinant human VEGF shifts T-helper polarity from Th1 (IFN-γ) to Th2 (IL-4) predominance. PBMC (A) isolated from healthy donors were stimulated with PMA and ionomycin in the presence of brefeldin-A, permeabolized and intracellularly

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: Evidence of systemic Th2 driven chronic inflammation in patients with metastatic melanoma

    doi: 10.1158/1078-0432.CCR-08-1980

    Figure Lengend Snippet: Co-culture with recombinant human VEGF shifts T-helper polarity from Th1 (IFN-γ) to Th2 (IL-4) predominance. PBMC (A) isolated from healthy donors were stimulated with PMA and ionomycin in the presence of brefeldin-A, permeabolized and intracellularly

    Article Snippet: The following anti-human monoclonal antibodies were used for intracellular staining for flow cytometry: anti-IFNγ FITC, anti-IL-13 PE, anti-IL-4 PE (R and D Systems Minneapolis, MN) and anti-FoxP3 Alexaflour 488 (Biolegend San Diego, CA).

    Techniques: Co-Culture Assay, Recombinant, Isolation