anti cd28  (Thermo Fisher)


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    Name:
    CD28 Monoclonal Antibody CD28 2
    Description:
    CD28 Monoclonal Antibody for Flow
    Catalog Number:
    11-0289-41
    Price:
    None
    Category:
    Antibodies Secondary Detection Reagents
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    Structured Review

    Thermo Fisher anti cd28
    SOCS2 deficiency does not affect peripheral Foxp3 + Treg phenotype. ( A ) CD4 + CD25 + lymphocytes purified with magnetic beads from WT and Socs2 −/− mice were labeled with CFSE and stimulated with plate-bound anti-CD3 (2 μg/ml) and <t>anti-CD28</t>
    CD28 Monoclonal Antibody for Flow
    https://www.bioz.com/result/anti cd28/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti cd28 - by Bioz Stars, 2021-05
    86/100 stars

    Images

    1) Product Images from "Regulation of Foxp3+ Inducible Regulatory T Cell Stability by SOCS2"

    Article Title: Regulation of Foxp3+ Inducible Regulatory T Cell Stability by SOCS2

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1201396

    SOCS2 deficiency does not affect peripheral Foxp3 + Treg phenotype. ( A ) CD4 + CD25 + lymphocytes purified with magnetic beads from WT and Socs2 −/− mice were labeled with CFSE and stimulated with plate-bound anti-CD3 (2 μg/ml) and anti-CD28
    Figure Legend Snippet: SOCS2 deficiency does not affect peripheral Foxp3 + Treg phenotype. ( A ) CD4 + CD25 + lymphocytes purified with magnetic beads from WT and Socs2 −/− mice were labeled with CFSE and stimulated with plate-bound anti-CD3 (2 μg/ml) and anti-CD28

    Techniques Used: Purification, Magnetic Beads, Mouse Assay, Labeling

    IL-4 promotes Socs2 −/− iTreg instability. WT and SOCS2-deficient iTregs were sorted from Socs2 −/− × Foxp3 eGFP and Foxp3 eGFP mice and further stimulated with plate-bound anti-CD3 (2 μg/ml) and anti-CD28 (1
    Figure Legend Snippet: IL-4 promotes Socs2 −/− iTreg instability. WT and SOCS2-deficient iTregs were sorted from Socs2 −/− × Foxp3 eGFP and Foxp3 eGFP mice and further stimulated with plate-bound anti-CD3 (2 μg/ml) and anti-CD28 (1

    Techniques Used: Mouse Assay

    SOCS2 regulates IL-4 signaling in iTregs. Foxp3 − T cells were isolated from Socs2 −/− × Foxp3 eGFP and Foxp3 eGFP mice and cultured with plate-bound anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) in the presence
    Figure Legend Snippet: SOCS2 regulates IL-4 signaling in iTregs. Foxp3 − T cells were isolated from Socs2 −/− × Foxp3 eGFP and Foxp3 eGFP mice and cultured with plate-bound anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) in the presence

    Techniques Used: Isolation, Mouse Assay, Cell Culture

    2) Product Images from "Sialic Acid-Dependent Inhibition of T cells by Exosomal Ganglioside GD3 in Ovarian Tumor Microenvironments"

    Article Title: Sialic Acid-Dependent Inhibition of T cells by Exosomal Ganglioside GD3 in Ovarian Tumor Microenvironments

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1801041

    GD3 liposomes arrest activation of T cell through the TCR in a dose dependent manner. (A) NDPBL were either left unactivated or activated for 2h with immobilized antibodies to CD3 and CD28 with various doses of PC or GD3/PC on liposomes. Activation was determined by counting the number of CD3+ cells with nuclear NFκB using confocal microscopy. (B) Percentage inhibition is shown. (C) NDPBL were either left unactivated or activated for 2h with PMA and ionomycin with 1 mM GD3/PC on liposomes. Activation was determined by counting the number of CD3+ cells with nuclear NFκB using confocal microscopy. Percentage inhibition is shown in parentheses. Mean ± SEM; n =3. NS = Not significant (p > 0.05) Mean ± SEM; n =3. ** p ≤ 0.01
    Figure Legend Snippet: GD3 liposomes arrest activation of T cell through the TCR in a dose dependent manner. (A) NDPBL were either left unactivated or activated for 2h with immobilized antibodies to CD3 and CD28 with various doses of PC or GD3/PC on liposomes. Activation was determined by counting the number of CD3+ cells with nuclear NFκB using confocal microscopy. (B) Percentage inhibition is shown. (C) NDPBL were either left unactivated or activated for 2h with PMA and ionomycin with 1 mM GD3/PC on liposomes. Activation was determined by counting the number of CD3+ cells with nuclear NFκB using confocal microscopy. Percentage inhibition is shown in parentheses. Mean ± SEM; n =3. NS = Not significant (p > 0.05) Mean ± SEM; n =3. ** p ≤ 0.01

    Techniques Used: Activation Assay, Confocal Microscopy, Inhibition

    Sialidase treatment of exosomes knocks down T cell arrest. NDPBL were either left unactivated (Unact) or activated for 2h with immobilized antibodies to CD3 and CD28 in media only (Act) or in media with exosomes that were untreated (Exo) or treated with 0.8 U/mL sialidase (Exo + Sia). A control group was also included where the cells were activated in the presence of the same concentration of sialidase without any exosomes present (Act + Sia). Activation was determined by monitoring the upregulation of CD69 on live T cells by flow cytometry following overnight incubation. (A) Representative experiment shown. (B) Compiled data from 5 experiments is shown. Numbers in parentheses indicate % inhibition. There was a 42% knockdown of the T cell arrest. Mean ± SEM. n = 3. NS = Not significant (p > 0.05), **p ≤ 0.01.
    Figure Legend Snippet: Sialidase treatment of exosomes knocks down T cell arrest. NDPBL were either left unactivated (Unact) or activated for 2h with immobilized antibodies to CD3 and CD28 in media only (Act) or in media with exosomes that were untreated (Exo) or treated with 0.8 U/mL sialidase (Exo + Sia). A control group was also included where the cells were activated in the presence of the same concentration of sialidase without any exosomes present (Act + Sia). Activation was determined by monitoring the upregulation of CD69 on live T cells by flow cytometry following overnight incubation. (A) Representative experiment shown. (B) Compiled data from 5 experiments is shown. Numbers in parentheses indicate % inhibition. There was a 42% knockdown of the T cell arrest. Mean ± SEM. n = 3. NS = Not significant (p > 0.05), **p ≤ 0.01.

    Techniques Used: Activated Clotting Time Assay, Concentration Assay, Activation Assay, Flow Cytometry, Cytometry, Incubation, Inhibition

    Antibody-mediated blockade of ganglioside GD3 diminishes exosome-mediated immunosuppression. NDPBL were either left unactivated (Unact) or activated with immobilized antibodies to CD3 and CD28 in media without (Act) or with exosomes (Exo) derived from ovarian tumor ascites fluid in the presence or absence of 10 mg of anti-GD3 antibody. Activation was monitored by determining nuclear translocation of NFkB in CD3+ cells after 2 hours by confocal microscopy (A) or intracellular IFN-g or IL-2 expression in CD3+ cells after 6 hours by flow cytometry (B-E). Representative experiment shown for IFN-g (B) and IL-2 (D). Mean ± SEM from 3 independent experiments shown for IFN-g (C) and IL-2 (E). Percentage inhibition is shown in parentheses. * p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
    Figure Legend Snippet: Antibody-mediated blockade of ganglioside GD3 diminishes exosome-mediated immunosuppression. NDPBL were either left unactivated (Unact) or activated with immobilized antibodies to CD3 and CD28 in media without (Act) or with exosomes (Exo) derived from ovarian tumor ascites fluid in the presence or absence of 10 mg of anti-GD3 antibody. Activation was monitored by determining nuclear translocation of NFkB in CD3+ cells after 2 hours by confocal microscopy (A) or intracellular IFN-g or IL-2 expression in CD3+ cells after 6 hours by flow cytometry (B-E). Representative experiment shown for IFN-g (B) and IL-2 (D). Mean ± SEM from 3 independent experiments shown for IFN-g (C) and IL-2 (E). Percentage inhibition is shown in parentheses. * p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

    Techniques Used: Activated Clotting Time Assay, Derivative Assay, Activation Assay, Translocation Assay, Confocal Microscopy, Expressing, Flow Cytometry, Cytometry, Inhibition

    Immunodepletion of GD3+ exosomes diminishes exosome-mediated T cell arrest. (A) NDPBL were either left unactivated (Unact) or activated for 2h with immobilized antibodies to CD3 and CD28 in media only (Act) or in media with total exosomes (Exo) or exosomes subjected to depletion using either anti-GD3 antibody coupled magnetic beads (GD3 Dep) or isotype control coupled magnetic beads (Iso Dep). Activation was determined by monitoring the upregulation of CD69 on live T cells by flow cytometry following overnight incubation. Representative experiment shown. (B) Compiled data from 5 experiments is shown. Numbers in parentheses indicate % inhibition. (C) Calculated % knockdown of inhibition is shown. Mean ± SEM. NS = Not significant (p > 0.05). * p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
    Figure Legend Snippet: Immunodepletion of GD3+ exosomes diminishes exosome-mediated T cell arrest. (A) NDPBL were either left unactivated (Unact) or activated for 2h with immobilized antibodies to CD3 and CD28 in media only (Act) or in media with total exosomes (Exo) or exosomes subjected to depletion using either anti-GD3 antibody coupled magnetic beads (GD3 Dep) or isotype control coupled magnetic beads (Iso Dep). Activation was determined by monitoring the upregulation of CD69 on live T cells by flow cytometry following overnight incubation. Representative experiment shown. (B) Compiled data from 5 experiments is shown. Numbers in parentheses indicate % inhibition. (C) Calculated % knockdown of inhibition is shown. Mean ± SEM. NS = Not significant (p > 0.05). * p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

    Techniques Used: Activated Clotting Time Assay, Magnetic Beads, Activation Assay, Flow Cytometry, Cytometry, Incubation, Inhibition

    Sialidase treatment of GD3 liposomes knocks down T cell arrest. NDPBL were either left unactivated or activated for 2h with immobilized antibodies to CD3 and CD28 in media only or in the presence of 1 mM PC or GD3/PC on liposomes that were untreated or treated with 0.8 U/mL sialidase. A control group was also included where the cells were activated in the presence of the same concentration of sialidase without any liposomes present. Activation was determined by counting the number of CD3+ cells with nuclear NFκB using confocal microscopy. Compiled data from 3 experiments is shown. There was a 97% knockdown of the T cell arrest. Mean ± SEM. n = 3. NS = Not significant (p > 0.05), ***p ≤ 0.001.
    Figure Legend Snippet: Sialidase treatment of GD3 liposomes knocks down T cell arrest. NDPBL were either left unactivated or activated for 2h with immobilized antibodies to CD3 and CD28 in media only or in the presence of 1 mM PC or GD3/PC on liposomes that were untreated or treated with 0.8 U/mL sialidase. A control group was also included where the cells were activated in the presence of the same concentration of sialidase without any liposomes present. Activation was determined by counting the number of CD3+ cells with nuclear NFκB using confocal microscopy. Compiled data from 3 experiments is shown. There was a 97% knockdown of the T cell arrest. Mean ± SEM. n = 3. NS = Not significant (p > 0.05), ***p ≤ 0.001.

    Techniques Used: Concentration Assay, Activation Assay, Confocal Microscopy

    3) Product Images from "Hair regrowth in alopecia areata patients following Stem Cell Educator therapy"

    Article Title: Hair regrowth in alopecia areata patients following Stem Cell Educator therapy

    Journal: BMC Medicine

    doi: 10.1186/s12916-015-0331-6

    Immune modulation of Stem Cell Educator therapy. Patient lymphocytes were isolated from peripheral blood by Ficoll-Hypaque technique (γ = 1.077) for flow cytometric analyses in AA patients at baseline and 4 weeks after Stem Cell Educator therapy. Isotype-matched IgG served as control. Histologic examination of alopecic skin (C and D) . (A) Flow analysis of intracellular cytokines demonstrating differential effects on key interleukins at 4 weeks post-treatment. (B) Flow analysis of costimulating molecules demonstrating an increase of CD28 expression at 4 weeks post-treatment. Data are representative of preparations from all AA participants (n = 9) who received Stem Cell Educator therapy. (C) Fresh skin tissues were collected from the scalps via biopsy for immunohistochemistry testing in participants with alopecia totalis before treatment and 12 weeks after receiving Stem Cell Educator therapy. TGF-β1 staining surrounds a hair follicle of AA participants after receiving Stem Cell Educator therapy, with vertical section of hair follicle (top panels) and horizontal section of hair follicle. Isotype-matched mouse IgG 1 served as a negative control for TGF-β1 immunostaining in a serial hair follicle section. Representative images were obtained from five experiments. Scale bar, 25 μm. (D) H E staining of scalp tissues. Scale bar, 25 μm.
    Figure Legend Snippet: Immune modulation of Stem Cell Educator therapy. Patient lymphocytes were isolated from peripheral blood by Ficoll-Hypaque technique (γ = 1.077) for flow cytometric analyses in AA patients at baseline and 4 weeks after Stem Cell Educator therapy. Isotype-matched IgG served as control. Histologic examination of alopecic skin (C and D) . (A) Flow analysis of intracellular cytokines demonstrating differential effects on key interleukins at 4 weeks post-treatment. (B) Flow analysis of costimulating molecules demonstrating an increase of CD28 expression at 4 weeks post-treatment. Data are representative of preparations from all AA participants (n = 9) who received Stem Cell Educator therapy. (C) Fresh skin tissues were collected from the scalps via biopsy for immunohistochemistry testing in participants with alopecia totalis before treatment and 12 weeks after receiving Stem Cell Educator therapy. TGF-β1 staining surrounds a hair follicle of AA participants after receiving Stem Cell Educator therapy, with vertical section of hair follicle (top panels) and horizontal section of hair follicle. Isotype-matched mouse IgG 1 served as a negative control for TGF-β1 immunostaining in a serial hair follicle section. Representative images were obtained from five experiments. Scale bar, 25 μm. (D) H E staining of scalp tissues. Scale bar, 25 μm.

    Techniques Used: Isolation, Flow Cytometry, Expressing, Immunohistochemistry, Staining, Negative Control, Immunostaining

    Ex vivo studies of the immune modulation of CB-SCs on T cells. (A) Phase contrast microscopy shows the formation of cell clusters in human peripheral blood-derived lymphocytes that were activated with Dynabeads coupled with anti-CD3, anti-CD28, and anti-CD137 antibodies, 50 U/ml rIL-2, and 5 ng/ml rIL-7 for 5 days, in absence (left panel) and presence (right panel) of CB-SCs. Co-culture with lymphocytes (top right panel) served as control. Original magnification, × 100. (B) Cell proliferation was analyzed with CellTrace™ CFSE Cell Proliferation Kit. Untreated lymphocytes (left panel) served as control. (C) Multi-color flow cytometry on CD8 + NKG2D + T cells. The gated CD8 + NKG2D + T cells were further analyzed for the expression of coinhibitory molecules BTLA and PD-1. Isotype-matched IgG Abs served as control for flow cytometry. Mean fluorescence intensity (MFI) was presented for CD8 + NKG2D + BTLA + PD-1 + T cells. Flow cytometry dot plots and the percentage of each population were representative of three independent experiments with similar results.
    Figure Legend Snippet: Ex vivo studies of the immune modulation of CB-SCs on T cells. (A) Phase contrast microscopy shows the formation of cell clusters in human peripheral blood-derived lymphocytes that were activated with Dynabeads coupled with anti-CD3, anti-CD28, and anti-CD137 antibodies, 50 U/ml rIL-2, and 5 ng/ml rIL-7 for 5 days, in absence (left panel) and presence (right panel) of CB-SCs. Co-culture with lymphocytes (top right panel) served as control. Original magnification, × 100. (B) Cell proliferation was analyzed with CellTrace™ CFSE Cell Proliferation Kit. Untreated lymphocytes (left panel) served as control. (C) Multi-color flow cytometry on CD8 + NKG2D + T cells. The gated CD8 + NKG2D + T cells were further analyzed for the expression of coinhibitory molecules BTLA and PD-1. Isotype-matched IgG Abs served as control for flow cytometry. Mean fluorescence intensity (MFI) was presented for CD8 + NKG2D + BTLA + PD-1 + T cells. Flow cytometry dot plots and the percentage of each population were representative of three independent experiments with similar results.

    Techniques Used: Ex Vivo, Microscopy, Derivative Assay, Co-Culture Assay, Flow Cytometry, Cytometry, Expressing, Fluorescence

    4) Product Images from "Monocyte:T cell interaction regulates human T cell activation through a CD28/CD46 crosstalk"

    Article Title: Monocyte:T cell interaction regulates human T cell activation through a CD28/CD46 crosstalk

    Journal: Immunology and cell biology

    doi: 10.1038/icb.2015.42

    Concomitant CD28 and CD46 ligations modulate cytokine production Purified CD4 + T cells were left unstimulated (US), or were activated by immobilized anti-CD3 alone or in addition of anti-CD28 and/or anti-CD46 antibodies, as indicated. (a) Expression of CD46 was determined after 2 days. Dashed line = isotype control. The MFI obtained for CD46 are indicated. The production of IL-10 (b) and IFNγ (c) in the culture supernatants of activated purified T cells was determined by ELISA (n=14). (d) The IL-10:IFNγ ratio is also represented for costimulated T cells. (e) Proliferation was monitored by thymidine incorporation. Mean +/− SEM is represented.
    Figure Legend Snippet: Concomitant CD28 and CD46 ligations modulate cytokine production Purified CD4 + T cells were left unstimulated (US), or were activated by immobilized anti-CD3 alone or in addition of anti-CD28 and/or anti-CD46 antibodies, as indicated. (a) Expression of CD46 was determined after 2 days. Dashed line = isotype control. The MFI obtained for CD46 are indicated. The production of IL-10 (b) and IFNγ (c) in the culture supernatants of activated purified T cells was determined by ELISA (n=14). (d) The IL-10:IFNγ ratio is also represented for costimulated T cells. (e) Proliferation was monitored by thymidine incorporation. Mean +/− SEM is represented.

    Techniques Used: Purification, Expressing, Enzyme-linked Immunosorbent Assay

    MMP inhibition results into increased surface CD46 expression on CD28/CD46 costimulated T cells CD4 + T cells were activated with immobilized antibodies as indicated in presence or absence of GM6001, a broad metalloproteinase inhibitor (10 μM). After 2 days, the cell surface expression of CD46 was determined by flow cytometry. Dashed line = isotype control. (a) shows the representative plots obtained for one donor, and (b) shows the data obtained for the different donors (n=8; mean +/− SEM).
    Figure Legend Snippet: MMP inhibition results into increased surface CD46 expression on CD28/CD46 costimulated T cells CD4 + T cells were activated with immobilized antibodies as indicated in presence or absence of GM6001, a broad metalloproteinase inhibitor (10 μM). After 2 days, the cell surface expression of CD46 was determined by flow cytometry. Dashed line = isotype control. (a) shows the representative plots obtained for one donor, and (b) shows the data obtained for the different donors (n=8; mean +/− SEM).

    Techniques Used: Inhibition, Expressing, Flow Cytometry, Cytometry

    5) Product Images from "Cytokine production in patients with recurrent acute tonsillitis: analysis of tonsil samples and blood"

    Article Title: Cytokine production in patients with recurrent acute tonsillitis: analysis of tonsil samples and blood

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-69981-1

    Cytokine release (pg/ml) from purified tonsillar (− T) or peripheral (− B) T cells stimulated with anti-CD3/CD28 Abs immobilized on beads. Data are presented as box plot; circles stand for individual patients, *Significant difference (p
    Figure Legend Snippet: Cytokine release (pg/ml) from purified tonsillar (− T) or peripheral (− B) T cells stimulated with anti-CD3/CD28 Abs immobilized on beads. Data are presented as box plot; circles stand for individual patients, *Significant difference (p

    Techniques Used: Purification

    6) Product Images from "T-cell function is partially maintained in the absence of class IA phosphoinositide 3-kinase signaling"

    Article Title: T-cell function is partially maintained in the absence of class IA phosphoinositide 3-kinase signaling

    Journal: Blood

    doi: 10.1182/blood-2006-07-038620

    Defective TCR-dependent proliferation and cytokine production in r1ΔT/r2n T cells . (A) Shown 60 hours after stimulation, CFSE-labeled purified T cells were gated on CD4 or CD8, and the histograms were overlaid between genotypes. Above each FACS plot are numbers showing the percentage of cells in each division, with the rightmost number indicating the nondivided population. (B) Thymidine incorporation assays of stimulated r1f/r2n (Cre − ) or r1ΔT/r2n (Cre + ) purified T cells. Stimuli used were anti-CD3 alone (i), anti-CD3 plus IL-2 (ii), anti-CD3 plus anti-CD28 (iii), and PMA plus onomycin (iv). Data are shown as the mean ± SEM of triplicate wells from a representative experiment. (C) Cell-size profile of 60-hour–stimulated Cre − or Cre + cells. The mean forward scatter of live cells is included on each dot plot. (D) ELISA quantification of IL-2 and IFN-γ in supernatants of r1f/r2n (Cre − ) or r1ΔT/r2n (Cre + ) CD4 + T cells stimulated for 48 hours. Data are shown as the mean ± SEM of 3 independent experiments comparing Cre − and Cre + T cells. (E) Intracellular stain for p85α in anti-CD3/anti-CD28–stimulated Cre − and Cre + purified T cells. Dot plot of forward scatter versus p85α is shown. Data are representative of at least 3 independent experiments.
    Figure Legend Snippet: Defective TCR-dependent proliferation and cytokine production in r1ΔT/r2n T cells . (A) Shown 60 hours after stimulation, CFSE-labeled purified T cells were gated on CD4 or CD8, and the histograms were overlaid between genotypes. Above each FACS plot are numbers showing the percentage of cells in each division, with the rightmost number indicating the nondivided population. (B) Thymidine incorporation assays of stimulated r1f/r2n (Cre − ) or r1ΔT/r2n (Cre + ) purified T cells. Stimuli used were anti-CD3 alone (i), anti-CD3 plus IL-2 (ii), anti-CD3 plus anti-CD28 (iii), and PMA plus onomycin (iv). Data are shown as the mean ± SEM of triplicate wells from a representative experiment. (C) Cell-size profile of 60-hour–stimulated Cre − or Cre + cells. The mean forward scatter of live cells is included on each dot plot. (D) ELISA quantification of IL-2 and IFN-γ in supernatants of r1f/r2n (Cre − ) or r1ΔT/r2n (Cre + ) CD4 + T cells stimulated for 48 hours. Data are shown as the mean ± SEM of 3 independent experiments comparing Cre − and Cre + T cells. (E) Intracellular stain for p85α in anti-CD3/anti-CD28–stimulated Cre − and Cre + purified T cells. Dot plot of forward scatter versus p85α is shown. Data are representative of at least 3 independent experiments.

    Techniques Used: Labeling, Purification, FACS, Enzyme-linked Immunosorbent Assay, Staining

    7) Product Images from "Burkholderia pseudomallei OMVs derived from infection mimicking conditions elicit similar protection to a live-attenuated vaccine"

    Article Title: Burkholderia pseudomallei OMVs derived from infection mimicking conditions elicit similar protection to a live-attenuated vaccine

    Journal: NPJ Vaccines

    doi: 10.1038/s41541-021-00281-z

    Immunization with M9 OMVs induces memory CD4 and CD8 T-cell responses. a – b Antigen-specific CD44 + CD4 + T cell and c CD8 + T-cell responses were measured in the spleens of mice ( n = 3 per group) immunized with M9 OMV, live-attenuated vaccine, or sham (saline). Splenocytes were restimulated with a-CD28 and heat-inactivated Bp82 grown in M9 (antigen). PMA/ionomycin-treated and unstimulated groups were used as controls. Cytokine-producing T cells were assessed by intracellular cytokine staining and flow cytometry. * p
    Figure Legend Snippet: Immunization with M9 OMVs induces memory CD4 and CD8 T-cell responses. a – b Antigen-specific CD44 + CD4 + T cell and c CD8 + T-cell responses were measured in the spleens of mice ( n = 3 per group) immunized with M9 OMV, live-attenuated vaccine, or sham (saline). Splenocytes were restimulated with a-CD28 and heat-inactivated Bp82 grown in M9 (antigen). PMA/ionomycin-treated and unstimulated groups were used as controls. Cytokine-producing T cells were assessed by intracellular cytokine staining and flow cytometry. * p

    Techniques Used: Mouse Assay, Staining, Flow Cytometry

    8) Product Images from "Affinity-Bead-Mediated Enrichment of CD8+ Lymphocytes from Peripheral Blood Progenitor Cell Products Using Acoustophoresis"

    Article Title: Affinity-Bead-Mediated Enrichment of CD8+ Lymphocytes from Peripheral Blood Progenitor Cell Products Using Acoustophoresis

    Journal: Micromachines

    doi: 10.3390/mi7060101

    CD3/CD28-mediated T cell proliferation of acoustically and magnetically sorted CD8+ cytotoxic T cells. Cells were stimulated in the presence of anti-CD3/CD28 and proliferation was measured on days 2, 3 and 4 of culture using CFSE staining ( n = 3). For each day the relative number of proliferating cells ( a ) as well as the proliferation index, i.e. , the average number of cell divisions all responding cells have undergone, are presented ( b ).
    Figure Legend Snippet: CD3/CD28-mediated T cell proliferation of acoustically and magnetically sorted CD8+ cytotoxic T cells. Cells were stimulated in the presence of anti-CD3/CD28 and proliferation was measured on days 2, 3 and 4 of culture using CFSE staining ( n = 3). For each day the relative number of proliferating cells ( a ) as well as the proliferation index, i.e. , the average number of cell divisions all responding cells have undergone, are presented ( b ).

    Techniques Used: Staining

    9) Product Images from "Anti-TNF drives regulatory T cell expansion by paradoxically promoting membrane TNF–TNF-RII binding in rheumatoid arthritis"

    Article Title: Anti-TNF drives regulatory T cell expansion by paradoxically promoting membrane TNF–TNF-RII binding in rheumatoid arthritis

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20151255

    Adalimumab increased the number of functionally suppressive T reg cells in PBMCs from RA patients but not healthy controls. (A, B, D, and E) 2 × 10 5 PBMCs from untreated or disease-modifying antirheumatic drug (DMARD)–treated RA patients and healthy controls (HC) were cultured with 10 µg/ml adalimumab, adalimumab F(ab’)2 ( n = 11), etanercept, or isotype control for 3 d. (A and B) Representative flow cytometry and cumulative data indicating the percentage and absolute number of CD4 + FoxP3 + from RA patients divided into untreated ( n = 13), on disease-modifying antirheumatic drug ( n = 13), and healthy controls ( n = 8) at time zero (T0) and at day 3 of the culture. (C) Adalimumab dose–response curve from RA PBMCs exposed to increasing concentrations of adalimumab for 3 d. Cumulative data depict percentage of CD4 + FoxP3 + T reg cells ( n = 7). (D) Percentage and absolute number of CD4 + RORC + cells in the cultures at day 3 ( n = 10). (E) The ratio of CD4 + FoxP3 + /CD4 + RORC + in the PBMC cultures from RA patients at day 3 ( n = 10). (F) After exposure to either anti-TNF agent for 3 d, CD4 + T cells were stained for IL-17 cytokine production. Representative flow cytometry shows IL-17 production from CD4 + in RA ( n = 18). (G) Classical T reg cell suppression assay: adalimumab- or etanercept-treated RA T reg cells were purified (CD4 + CD25 + CD127 − ) and co-cultured for 5 d with monocytes and untreated responder T cells (CD4 + CD25 − CD127 + ) at a 1:3 ratio in the presence of 1 µg/ml anti-CD3/CD28. Cumulative data indicate percentage of suppression of IL-17 ( n = 6) and IFN-γ ( n = 6) production. Data in A and B were obtained from five experimental repeats. C was obtained from two experimental repeats. D, E, and G were obtained from three experimental repeats. F is representative of six independent experiments. *, P
    Figure Legend Snippet: Adalimumab increased the number of functionally suppressive T reg cells in PBMCs from RA patients but not healthy controls. (A, B, D, and E) 2 × 10 5 PBMCs from untreated or disease-modifying antirheumatic drug (DMARD)–treated RA patients and healthy controls (HC) were cultured with 10 µg/ml adalimumab, adalimumab F(ab’)2 ( n = 11), etanercept, or isotype control for 3 d. (A and B) Representative flow cytometry and cumulative data indicating the percentage and absolute number of CD4 + FoxP3 + from RA patients divided into untreated ( n = 13), on disease-modifying antirheumatic drug ( n = 13), and healthy controls ( n = 8) at time zero (T0) and at day 3 of the culture. (C) Adalimumab dose–response curve from RA PBMCs exposed to increasing concentrations of adalimumab for 3 d. Cumulative data depict percentage of CD4 + FoxP3 + T reg cells ( n = 7). (D) Percentage and absolute number of CD4 + RORC + cells in the cultures at day 3 ( n = 10). (E) The ratio of CD4 + FoxP3 + /CD4 + RORC + in the PBMC cultures from RA patients at day 3 ( n = 10). (F) After exposure to either anti-TNF agent for 3 d, CD4 + T cells were stained for IL-17 cytokine production. Representative flow cytometry shows IL-17 production from CD4 + in RA ( n = 18). (G) Classical T reg cell suppression assay: adalimumab- or etanercept-treated RA T reg cells were purified (CD4 + CD25 + CD127 − ) and co-cultured for 5 d with monocytes and untreated responder T cells (CD4 + CD25 − CD127 + ) at a 1:3 ratio in the presence of 1 µg/ml anti-CD3/CD28. Cumulative data indicate percentage of suppression of IL-17 ( n = 6) and IFN-γ ( n = 6) production. Data in A and B were obtained from five experimental repeats. C was obtained from two experimental repeats. D, E, and G were obtained from three experimental repeats. F is representative of six independent experiments. *, P

    Techniques Used: Cell Culture, Flow Cytometry, Cytometry, Staining, Suppression Assay, Purification

    10) Product Images from "Calcineurin B in CD4+ T Cells Prevents Autoimmune Colitis by Negatively Regulating the JAK/STAT Pathway"

    Article Title: Calcineurin B in CD4+ T Cells Prevents Autoimmune Colitis by Negatively Regulating the JAK/STAT Pathway

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00261

    Normal suppressive activity of Treg cells from Cnb1 CD4 mice. (A) Expression patterns of the Treg-cell markers CD152 and CD73/CD39 in FoxP3 + CD4 + T cells obtained from colonic LP of Cnb1 fl/fl and Cnb1 CD4 mice. Representative dot-plots of the proportions of CD152 + and Foxp3 + gated on CD4 + T cells and CD73 + or CD39 + gated on Treg cells are shown. (B) IL-10 and TGFβ production by Foxp3 + and Foxp3 − CD4 + T cells in colonic-LP of Cnb1 fl/fl and Cnb1 CD4 mice. (C) Flow cytometry-based Treg-cell suppression assay. A total of 1 × 10 5 wild-type splenic-naïve CD4 + T cells labeled with CellTrace Violet were activated with anti-CD3 (5 µg/ml), anti-CD28 (2 µg/ml) antibodies, IL-2 (200 U/ml), and dendritic cells (2 × 10 4 ) and cultured with or without 1 × 10 5 Treg cells isolated from the spleens of Cnb1 fl/fl and Cnb1 CD4 mice. After 3 days, the proliferation of responder CD4 + T cells was analyzed using FlowJo and expressed as the percentage of proliferating cells. Cytokine production in the culture supernatants was assessed by ELISA. Data represent the means ± standard error of two independent experiments ( n = 2–3 mice per group, per experiment; cells plated in triplicate). The number of depicted cells ranges between 6,000 and 20,000. ** P
    Figure Legend Snippet: Normal suppressive activity of Treg cells from Cnb1 CD4 mice. (A) Expression patterns of the Treg-cell markers CD152 and CD73/CD39 in FoxP3 + CD4 + T cells obtained from colonic LP of Cnb1 fl/fl and Cnb1 CD4 mice. Representative dot-plots of the proportions of CD152 + and Foxp3 + gated on CD4 + T cells and CD73 + or CD39 + gated on Treg cells are shown. (B) IL-10 and TGFβ production by Foxp3 + and Foxp3 − CD4 + T cells in colonic-LP of Cnb1 fl/fl and Cnb1 CD4 mice. (C) Flow cytometry-based Treg-cell suppression assay. A total of 1 × 10 5 wild-type splenic-naïve CD4 + T cells labeled with CellTrace Violet were activated with anti-CD3 (5 µg/ml), anti-CD28 (2 µg/ml) antibodies, IL-2 (200 U/ml), and dendritic cells (2 × 10 4 ) and cultured with or without 1 × 10 5 Treg cells isolated from the spleens of Cnb1 fl/fl and Cnb1 CD4 mice. After 3 days, the proliferation of responder CD4 + T cells was analyzed using FlowJo and expressed as the percentage of proliferating cells. Cytokine production in the culture supernatants was assessed by ELISA. Data represent the means ± standard error of two independent experiments ( n = 2–3 mice per group, per experiment; cells plated in triplicate). The number of depicted cells ranges between 6,000 and 20,000. ** P

    Techniques Used: Activity Assay, Mouse Assay, Expressing, Flow Cytometry, Cytometry, Suppression Assay, Labeling, Cell Culture, Isolation, Enzyme-linked Immunosorbent Assay

    Increased JAK/STAT4 pathway in colonic Cnb1-deficient CD4 + T cells. (A) Table of some of the differentially expressed genes in CD44 low CD4 + T cells sorted from colonic-lamina propria (LP) of Cnb1 CD4 and Cnb1 fl/fl mice aged 7–10 weeks. (B) Quantitative RT-PCR analysis of mRNA expression of STAT4, STAT5b , and Il-12rb2 in CD44 low CD4 + T cells sorted from the colonic-LP of Cnb1 CD4 and Cnb1 fl/fl mice. (C) Relative expression of STAT4 and IL-12Rβ2 by CD44 low CD4 + T cells sorted from the colonic-LP of Cnb1 CD4 and Cnb1 fl/fl mice aged 8 weeks, as assessed by flow cytometry. Representative dot plots are shown. Values represent the means ± standard error of two independent experiments ( n = 2–3 mice per group, per experiment). (D) Representative confocal images of STAT4 immunofluorescent staining (red) in CD44 low CD4 + T cells sorted from the colonic-LP of Cnb1 CD4 and Cnb1 fl/fl mice aged 7–10 weeks old left untreated (UT) or stimulated by recombinant IL-12 (100 ng/ml) for 5 h. The total STAT4 expression and relative nuclear translocation were assessed by confocal microscopy. (E) Colonic-LP CD44 low CD4 + T cells from Cnb1 fl/fl and Cnb1 CD4 mice were stimulated with plate-bound anti-CD3 (5 µg/ml) and soluble anti-CD28 (2 µg/ml) alone or in combination with rIL-12 (100 ng/ml) for 8 h, and culture supernatants were analyzed by ELISA. (F) Cytokine production from CD11c + and CD11b + myeloid cells isolated from the mesenteric lymph node of Cnb1 CD4 and Cnb1 fl/fl mice was assessed at steady state and after LPS restimulation (10 µg/ml) by ELISA. * P
    Figure Legend Snippet: Increased JAK/STAT4 pathway in colonic Cnb1-deficient CD4 + T cells. (A) Table of some of the differentially expressed genes in CD44 low CD4 + T cells sorted from colonic-lamina propria (LP) of Cnb1 CD4 and Cnb1 fl/fl mice aged 7–10 weeks. (B) Quantitative RT-PCR analysis of mRNA expression of STAT4, STAT5b , and Il-12rb2 in CD44 low CD4 + T cells sorted from the colonic-LP of Cnb1 CD4 and Cnb1 fl/fl mice. (C) Relative expression of STAT4 and IL-12Rβ2 by CD44 low CD4 + T cells sorted from the colonic-LP of Cnb1 CD4 and Cnb1 fl/fl mice aged 8 weeks, as assessed by flow cytometry. Representative dot plots are shown. Values represent the means ± standard error of two independent experiments ( n = 2–3 mice per group, per experiment). (D) Representative confocal images of STAT4 immunofluorescent staining (red) in CD44 low CD4 + T cells sorted from the colonic-LP of Cnb1 CD4 and Cnb1 fl/fl mice aged 7–10 weeks old left untreated (UT) or stimulated by recombinant IL-12 (100 ng/ml) for 5 h. The total STAT4 expression and relative nuclear translocation were assessed by confocal microscopy. (E) Colonic-LP CD44 low CD4 + T cells from Cnb1 fl/fl and Cnb1 CD4 mice were stimulated with plate-bound anti-CD3 (5 µg/ml) and soluble anti-CD28 (2 µg/ml) alone or in combination with rIL-12 (100 ng/ml) for 8 h, and culture supernatants were analyzed by ELISA. (F) Cytokine production from CD11c + and CD11b + myeloid cells isolated from the mesenteric lymph node of Cnb1 CD4 and Cnb1 fl/fl mice was assessed at steady state and after LPS restimulation (10 µg/ml) by ELISA. * P

    Techniques Used: Mouse Assay, Quantitative RT-PCR, Expressing, Flow Cytometry, Cytometry, Staining, Recombinant, Translocation Assay, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Isolation

    11) Product Images from "Targeting the Vaginal Mucosa with Human Papillomavirus Pseudovirion Vaccines delivering SIV DNA"

    Article Title: Targeting the Vaginal Mucosa with Human Papillomavirus Pseudovirion Vaccines delivering SIV DNA

    Journal: Journal of Immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1101404

    SIV specific Memory T-cell responses to HPV PsVs (A) Antigen experienced CD95 + CD28 (+/-) CD8 + T- cells from PBMC’s were gated and the frequency of SIVGagCM9 + cells assessed post HPV vaccination. Animals treated with N9 foam are depicted in hatched
    Figure Legend Snippet: SIV specific Memory T-cell responses to HPV PsVs (A) Antigen experienced CD95 + CD28 (+/-) CD8 + T- cells from PBMC’s were gated and the frequency of SIVGagCM9 + cells assessed post HPV vaccination. Animals treated with N9 foam are depicted in hatched

    Techniques Used:

    12) Product Images from "GM-CSF Promotes the Expansion and Differentiation of Cord Blood Myeloid-Derived Suppressor Cells, Which Attenuate Xenogeneic Graft-vs.-Host Disease"

    Article Title: GM-CSF Promotes the Expansion and Differentiation of Cord Blood Myeloid-Derived Suppressor Cells, Which Attenuate Xenogeneic Graft-vs.-Host Disease

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00183

    MDSCs have suppressive effect for T cells and induce a polarization of helper T cells. (A) Healthy adult PBMCs were labeled with CFSE and stimulated with Dynabead Human T-Activator CD3 and CD28 in the presence of MDSCs at a 1:1, 1:0.5, and 1:0.25 (PBMCs: MDSCs) ratio. After 6 days, cells were then harvested, stained with anti-human CD3 (PE-Cy7), anti-human CD4 (APC) and anti-human CD8 (efluor450) antibodies. The cells were analyzed by FACSCanto II device. (B) CD4 + T cells were isolated from healthy adult PBMCs and 1 × 10 6 CD4 + T cells were stimulated with 0.5 μg/mL Dynabead Human T-Activator CD3 and CD28 in the presence or absence of 2 × 10 6 MDSCs at a 1:2 (CD4 + T cells: MDSCs) ratio in 12 well plates for 3 days. After 3 days, the cells were stained with fluorochrome-conjugated anti-human CD3 (PE-Cy7), anti-human CD4 (FITC), anti-human CD25 (APC), anti-human IL-17 (PerCP-Cy5.5) and anti-human FoxP3 (PE) antibodies. (C) Human IL-17 and IFN-γ was measured in the culture supernatants of (B) . Data are mean ± S.E.M. of three independent experiments, each performed in triplicate. * p
    Figure Legend Snippet: MDSCs have suppressive effect for T cells and induce a polarization of helper T cells. (A) Healthy adult PBMCs were labeled with CFSE and stimulated with Dynabead Human T-Activator CD3 and CD28 in the presence of MDSCs at a 1:1, 1:0.5, and 1:0.25 (PBMCs: MDSCs) ratio. After 6 days, cells were then harvested, stained with anti-human CD3 (PE-Cy7), anti-human CD4 (APC) and anti-human CD8 (efluor450) antibodies. The cells were analyzed by FACSCanto II device. (B) CD4 + T cells were isolated from healthy adult PBMCs and 1 × 10 6 CD4 + T cells were stimulated with 0.5 μg/mL Dynabead Human T-Activator CD3 and CD28 in the presence or absence of 2 × 10 6 MDSCs at a 1:2 (CD4 + T cells: MDSCs) ratio in 12 well plates for 3 days. After 3 days, the cells were stained with fluorochrome-conjugated anti-human CD3 (PE-Cy7), anti-human CD4 (FITC), anti-human CD25 (APC), anti-human IL-17 (PerCP-Cy5.5) and anti-human FoxP3 (PE) antibodies. (C) Human IL-17 and IFN-γ was measured in the culture supernatants of (B) . Data are mean ± S.E.M. of three independent experiments, each performed in triplicate. * p

    Techniques Used: Labeling, Staining, Isolation

    13) Product Images from "Manipulating glucose metabolism during different stages of viral pathogenesis can have either detrimental or beneficial effects"

    Article Title: Manipulating glucose metabolism during different stages of viral pathogenesis can have either detrimental or beneficial effects

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1700472

    2DG inhibits the metabolic reprogramming of CD4 T cells following activation (A) Naive CD4 T cells purified from C57BL/6 mice were cultured (500,000 cells/well) with 100U/ml IL-2, 1µg/ml anti-CD3/CD28 and in the presence or absence of 250µM 2DG for 3 days followed by extracellular flux analysis. Line graph showing changes in Extracellular acidification rates (ECAR) by CD4 T cells following addition of oligomycin and Histograms showing basal ECAR levels. (B) Naive CD4 T cells purified from C57BL/6 mice were cultured (100,000 cells/well) with 1µg/ml anti-CD3/CD28 and in the presence or absence of 250µM 2DG for 24 hours followed by gene expression analysis by QRT-PCR compared to beta-actin. Histogram representing expression of genes involved in glucose metabolism such as HK1, HK2 and Glut1 in naïve, activated and Activated in the presence of 2DG (2DG). (C) Naïve CD4 T cells were cultured (100,000 cells/well) with 1µg/ml anti-CD3/CD28 in the presence or absence of 250µM 2DG for 24 hours followed by measurement of Phosphorylation of S6 using flow cytometry. Representative FACS plots and histogram (MFI of S6P) of live CD4 T cells. Data represents means ± SEM from two independent experiments (n = 3/group) and the level of significance was determined by Student’s t test (unpaired) for A C and One-way ANOVA for B. P≤ 0.0001 (****), P≤0.001(***), P≤0.01(**), P≤0.05(*).
    Figure Legend Snippet: 2DG inhibits the metabolic reprogramming of CD4 T cells following activation (A) Naive CD4 T cells purified from C57BL/6 mice were cultured (500,000 cells/well) with 100U/ml IL-2, 1µg/ml anti-CD3/CD28 and in the presence or absence of 250µM 2DG for 3 days followed by extracellular flux analysis. Line graph showing changes in Extracellular acidification rates (ECAR) by CD4 T cells following addition of oligomycin and Histograms showing basal ECAR levels. (B) Naive CD4 T cells purified from C57BL/6 mice were cultured (100,000 cells/well) with 1µg/ml anti-CD3/CD28 and in the presence or absence of 250µM 2DG for 24 hours followed by gene expression analysis by QRT-PCR compared to beta-actin. Histogram representing expression of genes involved in glucose metabolism such as HK1, HK2 and Glut1 in naïve, activated and Activated in the presence of 2DG (2DG). (C) Naïve CD4 T cells were cultured (100,000 cells/well) with 1µg/ml anti-CD3/CD28 in the presence or absence of 250µM 2DG for 24 hours followed by measurement of Phosphorylation of S6 using flow cytometry. Representative FACS plots and histogram (MFI of S6P) of live CD4 T cells. Data represents means ± SEM from two independent experiments (n = 3/group) and the level of significance was determined by Student’s t test (unpaired) for A C and One-way ANOVA for B. P≤ 0.0001 (****), P≤0.001(***), P≤0.01(**), P≤0.05(*).

    Techniques Used: Activation Assay, Purification, Mouse Assay, Cell Culture, Expressing, Quantitative RT-PCR, Flow Cytometry, Cytometry, FACS

    Increasing glucose levels increases Th1 but not Treg differentiation Splenocytes from DO11.10 RAG2−/− mice were cultured (1 million cells) in the presence of 1 µg/ml of anti-CD3/CD28 antibody with either 100 U/ml of recombinant IL-2, 1ng/ml TGF-β (Treg differentiating conditions) or IL-12 (5ng/ml), anti-IL-4 (10 µg/ml) (Th1 differentiating conditions) with increasing concentrations of glucose(0.5mM–20mM) in glucose free conditions (A) Histogram showing frequency of IFN-γ during Th1 differentiation or (B) Foxp3 expression during Treg differentiation with increasing glucose concentrations (C) Representative FACS plots and histogram showing IFN-γ expression under Th1 differentiating conditions in the presence of glucose concentrations (5mM and 10mM). Cells were gated on live CD4+ T cells. Data represents means ± SEM from three independent experiments (n = 3/group). The level of significance was determined by Student’s t test (unpaired) P≤0.05(*).
    Figure Legend Snippet: Increasing glucose levels increases Th1 but not Treg differentiation Splenocytes from DO11.10 RAG2−/− mice were cultured (1 million cells) in the presence of 1 µg/ml of anti-CD3/CD28 antibody with either 100 U/ml of recombinant IL-2, 1ng/ml TGF-β (Treg differentiating conditions) or IL-12 (5ng/ml), anti-IL-4 (10 µg/ml) (Th1 differentiating conditions) with increasing concentrations of glucose(0.5mM–20mM) in glucose free conditions (A) Histogram showing frequency of IFN-γ during Th1 differentiation or (B) Foxp3 expression during Treg differentiation with increasing glucose concentrations (C) Representative FACS plots and histogram showing IFN-γ expression under Th1 differentiating conditions in the presence of glucose concentrations (5mM and 10mM). Cells were gated on live CD4+ T cells. Data represents means ± SEM from three independent experiments (n = 3/group). The level of significance was determined by Student’s t test (unpaired) P≤0.05(*).

    Techniques Used: Mouse Assay, Cell Culture, Recombinant, Expressing, FACS

    Effect of 2DG treatment on glycolysis and T cell differentiation Splenocytes from DO11.10 RAG2−/− mice were cultured (1 million cells) in the presence of 1 µg/ml of anti-CD3/CD28 antibody with either 100 U/ml of recombinant IL-2, 5ng/ml TGF-β (Treg differentiating conditions) or IL-12 (5ng/ml), anti-IL-4 (10 µg/ml) (Th1 differentiating conditions) with or without 2DG (250µM). After 5 days of culture, cells were analyzed for the expression of IFN-γ and Foxp3 on CD4 T cells. Representative FACS plots and histogram showing the frequency of Th1 and Treg. Cells were gated on live CD4+ Foxp3+ T cells (Treg) and live CD4+ IFN-γ + T cells (Th1). Data represents means ± SEMs of three independent experiments with n=3/group. Statistical significance was calculated by Student’s t test (unpaired) P≤ 0.0001 (****).
    Figure Legend Snippet: Effect of 2DG treatment on glycolysis and T cell differentiation Splenocytes from DO11.10 RAG2−/− mice were cultured (1 million cells) in the presence of 1 µg/ml of anti-CD3/CD28 antibody with either 100 U/ml of recombinant IL-2, 5ng/ml TGF-β (Treg differentiating conditions) or IL-12 (5ng/ml), anti-IL-4 (10 µg/ml) (Th1 differentiating conditions) with or without 2DG (250µM). After 5 days of culture, cells were analyzed for the expression of IFN-γ and Foxp3 on CD4 T cells. Representative FACS plots and histogram showing the frequency of Th1 and Treg. Cells were gated on live CD4+ Foxp3+ T cells (Treg) and live CD4+ IFN-γ + T cells (Th1). Data represents means ± SEMs of three independent experiments with n=3/group. Statistical significance was calculated by Student’s t test (unpaired) P≤ 0.0001 (****).

    Techniques Used: Cell Differentiation, Mouse Assay, Cell Culture, Recombinant, Expressing, FACS

    14) Product Images from "Induction and transcriptional regulation of the co-inhibitory gene module in T cells"

    Article Title: Induction and transcriptional regulation of the co-inhibitory gene module in T cells

    Journal: Nature

    doi: 10.1038/s41586-018-0206-z

    IL-27 induces multiple co-inhibitory receptors on CD4 + and CD8 + T cells. a) Naïve T cells from WT or IL27ra KO mice were stimulated in vitro with anti-CD3/CD28 in the presence or absence of IL-27. Expression of co-inhibitory receptors was determined by flow cytometry. Representative data of 3 biologically independent experiments are shown. b) Expression of PD-1, Tim-3, Lag-3, TIGIT, and IL-10 on CD8 + TILs obtained from WT and IL27ra KO mice bearing B16F10 melanoma was determined by flow cytometry. Thy1.1-IL-10 reporter mice crossed with WT and IL27ra KO mice were used for IL-10 expression analysis. Representative data of 3 biologically independent experiments are shown.
    Figure Legend Snippet: IL-27 induces multiple co-inhibitory receptors on CD4 + and CD8 + T cells. a) Naïve T cells from WT or IL27ra KO mice were stimulated in vitro with anti-CD3/CD28 in the presence or absence of IL-27. Expression of co-inhibitory receptors was determined by flow cytometry. Representative data of 3 biologically independent experiments are shown. b) Expression of PD-1, Tim-3, Lag-3, TIGIT, and IL-10 on CD8 + TILs obtained from WT and IL27ra KO mice bearing B16F10 melanoma was determined by flow cytometry. Thy1.1-IL-10 reporter mice crossed with WT and IL27ra KO mice were used for IL-10 expression analysis. Representative data of 3 biologically independent experiments are shown.

    Techniques Used: Mouse Assay, In Vitro, Expressing, Flow Cytometry, Cytometry

    Characterization of the role of Pdpn and Procr in CD8 + TILs a) Pdpn and Procr protein and mRNA expression was determined in T cells from WT and IL27ra KO stimulated with anti-CD3/CD28 in the presence or absence of IL-27. CD4 + cells were analyzed at 96hr and CD8 + cells at 72hr. Data are representative flow cytometry and qPCR data from biologically independent animals. mean s.e.m is shown. b) Representative flow cytometry data of 3 independent experiments showing Pdpn and Procr expression in PD-1 + Tim-3 + CD8 + and PD-1 - Tim-3 - CD8 + TILs obtained from WT and IL27ra KO mice bearing B16F10 melanoma. c) TILs from WT mice bearing B16F10 melanoma were stimulated with PMA and Ionomycin. Cytokine production in Procr + or Procr - CD8 + TILs is shown. Thy1.1-IL-10 reporter mice were used for IL-10 expression analysis. Data are from biologically independent animals. mean + s.e.m is shown. *p
    Figure Legend Snippet: Characterization of the role of Pdpn and Procr in CD8 + TILs a) Pdpn and Procr protein and mRNA expression was determined in T cells from WT and IL27ra KO stimulated with anti-CD3/CD28 in the presence or absence of IL-27. CD4 + cells were analyzed at 96hr and CD8 + cells at 72hr. Data are representative flow cytometry and qPCR data from biologically independent animals. mean s.e.m is shown. b) Representative flow cytometry data of 3 independent experiments showing Pdpn and Procr expression in PD-1 + Tim-3 + CD8 + and PD-1 - Tim-3 - CD8 + TILs obtained from WT and IL27ra KO mice bearing B16F10 melanoma. c) TILs from WT mice bearing B16F10 melanoma were stimulated with PMA and Ionomycin. Cytokine production in Procr + or Procr - CD8 + TILs is shown. Thy1.1-IL-10 reporter mice were used for IL-10 expression analysis. Data are from biologically independent animals. mean + s.e.m is shown. *p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Mouse Assay

    15) Product Images from "Beta-2 adrenergic receptors increase TREG cell suppression in an OVA-induced allergic asthma mouse model when mice are moderate aerobically exercised"

    Article Title: Beta-2 adrenergic receptors increase TREG cell suppression in an OVA-induced allergic asthma mouse model when mice are moderate aerobically exercised

    Journal: BMC Immunology

    doi: 10.1186/s12865-018-0244-1

    Treatment with effector specific membrane permeable cAMP analogues is sufficient to increase TREG suppressive function. TREG cells were treated with either PKA- or EPAC1- specific cyclic-AMP for 30 min. cAMP-treated TREG cells were co-cultured with untreated non-sensitized naive Th cells using artificial Th cell activation (anti-CD3 and anti-CD28) for 72 h. Th cell proliferation was assessed using 3H–thymidine incorporation. a non-sensitized TREG cells. ( n = 7–10) * P ≤ 0.02 between groups where indicated. b OVA-sensitized TREG cells. ( n = 7–10) * P ≤ 0.04 between groups where indicated. All data are shown as mean +/− SEM of triplicates of seven independent experiments
    Figure Legend Snippet: Treatment with effector specific membrane permeable cAMP analogues is sufficient to increase TREG suppressive function. TREG cells were treated with either PKA- or EPAC1- specific cyclic-AMP for 30 min. cAMP-treated TREG cells were co-cultured with untreated non-sensitized naive Th cells using artificial Th cell activation (anti-CD3 and anti-CD28) for 72 h. Th cell proliferation was assessed using 3H–thymidine incorporation. a non-sensitized TREG cells. ( n = 7–10) * P ≤ 0.02 between groups where indicated. b OVA-sensitized TREG cells. ( n = 7–10) * P ≤ 0.04 between groups where indicated. All data are shown as mean +/− SEM of triplicates of seven independent experiments

    Techniques Used: Cell Culture, Activation Assay

    β2-AR −/− TREG cells isolated from moderate aerobic exercised mice do not increase TREG suppressive function. Wildtype BALB/c mice were TREG cell depleted using anti-CD25 intravenous 24 h prior to receiving 5 × 10 5 wildtype or β2-AR −/− TREG cells. Mice underwent OVA-sensitization and exercise protocols (see Fig. 1 ). At the end of the protocol, TREG cells from each group were co-cultured with non-sensitized naive Th cells using artificial Th cell activation (anti-CD3 and anti-CD28) for 72 h. Th cell proliferation was assessed using 3H–thymidine incorporation. ( n = 8–14) * P ≤ 0.005 between groups where indicated. Two-way ANOVA: exercise treatment – p = 0.04, β2-AR treatment – n.s., Interaction – p = 0.04. All data are shown as mean +/− SEM of triplicates of two independent experiments
    Figure Legend Snippet: β2-AR −/− TREG cells isolated from moderate aerobic exercised mice do not increase TREG suppressive function. Wildtype BALB/c mice were TREG cell depleted using anti-CD25 intravenous 24 h prior to receiving 5 × 10 5 wildtype or β2-AR −/− TREG cells. Mice underwent OVA-sensitization and exercise protocols (see Fig. 1 ). At the end of the protocol, TREG cells from each group were co-cultured with non-sensitized naive Th cells using artificial Th cell activation (anti-CD3 and anti-CD28) for 72 h. Th cell proliferation was assessed using 3H–thymidine incorporation. ( n = 8–14) * P ≤ 0.005 between groups where indicated. Two-way ANOVA: exercise treatment – p = 0.04, β2-AR treatment – n.s., Interaction – p = 0.04. All data are shown as mean +/− SEM of triplicates of two independent experiments

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

    Treatment with formoterol is sufficient to increase TREG suppressive function. TREG cells isolated from all experimental mouse groups (S, E, SO, EO) were treated with a β2-adrenergic receptor agonist (formoterol) for 30 min. Formoterol-treated TREG cells were co-cultured with untreated non-sensitized naive Th cells using artificial Th cell activation (anti-CD3 and anti-CD28) for 72 h. Th cell proliferation was assessed using 3H–thymidine incorporation. a non-sensitized TREG cells (S and E). ( n = 7–10) * P
    Figure Legend Snippet: Treatment with formoterol is sufficient to increase TREG suppressive function. TREG cells isolated from all experimental mouse groups (S, E, SO, EO) were treated with a β2-adrenergic receptor agonist (formoterol) for 30 min. Formoterol-treated TREG cells were co-cultured with untreated non-sensitized naive Th cells using artificial Th cell activation (anti-CD3 and anti-CD28) for 72 h. Th cell proliferation was assessed using 3H–thymidine incorporation. a non-sensitized TREG cells (S and E). ( n = 7–10) * P

    Techniques Used: Isolation, Cell Culture, Activation Assay

    TREG cells lacking β2-AR expression exhibit decreased intracellular cAMP levels and TREG cell suppressive function. TREG cells (CD4 + CD25 + ) with (WT TREG) or without (β2-AR −/− TREG) β2-adrenergic receptors were negatively isolated and; ( a ) assessed for cyclic-AMP levels after lysing using RIA. ( n = 5 in triplicate) * P ≤ 0.05 between groups where indicated. b co-cultured with wildtype CD4 + CD25 − naive Th cells using artificial Th cell activation (anti-CD3 and anti-CD28) for 72 h. Th cell proliferation was assessed using 3H–thymidine incorporation. * P
    Figure Legend Snippet: TREG cells lacking β2-AR expression exhibit decreased intracellular cAMP levels and TREG cell suppressive function. TREG cells (CD4 + CD25 + ) with (WT TREG) or without (β2-AR −/− TREG) β2-adrenergic receptors were negatively isolated and; ( a ) assessed for cyclic-AMP levels after lysing using RIA. ( n = 5 in triplicate) * P ≤ 0.05 between groups where indicated. b co-cultured with wildtype CD4 + CD25 − naive Th cells using artificial Th cell activation (anti-CD3 and anti-CD28) for 72 h. Th cell proliferation was assessed using 3H–thymidine incorporation. * P

    Techniques Used: Expressing, Isolation, Cell Culture, Activation Assay

    16) Product Images from "HELIGMOSOMOIDES BAKERI INFECTION DECREASES SMAD7 EXPRESSION IN INTESTINAL CD4+ T CELLS WHICH ALLOWS TGFβ TO INDUCE IL10-PRODUCING REGULATORY T CELLS THAT BLOCK COLITIS"

    Article Title: HELIGMOSOMOIDES BAKERI INFECTION DECREASES SMAD7 EXPRESSION IN INTESTINAL CD4+ T CELLS WHICH ALLOWS TGFβ TO INDUCE IL10-PRODUCING REGULATORY T CELLS THAT BLOCK COLITIS

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1801392

    Colonic CD25 − CD4 + cells from Smad7 high expressing transgenic mice infected with Hpb do not readily convert to their regulatory phenotypes after TGFβ stimulation in vitro . (Panel A) Colonic T cells were isolated from Hpb -infected or uninfected C57BL6 SMAD7 high expression transgenic mice or from control WT mice. The CD4 + CD25 − T cells were sorted by FACS, and mRNA was extracted and converted to cDNA. Quantitative rt-PCR was used to assess the level of Smad7 mRNA expressed in these cells. Data were normalized relative to the expression of the housekeeping gene GAPDH. Smad7 mRNA expression was high in the Smad7 transgenic T cells relative to that of the control T cells. Also, Hpb infection did no decrease Smad7 expression in the transgenic T cells as it did in the WT T cells. (Panel B and C) Colonic CD25 − CD4+ T cells isolated from either uninfected or Hpb -infected Smad7 transgenic mice or WT control animals, were cultured for 72h with anti-CD3/CD28 and TGFβ. The cultured cells then were stained for Alexa Fluor 647-labeled CD25, and then fixed, permeabilized and stained for intracellular IL-10. Flow analysis shows that T cells from Hpb -infection WT mice are more prone to convert to their CD25 + /IL10 − and CD25 + /IL10 + regulatory phenotypes after stimulation with TGFβ compared to T cells from their uninfected controls. This is not the case with T cells from Smad7 transgenic mice. Data in panel A and C are mean ± SE calculated from the means of three independent experiments each containing three or four mice per group. The numbers in the panel B and C are the percentage of cells positive for the particular T cell phenotype.
    Figure Legend Snippet: Colonic CD25 − CD4 + cells from Smad7 high expressing transgenic mice infected with Hpb do not readily convert to their regulatory phenotypes after TGFβ stimulation in vitro . (Panel A) Colonic T cells were isolated from Hpb -infected or uninfected C57BL6 SMAD7 high expression transgenic mice or from control WT mice. The CD4 + CD25 − T cells were sorted by FACS, and mRNA was extracted and converted to cDNA. Quantitative rt-PCR was used to assess the level of Smad7 mRNA expressed in these cells. Data were normalized relative to the expression of the housekeeping gene GAPDH. Smad7 mRNA expression was high in the Smad7 transgenic T cells relative to that of the control T cells. Also, Hpb infection did no decrease Smad7 expression in the transgenic T cells as it did in the WT T cells. (Panel B and C) Colonic CD25 − CD4+ T cells isolated from either uninfected or Hpb -infected Smad7 transgenic mice or WT control animals, were cultured for 72h with anti-CD3/CD28 and TGFβ. The cultured cells then were stained for Alexa Fluor 647-labeled CD25, and then fixed, permeabilized and stained for intracellular IL-10. Flow analysis shows that T cells from Hpb -infection WT mice are more prone to convert to their CD25 + /IL10 − and CD25 + /IL10 + regulatory phenotypes after stimulation with TGFβ compared to T cells from their uninfected controls. This is not the case with T cells from Smad7 transgenic mice. Data in panel A and C are mean ± SE calculated from the means of three independent experiments each containing three or four mice per group. The numbers in the panel B and C are the percentage of cells positive for the particular T cell phenotype.

    Techniques Used: Expressing, Transgenic Assay, Mouse Assay, Infection, In Vitro, Isolation, FACS, Quantitative RT-PCR, Cell Culture, Staining, Labeling

    TGFβ readily converts Foxp3 − IL10 − CD4 + T cells from LP or MLN of Hpb -infected mice, as opposed to Foxp3 − IL10 − CD4 + T cells from uninfected mice, to Foxp3 + IL10 + CD4 + and Foxp3 − IL10 + CD4 + T cells. Foxp3 − IL10 − CD4 + T cells were isolated from dispersed colonic LPMC or MLN of Hpb -infected ( Hpb ) and uninfected (no Hpb ) mice using FACS. These cells were cultured in vitro in complete medium for 72h with anti-CD3/CD28 in the presence or absence of TGFβ (10ng/ml). The T cells then were subject to flow analysis to determine the percentage of the T cells that converted to Foxp3 + IL10 − CD4 + , Foxp3 + IL10 + CD4 + or Foxp3 − IL10 + CD4 + . Panel A and B show results using colonic LP T cells, whereas panels C and D show results using MLN T cells. Data in the tables are mean percentage ± SE calculated from the means of five independent experiments containing three or four mice per group. Also shown are representative FACS plots for each experimental condition.
    Figure Legend Snippet: TGFβ readily converts Foxp3 − IL10 − CD4 + T cells from LP or MLN of Hpb -infected mice, as opposed to Foxp3 − IL10 − CD4 + T cells from uninfected mice, to Foxp3 + IL10 + CD4 + and Foxp3 − IL10 + CD4 + T cells. Foxp3 − IL10 − CD4 + T cells were isolated from dispersed colonic LPMC or MLN of Hpb -infected ( Hpb ) and uninfected (no Hpb ) mice using FACS. These cells were cultured in vitro in complete medium for 72h with anti-CD3/CD28 in the presence or absence of TGFβ (10ng/ml). The T cells then were subject to flow analysis to determine the percentage of the T cells that converted to Foxp3 + IL10 − CD4 + , Foxp3 + IL10 + CD4 + or Foxp3 − IL10 + CD4 + . Panel A and B show results using colonic LP T cells, whereas panels C and D show results using MLN T cells. Data in the tables are mean percentage ± SE calculated from the means of five independent experiments containing three or four mice per group. Also shown are representative FACS plots for each experimental condition.

    Techniques Used: Infection, Mouse Assay, Isolation, FACS, Cell Culture, In Vitro

    CD25 is expressed on nearly all colonic LP CD4 + T cells, isolated from Hpb -infected mice, induced to express IL10 by exposure to TGFβ. Colonic Foxp3 − IL10 − CD4 + T cells from Hpb -infected ( Hpb ) mice were cultured in vitro in complete medium for 72h with anti-CD3/CD28 in the presence of TGFβ (10 ng/ml). The T cells then were subjected to flow analysis to determine the percentage of the T cells that displayed CD25 after conversion to Foxp3 + IL10 − CD4 + , Foxp3 + IL10 + CD4 + , or Foxp3 − IL10 + CD4 + . The representative FACS plot shows the various subsets of colonic T cells induced after TGFβ exposure (left panel). The figure on the right shows that the percentage of cells expressing CD25 was highest on the Foxp3 + IL10 + CD4 + and Foxp3 − IL10 + CD4 + T cell subsets. Data are means ± SE calculated from the means of three independent experiments each using pooled T cells from three or four mice. A or B vs. C or D, p
    Figure Legend Snippet: CD25 is expressed on nearly all colonic LP CD4 + T cells, isolated from Hpb -infected mice, induced to express IL10 by exposure to TGFβ. Colonic Foxp3 − IL10 − CD4 + T cells from Hpb -infected ( Hpb ) mice were cultured in vitro in complete medium for 72h with anti-CD3/CD28 in the presence of TGFβ (10 ng/ml). The T cells then were subjected to flow analysis to determine the percentage of the T cells that displayed CD25 after conversion to Foxp3 + IL10 − CD4 + , Foxp3 + IL10 + CD4 + , or Foxp3 − IL10 + CD4 + . The representative FACS plot shows the various subsets of colonic T cells induced after TGFβ exposure (left panel). The figure on the right shows that the percentage of cells expressing CD25 was highest on the Foxp3 + IL10 + CD4 + and Foxp3 − IL10 + CD4 + T cell subsets. Data are means ± SE calculated from the means of three independent experiments each using pooled T cells from three or four mice. A or B vs. C or D, p

    Techniques Used: Isolation, Infection, Mouse Assay, Cell Culture, In Vitro, FACS, Expressing

    Colonic LP CD4 + T cells from Hpb -infected mice induced to express IL10 by incubation with TGFβ will inhibit colitis when transferred into the Rag CD25 − CD4 + T cell transfer model of IBD. The experimental design is outlined in panel A . Colonic Foxp3 − IL10 − CD4 + T cells from Hpb -infected mice were cultured in vitro in complete medium for 72h with anti-CD3/CD28 and TGFβ to induce putative regulatory T cell subsets. The T cells then were subject to flow cytometry to isolate the T cells subsets that converted to Foxp3 + IL10 + or Foxp3- IL10+. Also, isolated was the Foxp3 − IL10 − T cell subset. Foxp3 − IL10 − , Foxp3 + IL10 + or Foxp3 − IL10 + CD4 + T cells were transferred (10 5 /mouse) by ip injection into Rag mice that also received splenic CD25 − CD4 + T cells (2 × 10 5 /mouse) from WT mice to promote colitis. A fourth group of Rag mice was reconstituted just with splenic CD25 − CD4 + T cells. Animals then were treated with piroxicam as described to induce colitis. At the end of the experiment, colonic tissue was sectioned and examined microscopically to score the severity of the colitis using a 4-point scale. B and C show severity of colitis in mice receiving just splenic CD25 − CD4 + T cells (No colonic T cell transfer) or CD25 − CD4 + T cells and one additional colonic T cell subset. B) As depicted in these representative images stained with H E and photographed at x40, an intense mucosa lymphocytic infiltration developed in mice receiving just splenic CD25 − CD4 + T cells (no colonic T cell transfer). This inflammation failed to develop in recipients of splenic CD25 − CD4 + T cells and TGFβ-induced, colonic Foxp3 + IL10 + or Foxp3 − IL10 + T cells. C) The table shows that recipients of splenic CD25 − CD4 + T cells along with TGFβ-induced, colonic Foxp3 + IL10 + or Foxp3 − IL10 + CD4 + T cells developed little colitis. Data are means ± SE from 3 separate experiments each containing four to five mice per group. No colonic T cell transfer or colonic Foxp3 − IL10 − recipients vs. the colonic Foxp3 + IL10 + or Foxp3 − IL10 + T cells recipients, p
    Figure Legend Snippet: Colonic LP CD4 + T cells from Hpb -infected mice induced to express IL10 by incubation with TGFβ will inhibit colitis when transferred into the Rag CD25 − CD4 + T cell transfer model of IBD. The experimental design is outlined in panel A . Colonic Foxp3 − IL10 − CD4 + T cells from Hpb -infected mice were cultured in vitro in complete medium for 72h with anti-CD3/CD28 and TGFβ to induce putative regulatory T cell subsets. The T cells then were subject to flow cytometry to isolate the T cells subsets that converted to Foxp3 + IL10 + or Foxp3- IL10+. Also, isolated was the Foxp3 − IL10 − T cell subset. Foxp3 − IL10 − , Foxp3 + IL10 + or Foxp3 − IL10 + CD4 + T cells were transferred (10 5 /mouse) by ip injection into Rag mice that also received splenic CD25 − CD4 + T cells (2 × 10 5 /mouse) from WT mice to promote colitis. A fourth group of Rag mice was reconstituted just with splenic CD25 − CD4 + T cells. Animals then were treated with piroxicam as described to induce colitis. At the end of the experiment, colonic tissue was sectioned and examined microscopically to score the severity of the colitis using a 4-point scale. B and C show severity of colitis in mice receiving just splenic CD25 − CD4 + T cells (No colonic T cell transfer) or CD25 − CD4 + T cells and one additional colonic T cell subset. B) As depicted in these representative images stained with H E and photographed at x40, an intense mucosa lymphocytic infiltration developed in mice receiving just splenic CD25 − CD4 + T cells (no colonic T cell transfer). This inflammation failed to develop in recipients of splenic CD25 − CD4 + T cells and TGFβ-induced, colonic Foxp3 + IL10 + or Foxp3 − IL10 + T cells. C) The table shows that recipients of splenic CD25 − CD4 + T cells along with TGFβ-induced, colonic Foxp3 + IL10 + or Foxp3 − IL10 + CD4 + T cells developed little colitis. Data are means ± SE from 3 separate experiments each containing four to five mice per group. No colonic T cell transfer or colonic Foxp3 − IL10 − recipients vs. the colonic Foxp3 + IL10 + or Foxp3 − IL10 + T cells recipients, p

    Techniques Used: Infection, Mouse Assay, Incubation, Cell Culture, In Vitro, Flow Cytometry, Isolation, Injection, Staining

    17) Product Images from "SLAP Deficiency Enhances Number and Function of Regulatory T Cells Preventing Chronic Autoimmune Arthritis in SKG Mice"

    Article Title: SLAP Deficiency Enhances Number and Function of Regulatory T Cells Preventing Chronic Autoimmune Arthritis in SKG Mice

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1003601

    Functional blockade of Tregs in DSSKO mice leads to the development of arthritis. A , Arthritis development in mice injected i.v. with a mixture of anti-CD25 Abs or control rat Ig (black arrows) on days 5, 7, and 9 after zymosan treatment. Data represent the average arthritis score (±SEM) from three independent experiments for SKG and DSSKO mice. Data for WT and SLAP knockout mice are from one experiment. B and C, CFSE-labeled WT CD4 + CD25 − peripheral T cells were stimulated with anti-CD3 and anti-CD28 in the presence of the indicated ratios of either freshly isolated ( B ) or activated ( C ) CD4 + CD25 + Tregs from WT, SLAP −/− , SKG, or DSSKO mice and proliferation determined by dilution of CFSE using flow cytometry. Data represent the average of three mice per genotype (±SEM) from three independent experiments.
    Figure Legend Snippet: Functional blockade of Tregs in DSSKO mice leads to the development of arthritis. A , Arthritis development in mice injected i.v. with a mixture of anti-CD25 Abs or control rat Ig (black arrows) on days 5, 7, and 9 after zymosan treatment. Data represent the average arthritis score (±SEM) from three independent experiments for SKG and DSSKO mice. Data for WT and SLAP knockout mice are from one experiment. B and C, CFSE-labeled WT CD4 + CD25 − peripheral T cells were stimulated with anti-CD3 and anti-CD28 in the presence of the indicated ratios of either freshly isolated ( B ) or activated ( C ) CD4 + CD25 + Tregs from WT, SLAP −/− , SKG, or DSSKO mice and proliferation determined by dilution of CFSE using flow cytometry. Data represent the average of three mice per genotype (±SEM) from three independent experiments.

    Techniques Used: Functional Assay, Mouse Assay, Injection, Knock-Out, Labeling, Isolation, Flow Cytometry, Cytometry

    Increased numbers of thymic and splenic Tregs in DSSKO mice compared with those in SKG mice. A and C, Representative dot plots of thymic ( A ) and splenic ( C ) Tregs from WT, SLAP −/− , SKG, and DSSKO mice. B and D , Frequency and absolute number of thymic ( B ) and splenic ( D ) Tregs in each group as shown in A and C, respectively. Data represent the average of 11 mice per genotype (±SEM) from 3 independent experiments. E , SLAP expression assessed by immunoprecipitation and Western blotting on thymocytes from WT and SLAP −/− mice, and WT nTregs (isolated from pooled spleen and lymph node peripheral lymphocytes from BALB/c mice), iTregs, and peripheral T cells before and after stimulation with anti-CD3 and anti-CD28. F , Analysis of Vβ usage in lymph node CD4 + Foxp3 + T cells from WT, SLAP −/− , SKG, and DSSKO mice. Data represent the average of nine mice per genotype (±SEM) from three independent experiments.
    Figure Legend Snippet: Increased numbers of thymic and splenic Tregs in DSSKO mice compared with those in SKG mice. A and C, Representative dot plots of thymic ( A ) and splenic ( C ) Tregs from WT, SLAP −/− , SKG, and DSSKO mice. B and D , Frequency and absolute number of thymic ( B ) and splenic ( D ) Tregs in each group as shown in A and C, respectively. Data represent the average of 11 mice per genotype (±SEM) from 3 independent experiments. E , SLAP expression assessed by immunoprecipitation and Western blotting on thymocytes from WT and SLAP −/− mice, and WT nTregs (isolated from pooled spleen and lymph node peripheral lymphocytes from BALB/c mice), iTregs, and peripheral T cells before and after stimulation with anti-CD3 and anti-CD28. F , Analysis of Vβ usage in lymph node CD4 + Foxp3 + T cells from WT, SLAP −/− , SKG, and DSSKO mice. Data represent the average of nine mice per genotype (±SEM) from three independent experiments.

    Techniques Used: Mouse Assay, Expressing, Immunoprecipitation, Western Blot, Isolation

    18) Product Images from "Calcium-dependent protein acyltransferase DHHC21 controls activation of CD4+ T cells"

    Article Title: Calcium-dependent protein acyltransferase DHHC21 controls activation of CD4+ T cells

    Journal: bioRxiv

    doi: 10.1101/2020.09.01.277947

    TCR signaling is impaired in Zdhhc21 dep CD4 + T cells. (A) CD4 + T cells from WT or Zdhhc21 dep mice were stimulated with cross-linked anti-CD3/CD28 antibodies for indicated times and indicated phospho-proteins (p-) and total proteins were analyzed by immunoblotting. β-Actin was used as a loading control. The results shown are representative of three independent experiments. (B) CD4 + T cells were loaded with Fura-2 calcium indicator and pre-incubated with anti-CD3/CD28 antibodies. IgG antibody was added to induce cross-linking after an observation period (upward arrow). Shown are representative traces from single WT (gray) and Zdhhc21 dep (red) cells. (C) Peak calcium release. CD4 + T cells were treated as described in (B). Data represent maximum calcium values averaged from four independent experiments (∼100 WT or Zdhhc21 dep cells per experiment). ± S.E.M. (D) Calcium response pattern. CD4 + T cells were treated as described in (B). Cells showing at least 10% increase in calcium values upon TCR stimulation were identified as responders. Data represent averaged percentage of responders from four independent experiments (∼100 WT or Zdhhc21 dep CD4 + T cells per experiment). ± S.E.M.
    Figure Legend Snippet: TCR signaling is impaired in Zdhhc21 dep CD4 + T cells. (A) CD4 + T cells from WT or Zdhhc21 dep mice were stimulated with cross-linked anti-CD3/CD28 antibodies for indicated times and indicated phospho-proteins (p-) and total proteins were analyzed by immunoblotting. β-Actin was used as a loading control. The results shown are representative of three independent experiments. (B) CD4 + T cells were loaded with Fura-2 calcium indicator and pre-incubated with anti-CD3/CD28 antibodies. IgG antibody was added to induce cross-linking after an observation period (upward arrow). Shown are representative traces from single WT (gray) and Zdhhc21 dep (red) cells. (C) Peak calcium release. CD4 + T cells were treated as described in (B). Data represent maximum calcium values averaged from four independent experiments (∼100 WT or Zdhhc21 dep cells per experiment). ± S.E.M. (D) Calcium response pattern. CD4 + T cells were treated as described in (B). Cells showing at least 10% increase in calcium values upon TCR stimulation were identified as responders. Data represent averaged percentage of responders from four independent experiments (∼100 WT or Zdhhc21 dep CD4 + T cells per experiment). ± S.E.M.

    Techniques Used: Mouse Assay, Incubation

    DHHC21 is required for T cell activation. (A) Surface expression of CD69 on CD4 + T cells from WT or Zdhhc21 dep mice. CD4 + T cells were stimulated with plate-bound anti-CD3/CD28 antibodies for 24 h and analyzed by flow cytometry. (B) Average percentage of CD69 + T cells from WT or Zdhhc21 dep mice. CD4 + T cells were stimulated with plate-bound anti-CD3/CD28 antibodies for 24 h and analyzed by flow cytometry. The graph shows mean ± SD values from three independent experiments. **, P
    Figure Legend Snippet: DHHC21 is required for T cell activation. (A) Surface expression of CD69 on CD4 + T cells from WT or Zdhhc21 dep mice. CD4 + T cells were stimulated with plate-bound anti-CD3/CD28 antibodies for 24 h and analyzed by flow cytometry. (B) Average percentage of CD69 + T cells from WT or Zdhhc21 dep mice. CD4 + T cells were stimulated with plate-bound anti-CD3/CD28 antibodies for 24 h and analyzed by flow cytometry. The graph shows mean ± SD values from three independent experiments. **, P

    Techniques Used: Activation Assay, Expressing, Mouse Assay, Flow Cytometry

    DHHC21 regulates CD4 + T activation and differentiation. Naïve CD4 + T cells from WT or Zdhhc21 dep mice were stimulated with plate-bound anti-CD3/CD28 antibodies and incubated under neutral (Th0) or polarizing (Th1, Th2, and Th17) conditions for 5 days, then CD4 + T cells were re-stimulated with plate-bound anti-CD3 antibodies for 6 h. (A) Effector cytokine production measured by ELISA. The graph shows mean ± SD values from at least three independent experiments. (B) mRNA expression of Th lineage-specific transcription factors determined by qRT-PCR normalized to 18S RNA. The graph shows mean ± SD values from at least three independent experiments. *, P
    Figure Legend Snippet: DHHC21 regulates CD4 + T activation and differentiation. Naïve CD4 + T cells from WT or Zdhhc21 dep mice were stimulated with plate-bound anti-CD3/CD28 antibodies and incubated under neutral (Th0) or polarizing (Th1, Th2, and Th17) conditions for 5 days, then CD4 + T cells were re-stimulated with plate-bound anti-CD3 antibodies for 6 h. (A) Effector cytokine production measured by ELISA. The graph shows mean ± SD values from at least three independent experiments. (B) mRNA expression of Th lineage-specific transcription factors determined by qRT-PCR normalized to 18S RNA. The graph shows mean ± SD values from at least three independent experiments. *, P

    Techniques Used: Activation Assay, Mouse Assay, Incubation, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

    DHHC21 mediates TCR-induced S-acylation of signaling proteins. (A) Overview of the acyl-resin assisted capture (Acyl-RAC) assay. Free cysteine thiols (-SH) are irreversibly blocked by S-methyl methanethiosulfonate (MMTS) following cell lysis. Thioester bonds between cysteines and acyl groups are then specifically cleaved by neutral hydroxylamine (HA). The newly formed free thiol groups are captured by the thiol-reactive sepharose and S-acylated proteins detected by immunoblotting. (B) TCR-induced protein S-acylation. CD4 + T cells from WT or Zdhhc21 dep mice were stimulated with cross-linked anti-CD3/CD28 antibodies for indicated times and protein S-acylation (acyl-) was determined using Acyl-RAC assay. Samples not treated with hydroxylamine (-HA) were used as a negative control. β-Actin, a known S-acylated protein ( 32 ), was used as a loading control.
    Figure Legend Snippet: DHHC21 mediates TCR-induced S-acylation of signaling proteins. (A) Overview of the acyl-resin assisted capture (Acyl-RAC) assay. Free cysteine thiols (-SH) are irreversibly blocked by S-methyl methanethiosulfonate (MMTS) following cell lysis. Thioester bonds between cysteines and acyl groups are then specifically cleaved by neutral hydroxylamine (HA). The newly formed free thiol groups are captured by the thiol-reactive sepharose and S-acylated proteins detected by immunoblotting. (B) TCR-induced protein S-acylation. CD4 + T cells from WT or Zdhhc21 dep mice were stimulated with cross-linked anti-CD3/CD28 antibodies for indicated times and protein S-acylation (acyl-) was determined using Acyl-RAC assay. Samples not treated with hydroxylamine (-HA) were used as a negative control. β-Actin, a known S-acylated protein ( 32 ), was used as a loading control.

    Techniques Used: Lysis, Mouse Assay, Negative Control

    19) Product Images from "NLRC3 negatively regulates CD4+ T cells and impacts protective immunity during Mycobacterium tuberculosis infection"

    Article Title: NLRC3 negatively regulates CD4+ T cells and impacts protective immunity during Mycobacterium tuberculosis infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007266

    NLRC3 suppresses activation of CD4 + T cells via negatively regulating NF-κB and ERK Signaling. Purified WT and Nlrc3 -/- CD4 + T cells were stimulated for 48 hr with anti-CD3 (1 μg/ml) plus anti-CD28 (1 μg/ml) in the presence or absence of the NF-κB inhibitor JSH-23 (20 μM), MEK1/2-inhibitor U0126 (40 μM) or the mix of the two. (A) Concentrations of IL-2 in supernatants were detected by ELISA. (B) The incorporation of thymidine was measured during the final 8 hr. (C) Concentrations of IFN-γ, IL-17, TNF-α and GM-CSF in supernatants were detected by ELISA. Data shown are the mean ±SD. * P
    Figure Legend Snippet: NLRC3 suppresses activation of CD4 + T cells via negatively regulating NF-κB and ERK Signaling. Purified WT and Nlrc3 -/- CD4 + T cells were stimulated for 48 hr with anti-CD3 (1 μg/ml) plus anti-CD28 (1 μg/ml) in the presence or absence of the NF-κB inhibitor JSH-23 (20 μM), MEK1/2-inhibitor U0126 (40 μM) or the mix of the two. (A) Concentrations of IL-2 in supernatants were detected by ELISA. (B) The incorporation of thymidine was measured during the final 8 hr. (C) Concentrations of IFN-γ, IL-17, TNF-α and GM-CSF in supernatants were detected by ELISA. Data shown are the mean ±SD. * P

    Techniques Used: Activation Assay, Purification, Enzyme-linked Immunosorbent Assay

    NLRC3 negatively regulates NF-κB and ERK Signaling in CD4 + T cells. (A-B) Purified WT or Nlrc3 -/- naïve CD4 + T cells were adoptively transferred into Rag2 -/- mice. Then recipient mice were infected with M . tuberculosis and were harvested at 3w.p.i.. Lungs were collected. (A) Immunoblot analysis of lung lysates. Each lane represents an individual mouse. (B) Densitometry quantification of band intensity for A. (C-D) Purified WT and Nlrc3 -/- CD4 + T cells were stimulated with anti-CD3 (1 μg/ml) plus anti-CD28 (1 μg/ml) in the presence or absence of the NF-κB inhibitor JSH-23 (20 μM) or MEK1/2-inhibitor U0126 (40 μM). (C) Lysates were probed for total and phosphorylated p65 (p-p65), p65, p-ERK, ERK and GAPDH. (D) Densitometry quantification of band intensity for C. Data shown in (B and D) are the mean ±SD. * P
    Figure Legend Snippet: NLRC3 negatively regulates NF-κB and ERK Signaling in CD4 + T cells. (A-B) Purified WT or Nlrc3 -/- naïve CD4 + T cells were adoptively transferred into Rag2 -/- mice. Then recipient mice were infected with M . tuberculosis and were harvested at 3w.p.i.. Lungs were collected. (A) Immunoblot analysis of lung lysates. Each lane represents an individual mouse. (B) Densitometry quantification of band intensity for A. (C-D) Purified WT and Nlrc3 -/- CD4 + T cells were stimulated with anti-CD3 (1 μg/ml) plus anti-CD28 (1 μg/ml) in the presence or absence of the NF-κB inhibitor JSH-23 (20 μM) or MEK1/2-inhibitor U0126 (40 μM). (C) Lysates were probed for total and phosphorylated p65 (p-p65), p65, p-ERK, ERK and GAPDH. (D) Densitometry quantification of band intensity for C. Data shown in (B and D) are the mean ±SD. * P

    Techniques Used: Purification, Mouse Assay, Infection

    T cell activation in vitro requires NLRC3. (A) Relative expression of Nlrc3 in purified macrophages (CD11b + Gr-1 - ), dendritic cells (CD11c + MHCII hi ), polymorphonuclear leukocytes (PMNs) (CD11b + Gr-1 + ), CD4 + T cells (CD3 + CD4 + ) and CD8 + T cells (CD3 + CD8 + ). (B) Purified naïve T cells isolated from WT and Nlrc3 -/- mice were stimulated with plate bound anti-CD3 (increasing concentrations) and anti-CD28 (1 μg/ml) for 48 hr and the incorporation of thymidine was measured during the final 8 hr. (C) Purified WT and Nlrc3 -/- naïve CD4 + T cells were labeled with CFSE and stimulated with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) for 3 d. (D) Concentrations of IL-2 in supernatants of purified WT and Nlrc3 -/- naïve CD4 + T cells stimulated for 0–80 h with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) were detected by ELISA. (E) Thymidine incorporation in purified WT and Nlrc3 -/- naive CD4 + T cells first primed with anti-CD3 and CD28 and then cultured with various concentrations of IL-2. (F) Purified WT and Nlrc3 -/- naïve CD4 + T cells were polarized in Th1 or Th17 culture conditions for 4 days. Data shown in (B, C, E, F) are the mean ±SD. * P
    Figure Legend Snippet: T cell activation in vitro requires NLRC3. (A) Relative expression of Nlrc3 in purified macrophages (CD11b + Gr-1 - ), dendritic cells (CD11c + MHCII hi ), polymorphonuclear leukocytes (PMNs) (CD11b + Gr-1 + ), CD4 + T cells (CD3 + CD4 + ) and CD8 + T cells (CD3 + CD8 + ). (B) Purified naïve T cells isolated from WT and Nlrc3 -/- mice were stimulated with plate bound anti-CD3 (increasing concentrations) and anti-CD28 (1 μg/ml) for 48 hr and the incorporation of thymidine was measured during the final 8 hr. (C) Purified WT and Nlrc3 -/- naïve CD4 + T cells were labeled with CFSE and stimulated with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) for 3 d. (D) Concentrations of IL-2 in supernatants of purified WT and Nlrc3 -/- naïve CD4 + T cells stimulated for 0–80 h with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) were detected by ELISA. (E) Thymidine incorporation in purified WT and Nlrc3 -/- naive CD4 + T cells first primed with anti-CD3 and CD28 and then cultured with various concentrations of IL-2. (F) Purified WT and Nlrc3 -/- naïve CD4 + T cells were polarized in Th1 or Th17 culture conditions for 4 days. Data shown in (B, C, E, F) are the mean ±SD. * P

    Techniques Used: Activation Assay, In Vitro, Expressing, Purification, Isolation, Mouse Assay, Labeling, Enzyme-linked Immunosorbent Assay, Cell Culture

    20) Product Images from "Impaired Circulating CD4+LAP+ Regulatory T Cells in Patients with Acute Coronary Syndrome and Its Mechanistic Study"

    Article Title: Impaired Circulating CD4+LAP+ Regulatory T Cells in Patients with Acute Coronary Syndrome and Its Mechanistic Study

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0088775

    The expression of GARP on CD4 + T cells in freshly isolated PBMCs and CD3/28-sitmulated PBMCs. Bood samples were collected from patients with CPS (n = 20), CSA (n = 17) and ACS (n = 37, AMI (n = 18), and UA (n = 19)). PBMCs were freshly isolated or stimulated with CD3/CD28 for 24 h. Then the cells were stained with anti-human CD4-FITC, anti-human GARP-PE and analyzed by flow cytometry using FACS Calibur (BD). (A) Representative dot plot shows the gated CD4 + T cells on the FSC/SSC. (B) Representative FACS images show GARP expression on CD4 + T cells in unstimulated PMBCs (upper panel) and stimulated PBMCs (lower panel) from one patient in each group. Comparison of the CD4 + GARP + T cells frequencies in unstimulated PBMCs (C) and stimulated PBMCs (D) among four groups. * p
    Figure Legend Snippet: The expression of GARP on CD4 + T cells in freshly isolated PBMCs and CD3/28-sitmulated PBMCs. Bood samples were collected from patients with CPS (n = 20), CSA (n = 17) and ACS (n = 37, AMI (n = 18), and UA (n = 19)). PBMCs were freshly isolated or stimulated with CD3/CD28 for 24 h. Then the cells were stained with anti-human CD4-FITC, anti-human GARP-PE and analyzed by flow cytometry using FACS Calibur (BD). (A) Representative dot plot shows the gated CD4 + T cells on the FSC/SSC. (B) Representative FACS images show GARP expression on CD4 + T cells in unstimulated PMBCs (upper panel) and stimulated PBMCs (lower panel) from one patient in each group. Comparison of the CD4 + GARP + T cells frequencies in unstimulated PBMCs (C) and stimulated PBMCs (D) among four groups. * p

    Techniques Used: Expressing, Isolation, Staining, Flow Cytometry, Cytometry, FACS

    The expression of GARP and LAP on CD4 + T cells in freshly isolated PBMCs and CD3/28-sitmulated PBMCs. Bood samples were collected from patients with ACS (AMI (n = 10) and UA (n = 10)) and controls (CSA (n = 17), CPS (n = 20), and PBMCs were freshly isolated or stimulted with CD3/CD28 for 24 h, then the cells were stained with anti-human CD4-FITC,anti-human LAP-APC, anti-human GARP-PE and analyzed the data by flow cytometry using FACS Calibur (BD).(A) Representative dot plot shows the gated CD4 T cells on the FSC/SSC. (B) Representative FACS pictures show the GARP and LAP expreesion on CD4 + T cells in unstimulated PMBCs (upper panel) and stimulated PBMCs (lower panel) from one patient in each group. (C) Statistical analysis of the percentage CD4 + LAP + GARP + T cells. Dark bars indicate data from unstimulated samples. Hatched bars indicate data from stimulated samples. * p
    Figure Legend Snippet: The expression of GARP and LAP on CD4 + T cells in freshly isolated PBMCs and CD3/28-sitmulated PBMCs. Bood samples were collected from patients with ACS (AMI (n = 10) and UA (n = 10)) and controls (CSA (n = 17), CPS (n = 20), and PBMCs were freshly isolated or stimulted with CD3/CD28 for 24 h, then the cells were stained with anti-human CD4-FITC,anti-human LAP-APC, anti-human GARP-PE and analyzed the data by flow cytometry using FACS Calibur (BD).(A) Representative dot plot shows the gated CD4 T cells on the FSC/SSC. (B) Representative FACS pictures show the GARP and LAP expreesion on CD4 + T cells in unstimulated PMBCs (upper panel) and stimulated PBMCs (lower panel) from one patient in each group. (C) Statistical analysis of the percentage CD4 + LAP + GARP + T cells. Dark bars indicate data from unstimulated samples. Hatched bars indicate data from stimulated samples. * p

    Techniques Used: Expressing, Isolation, Staining, Flow Cytometry, Cytometry, FACS

    21) Product Images from "A timer for analyzing temporally dynamic changes in transcription during differentiation in vivo"

    Article Title: A timer for analyzing temporally dynamic changes in transcription during differentiation in vivo

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201711048

    Foxp3-Tocky identifies newly generated Treg cells. (A) Use of Foxp3- Tocky mice to investigate in vivo dynamics of Foxp3 transcription. (B) Construct for the generation of Foxp3-Tocky BAC transgenic mice. (C) Splenic T cells were sored into blue − red+, blue + red + , and blue − red − and analyzed for intracellular Foxp3 proteins. (D) Foxp3- Tocky mice were crossed with Foxp3-IRES-GFP, and analyzed for the coexpression of GFP and Timer. (E) Timer-negative naive T cells and splenic Timer-positive Treg cells from Foxp3- Tocky mice were isolated and stimulated by anti-CD3 and anti-CD28 for 0, 22, or 50 h in the presence of IL-2 (and TGF-β for iTreg cells). Flow cytometry plots display raw blue versus red expression during the cultures. (F and G) Timer Angle versus Timer Intensity (F) or Timer Angle versus time in the data from E (F).
    Figure Legend Snippet: Foxp3-Tocky identifies newly generated Treg cells. (A) Use of Foxp3- Tocky mice to investigate in vivo dynamics of Foxp3 transcription. (B) Construct for the generation of Foxp3-Tocky BAC transgenic mice. (C) Splenic T cells were sored into blue − red+, blue + red + , and blue − red − and analyzed for intracellular Foxp3 proteins. (D) Foxp3- Tocky mice were crossed with Foxp3-IRES-GFP, and analyzed for the coexpression of GFP and Timer. (E) Timer-negative naive T cells and splenic Timer-positive Treg cells from Foxp3- Tocky mice were isolated and stimulated by anti-CD3 and anti-CD28 for 0, 22, or 50 h in the presence of IL-2 (and TGF-β for iTreg cells). Flow cytometry plots display raw blue versus red expression during the cultures. (F and G) Timer Angle versus Timer Intensity (F) or Timer Angle versus time in the data from E (F).

    Techniques Used: Generated, Mouse Assay, In Vivo, Construct, BAC Assay, Transgenic Assay, Isolation, Flow Cytometry, Cytometry, Expressing

    Cell division, costimulation, and IL-2 signaling do not affect Timer Angle progression. (A–F) CD4 + T cells from Nr4a3 -Tocky mice were labeled with a proliferation dye and activated for 72 h with anti-CD3. Cells were then analyzed based on dilution of proliferation dye and classified into number of cellular divisions. (A) Timer–blue versus Timer–red fluorescence in CD4 + T cells gated on dilution of proliferation dye. (B) Mean Timer Angle values in the cultures from A. (C and D) Splenocytes from Nr4a3 -Tocky mice were stimulated on anti-CD3–coated plates in the presence or absence of 100 U/ml rhIL-2 for 20 h. Timer–blue versus Timer–red fluorescence in CD4 + T cells from cultures (C). Mean Timer-Angle in cultures (D). (E and F) Splenocytes from Nr4a3 -Tocky mice were stimulated on plates coated with anti-CD28 alone, anti-CD3 alone, or anti-CD3 + anti-CD28 for 20 h. Timer–blue versus Timer–red fluorescence in CD4 + T cells from cultures (E). Mean Timer-Angle in cultures (F). n = 3 culture triplicates; error bars represent mean ± SEM. Data are representative of two independent experiments.
    Figure Legend Snippet: Cell division, costimulation, and IL-2 signaling do not affect Timer Angle progression. (A–F) CD4 + T cells from Nr4a3 -Tocky mice were labeled with a proliferation dye and activated for 72 h with anti-CD3. Cells were then analyzed based on dilution of proliferation dye and classified into number of cellular divisions. (A) Timer–blue versus Timer–red fluorescence in CD4 + T cells gated on dilution of proliferation dye. (B) Mean Timer Angle values in the cultures from A. (C and D) Splenocytes from Nr4a3 -Tocky mice were stimulated on anti-CD3–coated plates in the presence or absence of 100 U/ml rhIL-2 for 20 h. Timer–blue versus Timer–red fluorescence in CD4 + T cells from cultures (C). Mean Timer-Angle in cultures (D). (E and F) Splenocytes from Nr4a3 -Tocky mice were stimulated on plates coated with anti-CD28 alone, anti-CD3 alone, or anti-CD3 + anti-CD28 for 20 h. Timer–blue versus Timer–red fluorescence in CD4 + T cells from cultures (E). Mean Timer-Angle in cultures (F). n = 3 culture triplicates; error bars represent mean ± SEM. Data are representative of two independent experiments.

    Techniques Used: Mouse Assay, Labeling, Fluorescence

    22) Product Images from "Gut microbiota from multiple sclerosis patients enables spontaneous autoimmune encephalomyelitis in mice"

    Article Title: Gut microbiota from multiple sclerosis patients enables spontaneous autoimmune encephalomyelitis in mice

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1711233114

    Gut bacteria from healthy twins trigger an antiinflammatory T cell response. ( A ) IL-10 production of CD4 + T cells isolated from PBMCs of selected twin pairs. T cells were stimulated for 96 h with 1 µg/mL anti-CD3 and anti-CD28 antibodies. Levels
    Figure Legend Snippet: Gut bacteria from healthy twins trigger an antiinflammatory T cell response. ( A ) IL-10 production of CD4 + T cells isolated from PBMCs of selected twin pairs. T cells were stimulated for 96 h with 1 µg/mL anti-CD3 and anti-CD28 antibodies. Levels

    Techniques Used: Isolation

    Related Articles

    Ex Vivo:

    Article Title: CMIP is a negative regulator of T cell signaling
    Article Snippet: Purified T cells were isolated by negative selection using a Pan T Cell isolation kit (Miltenyi Biotec GmbH, Germany). .. Ex vivo T cell stimulationBefore stimulation, T cells were systematically synchronized at the G1 phase of the cell cycle by serum starvation in 2% fetal calf serum (FCS) for 6 h. Synchronized T cells were then activated for the indicated times with soluble anti-CD3 and anti-CD28 antibodies (eBiosciences, San Diego, CA, USA) at 1 µg/ml in RPMI complete medium supplemented with 10% FCS. ..

    Expressing:

    Article Title: The inhibitory effect against collagen-induced arthritis by Schistosoma japonicum infection is infection stage-dependent
    Article Snippet: After 2 hr incubation at room temperature and wash, the final color development was achieved by adding peroxidase substrate ABTS (2,2'-Azino-bis(3-Ethylbenzthiazoline-6-Sulfonic Acid) , Sigma) to each well at 100 μl/well and the absorbance was measured at 405 nm at appropriate time. .. Measurement of cytokine production by splenocytes To study cytokine expression by T lymphocytes, isolated spleen cells (5 × 106 cells/well) from mice that were immunized by CII for 8 weeks or from control mice were stimulated with 1 μg/ml anti-CD3ε (eBioscience) and 1 μg/ml anti-CD28 (eBioscience) at 37°C, 5%CO2 in 10% FCS/RPMI(GIBCO) culture medium for 72 hrs. .. The cytokine contents in the supernatants were analyzed by ELISA analysis as described by the e-Bioscience protocol http://www.ebioscience.com .

    Isolation:

    Article Title: The inhibitory effect against collagen-induced arthritis by Schistosoma japonicum infection is infection stage-dependent
    Article Snippet: After 2 hr incubation at room temperature and wash, the final color development was achieved by adding peroxidase substrate ABTS (2,2'-Azino-bis(3-Ethylbenzthiazoline-6-Sulfonic Acid) , Sigma) to each well at 100 μl/well and the absorbance was measured at 405 nm at appropriate time. .. Measurement of cytokine production by splenocytes To study cytokine expression by T lymphocytes, isolated spleen cells (5 × 106 cells/well) from mice that were immunized by CII for 8 weeks or from control mice were stimulated with 1 μg/ml anti-CD3ε (eBioscience) and 1 μg/ml anti-CD28 (eBioscience) at 37°C, 5%CO2 in 10% FCS/RPMI(GIBCO) culture medium for 72 hrs. .. The cytokine contents in the supernatants were analyzed by ELISA analysis as described by the e-Bioscience protocol http://www.ebioscience.com .

    Mouse Assay:

    Article Title: The inhibitory effect against collagen-induced arthritis by Schistosoma japonicum infection is infection stage-dependent
    Article Snippet: After 2 hr incubation at room temperature and wash, the final color development was achieved by adding peroxidase substrate ABTS (2,2'-Azino-bis(3-Ethylbenzthiazoline-6-Sulfonic Acid) , Sigma) to each well at 100 μl/well and the absorbance was measured at 405 nm at appropriate time. .. Measurement of cytokine production by splenocytes To study cytokine expression by T lymphocytes, isolated spleen cells (5 × 106 cells/well) from mice that were immunized by CII for 8 weeks or from control mice were stimulated with 1 μg/ml anti-CD3ε (eBioscience) and 1 μg/ml anti-CD28 (eBioscience) at 37°C, 5%CO2 in 10% FCS/RPMI(GIBCO) culture medium for 72 hrs. .. The cytokine contents in the supernatants were analyzed by ELISA analysis as described by the e-Bioscience protocol http://www.ebioscience.com .

    Purification:

    Article Title: Apaf1 plays a negative regulatory role in T cell responses by suppressing activation of antigen-stimulated T cells
    Article Snippet: For activation-induced apoptosis of peripheral lymph node (LN) T cells, LN cells were stained with FITC-labeled anti-Thy1.2 antibody (eBioscience) followed by positive selection procedure using anti-FITC magnetic beads (MACS; Miltenyi Biotec) and LS columns (Miltenyi Biotec). .. Purified T cells ( > 90% T cells, 3× 106 cells/well) were activated with plate-bound anti-CD3ε (0.3 μg/ml; clone 145-2C11, eBioscience) plus anti-CD28 antibodies (3 μg/ml; clone 37.51, eBioscience) for 48 hours in 6-well plates. .. After removal of apoptotic cells by staining the cells with Annexin V-APC followed by anti-APC magnetic beads (MACS) procedure, remaining cells (Annexin V-negative cells > 97.6%) were cultured (2 × 105 cells/200 μl/well) either in fresh medium alone, or conditioned medium prepared from the supernatants of the primary stimulation culture (anti-CD3ε plus anti-CD28 antibodies), or with anti-CD3ε antibody in fresh medium.

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    Thermo Fisher anti cd28 antibodies
    Generation of T cell-specific Apaf1-deficient mice. (A) Southern blot analysis of genomic DNA from Apaf1 f-Neo/+ thymocytes (left), Apaf1 f/+ thymocytes (right), and Apaf1 f/+ ES cells, in which Neo gene was removed by transient expression of FLPe recombinase (middle). DNA was digested with EcoRI and detected with the probe at exon 4. (See S1 Fig for detail.) (B) Western blot analysis of proteins from LN T cells and thymocytes of Apaf1 f/f mice and Lck- Cre - Apaf1 f/f mice. (C) Thymocytes from Apaf1 f/f mice (open columns) or Lck- Cre - Apaf1 f/f mice (closed columns) were stimulated with indicated doses of anti-Fas antibodies plus cycloheximide (αFas + CHX), dexamethasone (Dex), staurosporine (Stauro), γ-irradiation, or left untreated. Apoptotic cells were detected by flow cytometry. (D) Purified T cells from LN of Apaf1 f/f (open columns) or Lck- Cre - Apaf1 f/f (closed columns) were activated for 48 hours with anti-CD3ε antibody plus <t>anti-CD28</t> antibody. Activated cells, after removal of dead cells, were cultured in the presence of conditioned medium (CM), in the fresh medium for growth factor deprivation (Media), or re-stimulated with anti-CD3ε antibody in fresh medium for activation-induced cell death, for 20 hours. Apoptotic cells were detected by flow cytometry. Data show means + SD of triplicated samples. Experiments were repeated three times with similar results. *; p
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    Generation of T cell-specific Apaf1-deficient mice. (A) Southern blot analysis of genomic DNA from Apaf1 f-Neo/+ thymocytes (left), Apaf1 f/+ thymocytes (right), and Apaf1 f/+ ES cells, in which Neo gene was removed by transient expression of FLPe recombinase (middle). DNA was digested with EcoRI and detected with the probe at exon 4. (See S1 Fig for detail.) (B) Western blot analysis of proteins from LN T cells and thymocytes of Apaf1 f/f mice and Lck- Cre - Apaf1 f/f mice. (C) Thymocytes from Apaf1 f/f mice (open columns) or Lck- Cre - Apaf1 f/f mice (closed columns) were stimulated with indicated doses of anti-Fas antibodies plus cycloheximide (αFas + CHX), dexamethasone (Dex), staurosporine (Stauro), γ-irradiation, or left untreated. Apoptotic cells were detected by flow cytometry. (D) Purified T cells from LN of Apaf1 f/f (open columns) or Lck- Cre - Apaf1 f/f (closed columns) were activated for 48 hours with anti-CD3ε antibody plus anti-CD28 antibody. Activated cells, after removal of dead cells, were cultured in the presence of conditioned medium (CM), in the fresh medium for growth factor deprivation (Media), or re-stimulated with anti-CD3ε antibody in fresh medium for activation-induced cell death, for 20 hours. Apoptotic cells were detected by flow cytometry. Data show means + SD of triplicated samples. Experiments were repeated three times with similar results. *; p

    Journal: PLoS ONE

    Article Title: Apaf1 plays a negative regulatory role in T cell responses by suppressing activation of antigen-stimulated T cells

    doi: 10.1371/journal.pone.0195119

    Figure Lengend Snippet: Generation of T cell-specific Apaf1-deficient mice. (A) Southern blot analysis of genomic DNA from Apaf1 f-Neo/+ thymocytes (left), Apaf1 f/+ thymocytes (right), and Apaf1 f/+ ES cells, in which Neo gene was removed by transient expression of FLPe recombinase (middle). DNA was digested with EcoRI and detected with the probe at exon 4. (See S1 Fig for detail.) (B) Western blot analysis of proteins from LN T cells and thymocytes of Apaf1 f/f mice and Lck- Cre - Apaf1 f/f mice. (C) Thymocytes from Apaf1 f/f mice (open columns) or Lck- Cre - Apaf1 f/f mice (closed columns) were stimulated with indicated doses of anti-Fas antibodies plus cycloheximide (αFas + CHX), dexamethasone (Dex), staurosporine (Stauro), γ-irradiation, or left untreated. Apoptotic cells were detected by flow cytometry. (D) Purified T cells from LN of Apaf1 f/f (open columns) or Lck- Cre - Apaf1 f/f (closed columns) were activated for 48 hours with anti-CD3ε antibody plus anti-CD28 antibody. Activated cells, after removal of dead cells, were cultured in the presence of conditioned medium (CM), in the fresh medium for growth factor deprivation (Media), or re-stimulated with anti-CD3ε antibody in fresh medium for activation-induced cell death, for 20 hours. Apoptotic cells were detected by flow cytometry. Data show means + SD of triplicated samples. Experiments were repeated three times with similar results. *; p

    Article Snippet: Purified T cells ( > 90% T cells, 3× 106 cells/well) were activated with plate-bound anti-CD3ε (0.3 μg/ml; clone 145-2C11, eBioscience) plus anti-CD28 antibodies (3 μg/ml; clone 37.51, eBioscience) for 48 hours in 6-well plates.

    Techniques: Mouse Assay, Southern Blot, Expressing, Western Blot, Irradiation, Flow Cytometry, Cytometry, Purification, Cell Culture, Activation Assay

    Impact of CMIP expression and T cell activation on the total proteome. a Heat map showing 46 differentially expressed proteins in T cells obtained from transgenic and WT mice that were or were not subjected to 60 min of costimulation with anti-CD3/CD28 antibodies (1 μg/ml each). The column tree denotes mice grouped by hierarchical clustering. Rows correspond to 46 proteins whose differential expression was significant according to two-way ANOVA ( p

    Journal: Cellular and Molecular Immunology

    Article Title: CMIP is a negative regulator of T cell signaling

    doi: 10.1038/s41423-019-0266-5

    Figure Lengend Snippet: Impact of CMIP expression and T cell activation on the total proteome. a Heat map showing 46 differentially expressed proteins in T cells obtained from transgenic and WT mice that were or were not subjected to 60 min of costimulation with anti-CD3/CD28 antibodies (1 μg/ml each). The column tree denotes mice grouped by hierarchical clustering. Rows correspond to 46 proteins whose differential expression was significant according to two-way ANOVA ( p

    Article Snippet: Ex vivo T cell stimulationBefore stimulation, T cells were systematically synchronized at the G1 phase of the cell cycle by serum starvation in 2% fetal calf serum (FCS) for 6 h. Synchronized T cells were then activated for the indicated times with soluble anti-CD3 and anti-CD28 antibodies (eBiosciences, San Diego, CA, USA) at 1 µg/ml in RPMI complete medium supplemented with 10% FCS.

    Techniques: Expressing, Activation Assay, Transgenic Assay, Mouse Assay

    Transgenic T cells exhibit a hypophosphorylated protein profile with the downregulation of active Src. a Representative Western blot of protein lysates from transgenic and WT T cells after 60 min of activation by anti-CD3/CD28 antibodies incubated with anti-phosphotyrosine 4G10; blots were stripped and reprobed with anti-GAPDH antibody. b – e Western blots of protein lysates from transgenic and WT T cells several time points following anti-CD3/CD28 antibody activation (1 µg/ml each); blots were stripped and reprobed with an antibody raised against total specific protein. The results are representative of three independent experiments [pY 418 Src/total Src: one-way ANOVA, * p = 0.0163; pY 505 Lck/total Lck: Kruskal-Wallis test, * p = 0.0362; pY 528 Fyn/total GAPDH, one-way ANOVA, * p = 0.0204, Tg vs. WT (30 min), ** p = 0.0015; pYr 319 Zap70/total Zap70, one-way Anova, ** p = 0.0068]. f Immunofluorescence staining for pY 418 Src in transgenic and WT T cells isolated by negative immunoselection

    Journal: Cellular and Molecular Immunology

    Article Title: CMIP is a negative regulator of T cell signaling

    doi: 10.1038/s41423-019-0266-5

    Figure Lengend Snippet: Transgenic T cells exhibit a hypophosphorylated protein profile with the downregulation of active Src. a Representative Western blot of protein lysates from transgenic and WT T cells after 60 min of activation by anti-CD3/CD28 antibodies incubated with anti-phosphotyrosine 4G10; blots were stripped and reprobed with anti-GAPDH antibody. b – e Western blots of protein lysates from transgenic and WT T cells several time points following anti-CD3/CD28 antibody activation (1 µg/ml each); blots were stripped and reprobed with an antibody raised against total specific protein. The results are representative of three independent experiments [pY 418 Src/total Src: one-way ANOVA, * p = 0.0163; pY 505 Lck/total Lck: Kruskal-Wallis test, * p = 0.0362; pY 528 Fyn/total GAPDH, one-way ANOVA, * p = 0.0204, Tg vs. WT (30 min), ** p = 0.0015; pYr 319 Zap70/total Zap70, one-way Anova, ** p = 0.0068]. f Immunofluorescence staining for pY 418 Src in transgenic and WT T cells isolated by negative immunoselection

    Article Snippet: Ex vivo T cell stimulationBefore stimulation, T cells were systematically synchronized at the G1 phase of the cell cycle by serum starvation in 2% fetal calf serum (FCS) for 6 h. Synchronized T cells were then activated for the indicated times with soluble anti-CD3 and anti-CD28 antibodies (eBiosciences, San Diego, CA, USA) at 1 µg/ml in RPMI complete medium supplemented with 10% FCS.

    Techniques: Transgenic Assay, Western Blot, Activation Assay, Incubation, Immunofluorescence, Staining, Isolation

    Transgenic T cells exhibit higher levels of inactive Fyn and Lck in LRs and fail to be activated after CD3/CD28 costimulation. a Western blotting and TLC of raft (R) and nonraft (NR) fractions prepared at rest (0 min) and after the activation (30 min) of transgenic and WT mouse T cells. Rafts are enriched in Flotillin-1 and cholesterol. Samples were analyzed with anti-pY 528 Fyn and anti-pY 505 Lck antibodies, which recognize the inactive forms of Fyn and Lck, respectively, followed by antibodies recognizing total Fyn and Lck, respectively. Cholesterol was analyzed by TLC. b , c Inactive/total Fyn and inactive/total Lck in raft microdomains according to densitometric analysis of the bands shown in a

    Journal: Cellular and Molecular Immunology

    Article Title: CMIP is a negative regulator of T cell signaling

    doi: 10.1038/s41423-019-0266-5

    Figure Lengend Snippet: Transgenic T cells exhibit higher levels of inactive Fyn and Lck in LRs and fail to be activated after CD3/CD28 costimulation. a Western blotting and TLC of raft (R) and nonraft (NR) fractions prepared at rest (0 min) and after the activation (30 min) of transgenic and WT mouse T cells. Rafts are enriched in Flotillin-1 and cholesterol. Samples were analyzed with anti-pY 528 Fyn and anti-pY 505 Lck antibodies, which recognize the inactive forms of Fyn and Lck, respectively, followed by antibodies recognizing total Fyn and Lck, respectively. Cholesterol was analyzed by TLC. b , c Inactive/total Fyn and inactive/total Lck in raft microdomains according to densitometric analysis of the bands shown in a

    Article Snippet: Ex vivo T cell stimulationBefore stimulation, T cells were systematically synchronized at the G1 phase of the cell cycle by serum starvation in 2% fetal calf serum (FCS) for 6 h. Synchronized T cells were then activated for the indicated times with soluble anti-CD3 and anti-CD28 antibodies (eBiosciences, San Diego, CA, USA) at 1 µg/ml in RPMI complete medium supplemented with 10% FCS.

    Techniques: Transgenic Assay, Western Blot, Thin Layer Chromatography, Activation Assay

    CMIP inhibits clustering and activation of the raft signaling platform. a CMIP inhibits membrane clustering of Src kinases and CTB after CD3/CD28 stimulation. Immunofluorescence analysis of transgenic and WT T cells after 30 min of activation by anti-CD3/CD28 antibodies (1 μg/ml each) that were then fixed and immunostained for total Src (green) and CTB (red). Cellular nuclei were revealed by counterstaining with DAPI dye. Confocal analysis shows that Src kinases and CTB colocalize in WT but not transgenic T cells. b CMIP inhibits the clustering of LAT and CTB into LRs after CD3/CD28 stimulation. Fluorescence analysis of LAT and CTB after T cell activation performed as in a . c Negative Src, LAT and CTB controls: the specificity of each signal in WT T cells was assessed using IgG isotype control antibody instead of primary antibody. d CMIP inhibits LR clustering and T cell polarization. Transgenic and WT T cells were synchronized, stained with CTB, loaded into 8-well plates at 50,000 cells/well and activated with anti-CD3-coated beads (cells:beads, 2:1) and soluble anti-CD28 (1 μg/ml). Cells were kept at 37 °C. Data were acquired with a confocal microscope at 1 image/15 s. Images were extracted from movies ( 1 image/min) and analyzed by ImageJ software (magnification: ×63)

    Journal: Cellular and Molecular Immunology

    Article Title: CMIP is a negative regulator of T cell signaling

    doi: 10.1038/s41423-019-0266-5

    Figure Lengend Snippet: CMIP inhibits clustering and activation of the raft signaling platform. a CMIP inhibits membrane clustering of Src kinases and CTB after CD3/CD28 stimulation. Immunofluorescence analysis of transgenic and WT T cells after 30 min of activation by anti-CD3/CD28 antibodies (1 μg/ml each) that were then fixed and immunostained for total Src (green) and CTB (red). Cellular nuclei were revealed by counterstaining with DAPI dye. Confocal analysis shows that Src kinases and CTB colocalize in WT but not transgenic T cells. b CMIP inhibits the clustering of LAT and CTB into LRs after CD3/CD28 stimulation. Fluorescence analysis of LAT and CTB after T cell activation performed as in a . c Negative Src, LAT and CTB controls: the specificity of each signal in WT T cells was assessed using IgG isotype control antibody instead of primary antibody. d CMIP inhibits LR clustering and T cell polarization. Transgenic and WT T cells were synchronized, stained with CTB, loaded into 8-well plates at 50,000 cells/well and activated with anti-CD3-coated beads (cells:beads, 2:1) and soluble anti-CD28 (1 μg/ml). Cells were kept at 37 °C. Data were acquired with a confocal microscope at 1 image/15 s. Images were extracted from movies ( 1 image/min) and analyzed by ImageJ software (magnification: ×63)

    Article Snippet: Ex vivo T cell stimulationBefore stimulation, T cells were systematically synchronized at the G1 phase of the cell cycle by serum starvation in 2% fetal calf serum (FCS) for 6 h. Synchronized T cells were then activated for the indicated times with soluble anti-CD3 and anti-CD28 antibodies (eBiosciences, San Diego, CA, USA) at 1 µg/ml in RPMI complete medium supplemented with 10% FCS.

    Techniques: Activation Assay, CtB Assay, Immunofluorescence, Transgenic Assay, Fluorescence, Staining, Microscopy, Software

    Influence of CMIP induction on cytokine expression. a Expression of the CMIP transcript. Total RNA was extracted from synchronized T cells before and after CD3/CD28 stimulation (1 μg/ml each) at the indicated times. The expression of endogenous Cmip in WT (green) and Tg mice (blue) along with Tg Cmip (red) is shown. b Expression of CMIP protein under the same conditions. c IL-2, IFNγ, IL-4, and IL-10 transcripts were quantified by RT-qPCR. The results are representative of three independent experiments ( n = 5 mice in each group). [Tg vs. WT, mean with SEM; IL-2, 1 h: * p = 0.0278, 2 h: ** p = 0.0040, 4 h: ** p = 0.0040; IL-4, 1 h: ** p = 0.0040, 2 h: ** p = 0,0040, 4 h: ** p = 0,0040, 6 h: * p = 0.0278, 8 and 16 h: nonsignificant (ns); IFNγ, 1 h: * p = 0.0257, 2 h: ** p = 0.0040, 4 h: ** p = 0.0079, 6 h: * p = 0.0317, 8 h: * p = 0.0317 and 16 h: p = 0.532 (ns); IL-10, 1 h: * p = 0.0286, 2 h: * p = 0.0159, 4 h: p = 0.1508 (ns), 6 h: * p = 0.0159, 8 h: ** p = 0.0079, 16 h: ** p = 0.0079; Mann-Whitney tests]

    Journal: Cellular and Molecular Immunology

    Article Title: CMIP is a negative regulator of T cell signaling

    doi: 10.1038/s41423-019-0266-5

    Figure Lengend Snippet: Influence of CMIP induction on cytokine expression. a Expression of the CMIP transcript. Total RNA was extracted from synchronized T cells before and after CD3/CD28 stimulation (1 μg/ml each) at the indicated times. The expression of endogenous Cmip in WT (green) and Tg mice (blue) along with Tg Cmip (red) is shown. b Expression of CMIP protein under the same conditions. c IL-2, IFNγ, IL-4, and IL-10 transcripts were quantified by RT-qPCR. The results are representative of three independent experiments ( n = 5 mice in each group). [Tg vs. WT, mean with SEM; IL-2, 1 h: * p = 0.0278, 2 h: ** p = 0.0040, 4 h: ** p = 0.0040; IL-4, 1 h: ** p = 0.0040, 2 h: ** p = 0,0040, 4 h: ** p = 0,0040, 6 h: * p = 0.0278, 8 and 16 h: nonsignificant (ns); IFNγ, 1 h: * p = 0.0257, 2 h: ** p = 0.0040, 4 h: ** p = 0.0079, 6 h: * p = 0.0317, 8 h: * p = 0.0317 and 16 h: p = 0.532 (ns); IL-10, 1 h: * p = 0.0286, 2 h: * p = 0.0159, 4 h: p = 0.1508 (ns), 6 h: * p = 0.0159, 8 h: ** p = 0.0079, 16 h: ** p = 0.0079; Mann-Whitney tests]

    Article Snippet: Ex vivo T cell stimulationBefore stimulation, T cells were systematically synchronized at the G1 phase of the cell cycle by serum starvation in 2% fetal calf serum (FCS) for 6 h. Synchronized T cells were then activated for the indicated times with soluble anti-CD3 and anti-CD28 antibodies (eBiosciences, San Diego, CA, USA) at 1 µg/ml in RPMI complete medium supplemented with 10% FCS.

    Techniques: Expressing, Mouse Assay, Quantitative RT-PCR, MANN-WHITNEY

    Transgenic mice develop an altered T cell phenotype. a , b Representative flow cytometry analysis of naïve, effector and memory T cells in transgenic and WT T cells. Splenocytes from 12-week-old transgenic mice (Tg) and WT mice were gated for CD4 + cells ( a ) or CD8 + cells ( b ) and analyzed for CD44 and CD62L expression. T cell subpopulations were defined as naïve (CD44 low/verylow CD62L high ), memory (CD44 high CD62L high ), or effector T cells (CD44 high CD62L low ). The numbers inside and outside each small square indicate the percentage and the absolute number of cells, respectively. The total number of events is shown at the top. c and d Frequencies of naïve, effector and memory T cells. The results are representative of three independent experiments ( n = 5 each for WT and transgenic mice). The frequency of naïve T cells in both CD4 + and CD8 + compartments was significantly increased, whereas the level of memory CD4 + T cells was decreased in transgenic mice compared to WT mice (Tg vs. WT, mean with SEM: naïve CD4 + , ** p = 0.0079; naïve CD8 + , p = 0.0159; memory CD4 + , p = 0.0303, Mann-Whitney test). Although effector CD4+ and CD8 + subsets were decreased in Tg mice, this difference did not reach statistical significance. e Transgenic T cells exhibited a decreased proliferative capacity compared to WT T cells. T cells were isolated from Tg and WT mice and labeled with CFSE (1 μM). After synchronization, the cells were stimulated with anti-CD3/CD28 antibodies (1 μg/ml each). After 5 days, proliferation was analyzed by flow cytometry as the percentage of dividing cells. The addition of mouse recombinant IL2 (30 U/ml) at 24 and 72 h after stimulation restored the T cell proliferation rate, which was comparable between Tg and WT mice. Data are presented as the mean of four independent experiments. (Tg vs. WT at day 5, mean with SD: * p = 0.0286, Mann-Whitney test)

    Journal: Cellular and Molecular Immunology

    Article Title: CMIP is a negative regulator of T cell signaling

    doi: 10.1038/s41423-019-0266-5

    Figure Lengend Snippet: Transgenic mice develop an altered T cell phenotype. a , b Representative flow cytometry analysis of naïve, effector and memory T cells in transgenic and WT T cells. Splenocytes from 12-week-old transgenic mice (Tg) and WT mice were gated for CD4 + cells ( a ) or CD8 + cells ( b ) and analyzed for CD44 and CD62L expression. T cell subpopulations were defined as naïve (CD44 low/verylow CD62L high ), memory (CD44 high CD62L high ), or effector T cells (CD44 high CD62L low ). The numbers inside and outside each small square indicate the percentage and the absolute number of cells, respectively. The total number of events is shown at the top. c and d Frequencies of naïve, effector and memory T cells. The results are representative of three independent experiments ( n = 5 each for WT and transgenic mice). The frequency of naïve T cells in both CD4 + and CD8 + compartments was significantly increased, whereas the level of memory CD4 + T cells was decreased in transgenic mice compared to WT mice (Tg vs. WT, mean with SEM: naïve CD4 + , ** p = 0.0079; naïve CD8 + , p = 0.0159; memory CD4 + , p = 0.0303, Mann-Whitney test). Although effector CD4+ and CD8 + subsets were decreased in Tg mice, this difference did not reach statistical significance. e Transgenic T cells exhibited a decreased proliferative capacity compared to WT T cells. T cells were isolated from Tg and WT mice and labeled with CFSE (1 μM). After synchronization, the cells were stimulated with anti-CD3/CD28 antibodies (1 μg/ml each). After 5 days, proliferation was analyzed by flow cytometry as the percentage of dividing cells. The addition of mouse recombinant IL2 (30 U/ml) at 24 and 72 h after stimulation restored the T cell proliferation rate, which was comparable between Tg and WT mice. Data are presented as the mean of four independent experiments. (Tg vs. WT at day 5, mean with SD: * p = 0.0286, Mann-Whitney test)

    Article Snippet: Ex vivo T cell stimulationBefore stimulation, T cells were systematically synchronized at the G1 phase of the cell cycle by serum starvation in 2% fetal calf serum (FCS) for 6 h. Synchronized T cells were then activated for the indicated times with soluble anti-CD3 and anti-CD28 antibodies (eBiosciences, San Diego, CA, USA) at 1 µg/ml in RPMI complete medium supplemented with 10% FCS.

    Techniques: Transgenic Assay, Mouse Assay, Flow Cytometry, Expressing, MANN-WHITNEY, Isolation, Labeling, Recombinant

    The effects of CFA on the IL-4 production induced by Sj infection . Splenocytes from mice of uninfected (CTRL), 2 weeks of Sj infection, 7 to 10 weeks of Sj infection, 15 weeks of Sj infection, CIA, ESCIA (total 10 weeks of Sj infection), ASCIA (total 15 weeks of Sj infection) were stimulated by anti-CD3 and anti-CD28 antibodies for 72 hrs. The IL-4 contents in the supernatants were measured by ELISA. Shown are samples from individual mouse combined from two or three separately performed experiments. Asterisks* and ** represented P

    Journal: BMC Immunology

    Article Title: The inhibitory effect against collagen-induced arthritis by Schistosoma japonicum infection is infection stage-dependent

    doi: 10.1186/1471-2172-11-28

    Figure Lengend Snippet: The effects of CFA on the IL-4 production induced by Sj infection . Splenocytes from mice of uninfected (CTRL), 2 weeks of Sj infection, 7 to 10 weeks of Sj infection, 15 weeks of Sj infection, CIA, ESCIA (total 10 weeks of Sj infection), ASCIA (total 15 weeks of Sj infection) were stimulated by anti-CD3 and anti-CD28 antibodies for 72 hrs. The IL-4 contents in the supernatants were measured by ELISA. Shown are samples from individual mouse combined from two or three separately performed experiments. Asterisks* and ** represented P

    Article Snippet: Measurement of cytokine production by splenocytes To study cytokine expression by T lymphocytes, isolated spleen cells (5 × 106 cells/well) from mice that were immunized by CII for 8 weeks or from control mice were stimulated with 1 μg/ml anti-CD3ε (eBioscience) and 1 μg/ml anti-CD28 (eBioscience) at 37°C, 5%CO2 in 10% FCS/RPMI(GIBCO) culture medium for 72 hrs.

    Techniques: Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay

    IL-4 production is elevated in 7 to 10 weeks Sj infected DBA/1 mice . Splenocytes from DBA/1 mice infected with Sj either for 2 weeks or 7 to 10 weeks or not infected (CTRL) were stimulated by anti-CD3 and anti-CD28 antibodies for 72 hours in vitro. The supernatants were analyzed for IL-4 (A) and IFN-g (B). Shown are the combined results from two separate experiments. Significance was tested by one-way ANOVA with Bonferroni multiple comparison test with *** and ** as p

    Journal: BMC Immunology

    Article Title: The inhibitory effect against collagen-induced arthritis by Schistosoma japonicum infection is infection stage-dependent

    doi: 10.1186/1471-2172-11-28

    Figure Lengend Snippet: IL-4 production is elevated in 7 to 10 weeks Sj infected DBA/1 mice . Splenocytes from DBA/1 mice infected with Sj either for 2 weeks or 7 to 10 weeks or not infected (CTRL) were stimulated by anti-CD3 and anti-CD28 antibodies for 72 hours in vitro. The supernatants were analyzed for IL-4 (A) and IFN-g (B). Shown are the combined results from two separate experiments. Significance was tested by one-way ANOVA with Bonferroni multiple comparison test with *** and ** as p

    Article Snippet: Measurement of cytokine production by splenocytes To study cytokine expression by T lymphocytes, isolated spleen cells (5 × 106 cells/well) from mice that were immunized by CII for 8 weeks or from control mice were stimulated with 1 μg/ml anti-CD3ε (eBioscience) and 1 μg/ml anti-CD28 (eBioscience) at 37°C, 5%CO2 in 10% FCS/RPMI(GIBCO) culture medium for 72 hrs.

    Techniques: Infection, Mouse Assay, In Vitro

    Protective effects in ASCIA mice were associated with enhanced production of IL-4 and IL-10 and reduced production of IFN-γ . 56 days after CII immunization in ESCIA mice, ASCIA mice and CIA mice, splenocytes were stimulated by anti-CD3 and anti-CD28 antibodies for 72 hrs. The cytokine contents for IL-4 (A), IFN-g (B), IL-10 (C) and IL-17 (D) were measured by ELISA as described in the Materials and Methods. Shown are samples from individual mouse combined from two or three separately performed experiments. Asterisks* and ** represented P

    Journal: BMC Immunology

    Article Title: The inhibitory effect against collagen-induced arthritis by Schistosoma japonicum infection is infection stage-dependent

    doi: 10.1186/1471-2172-11-28

    Figure Lengend Snippet: Protective effects in ASCIA mice were associated with enhanced production of IL-4 and IL-10 and reduced production of IFN-γ . 56 days after CII immunization in ESCIA mice, ASCIA mice and CIA mice, splenocytes were stimulated by anti-CD3 and anti-CD28 antibodies for 72 hrs. The cytokine contents for IL-4 (A), IFN-g (B), IL-10 (C) and IL-17 (D) were measured by ELISA as described in the Materials and Methods. Shown are samples from individual mouse combined from two or three separately performed experiments. Asterisks* and ** represented P

    Article Snippet: Measurement of cytokine production by splenocytes To study cytokine expression by T lymphocytes, isolated spleen cells (5 × 106 cells/well) from mice that were immunized by CII for 8 weeks or from control mice were stimulated with 1 μg/ml anti-CD3ε (eBioscience) and 1 μg/ml anti-CD28 (eBioscience) at 37°C, 5%CO2 in 10% FCS/RPMI(GIBCO) culture medium for 72 hrs.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Generation of Aβ-specific Th1 cells retrovirally transduced to overexpress and secrete a biologically active BDNF. (A) A scheme showing the generation of BDNF-producing, Aβ-specific Th1 cells. Two month-old mice were first vaccinated with Aβ1–42. After 7 d, CD4 T cells were purified from draining lymph nodes and stimulated with Aβ in the presence of antigen-presenting cells. The CD4 T cells were then polarized to a Th1 phenotype and transduced with the pMP71-EGFP-PRE and pMP71-BDNF-T2A-EGFP-PRE retroviruses to generate Th1-GFP and Th1-BDNF T cells, respectively. (B) Representative flow cytometry histograms showing EGFP mean fluorescence intensity (MFI) in Th1-GFP and Th1-BDNF T cells. (C) Secreted levels of BDNF measured by ELISA in supernatants of Th1-GFP and Th1-BDNF cells, 48 h after activation with anti-CD3/anti-CD28 Dynabeads. (D) WB analysis of BDNF expression in Th1-GFP and Th1-BDNF whole-cell lysates. Th1 cells producing GFP and BDNF were activated with anti-CD3/anti-CD28 Dynabeads and the cells were collected 48 h later. Recombinant BDNF (rBDNF) was used as a control. (E) Representative images of Th1-GFP and Th1-BDNF cells immunolabeled with anti-BDNF (red) and nuclei-labeled with DAPI (blue). (F) WB analysis of non-phosphorylated and phosphorylated TrkB (707/706) and ERK1/2 in lysates of HEK293T TrkB-transduced cells treated with Th1-GFP and Th1-BDNF cell supernatants (2.5 ng/ml BDNF) or with recombinant BDNF (rBDNF, 2.5 ng/ml) for 15, 30, 45, or 60 min, as indicated. Tubulin served as an internal loading control.

    Journal: EBioMedicine

    Article Title: BDNF-producing, amyloid β-specific CD4 T cells as targeted drug-delivery vehicles in Alzheimer's disease

    doi: 10.1016/j.ebiom.2019.04.019

    Figure Lengend Snippet: Generation of Aβ-specific Th1 cells retrovirally transduced to overexpress and secrete a biologically active BDNF. (A) A scheme showing the generation of BDNF-producing, Aβ-specific Th1 cells. Two month-old mice were first vaccinated with Aβ1–42. After 7 d, CD4 T cells were purified from draining lymph nodes and stimulated with Aβ in the presence of antigen-presenting cells. The CD4 T cells were then polarized to a Th1 phenotype and transduced with the pMP71-EGFP-PRE and pMP71-BDNF-T2A-EGFP-PRE retroviruses to generate Th1-GFP and Th1-BDNF T cells, respectively. (B) Representative flow cytometry histograms showing EGFP mean fluorescence intensity (MFI) in Th1-GFP and Th1-BDNF T cells. (C) Secreted levels of BDNF measured by ELISA in supernatants of Th1-GFP and Th1-BDNF cells, 48 h after activation with anti-CD3/anti-CD28 Dynabeads. (D) WB analysis of BDNF expression in Th1-GFP and Th1-BDNF whole-cell lysates. Th1 cells producing GFP and BDNF were activated with anti-CD3/anti-CD28 Dynabeads and the cells were collected 48 h later. Recombinant BDNF (rBDNF) was used as a control. (E) Representative images of Th1-GFP and Th1-BDNF cells immunolabeled with anti-BDNF (red) and nuclei-labeled with DAPI (blue). (F) WB analysis of non-phosphorylated and phosphorylated TrkB (707/706) and ERK1/2 in lysates of HEK293T TrkB-transduced cells treated with Th1-GFP and Th1-BDNF cell supernatants (2.5 ng/ml BDNF) or with recombinant BDNF (rBDNF, 2.5 ng/ml) for 15, 30, 45, or 60 min, as indicated. Tubulin served as an internal loading control.

    Article Snippet: Th1 cells were stimulated for 48 h at a density of 1 × 106 cells/ml with 25 ul of anti-CD3/anti-CD28 Dynabeads (Thermo Fisher Scientific Inc., Waltham, MA).

    Techniques: Mouse Assay, Purification, Transduction, Flow Cytometry, Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot, Expressing, Recombinant, Immunolabeling, Labeling