cd28 functional grade monoclonal antibody  (Thermo Fisher)


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    Name:
    CD28 Functional Grade Monoclonal Antibody 37 51
    Description:
    CD28 Functional Grade Monoclonal Antibody for Flow FN
    Catalog Number:
    16-0281-81
    Price:
    None
    Category:
    Antibodies Secondary Detection Reagents
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    Structured Review

    Thermo Fisher cd28 functional grade monoclonal antibody
    SHP2 inhibition enhances the activation of CD8 + cytotoxic T lymphocytes in vitro . (A)–(C) Murine splenic CD8 + T cells were incubated with various concentrations of SHP099 for 24 h in the presence of plate-bound 0.5 μg/mL murine anti-CD3 and 0.5 μg/mL murine <t>anti-CD28.</t> (A) and (B) Flow cytomytry assay for detecting GZMB and PRF in CD8 + T cells. (C) qPCR assay for mRNA expressions of Ifn-γ, Prf, GzmB, FasL and Tnf-α in CD8 + T cells. (D) Human peripheral blood mononuclear cells (PBMCs) were incubated with 10 μmol/L SHP099 for 24 h in the presence of plate-bound 0.25 μg/mL human anti-CD3 and 1 μg/mL human anti-CD28. Relative mRNA levels of Ifn-γ, GzmB and Prf were measured by qPCR assay. (E) MTT assay for murine splenic CD8 + T cell viability. (F)–(I) Murine splenic cells were incubated with 10 μmol/L SHP099 for 24 h in the presence of 10 μg/mL Con A. Relative mRNA expressions of Ifn-γ, Prf, Tnf-α and FasL were measured by qPCR assay. (J) Human PBMCs were incubated with 10 μmol/L SHP099 for 24 h in the presence of 10 μg/mL Con A. Relative mRNA levels of Ifn-γ, GzmB and Prf were measured by qPCR assay. Data are presented as mean ± SEM of three different experiments. * P
    CD28 Functional Grade Monoclonal Antibody for Flow FN
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    1) Product Images from "SHP2 inhibition triggers anti-tumor immunity and synergizes with PD-1 blockade"

    Article Title: SHP2 inhibition triggers anti-tumor immunity and synergizes with PD-1 blockade

    Journal: Acta Pharmaceutica Sinica. B

    doi: 10.1016/j.apsb.2018.08.009

    SHP2 inhibition enhances the activation of CD8 + cytotoxic T lymphocytes in vitro . (A)–(C) Murine splenic CD8 + T cells were incubated with various concentrations of SHP099 for 24 h in the presence of plate-bound 0.5 μg/mL murine anti-CD3 and 0.5 μg/mL murine anti-CD28. (A) and (B) Flow cytomytry assay for detecting GZMB and PRF in CD8 + T cells. (C) qPCR assay for mRNA expressions of Ifn-γ, Prf, GzmB, FasL and Tnf-α in CD8 + T cells. (D) Human peripheral blood mononuclear cells (PBMCs) were incubated with 10 μmol/L SHP099 for 24 h in the presence of plate-bound 0.25 μg/mL human anti-CD3 and 1 μg/mL human anti-CD28. Relative mRNA levels of Ifn-γ, GzmB and Prf were measured by qPCR assay. (E) MTT assay for murine splenic CD8 + T cell viability. (F)–(I) Murine splenic cells were incubated with 10 μmol/L SHP099 for 24 h in the presence of 10 μg/mL Con A. Relative mRNA expressions of Ifn-γ, Prf, Tnf-α and FasL were measured by qPCR assay. (J) Human PBMCs were incubated with 10 μmol/L SHP099 for 24 h in the presence of 10 μg/mL Con A. Relative mRNA levels of Ifn-γ, GzmB and Prf were measured by qPCR assay. Data are presented as mean ± SEM of three different experiments. * P
    Figure Legend Snippet: SHP2 inhibition enhances the activation of CD8 + cytotoxic T lymphocytes in vitro . (A)–(C) Murine splenic CD8 + T cells were incubated with various concentrations of SHP099 for 24 h in the presence of plate-bound 0.5 μg/mL murine anti-CD3 and 0.5 μg/mL murine anti-CD28. (A) and (B) Flow cytomytry assay for detecting GZMB and PRF in CD8 + T cells. (C) qPCR assay for mRNA expressions of Ifn-γ, Prf, GzmB, FasL and Tnf-α in CD8 + T cells. (D) Human peripheral blood mononuclear cells (PBMCs) were incubated with 10 μmol/L SHP099 for 24 h in the presence of plate-bound 0.25 μg/mL human anti-CD3 and 1 μg/mL human anti-CD28. Relative mRNA levels of Ifn-γ, GzmB and Prf were measured by qPCR assay. (E) MTT assay for murine splenic CD8 + T cell viability. (F)–(I) Murine splenic cells were incubated with 10 μmol/L SHP099 for 24 h in the presence of 10 μg/mL Con A. Relative mRNA expressions of Ifn-γ, Prf, Tnf-α and FasL were measured by qPCR assay. (J) Human PBMCs were incubated with 10 μmol/L SHP099 for 24 h in the presence of 10 μg/mL Con A. Relative mRNA levels of Ifn-γ, GzmB and Prf were measured by qPCR assay. Data are presented as mean ± SEM of three different experiments. * P

    Techniques Used: Inhibition, Activation Assay, In Vitro, Incubation, Flow Cytometry, Real-time Polymerase Chain Reaction, MTT Assay

    2) Product Images from "Mitochondrial Superoxide Signaling Contributes to Norepinephrine-Mediated T-Lymphocyte Cytokine Profiles"

    Article Title: Mitochondrial Superoxide Signaling Contributes to Norepinephrine-Mediated T-Lymphocyte Cytokine Profiles

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0164609

    Mitochondrial O 2 ●- partially regulates CD4+ T-lymphocyte cytokine profiles. CD4+ T-lymphocytes were isolated, purified, and activated via CD3/CD28 stimulation in the presence of NE, Tempol (1 μM), and/or MitoTempol (1 μM) for 96 hours. Media was harvested for cytokine analysis and results normalized to cell number. N = 5. *p
    Figure Legend Snippet: Mitochondrial O 2 ●- partially regulates CD4+ T-lymphocyte cytokine profiles. CD4+ T-lymphocytes were isolated, purified, and activated via CD3/CD28 stimulation in the presence of NE, Tempol (1 μM), and/or MitoTempol (1 μM) for 96 hours. Media was harvested for cytokine analysis and results normalized to cell number. N = 5. *p

    Techniques Used: Isolation, Purification

    Inhibiting NE transport increases intracellular O 2 ●- levels. T-lymphocytes were isolated, purified, and activated via CD3/CD28 stimulation in the presence of NE and/or the norepinephrine transporter inhibitor atomoxetine. Quantification of DHE oxidation in CD4+ (upper) and CD8+ (lower) T-lymphocytes 96 hours post-activation. N = 4. *p
    Figure Legend Snippet: Inhibiting NE transport increases intracellular O 2 ●- levels. T-lymphocytes were isolated, purified, and activated via CD3/CD28 stimulation in the presence of NE and/or the norepinephrine transporter inhibitor atomoxetine. Quantification of DHE oxidation in CD4+ (upper) and CD8+ (lower) T-lymphocytes 96 hours post-activation. N = 4. *p

    Techniques Used: Isolation, Purification, Activation Assay

    NE provokes inverse effects on T-lymphocyte growth and O 2 ●- levels. T-lymphocytes were isolated, purified, and activated via CD3/CD28 stimulation in the presence of 1 μM NE. A . T-lymphocyte growth curves at various time points of ex vivo culture. N = 4. B . Quantification of DHE oxidation in CD4+ and CD8+ T-lymphocytes at various time points post-activation. N = 4. C . Representative EPR spectra showing amplitude (Amp) for T-lymphocytes stimulated in the presence or absence of 1 μM NE for 96 hours. N = 3. *p
    Figure Legend Snippet: NE provokes inverse effects on T-lymphocyte growth and O 2 ●- levels. T-lymphocytes were isolated, purified, and activated via CD3/CD28 stimulation in the presence of 1 μM NE. A . T-lymphocyte growth curves at various time points of ex vivo culture. N = 4. B . Quantification of DHE oxidation in CD4+ and CD8+ T-lymphocytes at various time points post-activation. N = 4. C . Representative EPR spectra showing amplitude (Amp) for T-lymphocytes stimulated in the presence or absence of 1 μM NE for 96 hours. N = 3. *p

    Techniques Used: Isolation, Purification, Ex Vivo, Activation Assay, Electron Paramagnetic Resonance

    Mitochondrial O 2 ●- is increased in T-lymphocytes treated with NE. T-lymphocytes were isolated, purified, and activated via CD3/CD28 stimulation in the presence of 1 μM NE. A . Quantification of DHE oxidation in CD4+ and CD8+ T-lymphocytes 96 hours post-activation. Cells were incubated with DPI 1 hour prior to and during the incubation with DHE. N = 3. *p
    Figure Legend Snippet: Mitochondrial O 2 ●- is increased in T-lymphocytes treated with NE. T-lymphocytes were isolated, purified, and activated via CD3/CD28 stimulation in the presence of 1 μM NE. A . Quantification of DHE oxidation in CD4+ and CD8+ T-lymphocytes 96 hours post-activation. Cells were incubated with DPI 1 hour prior to and during the incubation with DHE. N = 3. *p

    Techniques Used: Isolation, Purification, Activation Assay, Incubation

    NE-mediated changes in O 2 ●- are mediated via various adrenergic receptors. T-lymphocytes were isolated, purified, and cells were activated via CD3/CD28 stimulation in the presence of 1 μM NE or the respective antagonist for 96 hours prior to analysis. Quantification of DHE oxidation in CD4+ (upper) and CD8+ (lower) T-lymphocytes activated in the presence of the treatments for 96 hours. N = 4. *p
    Figure Legend Snippet: NE-mediated changes in O 2 ●- are mediated via various adrenergic receptors. T-lymphocytes were isolated, purified, and cells were activated via CD3/CD28 stimulation in the presence of 1 μM NE or the respective antagonist for 96 hours prior to analysis. Quantification of DHE oxidation in CD4+ (upper) and CD8+ (lower) T-lymphocytes activated in the presence of the treatments for 96 hours. N = 4. *p

    Techniques Used: Isolation, Purification

    Mitochondrial O 2 ●- partially regulates CD8+ T-lymphocyte cytokine profiles. CD8+ T-lymphocytes were isolated, purified, and activated via CD3/CD28 stimulation in the presence of NE, Tempol (1 μM), and/or MitoTempol (1 μM) for 96 hours. Media was harvested for cytokine analysis and results normalized to cell number. N = 5. *p
    Figure Legend Snippet: Mitochondrial O 2 ●- partially regulates CD8+ T-lymphocyte cytokine profiles. CD8+ T-lymphocytes were isolated, purified, and activated via CD3/CD28 stimulation in the presence of NE, Tempol (1 μM), and/or MitoTempol (1 μM) for 96 hours. Media was harvested for cytokine analysis and results normalized to cell number. N = 5. *p

    Techniques Used: Isolation, Purification

    T-lymphocyte mitochondrial reserve respiratory capacity is decreased with NE. T-lymphocytes were isolated and purified. Cells were either immediately analyzed in a Seahorse Bioscience XFp extracellular flux analyzer with a 30-minute acute treatment of 1 μM NE, or activated via CD3/CD28 stimulation in the presence of 1 μM NE for 96 hours prior to analysis. A . Cumulative average data of oxygen consumption rate (OCR) measured over-time with acute treatment of NE. N = 9. B . Cumulative average data of OCR measured over-time in activated T-lymphocytes cultured in the presence of NE. N = 9. *p
    Figure Legend Snippet: T-lymphocyte mitochondrial reserve respiratory capacity is decreased with NE. T-lymphocytes were isolated and purified. Cells were either immediately analyzed in a Seahorse Bioscience XFp extracellular flux analyzer with a 30-minute acute treatment of 1 μM NE, or activated via CD3/CD28 stimulation in the presence of 1 μM NE for 96 hours prior to analysis. A . Cumulative average data of oxygen consumption rate (OCR) measured over-time with acute treatment of NE. N = 9. B . Cumulative average data of OCR measured over-time in activated T-lymphocytes cultured in the presence of NE. N = 9. *p

    Techniques Used: Isolation, Purification, Cell Culture

    3) Product Images from "Role of caveolin-3 in lymphocyte activation"

    Article Title: Role of caveolin-3 in lymphocyte activation

    Journal: Life sciences

    doi: 10.1016/j.lfs.2014.11.017

    T-cell activation and cytokine production is altered in Cav-3 KO mice. Lymphocytes isolated from Cav-3 KO and WT mice were stimulated by anti-CD28 and CD3 antibodies to induce activation of T-cells. Lymphocytes from Cav-3 KO mice exhibited a decrease
    Figure Legend Snippet: T-cell activation and cytokine production is altered in Cav-3 KO mice. Lymphocytes isolated from Cav-3 KO and WT mice were stimulated by anti-CD28 and CD3 antibodies to induce activation of T-cells. Lymphocytes from Cav-3 KO mice exhibited a decrease

    Techniques Used: Activation Assay, Mouse Assay, Isolation

    4) Product Images from "Regulatory T Cell Migration Is Dependent on Glucokinase-Mediated Glycolysis"

    Article Title: Regulatory T Cell Migration Is Dependent on Glucokinase-Mediated Glycolysis

    Journal: Immunity

    doi: 10.1016/j.immuni.2017.10.017

    Pro-migratory Stimuli Induce Metabolic Reprogramming of Treg Cells (A and B) Expression of the indicated enzymes in Treg cells was measured 4 hr after antibody stimulation by western blotting. In (B) the mean relative expression measured by densitometric analysis in 3 independent experiments ± SD is shown. (C) Expression of the indicated enzymes by CD28- or LFA-1-stimulated Treg cells measured by western blotting at the indicated time points. (D–G) Relative mRNA expression of GCK (D) and GCKR (E) and cellular protein expression (F and G) by antibody-stimulated Treg cells was measured by RT-PCR and confocal microscopy, respectively. In (G) the mean MFI ± SEM measured using ImageJ software is shown. N = 3. Scale bar 20 μm. (H) AZD1656 (GCK activator, 1 μM) and vehicle-treated Treg cells (2 hr in insulin-free medium) were labeled with different intravital fluorescent dyes, and co-injected into syngeneic recipients that had received IFN-γ i.p. 48 hr earlier. Cells were recovered from the peritoneum or spleen after 24 hr and analyzed by flow cytometry. Representative dot plots from 2 independent experiments are shown. The bar graphs indicate mean absolute number of labeled cells retrieved ± SD (n = 4, N = 2). (I and J) expression of PCNA by Treg cells stimulated with allogeneic DCs following treatment with either AZD1656 (I) or Clotrimazole (CLT, 1 μM, 2 hours, J) or vehicle alone was measured by flow cytometry. NS, non-stimulated control cells. Representative histograms from 3 independent experiments are shown. (n = 3, N = 3). (K) ECAR of Treg cells activated with recombinant ICAM-1 or Fc control. CLT or vehicle as well as other glycolysis-affecting drugs were added as indicated. (L) CLT- or vehicle-treated Treg cells underwent CD28 or isotype-matched antibody stimulation, labeled with different intravital fluorescent dyes, and injected i.v. in syngeneic recipients that had received IFN-γ i.p. 48 hr earlier. Cells were recovered from the peritoneum (P) or spleen (S) after 24 hr and analyzed by flow cytometry. Representative dot plots from 2 independent experiments are shown. The column graphs indicate mean absolute number of labeled cells retrieved ± SD (n = 3, N = 2). ∗ p
    Figure Legend Snippet: Pro-migratory Stimuli Induce Metabolic Reprogramming of Treg Cells (A and B) Expression of the indicated enzymes in Treg cells was measured 4 hr after antibody stimulation by western blotting. In (B) the mean relative expression measured by densitometric analysis in 3 independent experiments ± SD is shown. (C) Expression of the indicated enzymes by CD28- or LFA-1-stimulated Treg cells measured by western blotting at the indicated time points. (D–G) Relative mRNA expression of GCK (D) and GCKR (E) and cellular protein expression (F and G) by antibody-stimulated Treg cells was measured by RT-PCR and confocal microscopy, respectively. In (G) the mean MFI ± SEM measured using ImageJ software is shown. N = 3. Scale bar 20 μm. (H) AZD1656 (GCK activator, 1 μM) and vehicle-treated Treg cells (2 hr in insulin-free medium) were labeled with different intravital fluorescent dyes, and co-injected into syngeneic recipients that had received IFN-γ i.p. 48 hr earlier. Cells were recovered from the peritoneum or spleen after 24 hr and analyzed by flow cytometry. Representative dot plots from 2 independent experiments are shown. The bar graphs indicate mean absolute number of labeled cells retrieved ± SD (n = 4, N = 2). (I and J) expression of PCNA by Treg cells stimulated with allogeneic DCs following treatment with either AZD1656 (I) or Clotrimazole (CLT, 1 μM, 2 hours, J) or vehicle alone was measured by flow cytometry. NS, non-stimulated control cells. Representative histograms from 3 independent experiments are shown. (n = 3, N = 3). (K) ECAR of Treg cells activated with recombinant ICAM-1 or Fc control. CLT or vehicle as well as other glycolysis-affecting drugs were added as indicated. (L) CLT- or vehicle-treated Treg cells underwent CD28 or isotype-matched antibody stimulation, labeled with different intravital fluorescent dyes, and injected i.v. in syngeneic recipients that had received IFN-γ i.p. 48 hr earlier. Cells were recovered from the peritoneum (P) or spleen (S) after 24 hr and analyzed by flow cytometry. Representative dot plots from 2 independent experiments are shown. The column graphs indicate mean absolute number of labeled cells retrieved ± SD (n = 3, N = 2). ∗ p

    Techniques Used: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Confocal Microscopy, Software, Labeling, Injection, Flow Cytometry, Cytometry, Recombinant

    mTORC2 Controls Metabolic Reprogramming Induced by Pro-migratory Stimuli (A) Phosphorylation of AKT at Thr 308 and Ser 473 in Treg cells activated with CD28- or IsC-antibody ligation was measured by immunoblotting. (B) Treg cells were virally transfected with Rictor-specific or GCK-specific or non-sense (PLKO.1) sh-RNAs, as described in STAR Methods . Expression of GCK was measured by immunoblotting 24 hr later. (C–E) Expression of GCK and GCKR by control or Rictor-deficient Treg cells following CD28 or LFA-1 activation for 45 min. Bar graphs (D and E) show the mean protein expression (Total cell fluorescence) measured in 3 independent experiments by ImageJ software ± SD. Scale bar, 40 μm. (F–I) ECAR of CD28- or LFA-1-stimulated Rictor- and GCK-deficient and control T cells was measured with an extracellular flux analyzer. A glycolysis stress assay was performed by adding the indicated compounds at the time points indicated by the green lines. The basal and maximal glycolysis and the glycolytic reserve are shown in (G), (H), and (I), respectively (±SEM). N = 2. ∗ p
    Figure Legend Snippet: mTORC2 Controls Metabolic Reprogramming Induced by Pro-migratory Stimuli (A) Phosphorylation of AKT at Thr 308 and Ser 473 in Treg cells activated with CD28- or IsC-antibody ligation was measured by immunoblotting. (B) Treg cells were virally transfected with Rictor-specific or GCK-specific or non-sense (PLKO.1) sh-RNAs, as described in STAR Methods . Expression of GCK was measured by immunoblotting 24 hr later. (C–E) Expression of GCK and GCKR by control or Rictor-deficient Treg cells following CD28 or LFA-1 activation for 45 min. Bar graphs (D and E) show the mean protein expression (Total cell fluorescence) measured in 3 independent experiments by ImageJ software ± SD. Scale bar, 40 μm. (F–I) ECAR of CD28- or LFA-1-stimulated Rictor- and GCK-deficient and control T cells was measured with an extracellular flux analyzer. A glycolysis stress assay was performed by adding the indicated compounds at the time points indicated by the green lines. The basal and maximal glycolysis and the glycolytic reserve are shown in (G), (H), and (I), respectively (±SEM). N = 2. ∗ p

    Techniques Used: Ligation, Transfection, Expressing, Activation Assay, Fluorescence, Software

    CD28-Induced Migration and Metabolic Reprogramming Require PI3K but Not mTORC-1 Activation WT and Cd28 Y170F mice received an i.p. injection of Zymosan. Samples were collected either before or 72 hr after the injection. The presence of Treg cells in the indicated tissues was measured by flow cytometry. (A) Representative dot plots from 2 independent experiments. (B) The mean percentage of cells measured in two experiments of identical design ± SD. (C and D) Ratio of cells retrieved in the indicated tissues over time ± SD (n = 3). (E) ECAR (±SD) of antibody-stimulated Cd28 Y170F and WT Treg cells was compared using fluxometry. n = 4, N = 2. (F) Migration of antibody-stimulated Cd28 Y170F and WT Treg cells through IFN-γ-treated EC monolayers. Results are expressed as mean percentage of migrated cells after 24 hr ± SD. N = 4. (G–J) Equal numbers of antibody-stimulated PKH26-labeled WT or Cd28 Y170F Treg cells were injected i.v. into syngeneic mice treated with IFN-γ i.p. 48 hr earlier. Cells were harvested from the indicated tissues 24 hr later, counter-stained for Foxp3, and analyzed by flow cytometry. Representative dot plots of 2 independent experiments are shown in (G) and (I). The bar graphs in (H) and (J) indicate mean absolute numbers of labeled cells (n = 4, N = 2) ± SD. ∗ p
    Figure Legend Snippet: CD28-Induced Migration and Metabolic Reprogramming Require PI3K but Not mTORC-1 Activation WT and Cd28 Y170F mice received an i.p. injection of Zymosan. Samples were collected either before or 72 hr after the injection. The presence of Treg cells in the indicated tissues was measured by flow cytometry. (A) Representative dot plots from 2 independent experiments. (B) The mean percentage of cells measured in two experiments of identical design ± SD. (C and D) Ratio of cells retrieved in the indicated tissues over time ± SD (n = 3). (E) ECAR (±SD) of antibody-stimulated Cd28 Y170F and WT Treg cells was compared using fluxometry. n = 4, N = 2. (F) Migration of antibody-stimulated Cd28 Y170F and WT Treg cells through IFN-γ-treated EC monolayers. Results are expressed as mean percentage of migrated cells after 24 hr ± SD. N = 4. (G–J) Equal numbers of antibody-stimulated PKH26-labeled WT or Cd28 Y170F Treg cells were injected i.v. into syngeneic mice treated with IFN-γ i.p. 48 hr earlier. Cells were harvested from the indicated tissues 24 hr later, counter-stained for Foxp3, and analyzed by flow cytometry. Representative dot plots of 2 independent experiments are shown in (G) and (I). The bar graphs in (H) and (J) indicate mean absolute numbers of labeled cells (n = 4, N = 2) ± SD. ∗ p

    Techniques Used: Migration, Activation Assay, Mouse Assay, Injection, Flow Cytometry, Cytometry, Labeling, Staining

    Rictor-Deficient Treg Cells Display Impaired Motility (A–E) CD28-stimulated or IsC-treated PLKO.1, Rictor- or GCK-deficient Treg cells were labeled with PKH26 and co-injected i.v. with identical numbers of IsC-treated CFSE-labeled cells in syngeneic recipients treated with IFN-γ 48 hr earlier. The presence of differently labeled cells in the indicated organs was assessed by flow cytometry 24 hr later. Representative dot plots from 3 independent experiments are shown on top in (A), (B), and (C). The bar graphs in (A), (B), and (D) indicate mean absolute number of labeled Treg cells recovered in the indicated tissues from 4 different recipients ± SD (N = 3). The bar graph in (E) shows the ratio of Treg cells recovered in the lung and the spleen (n = 4) ± SD. (F and K) Control and ShGCK Treg cell migration through 5 or 3 μm pore bare-filter transwells in response to CCL22 (F) or CXCL10 (K). Results are expressed as percentage of migrated cells at the indicated time points ± SD (n = 3). (H–J) BALB/c-derived skin was grafted onto C57BL/6 recipients who had received mock-transduced, Rictor- or GCK-depleted Treg cells or no cells 24 hr earlier. Graft rejection was monitored daily (H). Some grafts were removed 5 days post-grafting and the presence of fluorescently labeled Treg cells in the indicated tissue was assessed by widefield fluorescence microscopy. Representative images of grafts and spleens from 2 independent experiments are shown in (I). The bar graphs (J) indicate the mean number of labeled cells detected in at least 10 10× tissue images from each animal ± SD (n = 8). ∗ p
    Figure Legend Snippet: Rictor-Deficient Treg Cells Display Impaired Motility (A–E) CD28-stimulated or IsC-treated PLKO.1, Rictor- or GCK-deficient Treg cells were labeled with PKH26 and co-injected i.v. with identical numbers of IsC-treated CFSE-labeled cells in syngeneic recipients treated with IFN-γ 48 hr earlier. The presence of differently labeled cells in the indicated organs was assessed by flow cytometry 24 hr later. Representative dot plots from 3 independent experiments are shown on top in (A), (B), and (C). The bar graphs in (A), (B), and (D) indicate mean absolute number of labeled Treg cells recovered in the indicated tissues from 4 different recipients ± SD (N = 3). The bar graph in (E) shows the ratio of Treg cells recovered in the lung and the spleen (n = 4) ± SD. (F and K) Control and ShGCK Treg cell migration through 5 or 3 μm pore bare-filter transwells in response to CCL22 (F) or CXCL10 (K). Results are expressed as percentage of migrated cells at the indicated time points ± SD (n = 3). (H–J) BALB/c-derived skin was grafted onto C57BL/6 recipients who had received mock-transduced, Rictor- or GCK-depleted Treg cells or no cells 24 hr earlier. Graft rejection was monitored daily (H). Some grafts were removed 5 days post-grafting and the presence of fluorescently labeled Treg cells in the indicated tissue was assessed by widefield fluorescence microscopy. Representative images of grafts and spleens from 2 independent experiments are shown in (I). The bar graphs (J) indicate the mean number of labeled cells detected in at least 10 10× tissue images from each animal ± SD (n = 8). ∗ p

    Techniques Used: Labeling, Injection, Flow Cytometry, Cytometry, Migration, Derivative Assay, Fluorescence, Microscopy

    5) Product Images from "Nod2 Activates NF-kB in CD4+ T Cells but Its Expression Is Dispensable for T Cell-Induced Colitis"

    Article Title: Nod2 Activates NF-kB in CD4+ T Cells but Its Expression Is Dispensable for T Cell-Induced Colitis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082623

    Cytokine secretion is not modulated by MDP stimulation of CD4 + T cells in vitro . Splenic CD4 + T cells isolated from C57BL/6 mice were cultured with anti-CD3 (10µg/mL) and anti-CD28 (2µg/mL) monoclonal antibodies in presence or absence of MDP (10µg/mL) and PAM3CSK4 (10µg/mL) for 48h. Cytokine secretion was quantified by cytometric bead array. Results show the mean ± SEM and are representative of three independent experiments.
    Figure Legend Snippet: Cytokine secretion is not modulated by MDP stimulation of CD4 + T cells in vitro . Splenic CD4 + T cells isolated from C57BL/6 mice were cultured with anti-CD3 (10µg/mL) and anti-CD28 (2µg/mL) monoclonal antibodies in presence or absence of MDP (10µg/mL) and PAM3CSK4 (10µg/mL) for 48h. Cytokine secretion was quantified by cytometric bead array. Results show the mean ± SEM and are representative of three independent experiments.

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

    Nod2 is expressed in CD4 + T cells and inducible after TCR ligation. (A) Splenic CD4 + T cells isolated from C57BL/6 or NOD2 -/- mice were cultured alone or in presence of anti-CD3 (1-10µg/mL) and anti-CD28 (2µg/mL) monoclonal antibodies for 24 hours. Nod2 expression was assessed by western blot. (B) Naïve CD4 + CD45RB high , effector CD4 + CD45RB low Foxp3 - and regulatory CD4 + CD45RB low Foxp3 + splenic T cell subsets were isolated from Foxp3-GFP mice and Nod2 mRNA level was assessed by RT-qPCR. (C) Lamina propria lymphocytes were isolated from the cecum of NOD2-GFP mice and analyzed by flow cytometry. The histograms show the level of GFP expression. Cells were gated on total viable TCRβ + CD4 + cells (left panel), on TCRβ + CD4 + CD44 - cells (middle panel), or TCRβ + CD4 + CD44 + cells (right panel).
    Figure Legend Snippet: Nod2 is expressed in CD4 + T cells and inducible after TCR ligation. (A) Splenic CD4 + T cells isolated from C57BL/6 or NOD2 -/- mice were cultured alone or in presence of anti-CD3 (1-10µg/mL) and anti-CD28 (2µg/mL) monoclonal antibodies for 24 hours. Nod2 expression was assessed by western blot. (B) Naïve CD4 + CD45RB high , effector CD4 + CD45RB low Foxp3 - and regulatory CD4 + CD45RB low Foxp3 + splenic T cell subsets were isolated from Foxp3-GFP mice and Nod2 mRNA level was assessed by RT-qPCR. (C) Lamina propria lymphocytes were isolated from the cecum of NOD2-GFP mice and analyzed by flow cytometry. The histograms show the level of GFP expression. Cells were gated on total viable TCRβ + CD4 + cells (left panel), on TCRβ + CD4 + CD44 - cells (middle panel), or TCRβ + CD4 + CD44 + cells (right panel).

    Techniques Used: Ligation, Isolation, Mouse Assay, Cell Culture, Expressing, Western Blot, Quantitative RT-PCR, Flow Cytometry, Cytometry

    6) Product Images from "MCPIP1 Down-Regulates IL-2 Expression through an ARE-Independent Pathway"

    Article Title: MCPIP1 Down-Regulates IL-2 Expression through an ARE-Independent Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0049841

    MCPIP1 negatively regulate IL-2 expression in the mouse CD4 + T lymphocytes. (A–B). Overexpression of MCPIP1 inhibits IL-2 expression. Purified CD4 + T lymphocytes were transiently transfected with mMCPIP1-flag or control plasmids by electroporation. After incubated for 4 h, cells were challenged with anti-CD3 and anti-CD28 Abs for 12 hours. Cells and cultured medium were harvested and IL-2 mRNA level was measured by Q-PCR (A) and protein level was detected by ELISA (B). (C–D). Knockdown of MCPIP1 promotes IL-2 expression. Purified CD4 + T lymphocytes were transiently transfected with MCPIP1-siRNA or control-siRNA by electroporation. After incubated for 4 h, cells were challenged with anti-CD3 and anti-CD28 Abs for 12 hours. Cells and cultured media were harvested and IL-2 mRNA level was measured by Q-PCR (C) and protein level was detected by ELISA (D). (E). MCPIP1 destabilizes IL-2 mRNA in mouse CD4 + T lymphocytes . Purified CD4 + T lymphocytes were transiently transfected with MCPIP1-siRNA or control-siRNA by electroporation. After incubated for 4 h, cells were challenged with anti-CD3 and anti-CD28 Abs. After 6 hours, actinomycin D (10 µg/ml) was added to stop transcription, and total RNA was harvested after 0, 15, 30, or 45 min. IL-2 mRNA levels were measured by Q-PCR and normalized to β-actin mRNA. The normalized level of IL-2 mRNA at time 0 was set at 100. Data were presented as mean ± S.D of three independent experiments. *P
    Figure Legend Snippet: MCPIP1 negatively regulate IL-2 expression in the mouse CD4 + T lymphocytes. (A–B). Overexpression of MCPIP1 inhibits IL-2 expression. Purified CD4 + T lymphocytes were transiently transfected with mMCPIP1-flag or control plasmids by electroporation. After incubated for 4 h, cells were challenged with anti-CD3 and anti-CD28 Abs for 12 hours. Cells and cultured medium were harvested and IL-2 mRNA level was measured by Q-PCR (A) and protein level was detected by ELISA (B). (C–D). Knockdown of MCPIP1 promotes IL-2 expression. Purified CD4 + T lymphocytes were transiently transfected with MCPIP1-siRNA or control-siRNA by electroporation. After incubated for 4 h, cells were challenged with anti-CD3 and anti-CD28 Abs for 12 hours. Cells and cultured media were harvested and IL-2 mRNA level was measured by Q-PCR (C) and protein level was detected by ELISA (D). (E). MCPIP1 destabilizes IL-2 mRNA in mouse CD4 + T lymphocytes . Purified CD4 + T lymphocytes were transiently transfected with MCPIP1-siRNA or control-siRNA by electroporation. After incubated for 4 h, cells were challenged with anti-CD3 and anti-CD28 Abs. After 6 hours, actinomycin D (10 µg/ml) was added to stop transcription, and total RNA was harvested after 0, 15, 30, or 45 min. IL-2 mRNA levels were measured by Q-PCR and normalized to β-actin mRNA. The normalized level of IL-2 mRNA at time 0 was set at 100. Data were presented as mean ± S.D of three independent experiments. *P

    Techniques Used: Expressing, Over Expression, Purification, Transfection, Electroporation, Incubation, Cell Culture, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    MCPIP1 binds to the 3′UTR of IL-2 mRNA. (A). EL-4 cells were transfected with mMCPIP1- flag or control plasmids. After 24 h, cells were stimulated with PMA (5 ng/ml) and ionomycin (500 ng/ml) for 3 h. Cell lysates were subjected to RIP with anti-flag antibody or IgG. Immumoprecipitated RNA was reverse transcribed to cDNA and then quantified using Sybr Green Q-PCR. (B). Purified mouse T cells were stimulated with anti-CD3 (1 µg/ml) and anti-CD28 (3 µg/ml) antibodies for 6 h. Cell lysates were subjected to RIP with anti-MCPIP1 antibody or IgG. Immumoprecipitated RNA was reverse transcribed to cDNA and then quantified using Sybr Green Q-PCR. (C). MCPIP1-flag expression by Western blot after biotin pulldown assay with biotinylated transcripts - full-length, stem-loop mutant1 and Δ83-166 of IL-2 3′UTR or IL-6 3′UTR as positive control and β-actin 3′UTR as negative control.Data were presented as mean±S.D of three independent experiments. *P
    Figure Legend Snippet: MCPIP1 binds to the 3′UTR of IL-2 mRNA. (A). EL-4 cells were transfected with mMCPIP1- flag or control plasmids. After 24 h, cells were stimulated with PMA (5 ng/ml) and ionomycin (500 ng/ml) for 3 h. Cell lysates were subjected to RIP with anti-flag antibody or IgG. Immumoprecipitated RNA was reverse transcribed to cDNA and then quantified using Sybr Green Q-PCR. (B). Purified mouse T cells were stimulated with anti-CD3 (1 µg/ml) and anti-CD28 (3 µg/ml) antibodies for 6 h. Cell lysates were subjected to RIP with anti-MCPIP1 antibody or IgG. Immumoprecipitated RNA was reverse transcribed to cDNA and then quantified using Sybr Green Q-PCR. (C). MCPIP1-flag expression by Western blot after biotin pulldown assay with biotinylated transcripts - full-length, stem-loop mutant1 and Δ83-166 of IL-2 3′UTR or IL-6 3′UTR as positive control and β-actin 3′UTR as negative control.Data were presented as mean±S.D of three independent experiments. *P

    Techniques Used: Transfection, SYBR Green Assay, Polymerase Chain Reaction, Purification, Expressing, Western Blot, Positive Control, Negative Control

    MCPIP1 proteins are induced by anti-CD3 and anti-CD28 Abs in mouse primary CD4 + T lymphocytes. (A). Detection of MCPIP1 level by real-time PCR and Western blot. Purified CD4 + T lymphocytes were stimulated by anti-CD3 and anti-CD28 Abs and harvested at the indicated time points. Samples were collected and subjected to Q-PCR analysis. Data were normalized to β-actin expression. Cell lysates were prepared and MCPIP1 protein level was evaluated by Western blot. (B). Detection of MCPIP2, MCPIP3 and MCPIP4 level by real-time PCR. Purified CD4 + T lymphocytes were stimulated by anti-CD3 and anti-CD28 Abs and harvested at the indicated time points. Samples were collected and subjected to Q-PCR analysis. Data were normalized to β-actin expression. Data were presented as mean ± S.D of three representative independent experiments.
    Figure Legend Snippet: MCPIP1 proteins are induced by anti-CD3 and anti-CD28 Abs in mouse primary CD4 + T lymphocytes. (A). Detection of MCPIP1 level by real-time PCR and Western blot. Purified CD4 + T lymphocytes were stimulated by anti-CD3 and anti-CD28 Abs and harvested at the indicated time points. Samples were collected and subjected to Q-PCR analysis. Data were normalized to β-actin expression. Cell lysates were prepared and MCPIP1 protein level was evaluated by Western blot. (B). Detection of MCPIP2, MCPIP3 and MCPIP4 level by real-time PCR. Purified CD4 + T lymphocytes were stimulated by anti-CD3 and anti-CD28 Abs and harvested at the indicated time points. Samples were collected and subjected to Q-PCR analysis. Data were normalized to β-actin expression. Data were presented as mean ± S.D of three representative independent experiments.

    Techniques Used: Real-time Polymerase Chain Reaction, Western Blot, Purification, Polymerase Chain Reaction, Expressing

    MCPIP1 negatively regulates IL-2 gene expression in human peripheral CD4 + T lymphocytes. (A–B). Overexpression of MCPIP1 inhibits IL-2 expression. Human peripheral mononuclear cells (PBMC) were obtained from healthy subjects by Ficoll-Hypaque density centrifugation. Purified CD4 + T lymphocytes were transiently transfected with hMCPIP1-flag or control plasmids by electroporation. After incubated for 4 h, cells were challenged with anti-CD3 and anti-CD28 for 12 hours. Cells and cultured media were harvested and IL-2 mRNA level was measured by Q-PCR (A) and protein level was detected by ELISA (B). (C-D).MCPIP1 Knockdown promotes IL-2 expression stimulated by anti-CD3 and anti-CD28 Abs. Purified CD4 + T lymphocytes were transiently transfected with control-siRNA or MCPIP1-siRNA by electroporation. After incubated for 4 h, cells were challenged with anti-CD3 and anti-CD28 Abs for 12 hours. Cells and cultured media were harvested and IL-2 mRNA level was measured by Q-PCR (C) and protein level was detected by ELISA (D). (E). MCPIP1 destabilizes IL-2 mRNA in human CD4 + T lymphocytes . Purified CD4 + T lymphocytes were transiently transfected with MCPIP1-siRNA or control-siRNA by electroporation. After incubated for 4 h, cells were challenged with anti-CD3 and anti-CD28 Abs. 6 h later, actinomycin D (10 µg/ml) was added to stop transcription, and total RNA was harvested after 0, 15, 30, or 45 min. IL-2 mRNA levels were measured by Q-PCR and normalized to β-actin mRNA. The normalized level of IL-2 mRNA at time 0 was set at 100. Data were presented as mean ± S.D of three independent experiments. *P
    Figure Legend Snippet: MCPIP1 negatively regulates IL-2 gene expression in human peripheral CD4 + T lymphocytes. (A–B). Overexpression of MCPIP1 inhibits IL-2 expression. Human peripheral mononuclear cells (PBMC) were obtained from healthy subjects by Ficoll-Hypaque density centrifugation. Purified CD4 + T lymphocytes were transiently transfected with hMCPIP1-flag or control plasmids by electroporation. After incubated for 4 h, cells were challenged with anti-CD3 and anti-CD28 for 12 hours. Cells and cultured media were harvested and IL-2 mRNA level was measured by Q-PCR (A) and protein level was detected by ELISA (B). (C-D).MCPIP1 Knockdown promotes IL-2 expression stimulated by anti-CD3 and anti-CD28 Abs. Purified CD4 + T lymphocytes were transiently transfected with control-siRNA or MCPIP1-siRNA by electroporation. After incubated for 4 h, cells were challenged with anti-CD3 and anti-CD28 Abs for 12 hours. Cells and cultured media were harvested and IL-2 mRNA level was measured by Q-PCR (C) and protein level was detected by ELISA (D). (E). MCPIP1 destabilizes IL-2 mRNA in human CD4 + T lymphocytes . Purified CD4 + T lymphocytes were transiently transfected with MCPIP1-siRNA or control-siRNA by electroporation. After incubated for 4 h, cells were challenged with anti-CD3 and anti-CD28 Abs. 6 h later, actinomycin D (10 µg/ml) was added to stop transcription, and total RNA was harvested after 0, 15, 30, or 45 min. IL-2 mRNA levels were measured by Q-PCR and normalized to β-actin mRNA. The normalized level of IL-2 mRNA at time 0 was set at 100. Data were presented as mean ± S.D of three independent experiments. *P

    Techniques Used: Expressing, Over Expression, Centrifugation, Purification, Transfection, Electroporation, Incubation, Cell Culture, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    7) Product Images from "Alkylglycerols Modulate the Proliferation and Differentiation of Non-Specific Agonist and Specific Antigen-Stimulated Splenic Lymphocytes"

    Article Title: Alkylglycerols Modulate the Proliferation and Differentiation of Non-Specific Agonist and Specific Antigen-Stimulated Splenic Lymphocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096207

    AKGs modulate Th1/Th2 cytokine production of naïve T cells induced by anti-CD3 and anti-CD28. (A–D) CD4+CD62L+ T cells were stimulated with anti-CD3 and anti-CD28 (each 1 µg/ml), and cultured with DMSO, 100 nM batyl alcohol or 100 nM chimyl alcohol for 7 days. The supernatants were collected and analyzed for cytokines by ELISA. (A): TNF-α; (B): IFN-γ; (C): IL-4; (D): IL-10. The data shown represented the mean±SE for three independent experiments. *, P
    Figure Legend Snippet: AKGs modulate Th1/Th2 cytokine production of naïve T cells induced by anti-CD3 and anti-CD28. (A–D) CD4+CD62L+ T cells were stimulated with anti-CD3 and anti-CD28 (each 1 µg/ml), and cultured with DMSO, 100 nM batyl alcohol or 100 nM chimyl alcohol for 7 days. The supernatants were collected and analyzed for cytokines by ELISA. (A): TNF-α; (B): IFN-γ; (C): IL-4; (D): IL-10. The data shown represented the mean±SE for three independent experiments. *, P

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

    AKGs shaped the differentiation of naïve T cells induced by anti-CD3 and anti-CD28. (A) CD4+CD62L+ T cells were stimulated with anti-CD3 and anti-CD28 (each 1 µg/ml), and cultured with DMSO, 100 nM batyl alcohol or 100 nM chimyl alcohol for 7 days. Th1 transcription factor T-BET and Th2 transcription factor GATA-3 were analyzed by flow cytometry, and histogram plots showed the expression of indicated proteins in DMSO (shaded histograms), batyl or chimyl alcohol (red lined histograms) treated cells. The T-BET+ or GATA-3+ T cells were gated, and the p values for T-BET and GATA-3 expression differences between control and treated cells were indicated. The black lined histograms indicate isotype control. (B) The graph showed the mean fluorescent intensity (MFI) of T-BET and GATA3 within gates in CD3/CD28–stimulated T cells of DMSO, batyl or chimyl alcohol treatment. The data shown represented the mean±SE for at least three independent experiments. *, P
    Figure Legend Snippet: AKGs shaped the differentiation of naïve T cells induced by anti-CD3 and anti-CD28. (A) CD4+CD62L+ T cells were stimulated with anti-CD3 and anti-CD28 (each 1 µg/ml), and cultured with DMSO, 100 nM batyl alcohol or 100 nM chimyl alcohol for 7 days. Th1 transcription factor T-BET and Th2 transcription factor GATA-3 were analyzed by flow cytometry, and histogram plots showed the expression of indicated proteins in DMSO (shaded histograms), batyl or chimyl alcohol (red lined histograms) treated cells. The T-BET+ or GATA-3+ T cells were gated, and the p values for T-BET and GATA-3 expression differences between control and treated cells were indicated. The black lined histograms indicate isotype control. (B) The graph showed the mean fluorescent intensity (MFI) of T-BET and GATA3 within gates in CD3/CD28–stimulated T cells of DMSO, batyl or chimyl alcohol treatment. The data shown represented the mean±SE for at least three independent experiments. *, P

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

    AKGs boosted the proliferation of T cells induced by anti-CD3 and anti-CD28 or by antigen –bearing DCs. (A–B) Naïve CD4+CD62L+ T cells were stimulated with anti-CD3 and anti-CD28 (each 1 µg/ml), and cultured with different concentrations of batyl alcohol (A) or chimyl alcohol (B) for 72 h. Cells were treated with [ 3 H]thymidine (0.2 µCi/well) within the last 4 h of culture, and [ 3 H]thymidine incorporation was measured after cell harvest. (C) Whole CD4+ T cells (from HBsAg immunized mice) were stimulated with HBsAg –bearing DCs, and cultured with batyl alcohol or chimyl alcohol (100 nM) for 72 h. [ 3 H]thymidine was determined as in A and B. The data shown represented the mean±SE for three independent experiments. *, P
    Figure Legend Snippet: AKGs boosted the proliferation of T cells induced by anti-CD3 and anti-CD28 or by antigen –bearing DCs. (A–B) Naïve CD4+CD62L+ T cells were stimulated with anti-CD3 and anti-CD28 (each 1 µg/ml), and cultured with different concentrations of batyl alcohol (A) or chimyl alcohol (B) for 72 h. Cells were treated with [ 3 H]thymidine (0.2 µCi/well) within the last 4 h of culture, and [ 3 H]thymidine incorporation was measured after cell harvest. (C) Whole CD4+ T cells (from HBsAg immunized mice) were stimulated with HBsAg –bearing DCs, and cultured with batyl alcohol or chimyl alcohol (100 nM) for 72 h. [ 3 H]thymidine was determined as in A and B. The data shown represented the mean±SE for three independent experiments. *, P

    Techniques Used: Cell Culture, Mouse Assay

    8) Product Images from "NF1 heterozygosity fosters de novo tumorigenesis but impairs malignant transformation"

    Article Title: NF1 heterozygosity fosters de novo tumorigenesis but impairs malignant transformation

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07452-y

    Impact of Nf1 heterozygosity on T cells proliferation and function. a , b Proliferation of peripheral T cells from Nf1 +/+ and Nf1 +/− mice stimulated by various concentrations of a anti-CD3e antibody alone or with b CD28 costimulation. Proliferation was assessed in biological duplicate and technical triplicate by 3 H-thymidine incorporation during the last 20 h of culture. c Delayed-type hypersensitivity (DTH) assay: On day 0, Nf1 +/+ and Nf1 +/− mice were sensitized on abdominal skin by topical application of oxazolone. On day 6, Nf1 +/+ and Nf1 +/− mice were challenged by topical application on the left and right ear with oxazolone and control solvent, respectively. DTH was assessed daily through by measuring ear thickness (Δ). Experiments were performed in biological triplicates. d Two days after challenge, cellularity of draining lymph nodes (DLNs) of Nf1 +/+ and Nf1 +/− mice were determined. e Spontaneous activation of cells from DLNs (T cells) was measured by 3 H-thymidine incorporation after culturing for 3 days without stimuli. f Fraction of CD4 + cells that are activated. g Fraction of CD8 + cells that are activated. h Fraction of B cells + that are activated. i Fraction of Treg + cells in total CD4 + subset. Panels d – i were performed in biological triplicates. Statistical paired t -test performed; * P
    Figure Legend Snippet: Impact of Nf1 heterozygosity on T cells proliferation and function. a , b Proliferation of peripheral T cells from Nf1 +/+ and Nf1 +/− mice stimulated by various concentrations of a anti-CD3e antibody alone or with b CD28 costimulation. Proliferation was assessed in biological duplicate and technical triplicate by 3 H-thymidine incorporation during the last 20 h of culture. c Delayed-type hypersensitivity (DTH) assay: On day 0, Nf1 +/+ and Nf1 +/− mice were sensitized on abdominal skin by topical application of oxazolone. On day 6, Nf1 +/+ and Nf1 +/− mice were challenged by topical application on the left and right ear with oxazolone and control solvent, respectively. DTH was assessed daily through by measuring ear thickness (Δ). Experiments were performed in biological triplicates. d Two days after challenge, cellularity of draining lymph nodes (DLNs) of Nf1 +/+ and Nf1 +/− mice were determined. e Spontaneous activation of cells from DLNs (T cells) was measured by 3 H-thymidine incorporation after culturing for 3 days without stimuli. f Fraction of CD4 + cells that are activated. g Fraction of CD8 + cells that are activated. h Fraction of B cells + that are activated. i Fraction of Treg + cells in total CD4 + subset. Panels d – i were performed in biological triplicates. Statistical paired t -test performed; * P

    Techniques Used: Mouse Assay, Activation Assay

    9) Product Images from "NF1 heterozygosity fosters de novo tumorigenesis but impairs malignant transformation"

    Article Title: NF1 heterozygosity fosters de novo tumorigenesis but impairs malignant transformation

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07452-y

    Impact of Nf1 heterozygosity on T cells proliferation and function. a , b Proliferation of peripheral T cells from Nf1 +/+ and Nf1 +/− mice stimulated by various concentrations of a anti-CD3e antibody alone or with b CD28 costimulation. Proliferation was assessed in biological duplicate and technical triplicate by 3 H-thymidine incorporation during the last 20 h of culture. c Delayed-type hypersensitivity (DTH) assay: On day 0, Nf1 +/+ and Nf1 +/− mice were sensitized on abdominal skin by topical application of oxazolone. On day 6, Nf1 +/+ and Nf1 +/− mice were challenged by topical application on the left and right ear with oxazolone and control solvent, respectively. DTH was assessed daily through by measuring ear thickness (Δ). Experiments were performed in biological triplicates. d Two days after challenge, cellularity of draining lymph nodes (DLNs) of Nf1 +/+ and Nf1 +/− mice were determined. e Spontaneous activation of cells from DLNs (T cells) was measured by 3 H-thymidine incorporation after culturing for 3 days without stimuli. f Fraction of CD4 + cells that are activated. g Fraction of CD8 + cells that are activated. h Fraction of B cells + that are activated. i Fraction of Treg + cells in total CD4 + subset. Panels d – i were performed in biological triplicates. Statistical paired t -test performed; * P
    Figure Legend Snippet: Impact of Nf1 heterozygosity on T cells proliferation and function. a , b Proliferation of peripheral T cells from Nf1 +/+ and Nf1 +/− mice stimulated by various concentrations of a anti-CD3e antibody alone or with b CD28 costimulation. Proliferation was assessed in biological duplicate and technical triplicate by 3 H-thymidine incorporation during the last 20 h of culture. c Delayed-type hypersensitivity (DTH) assay: On day 0, Nf1 +/+ and Nf1 +/− mice were sensitized on abdominal skin by topical application of oxazolone. On day 6, Nf1 +/+ and Nf1 +/− mice were challenged by topical application on the left and right ear with oxazolone and control solvent, respectively. DTH was assessed daily through by measuring ear thickness (Δ). Experiments were performed in biological triplicates. d Two days after challenge, cellularity of draining lymph nodes (DLNs) of Nf1 +/+ and Nf1 +/− mice were determined. e Spontaneous activation of cells from DLNs (T cells) was measured by 3 H-thymidine incorporation after culturing for 3 days without stimuli. f Fraction of CD4 + cells that are activated. g Fraction of CD8 + cells that are activated. h Fraction of B cells + that are activated. i Fraction of Treg + cells in total CD4 + subset. Panels d – i were performed in biological triplicates. Statistical paired t -test performed; * P

    Techniques Used: Mouse Assay, Activation Assay

    Related Articles

    Purification:

    Article Title: NF1 heterozygosity fosters de novo tumorigenesis but impairs malignant transformation
    Article Snippet: T cell proliferation assay Following the manufacturer’s recommendations, CD3+ T cells were purified from the lymph nodes of Nf1 +/+ and Nf1 +/− mice (age 3–6 months) using pan-T-cell isolation kit (Miltenyi Biotec, Auburn, CA). .. Purified CD3+ T cells (2 × 105 /well) were cultured for 2 days in 96-well plates (in triplicate) precoated with indicated doses of anti-CD3 Ab (eBioscience; cat. no. 16-0031-86) alone or anti-CD28 mAb (eBioscience; cat. no. 16-0281-85). .. After pulsing with 3 H-thymidine (1 μCi/well [0.037 MBq/well]) for 20–22 h, cells were collected and evaluated for 3 H radioactivity.

    Cell Culture:

    Article Title: NF1 heterozygosity fosters de novo tumorigenesis but impairs malignant transformation
    Article Snippet: T cell proliferation assay Following the manufacturer’s recommendations, CD3+ T cells were purified from the lymph nodes of Nf1 +/+ and Nf1 +/− mice (age 3–6 months) using pan-T-cell isolation kit (Miltenyi Biotec, Auburn, CA). .. Purified CD3+ T cells (2 × 105 /well) were cultured for 2 days in 96-well plates (in triplicate) precoated with indicated doses of anti-CD3 Ab (eBioscience; cat. no. 16-0031-86) alone or anti-CD28 mAb (eBioscience; cat. no. 16-0281-85). .. After pulsing with 3 H-thymidine (1 μCi/well [0.037 MBq/well]) for 20–22 h, cells were collected and evaluated for 3 H radioactivity.

    Activation Assay:

    Article Title: Regulatory T Cell Migration Is Dependent on Glucokinase-Mediated Glycolysis
    Article Snippet: For use in functional assays, Treg cells were used 6-8 days after stimulation. .. Antibody-mediated T cell activation Activated T cells were obtained by polyclonal stimulation of LN cells with plate-bound anti-CD3 (1 μg/ml, eBiosciences, Cat# 16-0032-85) and plate-bound anti-CD28 (5 μg/ml, eBiosciences, Cat# 16-0281-86) in complete T cell medium supplemented with 20 U/ml recombinant IL-2 (Roche, West Sussex, UK) for 7 days at 37°C. ..

    Recombinant:

    Article Title: Regulatory T Cell Migration Is Dependent on Glucokinase-Mediated Glycolysis
    Article Snippet: For use in functional assays, Treg cells were used 6-8 days after stimulation. .. Antibody-mediated T cell activation Activated T cells were obtained by polyclonal stimulation of LN cells with plate-bound anti-CD3 (1 μg/ml, eBiosciences, Cat# 16-0032-85) and plate-bound anti-CD28 (5 μg/ml, eBiosciences, Cat# 16-0281-86) in complete T cell medium supplemented with 20 U/ml recombinant IL-2 (Roche, West Sussex, UK) for 7 days at 37°C. ..

    other:

    Article Title: Nod2 Activates NF-kB in CD4+ T Cells but Its Expression Is Dispensable for T Cell-Induced Colitis
    Article Snippet: Material and reagents The following antibodies were used for the experiments: anti-CD3ε (100331, BioLegend, San Diego, CA, USA), anti-CD3ε-FITC (11-0031-82, eBioscience, San Diego, CA, USA), anti-CD4-APC (17-0041-82, eBioscience), anti-CD4-A780 (47-0042-82, eBioscience), anti-CD8α (553027, BD Biosciences, San Jose, CA, USA), anti-CD8α-APC (17-0081-81, eBioscience), anti-TCRβ-APC-eFluor780 (47-5961-82, eBioscience), anti-CD44-PE (12-0441-82, eBioscience), anti-CD11b (553308, BD Biosciences), anti-CD25-APC (17-0251-82, eBioscience), anti-CD28 (16-0281-85, eBioscience), anti-CD45RB-PE (12-0455-82, eBioscience), anti-Foxp3-PE-Cy7 (25-5773-82, eBioscience), anti-B220 (553084, BD Biosciences), anti-Nod2 (14-5858, eBioscience), anti-c-Rel (sc-71, Santa Cruz Biotechnology, Heidelberg, Germany), anti-p65 (3034, Cell Signaling, Danvers, MA, USA), anti-β-actin (sc-130656, Santa Cruz Biotechnology), anti-RNA polymerase II (sc-899, Santa Cruz Biotechnology).

    Staining:

    Article Title: IL-31 plays dual roles in lung inflammation in an OVA-induced murine asthma model
    Article Snippet: T cell proliferation Spleen CD4+ T cells were isolated from WT and IL-31RA KO mice by negative selection via incubation with hybridoma supernatants (αB220, αFCγ, αCD8, αMHCII) followed by magnetic bead purification (Life Technologies). .. T cells at 2 million cells/ml were washed and resuspended in 2 μM CFSE (Invitrogen) staining solution for 10 min, and CFSE fluorescence was measured by flow cytometry at 96 h after culture with 1 μl/ml anti-CD3 (CAT#85-16-0032-81)/anti-CD28 (CAT85-16-0281-81) (both eBioscience). .. T cell activation and differentiation CD4+ T cells were resuspended at 2 million cells/ml and stimulated with anti-CD3/anti-CD28 (1 μl/ml) under neutral or Th1- (50 ng/ml IL-12, 125 ng/ml anti-IL-4), Th2- (40 ng/ml IL-4, 50 ng/ml anti-IFN-γ), Th17- (50 ng/ml IL-6, 2 ng/ml TGF-β1) polarizing conditions.

    Fluorescence:

    Article Title: IL-31 plays dual roles in lung inflammation in an OVA-induced murine asthma model
    Article Snippet: T cell proliferation Spleen CD4+ T cells were isolated from WT and IL-31RA KO mice by negative selection via incubation with hybridoma supernatants (αB220, αFCγ, αCD8, αMHCII) followed by magnetic bead purification (Life Technologies). .. T cells at 2 million cells/ml were washed and resuspended in 2 μM CFSE (Invitrogen) staining solution for 10 min, and CFSE fluorescence was measured by flow cytometry at 96 h after culture with 1 μl/ml anti-CD3 (CAT#85-16-0032-81)/anti-CD28 (CAT85-16-0281-81) (both eBioscience). .. T cell activation and differentiation CD4+ T cells were resuspended at 2 million cells/ml and stimulated with anti-CD3/anti-CD28 (1 μl/ml) under neutral or Th1- (50 ng/ml IL-12, 125 ng/ml anti-IL-4), Th2- (40 ng/ml IL-4, 50 ng/ml anti-IFN-γ), Th17- (50 ng/ml IL-6, 2 ng/ml TGF-β1) polarizing conditions.

    Flow Cytometry:

    Article Title: IL-31 plays dual roles in lung inflammation in an OVA-induced murine asthma model
    Article Snippet: T cell proliferation Spleen CD4+ T cells were isolated from WT and IL-31RA KO mice by negative selection via incubation with hybridoma supernatants (αB220, αFCγ, αCD8, αMHCII) followed by magnetic bead purification (Life Technologies). .. T cells at 2 million cells/ml were washed and resuspended in 2 μM CFSE (Invitrogen) staining solution for 10 min, and CFSE fluorescence was measured by flow cytometry at 96 h after culture with 1 μl/ml anti-CD3 (CAT#85-16-0032-81)/anti-CD28 (CAT85-16-0281-81) (both eBioscience). .. T cell activation and differentiation CD4+ T cells were resuspended at 2 million cells/ml and stimulated with anti-CD3/anti-CD28 (1 μl/ml) under neutral or Th1- (50 ng/ml IL-12, 125 ng/ml anti-IL-4), Th2- (40 ng/ml IL-4, 50 ng/ml anti-IFN-γ), Th17- (50 ng/ml IL-6, 2 ng/ml TGF-β1) polarizing conditions.

    Cytometry:

    Article Title: IL-31 plays dual roles in lung inflammation in an OVA-induced murine asthma model
    Article Snippet: T cell proliferation Spleen CD4+ T cells were isolated from WT and IL-31RA KO mice by negative selection via incubation with hybridoma supernatants (αB220, αFCγ, αCD8, αMHCII) followed by magnetic bead purification (Life Technologies). .. T cells at 2 million cells/ml were washed and resuspended in 2 μM CFSE (Invitrogen) staining solution for 10 min, and CFSE fluorescence was measured by flow cytometry at 96 h after culture with 1 μl/ml anti-CD3 (CAT#85-16-0032-81)/anti-CD28 (CAT85-16-0281-81) (both eBioscience). .. T cell activation and differentiation CD4+ T cells were resuspended at 2 million cells/ml and stimulated with anti-CD3/anti-CD28 (1 μl/ml) under neutral or Th1- (50 ng/ml IL-12, 125 ng/ml anti-IL-4), Th2- (40 ng/ml IL-4, 50 ng/ml anti-IFN-γ), Th17- (50 ng/ml IL-6, 2 ng/ml TGF-β1) polarizing conditions.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: IL-31 plays dual roles in lung inflammation in an OVA-induced murine asthma model
    Article Snippet: T cell proliferation Spleen CD4+ T cells were isolated from WT and IL-31RA KO mice by negative selection via incubation with hybridoma supernatants (αB220, αFCγ, αCD8, αMHCII) followed by magnetic bead purification (Life Technologies). .. T cells at 2 million cells/ml were washed and resuspended in 2 μM CFSE (Invitrogen) staining solution for 10 min, and CFSE fluorescence was measured by flow cytometry at 96 h after culture with 1 μl/ml anti-CD3 (CAT#85-16-0032-81)/anti-CD28 (CAT85-16-0281-81) (both eBioscience). .. T cell activation and differentiation CD4+ T cells were resuspended at 2 million cells/ml and stimulated with anti-CD3/anti-CD28 (1 μl/ml) under neutral or Th1- (50 ng/ml IL-12, 125 ng/ml anti-IL-4), Th2- (40 ng/ml IL-4, 50 ng/ml anti-IFN-γ), Th17- (50 ng/ml IL-6, 2 ng/ml TGF-β1) polarizing conditions.

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    Thermo Fisher mouse anti human cd28 mab
    Preparation and phenotypic analyses of AAPC-beads. ( a ) Magnetic Dynal beads were incubated with 1 mL of PBS containing a series of H-2K b /OVA 257–264 -Ig dimer quantities at 4 °C overnight with rotation, blocked with BSA, and then stained with PE-labeled anti-mouse IgG1 mAb. ( b ) The magnetic beads were incubated with the indicated amounts of H-2K b /OVA 257–264 -Ig dimers overnight followed by BSA blocking first and PE-labeled anti-H-2K b mAb (clone AF6–88.5) staining later. ( c ) Phenotypic analyses of H-2K b -Ig-beads, <t>Anti-CD28-beads,</t> and two-signal AAPC-beads (H-2K b -Ig/anti-CD28-beads). Gray shaded diagrams represent the isotype control, and solid black lines indicate PE-anti-mouse IgG1 or FITC-anti-hamster IgG staining. ( d) Schematic representation of AAPC-beads co-coupled with H-2K b /OVA 257–264 -Ig dimer and anti-CD28 mAb.
    Mouse Anti Human Cd28 Mab, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti human cd28 mab/product/Thermo Fisher
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti human cd28 mab - by Bioz Stars, 2021-05
    93/100 stars
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    95
    Thermo Fisher anti cd28
    Natural killer (NK) cells reduce the proportion of Th17 and Th1 cells in vitro . Memory CD4 + T cells and NK cells were isolated from healthy subject PBMC. Memory CD4 + T cells were CD3 + CD4 + CD45RO + CD45RA − as shown in representative dot plots (A,B) . NK cells were CD3 − CD56 + as shown in a representative dot plot (C) . Memory CD4 + T cells were cultured without (D,E) , or with NK cells at a 1:1 ratio (F,G) for 4 days with anti-CD3, <t>anti-CD28,</t> and Th17 polarizing factors for 4 days. Plots are shown for CD4 × CD56, which was used to gate CD4 + helper T (Th) cells. Expression of IL-17 and IFN-γ by CD4 + T cells was assessed by intracellular flow cytometry (Th17 = CD3 + CD4 + IL-17A + IFN-γ − , Th1 = CD3 + CD4 + IL-17A − IFN-γ + , and Th1/17 = CD3 + CD4 + IL-17A + IFN-γ + ). Data pooled from 12 experiments showing the proportion (H) and absolute number of Th cells (I) . A time-course analysis for Th17 cells (J) and Th1 cells (K) was performed for 5 days using intracellular cytokine staining. Open diamond = T cells and closed square = T cells + NK cells.
    Anti Cd28, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti cd28/product/Thermo Fisher
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti cd28 - by Bioz Stars, 2021-05
    95/100 stars
      Buy from Supplier

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    CD283 TLR3 Functional Grade Monoclonal Antibody for Flow Neu FN
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    CD2 Functional Grade Monoclonal Antibody for Flow Neu FN
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    Preparation and phenotypic analyses of AAPC-beads. ( a ) Magnetic Dynal beads were incubated with 1 mL of PBS containing a series of H-2K b /OVA 257–264 -Ig dimer quantities at 4 °C overnight with rotation, blocked with BSA, and then stained with PE-labeled anti-mouse IgG1 mAb. ( b ) The magnetic beads were incubated with the indicated amounts of H-2K b /OVA 257–264 -Ig dimers overnight followed by BSA blocking first and PE-labeled anti-H-2K b mAb (clone AF6–88.5) staining later. ( c ) Phenotypic analyses of H-2K b -Ig-beads, Anti-CD28-beads, and two-signal AAPC-beads (H-2K b -Ig/anti-CD28-beads). Gray shaded diagrams represent the isotype control, and solid black lines indicate PE-anti-mouse IgG1 or FITC-anti-hamster IgG staining. ( d) Schematic representation of AAPC-beads co-coupled with H-2K b /OVA 257–264 -Ig dimer and anti-CD28 mAb.

    Journal: Scientific Reports

    Article Title: Frequency and reactivity of antigen-specific T cells were concurrently measured through the combination of artificial antigen-presenting cell, MACS and ELISPOT

    doi: 10.1038/s41598-017-16549-1

    Figure Lengend Snippet: Preparation and phenotypic analyses of AAPC-beads. ( a ) Magnetic Dynal beads were incubated with 1 mL of PBS containing a series of H-2K b /OVA 257–264 -Ig dimer quantities at 4 °C overnight with rotation, blocked with BSA, and then stained with PE-labeled anti-mouse IgG1 mAb. ( b ) The magnetic beads were incubated with the indicated amounts of H-2K b /OVA 257–264 -Ig dimers overnight followed by BSA blocking first and PE-labeled anti-H-2K b mAb (clone AF6–88.5) staining later. ( c ) Phenotypic analyses of H-2K b -Ig-beads, Anti-CD28-beads, and two-signal AAPC-beads (H-2K b -Ig/anti-CD28-beads). Gray shaded diagrams represent the isotype control, and solid black lines indicate PE-anti-mouse IgG1 or FITC-anti-hamster IgG staining. ( d) Schematic representation of AAPC-beads co-coupled with H-2K b /OVA 257–264 -Ig dimer and anti-CD28 mAb.

    Article Snippet: Purified hamster anti-mouse CD28 mAb and mouse anti-human CD28 mAb (Functional grade), PE-anti-mouse CD4 and FITC-anti-mouse CD8a, APC-anti-mouse CD3e and PE-anti-mouse Vα2 TCR, and PE-mouse anti-human HLA-ABC mAb (W6/32) were from eBioscience (San Diego, CA, USA).

    Techniques: Incubation, Staining, Labeling, Magnetic Beads, Blocking Assay

    Natural killer (NK) cells reduce the proportion of Th17 and Th1 cells in vitro . Memory CD4 + T cells and NK cells were isolated from healthy subject PBMC. Memory CD4 + T cells were CD3 + CD4 + CD45RO + CD45RA − as shown in representative dot plots (A,B) . NK cells were CD3 − CD56 + as shown in a representative dot plot (C) . Memory CD4 + T cells were cultured without (D,E) , or with NK cells at a 1:1 ratio (F,G) for 4 days with anti-CD3, anti-CD28, and Th17 polarizing factors for 4 days. Plots are shown for CD4 × CD56, which was used to gate CD4 + helper T (Th) cells. Expression of IL-17 and IFN-γ by CD4 + T cells was assessed by intracellular flow cytometry (Th17 = CD3 + CD4 + IL-17A + IFN-γ − , Th1 = CD3 + CD4 + IL-17A − IFN-γ + , and Th1/17 = CD3 + CD4 + IL-17A + IFN-γ + ). Data pooled from 12 experiments showing the proportion (H) and absolute number of Th cells (I) . A time-course analysis for Th17 cells (J) and Th1 cells (K) was performed for 5 days using intracellular cytokine staining. Open diamond = T cells and closed square = T cells + NK cells.

    Journal: Frontiers in Immunology

    Article Title: Natural Killer Cells Regulate Th17 Cells After Autologous Hematopoietic Stem Cell Transplantation for Relapsing Remitting Multiple Sclerosis

    doi: 10.3389/fimmu.2018.00834

    Figure Lengend Snippet: Natural killer (NK) cells reduce the proportion of Th17 and Th1 cells in vitro . Memory CD4 + T cells and NK cells were isolated from healthy subject PBMC. Memory CD4 + T cells were CD3 + CD4 + CD45RO + CD45RA − as shown in representative dot plots (A,B) . NK cells were CD3 − CD56 + as shown in a representative dot plot (C) . Memory CD4 + T cells were cultured without (D,E) , or with NK cells at a 1:1 ratio (F,G) for 4 days with anti-CD3, anti-CD28, and Th17 polarizing factors for 4 days. Plots are shown for CD4 × CD56, which was used to gate CD4 + helper T (Th) cells. Expression of IL-17 and IFN-γ by CD4 + T cells was assessed by intracellular flow cytometry (Th17 = CD3 + CD4 + IL-17A + IFN-γ − , Th1 = CD3 + CD4 + IL-17A − IFN-γ + , and Th1/17 = CD3 + CD4 + IL-17A + IFN-γ + ). Data pooled from 12 experiments showing the proportion (H) and absolute number of Th cells (I) . A time-course analysis for Th17 cells (J) and Th1 cells (K) was performed for 5 days using intracellular cytokine staining. Open diamond = T cells and closed square = T cells + NK cells.

    Article Snippet: Complete or depleted samples were then incubated with functional grade anti-CD3 (OKT3 1.0 µg/ml; eBioscience), anti-CD28 (CD28.2; 1.0 µg/ml; eBioscience), and neutralizing anti-IFN-γ and anti-IL-4 antibodies (5 µg/ml, R & D systems) for 4 days, then intracellular cytokine stained for IL-17A and IFN-γ, and supernatants analyzed for IL-17A using uncoated enzyme-linked immunosorbent assay (ELISA) kit for human IL-17A (eBioscience) and human IFN-γ (eBioscience).

    Techniques: In Vitro, Isolation, Cell Culture, Expressing, Flow Cytometry, Cytometry, Staining

    Natural killer (NK) cells augment IL-17A and RORC levels in memory CD4 T cells. Purified memory CD4 + T cells from healthy subject PBMCs were activated with anti-CD3, anti-CD28, and Th17 polarizing factors without (open diamond) or with NK cells (closed square). Cytokines IL-17A (A) and IFN-γ (B) were measured in the supernatant by enzyme-linked immunosorbent assay. Expression of RORC (C) and IL17A (D) mRNA was measured by qPCR at the indicated time points. Data are representative of three samples. Groups included non-activated memory CD4 + T cells (T nil; closed circles), activated memory CD4 + T cells (T; open circle), activated memory CD4 + T cells with NK cells (T NK; closed squares), and NK cells cultured alone with IL-2 (NK IL-2; open squares). Representative plots of IL-17A and IFN-γ expression in NK cells (CD3 − CD56 + ) are shown (E,F) .

    Journal: Frontiers in Immunology

    Article Title: Natural Killer Cells Regulate Th17 Cells After Autologous Hematopoietic Stem Cell Transplantation for Relapsing Remitting Multiple Sclerosis

    doi: 10.3389/fimmu.2018.00834

    Figure Lengend Snippet: Natural killer (NK) cells augment IL-17A and RORC levels in memory CD4 T cells. Purified memory CD4 + T cells from healthy subject PBMCs were activated with anti-CD3, anti-CD28, and Th17 polarizing factors without (open diamond) or with NK cells (closed square). Cytokines IL-17A (A) and IFN-γ (B) were measured in the supernatant by enzyme-linked immunosorbent assay. Expression of RORC (C) and IL17A (D) mRNA was measured by qPCR at the indicated time points. Data are representative of three samples. Groups included non-activated memory CD4 + T cells (T nil; closed circles), activated memory CD4 + T cells (T; open circle), activated memory CD4 + T cells with NK cells (T NK; closed squares), and NK cells cultured alone with IL-2 (NK IL-2; open squares). Representative plots of IL-17A and IFN-γ expression in NK cells (CD3 − CD56 + ) are shown (E,F) .

    Article Snippet: Complete or depleted samples were then incubated with functional grade anti-CD3 (OKT3 1.0 µg/ml; eBioscience), anti-CD28 (CD28.2; 1.0 µg/ml; eBioscience), and neutralizing anti-IFN-γ and anti-IL-4 antibodies (5 µg/ml, R & D systems) for 4 days, then intracellular cytokine stained for IL-17A and IFN-γ, and supernatants analyzed for IL-17A using uncoated enzyme-linked immunosorbent assay (ELISA) kit for human IL-17A (eBioscience) and human IFN-γ (eBioscience).

    Techniques: Purification, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction, Cell Culture

    Natural killer (NK) cells are cytotoxic toward helper T (Th) cells through necrosis but not apoptosis. Memory CD4 + T cells from healthy subject PBMC were activated with anti-CD3, anti-CD28, and Th17 polarizing factors, either in the absence or presence of NK cells for 4 days. Samples were stained with CD3, CD56, 7AAD, and annexin V. Representative plots of T cells (CD4 + CD56 − ) cultured without (A) or with NK cells (B) are shown. The average proportion of necrotic (7-AAD + annexinV + ) T cells is shown (C) . Open bars = memory CD4 T cells, closed bars = memory CD4 + T cells plus NK cells. N = 8 samples. A time-course analysis of apoptotic (7-AAD − annexin V + ) T cells is shown (D) . Open circles indicate memory CD4 T cells alone and filled squares indicate T cells plus NK cells.

    Journal: Frontiers in Immunology

    Article Title: Natural Killer Cells Regulate Th17 Cells After Autologous Hematopoietic Stem Cell Transplantation for Relapsing Remitting Multiple Sclerosis

    doi: 10.3389/fimmu.2018.00834

    Figure Lengend Snippet: Natural killer (NK) cells are cytotoxic toward helper T (Th) cells through necrosis but not apoptosis. Memory CD4 + T cells from healthy subject PBMC were activated with anti-CD3, anti-CD28, and Th17 polarizing factors, either in the absence or presence of NK cells for 4 days. Samples were stained with CD3, CD56, 7AAD, and annexin V. Representative plots of T cells (CD4 + CD56 − ) cultured without (A) or with NK cells (B) are shown. The average proportion of necrotic (7-AAD + annexinV + ) T cells is shown (C) . Open bars = memory CD4 T cells, closed bars = memory CD4 + T cells plus NK cells. N = 8 samples. A time-course analysis of apoptotic (7-AAD − annexin V + ) T cells is shown (D) . Open circles indicate memory CD4 T cells alone and filled squares indicate T cells plus NK cells.

    Article Snippet: Complete or depleted samples were then incubated with functional grade anti-CD3 (OKT3 1.0 µg/ml; eBioscience), anti-CD28 (CD28.2; 1.0 µg/ml; eBioscience), and neutralizing anti-IFN-γ and anti-IL-4 antibodies (5 µg/ml, R & D systems) for 4 days, then intracellular cytokine stained for IL-17A and IFN-γ, and supernatants analyzed for IL-17A using uncoated enzyme-linked immunosorbent assay (ELISA) kit for human IL-17A (eBioscience) and human IFN-γ (eBioscience).

    Techniques: Staining, Cell Culture

    Natural killer (NK) cells support IL-17A expression by helper T (Th) cells by CD58 co-stimulation. A representative plot of CD58 expression by CD3 − CD56 + NK cells is shown from healthy subject peripheral blood mononuclear cell (PBMC) (A) . Memory CD4 + T cells from healthy subjects PBMC were activated with anti-CD3, anti-CD28, and Th17 polarizing factors with NK cells in the presence or absence of CD58 neutralizing or isotype control antibody for 4 days. The cytokine IL-17A was assessed by enzyme-linked immunosorbent assay from cell culture supernatants (B) . Graph indicates mean IL-17A concentration for N = 3 samples.

    Journal: Frontiers in Immunology

    Article Title: Natural Killer Cells Regulate Th17 Cells After Autologous Hematopoietic Stem Cell Transplantation for Relapsing Remitting Multiple Sclerosis

    doi: 10.3389/fimmu.2018.00834

    Figure Lengend Snippet: Natural killer (NK) cells support IL-17A expression by helper T (Th) cells by CD58 co-stimulation. A representative plot of CD58 expression by CD3 − CD56 + NK cells is shown from healthy subject peripheral blood mononuclear cell (PBMC) (A) . Memory CD4 + T cells from healthy subjects PBMC were activated with anti-CD3, anti-CD28, and Th17 polarizing factors with NK cells in the presence or absence of CD58 neutralizing or isotype control antibody for 4 days. The cytokine IL-17A was assessed by enzyme-linked immunosorbent assay from cell culture supernatants (B) . Graph indicates mean IL-17A concentration for N = 3 samples.

    Article Snippet: Complete or depleted samples were then incubated with functional grade anti-CD3 (OKT3 1.0 µg/ml; eBioscience), anti-CD28 (CD28.2; 1.0 µg/ml; eBioscience), and neutralizing anti-IFN-γ and anti-IL-4 antibodies (5 µg/ml, R & D systems) for 4 days, then intracellular cytokine stained for IL-17A and IFN-γ, and supernatants analyzed for IL-17A using uncoated enzyme-linked immunosorbent assay (ELISA) kit for human IL-17A (eBioscience) and human IFN-γ (eBioscience).

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Concentration Assay

    Changes in blood natural killer (NK) cell populations correlate with changes in helper T (Th) cells populations following aHSCT in MS patients. MS patient peripheral blood mononuclear cell (PBMC) samples from baseline (BL) and 12 month post-aHSCT were activated in vitro with anti-CD3, anti-CD28, and Th17 polarizing factors for 4 days. Th17 and Th1 cells were assessed by analysis of cytokine production by intracellular flow cytometry (CD3 + CD4 + IL-17A + IFN-γ − or CD3 + CD4 + IL-17A − IFN-γ + , respectively). The change in frequency of Th17 cells (A) or Th1 cells (B) was plotted against the change in NK cell frequency, and linear regression was performed on the data points. N = 7 patients.

    Journal: Frontiers in Immunology

    Article Title: Natural Killer Cells Regulate Th17 Cells After Autologous Hematopoietic Stem Cell Transplantation for Relapsing Remitting Multiple Sclerosis

    doi: 10.3389/fimmu.2018.00834

    Figure Lengend Snippet: Changes in blood natural killer (NK) cell populations correlate with changes in helper T (Th) cells populations following aHSCT in MS patients. MS patient peripheral blood mononuclear cell (PBMC) samples from baseline (BL) and 12 month post-aHSCT were activated in vitro with anti-CD3, anti-CD28, and Th17 polarizing factors for 4 days. Th17 and Th1 cells were assessed by analysis of cytokine production by intracellular flow cytometry (CD3 + CD4 + IL-17A + IFN-γ − or CD3 + CD4 + IL-17A − IFN-γ + , respectively). The change in frequency of Th17 cells (A) or Th1 cells (B) was plotted against the change in NK cell frequency, and linear regression was performed on the data points. N = 7 patients.

    Article Snippet: Complete or depleted samples were then incubated with functional grade anti-CD3 (OKT3 1.0 µg/ml; eBioscience), anti-CD28 (CD28.2; 1.0 µg/ml; eBioscience), and neutralizing anti-IFN-γ and anti-IL-4 antibodies (5 µg/ml, R & D systems) for 4 days, then intracellular cytokine stained for IL-17A and IFN-γ, and supernatants analyzed for IL-17A using uncoated enzyme-linked immunosorbent assay (ELISA) kit for human IL-17A (eBioscience) and human IFN-γ (eBioscience).

    Techniques: Mass Spectrometry, In Vitro, Flow Cytometry, Cytometry

    CD56 + cells suppress Th17 and Th1 cell responses in aHSCT. CD56 + cells were depleted from MS patient PBMC samples collected at 12 months post-aHSCT. Representative plots are shown for complete (A,B) and CD56-depleted samples (C,D) . Cells were activated in vitro with anti-CD3, anti-CD28, and Th17 polarizing factors (Act) for 4 days. The proportions of Th17 and Th1 cells were assessed by analysis of cytokine production by intracellular flow cytometry. Representative plots are shown for complete samples (B) and CD56-depleted samples (D) . The average proportion of Th17 (E) or Th1 cells (F) is shown. N = 3 patients.

    Journal: Frontiers in Immunology

    Article Title: Natural Killer Cells Regulate Th17 Cells After Autologous Hematopoietic Stem Cell Transplantation for Relapsing Remitting Multiple Sclerosis

    doi: 10.3389/fimmu.2018.00834

    Figure Lengend Snippet: CD56 + cells suppress Th17 and Th1 cell responses in aHSCT. CD56 + cells were depleted from MS patient PBMC samples collected at 12 months post-aHSCT. Representative plots are shown for complete (A,B) and CD56-depleted samples (C,D) . Cells were activated in vitro with anti-CD3, anti-CD28, and Th17 polarizing factors (Act) for 4 days. The proportions of Th17 and Th1 cells were assessed by analysis of cytokine production by intracellular flow cytometry. Representative plots are shown for complete samples (B) and CD56-depleted samples (D) . The average proportion of Th17 (E) or Th1 cells (F) is shown. N = 3 patients.

    Article Snippet: Complete or depleted samples were then incubated with functional grade anti-CD3 (OKT3 1.0 µg/ml; eBioscience), anti-CD28 (CD28.2; 1.0 µg/ml; eBioscience), and neutralizing anti-IFN-γ and anti-IL-4 antibodies (5 µg/ml, R & D systems) for 4 days, then intracellular cytokine stained for IL-17A and IFN-γ, and supernatants analyzed for IL-17A using uncoated enzyme-linked immunosorbent assay (ELISA) kit for human IL-17A (eBioscience) and human IFN-γ (eBioscience).

    Techniques: Mass Spectrometry, In Vitro, Activated Clotting Time Assay, Flow Cytometry, Cytometry

    Activated memory CD4 + T cells express MICA and are sensitive to NKG2D-mediated natural killer (NK) cell cytotoxicity. Memory CD4 + T cells were obtained from healthy subject PBMC, as described in Figure 5 , and activated with anti-CD3, anti-CD28, and Th17 polarizing factors for 4 days. Expression of CD4, MICA (Zenon labeled), IL-17A, and IFN-γ were assessed by intracellular cytokine staining and flow cytometry. Representative plots of FSC × SSC (A) , CD4 × SSC (B) , and IL-17A × IFN-γ (C) are shown. MICA expression on Th17 (D) , Th1 (E) , and the Th0 cells (F) is shown. Open histogram indicate MICA stained cells and closed histograms indicate an isotype control. The average mean fluorescent intensity of MICA minus the isotype control is shown [ΔMFI; (G) ]. Expression of CD56 and NKG2D by NK cells was assessed by flow cytometry and a representative plot is shown (H) . NK cells were cultured with memory CD4 + T cells and activated with anti-CD3, anti-CD28, and Th17 polarizing factors, at the same time treated without antibody, (NIL), anti-NKG2D neutralizing antibody, or isotype control antibody (I) . N = 7 samples.

    Journal: Frontiers in Immunology

    Article Title: Natural Killer Cells Regulate Th17 Cells After Autologous Hematopoietic Stem Cell Transplantation for Relapsing Remitting Multiple Sclerosis

    doi: 10.3389/fimmu.2018.00834

    Figure Lengend Snippet: Activated memory CD4 + T cells express MICA and are sensitive to NKG2D-mediated natural killer (NK) cell cytotoxicity. Memory CD4 + T cells were obtained from healthy subject PBMC, as described in Figure 5 , and activated with anti-CD3, anti-CD28, and Th17 polarizing factors for 4 days. Expression of CD4, MICA (Zenon labeled), IL-17A, and IFN-γ were assessed by intracellular cytokine staining and flow cytometry. Representative plots of FSC × SSC (A) , CD4 × SSC (B) , and IL-17A × IFN-γ (C) are shown. MICA expression on Th17 (D) , Th1 (E) , and the Th0 cells (F) is shown. Open histogram indicate MICA stained cells and closed histograms indicate an isotype control. The average mean fluorescent intensity of MICA minus the isotype control is shown [ΔMFI; (G) ]. Expression of CD56 and NKG2D by NK cells was assessed by flow cytometry and a representative plot is shown (H) . NK cells were cultured with memory CD4 + T cells and activated with anti-CD3, anti-CD28, and Th17 polarizing factors, at the same time treated without antibody, (NIL), anti-NKG2D neutralizing antibody, or isotype control antibody (I) . N = 7 samples.

    Article Snippet: Complete or depleted samples were then incubated with functional grade anti-CD3 (OKT3 1.0 µg/ml; eBioscience), anti-CD28 (CD28.2; 1.0 µg/ml; eBioscience), and neutralizing anti-IFN-γ and anti-IL-4 antibodies (5 µg/ml, R & D systems) for 4 days, then intracellular cytokine stained for IL-17A and IFN-γ, and supernatants analyzed for IL-17A using uncoated enzyme-linked immunosorbent assay (ELISA) kit for human IL-17A (eBioscience) and human IFN-γ (eBioscience).

    Techniques: Expressing, Labeling, Staining, Flow Cytometry, Cytometry, Cell Culture

    Flow cytometry showing induction of FoxP3+ CD4+Tregs and their expansion in vitro . Mouse CD4 T cells were isolated from spleen and lymph nodes, stimulated with anti-CD3 and anti-CD28 mAbs for 4-5 days in the presence of anti-IL-4, anti-IFNγ, IL-2 and TGF-β. The cells were then expanded in the presence of IL-2 and TGF-β for another 4 days. The data shown are representative of more than 10 experiments carried out.

    Journal: bioRxiv

    Article Title: Regulatory T cell activation triggers specific changes in glycosylation associated with Siglec-1-dependent inflammatory responses

    doi: 10.1101/2020.07.29.226399

    Figure Lengend Snippet: Flow cytometry showing induction of FoxP3+ CD4+Tregs and their expansion in vitro . Mouse CD4 T cells were isolated from spleen and lymph nodes, stimulated with anti-CD3 and anti-CD28 mAbs for 4-5 days in the presence of anti-IL-4, anti-IFNγ, IL-2 and TGF-β. The cells were then expanded in the presence of IL-2 and TGF-β for another 4 days. The data shown are representative of more than 10 experiments carried out.

    Article Snippet: Generation and culture of TregsRPMI 1640 medium with L-glutamine (Gibco™), FBS (Gibco™), Penicillin-Streptomycin (Gibco™), 2-mercaptoethanol (Gibco™), functional grade anti-mouse CD28 (eBioscience™, clone: 37.51), functional grade anti-mouse IL-4 (eBioscience™, clone: 11B11), functional grade anti-mouse INF-γ (eBioscience™, clone: XMG1.2) were from ThermoFisher, Loughborough, UK.

    Techniques: Flow Cytometry, In Vitro, Isolation

    Effects of SHP1 and/or SHP2 KO on PD-1 and BTLA signaling. (A) Cartoons depicting a Raji-Jurkat co-culture assay to probe PD-1 or BTLA signaling in a SHP1 KO background. PD-1 (WT)-mGFP or PD-1 (FF)-mGFP transduced SHP1 KO Jurkat T cells were stimulated with PD-L1 or HVEM positive Raji B cells preloaded with SEE antigen. (B) Shown on the left are representative immunoblots showing the phosphorylations of CD3ζ (anti-pY142)and CD28 (co-IP’ed p85) for the indicated co-cultures with the cells lysed at the indicated time points after Raji-Jurkat contact (see Methods ). Shown on the right are quantification graphs of CD28 and CD3ζ phosphorylations, incorporating results from three independent experiments. Data were normalized to the highest phosphorylation under PD-1 FF or BTLA FFFF condition, respectively. (C) Bar graphs summarizing the levels of secreted IL-2 of the indicated Raji-Jurkat co-cultures (see Methods ). ( D-F ) Same as A-C except using SHP2 KO Jurkat cells. ( G-I ) Same as A-C except using SHP1/SHP2 double KO Jurkat cells. Data in this figure are presented as means ± SD from three independent measurements. ****P

    Journal: bioRxiv

    Article Title: BTLA and PD-1 employ distinct phosphatases to differentially repress T cell signaling

    doi: 10.1101/669812

    Figure Lengend Snippet: Effects of SHP1 and/or SHP2 KO on PD-1 and BTLA signaling. (A) Cartoons depicting a Raji-Jurkat co-culture assay to probe PD-1 or BTLA signaling in a SHP1 KO background. PD-1 (WT)-mGFP or PD-1 (FF)-mGFP transduced SHP1 KO Jurkat T cells were stimulated with PD-L1 or HVEM positive Raji B cells preloaded with SEE antigen. (B) Shown on the left are representative immunoblots showing the phosphorylations of CD3ζ (anti-pY142)and CD28 (co-IP’ed p85) for the indicated co-cultures with the cells lysed at the indicated time points after Raji-Jurkat contact (see Methods ). Shown on the right are quantification graphs of CD28 and CD3ζ phosphorylations, incorporating results from three independent experiments. Data were normalized to the highest phosphorylation under PD-1 FF or BTLA FFFF condition, respectively. (C) Bar graphs summarizing the levels of secreted IL-2 of the indicated Raji-Jurkat co-cultures (see Methods ). ( D-F ) Same as A-C except using SHP2 KO Jurkat cells. ( G-I ) Same as A-C except using SHP1/SHP2 double KO Jurkat cells. Data in this figure are presented as means ± SD from three independent measurements. ****P

    Article Snippet: CD28 monoclonal antibody (#16-0289-85), GFP polyclonal antibody (#A6455), PD-1 PE (#12-9969-42) and Dynabeads Protein G (#10004) were purchased from Thermo Fisher Scientific.

    Techniques: Co-culture Assay, Western Blot, Co-Immunoprecipitation Assay

    PD-1 inhibits CD28 phosphorylation while BTLA inhibits both TCR and CD28 phosphorylation. (A) Cartoon illustrating an intact cell assay in which mock transduced, PD-1 (WT)-mGFP or PD-1 (FF)-mGFP transduced Jurkat T cells were stimulated with PD-L1 transduced Raji B cells preloaded with SEE antigen. (B) Flow cytometry histograms showing PD-1 and CD28 surface expressions in the indicated Jurkat T cells. a. u. denotes arbitrary units. (C) Bar graphs summarizing IL-2 levels in the medium of the indicated Jurkat-Raji co-cultures (see Methods). ( D-F ) Same as A-C except PD-1 (WT)-mGFP, PD-1 (FF)-mGFP, and PD-L1 were replaced with BTLA (WT)-mGFP, BTLA (FFFF) -mGFP and HVEM respectively. (G, H) Shown on the left are representative immunoblots showing the phosphorylation of CD3ζ (anti-pY142) and CD28 (co-IP’ed p85) in the indicated co-cultures, with the cells lysed at the indicated times after the initial contact (see Methods). GAPDH IB indicates the input of each sample. Non-specific bands were labeled by asterisks. WCL, whole cell lysate. Shown on the right are quantification graphs of CD28 and CD3ζ phosphorylations. Data were normalized to the highest phosphorylation level under PD-1 FF or BTLA FFFF condition, respectively. Data in this figure are presented as means ± SD from three independent measurements. ****P

    Journal: bioRxiv

    Article Title: BTLA and PD-1 employ distinct phosphatases to differentially repress T cell signaling

    doi: 10.1101/669812

    Figure Lengend Snippet: PD-1 inhibits CD28 phosphorylation while BTLA inhibits both TCR and CD28 phosphorylation. (A) Cartoon illustrating an intact cell assay in which mock transduced, PD-1 (WT)-mGFP or PD-1 (FF)-mGFP transduced Jurkat T cells were stimulated with PD-L1 transduced Raji B cells preloaded with SEE antigen. (B) Flow cytometry histograms showing PD-1 and CD28 surface expressions in the indicated Jurkat T cells. a. u. denotes arbitrary units. (C) Bar graphs summarizing IL-2 levels in the medium of the indicated Jurkat-Raji co-cultures (see Methods). ( D-F ) Same as A-C except PD-1 (WT)-mGFP, PD-1 (FF)-mGFP, and PD-L1 were replaced with BTLA (WT)-mGFP, BTLA (FFFF) -mGFP and HVEM respectively. (G, H) Shown on the left are representative immunoblots showing the phosphorylation of CD3ζ (anti-pY142) and CD28 (co-IP’ed p85) in the indicated co-cultures, with the cells lysed at the indicated times after the initial contact (see Methods). GAPDH IB indicates the input of each sample. Non-specific bands were labeled by asterisks. WCL, whole cell lysate. Shown on the right are quantification graphs of CD28 and CD3ζ phosphorylations. Data were normalized to the highest phosphorylation level under PD-1 FF or BTLA FFFF condition, respectively. Data in this figure are presented as means ± SD from three independent measurements. ****P

    Article Snippet: CD28 monoclonal antibody (#16-0289-85), GFP polyclonal antibody (#A6455), PD-1 PE (#12-9969-42) and Dynabeads Protein G (#10004) were purchased from Thermo Fisher Scientific.

    Techniques: Flow Cytometry, Western Blot, Co-Immunoprecipitation Assay, Labeling

    Proposed model for PD-1 and BTLA signaling. (A) PD-1 recruits SHP2, but not SHP1 to dephosphorylate CD28 (panel A). (B) BTLA recruits SHP1, and to a lesser extent, SHP2. The BTLA:SHP1 complex potently dephosphorylates both TCR and CD28, where the BTLA:SHP2 complex dephosphorylates CD28, with limited impact on TCR phosphorylation.

    Journal: bioRxiv

    Article Title: BTLA and PD-1 employ distinct phosphatases to differentially repress T cell signaling

    doi: 10.1101/669812

    Figure Lengend Snippet: Proposed model for PD-1 and BTLA signaling. (A) PD-1 recruits SHP2, but not SHP1 to dephosphorylate CD28 (panel A). (B) BTLA recruits SHP1, and to a lesser extent, SHP2. The BTLA:SHP1 complex potently dephosphorylates both TCR and CD28, where the BTLA:SHP2 complex dephosphorylates CD28, with limited impact on TCR phosphorylation.

    Article Snippet: CD28 monoclonal antibody (#16-0289-85), GFP polyclonal antibody (#A6455), PD-1 PE (#12-9969-42) and Dynabeads Protein G (#10004) were purchased from Thermo Fisher Scientific.

    Techniques: