anti cd28 antibodies  (Thermo Fisher)


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    Name:
    Dynabeads Human T Activator CD3 CD28 for T Cell Expansion and Activation
    Description:
    Dynabeads Human T Activator CD3 CD28 are for the activation and expansion of human T cells Advantages of Dynabeads Human T Activator CD3 CD28 • Activation of T cells without the need for feeder cells• Activated cells that retain in vivo like function• Maximum reproducibility without contamination by soluble antibodies or mitogens• From 100 to 1 000 fold T cell expansionAbout Dynabeads Human T Activator CD3 CD28Dynabeads Human T Activator CD3 CD28 offer a simple method for activation and expansion of T cells that does not require feeder cells antigen presenting cells or antigen The uniform 4 5 µm diameter inert superparamagnetic beads are similar in size to antigen presenting cells and are covalently coupled to anti CD3 and anti CD28 antibodies These two antibodies provide primary and co stimulatory signals optimized for efficient T cell activation and expansion Expansion of the T cell population can be stimulated using recombinant IL 2 and after activation or expansion the magnetic beads can be easily removed using a DynaMag magnet For expansion of antigen specific T cells from T cell clones or T cell lines we recommend using Dynabeads Human T Activator CD3 CD28 CD137 Starting samplesStart with mononuclear cells MNCs peripheral blood mononuclear cells PBMCs from either whole blood or buffy coat or T cell subsets including CD3 T cells CD4 or CD8 T cells or regulatory T cells Downstream applicationsThe activated T cells can be analyzed shortly after activation for transfection transduction or to study TCR signaling proteomics gene expression etc T cells can be left in culture to differentiate into T helper cell subsets or expansion of polyclonal antigen specific T cells Learn more about Dynabeads products• Find Dynabeads products for a whole range of applications • Find magnets for Dynabeads separations
    Catalog Number:
    11131D
    Price:
    None
    Category:
    Beads Microspheres
    Applications:
    Cell Activation & Expansion|Cell Analysis|Cell Isolation|Human Cell Activation & Expansion|Human Cell Isolation|Cell Isolation & Expansion
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    Structured Review

    Thermo Fisher anti cd28 antibodies
    Effects of TCRA, TCRB , and 3’-UTR variants on nanoluciferase reporter translation. a , Schematic of the nanoluciferase reporters stably expressed in Jurkat cells. WT, intact 3’-UTR from either TCRA or TCRB mRNA; ΔPAR , 3’-UTR of TCRA or TCRB with the eIF3 PAR-CLIP site deleted (nucleotides 102-338 in the 3’-UTR of TCRA mRNA or nucleotides 16-161 in the 3’-UTR of TCRB mRNA); R* PAR , reversed PARCLIP sequence in the 3’-UTR of TCRA or TCRB mRNA. b , Luciferase activity in <t>anti-CD3/anti-CD28</t> activated Jurkat cells, relative to non-activated controls ( n = 3, with mean and standard deviations shown). P
    Dynabeads Human T Activator CD3 CD28 are for the activation and expansion of human T cells Advantages of Dynabeads Human T Activator CD3 CD28 • Activation of T cells without the need for feeder cells• Activated cells that retain in vivo like function• Maximum reproducibility without contamination by soluble antibodies or mitogens• From 100 to 1 000 fold T cell expansionAbout Dynabeads Human T Activator CD3 CD28Dynabeads Human T Activator CD3 CD28 offer a simple method for activation and expansion of T cells that does not require feeder cells antigen presenting cells or antigen The uniform 4 5 µm diameter inert superparamagnetic beads are similar in size to antigen presenting cells and are covalently coupled to anti CD3 and anti CD28 antibodies These two antibodies provide primary and co stimulatory signals optimized for efficient T cell activation and expansion Expansion of the T cell population can be stimulated using recombinant IL 2 and after activation or expansion the magnetic beads can be easily removed using a DynaMag magnet For expansion of antigen specific T cells from T cell clones or T cell lines we recommend using Dynabeads Human T Activator CD3 CD28 CD137 Starting samplesStart with mononuclear cells MNCs peripheral blood mononuclear cells PBMCs from either whole blood or buffy coat or T cell subsets including CD3 T cells CD4 or CD8 T cells or regulatory T cells Downstream applicationsThe activated T cells can be analyzed shortly after activation for transfection transduction or to study TCR signaling proteomics gene expression etc T cells can be left in culture to differentiate into T helper cell subsets or expansion of polyclonal antigen specific T cells Learn more about Dynabeads products• Find Dynabeads products for a whole range of applications • Find magnets for Dynabeads separations
    https://www.bioz.com/result/anti cd28 antibodies/product/Thermo Fisher
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    anti cd28 antibodies - by Bioz Stars, 2021-05
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    Images

    1) Product Images from "Dynamics of T cell activation mediated by eIF3 interactions with T cell receptor mRNAs"

    Article Title: Dynamics of T cell activation mediated by eIF3 interactions with T cell receptor mRNAs

    Journal: bioRxiv

    doi: 10.1101/2019.12.20.885558

    Effects of TCRA, TCRB , and 3’-UTR variants on nanoluciferase reporter translation. a , Schematic of the nanoluciferase reporters stably expressed in Jurkat cells. WT, intact 3’-UTR from either TCRA or TCRB mRNA; ΔPAR , 3’-UTR of TCRA or TCRB with the eIF3 PAR-CLIP site deleted (nucleotides 102-338 in the 3’-UTR of TCRA mRNA or nucleotides 16-161 in the 3’-UTR of TCRB mRNA); R* PAR , reversed PARCLIP sequence in the 3’-UTR of TCRA or TCRB mRNA. b , Luciferase activity in anti-CD3/anti-CD28 activated Jurkat cells, relative to non-activated controls ( n = 3, with mean and standard deviations shown). P
    Figure Legend Snippet: Effects of TCRA, TCRB , and 3’-UTR variants on nanoluciferase reporter translation. a , Schematic of the nanoluciferase reporters stably expressed in Jurkat cells. WT, intact 3’-UTR from either TCRA or TCRB mRNA; ΔPAR , 3’-UTR of TCRA or TCRB with the eIF3 PAR-CLIP site deleted (nucleotides 102-338 in the 3’-UTR of TCRA mRNA or nucleotides 16-161 in the 3’-UTR of TCRB mRNA); R* PAR , reversed PARCLIP sequence in the 3’-UTR of TCRA or TCRB mRNA. b , Luciferase activity in anti-CD3/anti-CD28 activated Jurkat cells, relative to non-activated controls ( n = 3, with mean and standard deviations shown). P

    Techniques Used: Stable Transfection, Cross-linking Immunoprecipitation, Sequencing, Luciferase, Activity Assay

    TCR levels in activated primary human T cells with eIF3 3’-UTR PARCLIP sites deleted. a , Western blots measuring TCRA protein levels as a function of time after anti-CD3/anti-CD28 activation. Cell lines used are labeled on the left: TCRA ΔPAR (TCRA gRNA 1+2), TCRB Δ PAR ( TCRB gRNA 1+2), and SC (scrambled gRNA). HSP90 was used as a loading control. Schematics of TCRA and TCRB mRNAs with and without eIF3 PAR-CLIP sites are shown above. SC control cells have the wild-type (WT) 3’-UTRs for TCRA and TCRB mRNAs. b , The number of T cells with one or more TCR clusters measured by anti-TCRA/anti-TCRB protein staining and epifluorescence microscopy as a function of time after anti-CD3/anti-CD28 activation. A total of 100 cells from each donor were imaged for TCRA ΔPAR (n = 2 donors, stained with anti-TCRA antibody), TCRB ΔPAR (n = 2 donors, stained with anti-TCRB antibody), and SC cell lines ( n = 2 donors, each stained separately with anti-TCRA and anti-TCRB antibodies). Values are mean ± standard deviation. P
    Figure Legend Snippet: TCR levels in activated primary human T cells with eIF3 3’-UTR PARCLIP sites deleted. a , Western blots measuring TCRA protein levels as a function of time after anti-CD3/anti-CD28 activation. Cell lines used are labeled on the left: TCRA ΔPAR (TCRA gRNA 1+2), TCRB Δ PAR ( TCRB gRNA 1+2), and SC (scrambled gRNA). HSP90 was used as a loading control. Schematics of TCRA and TCRB mRNAs with and without eIF3 PAR-CLIP sites are shown above. SC control cells have the wild-type (WT) 3’-UTRs for TCRA and TCRB mRNAs. b , The number of T cells with one or more TCR clusters measured by anti-TCRA/anti-TCRB protein staining and epifluorescence microscopy as a function of time after anti-CD3/anti-CD28 activation. A total of 100 cells from each donor were imaged for TCRA ΔPAR (n = 2 donors, stained with anti-TCRA antibody), TCRB ΔPAR (n = 2 donors, stained with anti-TCRB antibody), and SC cell lines ( n = 2 donors, each stained separately with anti-TCRA and anti-TCRB antibodies). Values are mean ± standard deviation. P

    Techniques Used: Western Blot, Activation Assay, Labeling, Cross-linking Immunoprecipitation, Staining, Epifluorescence Microscopy, Standard Deviation

    Phenotypic analysis of primary human T cells. a, Flow cytometric analysis measuring T cell activation markers CD69 (early activation marker) and CD25 (midactivation marker) in TCRA ΔPAR, TCRB ΔPAR, ΔTCR and SC CD8+ T cells, quantifying mean percent cells expressing CD69+ only (left) and CD69+ CD25+ (right) from ( n = 2 donors). b , Density plots showing cell populations expressing CD69+ only, CD69+ CD25+ or CD25+ only from one of the donors. Cells sorted as shown in Extended Data Fig. 9 . c , Quantification of secreted IL2 and IFNγ from cell lines TCRA ΔPAR, TCRB ΔPAR, ΔTCR and SC cell populations from ( n = 2 donors) at different time points after stimulation with anti-CD3/anti-CD28 antibodies, as determined by ELISA. For both figures a, and c, Values are mean ± standard deviation, ****P
    Figure Legend Snippet: Phenotypic analysis of primary human T cells. a, Flow cytometric analysis measuring T cell activation markers CD69 (early activation marker) and CD25 (midactivation marker) in TCRA ΔPAR, TCRB ΔPAR, ΔTCR and SC CD8+ T cells, quantifying mean percent cells expressing CD69+ only (left) and CD69+ CD25+ (right) from ( n = 2 donors). b , Density plots showing cell populations expressing CD69+ only, CD69+ CD25+ or CD25+ only from one of the donors. Cells sorted as shown in Extended Data Fig. 9 . c , Quantification of secreted IL2 and IFNγ from cell lines TCRA ΔPAR, TCRB ΔPAR, ΔTCR and SC cell populations from ( n = 2 donors) at different time points after stimulation with anti-CD3/anti-CD28 antibodies, as determined by ELISA. For both figures a, and c, Values are mean ± standard deviation, ****P

    Techniques Used: Activation Assay, Marker, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Regulatory pathways leading to dynamics of TCR protein expression levels. a, Western blots of TCRA protein levels in primary human T cells using different activation modes. b, Western blot of TCRA protein levels in Jurkat cells activated with anti-CD3/anti-CD28 antibodies. An anti-Phospho-GSK-3β antibody was used to measure AKT activity. c, Western blot of TCRA protein levels in Jurkat cells activated with anti-CD3/anti-CD28 antibodies in the presence of AKT inhibitor AZD5363. In all panels HSP90 was used as a loading control. Experiments with AKT inhibitors were carried out in duplicate.
    Figure Legend Snippet: Regulatory pathways leading to dynamics of TCR protein expression levels. a, Western blots of TCRA protein levels in primary human T cells using different activation modes. b, Western blot of TCRA protein levels in Jurkat cells activated with anti-CD3/anti-CD28 antibodies. An anti-Phospho-GSK-3β antibody was used to measure AKT activity. c, Western blot of TCRA protein levels in Jurkat cells activated with anti-CD3/anti-CD28 antibodies in the presence of AKT inhibitor AZD5363. In all panels HSP90 was used as a loading control. Experiments with AKT inhibitors were carried out in duplicate.

    Techniques Used: Expressing, Western Blot, Activation Assay, Activity Assay

    2) Product Images from "Identification of Naturally Occurring Fatty Acids of the Myelin Sheath That Resolve Neuroinflammation"

    Article Title: Identification of Naturally Occurring Fatty Acids of the Myelin Sheath That Resolve Neuroinflammation

    Journal: Science translational medicine

    doi: 10.1126/scitranslmed.3003831

    POPS, PGPC, azPC, and azPC ester induce apoptotic signaling pathways and T-cell apoptosis. ( A ) Annexin V and 7AAD staining of CD3 + T cells purified from wild-type B6 mice and stimulated with plate-bound anti-CD3 and anti-CD28 antibodies for 48 h with
    Figure Legend Snippet: POPS, PGPC, azPC, and azPC ester induce apoptotic signaling pathways and T-cell apoptosis. ( A ) Annexin V and 7AAD staining of CD3 + T cells purified from wild-type B6 mice and stimulated with plate-bound anti-CD3 and anti-CD28 antibodies for 48 h with

    Techniques Used: Staining, Purification, Mouse Assay

    3) Product Images from "Ubc13 maintains the suppressive function of regulatory T cells and prevents their conversion into effector-like T cells"

    Article Title: Ubc13 maintains the suppressive function of regulatory T cells and prevents their conversion into effector-like T cells

    Journal: Nature Immunology

    doi: 10.1038/ni.2267

    The T reg -specific function of Ubc13 involves its downstream target IKK ( a ) Flow cytometric analysis of RelA phosphorylation in sorted T reg cells from WT or Ube2n Treg-KO mice, stimulated with anti-CD3 and anti-CD28 for 15 min using a crosslinking method 45 . ( b ) Flow cytometric analysis of CD44 hi CD62L lo memory-like T cells (gated on CD3 + CD4 + cells) within different lymphoid organs of the indicated mice (6–8 weeks old). Data are presented as mean ± SD value and representative of three independent experiments. *p=0.05 and **p=0.01 (two-tailed unpaired t-test). ( c ) Flow cytometric analysis of CD4 + T cells derived from the indicated lymphoid organs of WT or IKK2 Treg-KO mice (7 weeks old), measuring the percentage (numbers in quadrangles) of naïve (CD44 lo CD62L hi ) and memory-like (CD44 hi CD62L lo ) T cells. Data are representative of three experiments. ( d, e ) Flow cytometric analysis of the frequency ( d ) and absolute number ( e ) of CD44 hi CD62 lo memory-like CD4 + T cells in the indicated lymphoid organs of WT and IKK2 Treg-KO littermates (7 weeks old). Data are representative of two independent experiments.
    Figure Legend Snippet: The T reg -specific function of Ubc13 involves its downstream target IKK ( a ) Flow cytometric analysis of RelA phosphorylation in sorted T reg cells from WT or Ube2n Treg-KO mice, stimulated with anti-CD3 and anti-CD28 for 15 min using a crosslinking method 45 . ( b ) Flow cytometric analysis of CD44 hi CD62L lo memory-like T cells (gated on CD3 + CD4 + cells) within different lymphoid organs of the indicated mice (6–8 weeks old). Data are presented as mean ± SD value and representative of three independent experiments. *p=0.05 and **p=0.01 (two-tailed unpaired t-test). ( c ) Flow cytometric analysis of CD4 + T cells derived from the indicated lymphoid organs of WT or IKK2 Treg-KO mice (7 weeks old), measuring the percentage (numbers in quadrangles) of naïve (CD44 lo CD62L hi ) and memory-like (CD44 hi CD62L lo ) T cells. Data are representative of three experiments. ( d, e ) Flow cytometric analysis of the frequency ( d ) and absolute number ( e ) of CD44 hi CD62 lo memory-like CD4 + T cells in the indicated lymphoid organs of WT and IKK2 Treg-KO littermates (7 weeks old). Data are representative of two independent experiments.

    Techniques Used: Flow Cytometry, Mouse Assay, Two Tailed Test, Derivative Assay

    4) Product Images from "Microbial Anti-Inflammatory Molecule (MAM) from Faecalibacterium prausnitzii Shows a Protective Effect on DNBS and DSS-Induced Colitis Model in Mice through Inhibition of NF-κB Pathway"

    Article Title: Microbial Anti-Inflammatory Molecule (MAM) from Faecalibacterium prausnitzii Shows a Protective Effect on DNBS and DSS-Induced Colitis Model in Mice through Inhibition of NF-κB Pathway

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.00114

    Cytokines secreted by reactivated lymphocytes from MLN in DSS-induced colitis model . Mice were orally administered with LL-pILMAM, LL-pILEMPTY or PBS 7 days before and during colitis induction. Colitis was induced by adding DSS in drinking water during 7 days (D0-D7). Then DSS was removed from drinking water and mice allowed to recover during 5 days (D7-D12). At D12 mice were killed, MLN withdrawn, cells were isolated and lymphocytes reactivated with antiCD3/anti-CD28 antibodies. Cytokine concentration in medium was monitored 48 h after reactivation by ELISA. * P
    Figure Legend Snippet: Cytokines secreted by reactivated lymphocytes from MLN in DSS-induced colitis model . Mice were orally administered with LL-pILMAM, LL-pILEMPTY or PBS 7 days before and during colitis induction. Colitis was induced by adding DSS in drinking water during 7 days (D0-D7). Then DSS was removed from drinking water and mice allowed to recover during 5 days (D7-D12). At D12 mice were killed, MLN withdrawn, cells were isolated and lymphocytes reactivated with antiCD3/anti-CD28 antibodies. Cytokine concentration in medium was monitored 48 h after reactivation by ELISA. * P

    Techniques Used: Mouse Assay, Isolation, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Cytokines secreted by reactivated lymphocytes from MLN in a DNBS-induced colitis model . NF-κB luciferase mice were orally administered with PBS, LL-pILEMPTY or LL-pILMAM 7 days before DNBS intrarectal injection (D0) and until sacrifice (D4). At D4 MLN were withdrawn and isolated lymphocytes reactivated by anti-CD3/anti-CD28 antibodies. Cytokine concentration in medium was monitored 48 h after reactivation by ELISA. * P
    Figure Legend Snippet: Cytokines secreted by reactivated lymphocytes from MLN in a DNBS-induced colitis model . NF-κB luciferase mice were orally administered with PBS, LL-pILEMPTY or LL-pILMAM 7 days before DNBS intrarectal injection (D0) and until sacrifice (D4). At D4 MLN were withdrawn and isolated lymphocytes reactivated by anti-CD3/anti-CD28 antibodies. Cytokine concentration in medium was monitored 48 h after reactivation by ELISA. * P

    Techniques Used: Luciferase, Mouse Assay, Injection, Isolation, Concentration Assay, Enzyme-linked Immunosorbent Assay

    5) Product Images from "Kinase Control of Latent HIV-1 Infection: PIM-1 Kinase as a Major Contributor to HIV-1 Reactivation"

    Article Title: Kinase Control of Latent HIV-1 Infection: PIM-1 Kinase as a Major Contributor to HIV-1 Reactivation

    Journal: Journal of Virology

    doi: 10.1128/JVI.02682-13

    ). Active infection events were indicated by GFP fluorescence (Donor 1) or by p24 staining (Donor 2). Over low-level background infection (control), HIV-1 reactivation was triggered using a CD3/CD28 MAb combination. Cyclosporine (Cs) prevented and PIMi IV markedly inhibited CD3/CD28 MAb induced reactivation. The percentage of GFP-positive cells is indicated. (B) To test whether PIMi IV inhibits anti-CD3/CD28 MAb-mediated T cell activation, primary T cells were left untreated (control) or CD3/CD28 MAb stimulated in the absence or presence of 10 μM PIMi IV. T cell activation was determined as the induction of CD25/IL-2 receptor-α chain expression by flow cytometric analysis. The experiment is representative of results from a total of four healthy donors tested. α, anti; SSC, side scatter.
    Figure Legend Snippet: ). Active infection events were indicated by GFP fluorescence (Donor 1) or by p24 staining (Donor 2). Over low-level background infection (control), HIV-1 reactivation was triggered using a CD3/CD28 MAb combination. Cyclosporine (Cs) prevented and PIMi IV markedly inhibited CD3/CD28 MAb induced reactivation. The percentage of GFP-positive cells is indicated. (B) To test whether PIMi IV inhibits anti-CD3/CD28 MAb-mediated T cell activation, primary T cells were left untreated (control) or CD3/CD28 MAb stimulated in the absence or presence of 10 μM PIMi IV. T cell activation was determined as the induction of CD25/IL-2 receptor-α chain expression by flow cytometric analysis. The experiment is representative of results from a total of four healthy donors tested. α, anti; SSC, side scatter.

    Techniques Used: Infection, Fluorescence, Staining, Activation Assay, Expressing, Flow Cytometry

    6) Product Images from "Natural Killer Cells Regulate Th17 Cells After Autologous Hematopoietic Stem Cell Transplantation for Relapsing Remitting Multiple Sclerosis"

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

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00834

    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.
    Figure Legend 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.

    Techniques Used: 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) .
    Figure Legend 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) .

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

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

    7) Product Images from "Existence of Circulating Mitochondria in Human and Animal Peripheral Blood"

    Article Title: Existence of Circulating Mitochondria in Human and Animal Peripheral Blood

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21062122

    Immune modulation of peripheral blood (PB)-derived mitochondria on PBMC. Health donor-derived PBMC ( N = 4) were treated with PB-derived mitochondria ( N = 4) in duplicate for 3 days in the presence of T-cell activator CD3/CD28 Dynabeads. ( A ) Phase contrast image show the cluster formation with different sizes. Untreated PBMC served as control (left panel). ( B ) Increase the PBMC proliferation by PB-derived mitochondria. ( C ) Improve the percentage of the activated CD4 + HLA-DR + T cells by PB-derived mitochondria. ( D ) Improve the percentage of the activated CD8 + HLA-DR + T cells by PB-derived mitochondria. ( E ) Suppression of inflammatory cytokine IFN-γ production in the presence of PB-derived mitochondria at 200 µg/mL. ( F ) Decrease the level of cytokine IL-12 in CD3/CD28 Dynabead-activated PBMC after the treatment with PB-derived mitochondria at 200 µg/mL. Data were given as mean ± SD (standard deviation, N = 4).
    Figure Legend Snippet: Immune modulation of peripheral blood (PB)-derived mitochondria on PBMC. Health donor-derived PBMC ( N = 4) were treated with PB-derived mitochondria ( N = 4) in duplicate for 3 days in the presence of T-cell activator CD3/CD28 Dynabeads. ( A ) Phase contrast image show the cluster formation with different sizes. Untreated PBMC served as control (left panel). ( B ) Increase the PBMC proliferation by PB-derived mitochondria. ( C ) Improve the percentage of the activated CD4 + HLA-DR + T cells by PB-derived mitochondria. ( D ) Improve the percentage of the activated CD8 + HLA-DR + T cells by PB-derived mitochondria. ( E ) Suppression of inflammatory cytokine IFN-γ production in the presence of PB-derived mitochondria at 200 µg/mL. ( F ) Decrease the level of cytokine IL-12 in CD3/CD28 Dynabead-activated PBMC after the treatment with PB-derived mitochondria at 200 µg/mL. Data were given as mean ± SD (standard deviation, N = 4).

    Techniques Used: Derivative Assay, Standard Deviation

    Intracellular cytokine analysis by flow cytometry. Health donor-derived PBMC ( N = 4) were treated with PB-derived mitochondria at 200 µg/mL ( N = 4) in duplicate for 3 days in the presence of T-cell activator CD3/CD28 Dynabeads. ( A – H ) There were no marked differences in the percentages of IL-1β ( A ), IL-4 ( B ), IL-5 ( C ), IL-10 ( D ), IL-13 ( E ), TGF-β1 ( G ), and TNF-α ( H )-producing cells after the treatment with PB-derived mitochondria. ( F ) Downregulation of the percentage of IL-17A-positive cells after the treatment with PB-derived mitochondria. Data were given as mean ± SD (standard deviation, N = 4).
    Figure Legend Snippet: Intracellular cytokine analysis by flow cytometry. Health donor-derived PBMC ( N = 4) were treated with PB-derived mitochondria at 200 µg/mL ( N = 4) in duplicate for 3 days in the presence of T-cell activator CD3/CD28 Dynabeads. ( A – H ) There were no marked differences in the percentages of IL-1β ( A ), IL-4 ( B ), IL-5 ( C ), IL-10 ( D ), IL-13 ( E ), TGF-β1 ( G ), and TNF-α ( H )-producing cells after the treatment with PB-derived mitochondria. ( F ) Downregulation of the percentage of IL-17A-positive cells after the treatment with PB-derived mitochondria. Data were given as mean ± SD (standard deviation, N = 4).

    Techniques Used: Flow Cytometry, Derivative Assay, Standard Deviation

    8) Product Images from "Regulation of CUG-binding Protein 1 (CUGBP1) Binding to Target Transcripts upon T Cell Activation"

    Article Title: Regulation of CUG-binding Protein 1 (CUGBP1) Binding to Target Transcripts upon T Cell Activation

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.291658

    CUGBP1 was phosphorylated upon T cell activation. Cytoplasmic lysates were prepared from resting T cells and T cells that were stimulated for 6 h with anti-CD3 and anti-CD28 antibodies. The lysates were immunoprecipitated using an anti-CUGBP1 antibody,
    Figure Legend Snippet: CUGBP1 was phosphorylated upon T cell activation. Cytoplasmic lysates were prepared from resting T cells and T cells that were stimulated for 6 h with anti-CD3 and anti-CD28 antibodies. The lysates were immunoprecipitated using an anti-CUGBP1 antibody,

    Techniques Used: Activation Assay, Immunoprecipitation

    9) Product Images from "Mammalian nitrilase 1 homologue Nit1 is a negative regulator in T cells"

    Article Title: Mammalian nitrilase 1 homologue Nit1 is a negative regulator in T cells

    Journal: International Immunology

    doi: 10.1093/intimm/dxp038

    Analysis of lymphocyte proliferation responses. (A) Total splenocytes and lymph node cells were stimulated with anti-CD3 plus anti-CD28 antibodies and proliferation determined by [ 3 H] thymidine incorporation assays. (B) Purified T cells were stimulated with anti-CD3 plus anti-CD28 antibodies and proliferation determined by [ 3 H] thymidine incorporation assays (left). Growth curves were generated by counting cell numbers at times indicated (right).
    Figure Legend Snippet: Analysis of lymphocyte proliferation responses. (A) Total splenocytes and lymph node cells were stimulated with anti-CD3 plus anti-CD28 antibodies and proliferation determined by [ 3 H] thymidine incorporation assays. (B) Purified T cells were stimulated with anti-CD3 plus anti-CD28 antibodies and proliferation determined by [ 3 H] thymidine incorporation assays (left). Growth curves were generated by counting cell numbers at times indicated (right).

    Techniques Used: Purification, Generated

    mRNA and protein turnover of Cyc E and CDC2. (A) RNA was extracted from resting (0 h) and activated (24 h) T cells, and semi-quantitative RT–PCR performed to determine the relative levels of mRNA of Cyc E and CDC2. Density was determined using ImageJ (National Institutes of Health) and normalized against β-actin. Numbers indicated fold over the level of β-actin in activated (24 h) Nit1 −/− T cells, which is set as 1. (B) T cells were stimulated with anti-CD3 plus anti-CD28 antibodies, and at 24 h after stimulation, cycloheximide was added, cells were harvested at the indicated times and the levels of the Cyc E and CDC2 proteins analyzed by western blotting.
    Figure Legend Snippet: mRNA and protein turnover of Cyc E and CDC2. (A) RNA was extracted from resting (0 h) and activated (24 h) T cells, and semi-quantitative RT–PCR performed to determine the relative levels of mRNA of Cyc E and CDC2. Density was determined using ImageJ (National Institutes of Health) and normalized against β-actin. Numbers indicated fold over the level of β-actin in activated (24 h) Nit1 −/− T cells, which is set as 1. (B) T cells were stimulated with anti-CD3 plus anti-CD28 antibodies, and at 24 h after stimulation, cycloheximide was added, cells were harvested at the indicated times and the levels of the Cyc E and CDC2 proteins analyzed by western blotting.

    Techniques Used: Quantitative RT-PCR, Western Blot

    Analysis of T cell activation markers. Freshly isolated, resting T cells (0 h) and purified T cells stimulated with anti-CD3 plus anti-CD28 antibodies (18 h) were stained for CD25, CD28, CD44 and CD69 and analyzed by flow cytometry. The expression levels are depicted as flow cytometric histograms. The numbers indicate percentages of T cells that express each activation marker. The IL-2 levels were determined by intracellular staining of unstimulated (0 h) or T cells that have been stimulated with anti-CD3 plus anti-CD28 antibodies for (6 h), followed by flow cytometry. The numbers indicate mean fluorescent intensities.
    Figure Legend Snippet: Analysis of T cell activation markers. Freshly isolated, resting T cells (0 h) and purified T cells stimulated with anti-CD3 plus anti-CD28 antibodies (18 h) were stained for CD25, CD28, CD44 and CD69 and analyzed by flow cytometry. The expression levels are depicted as flow cytometric histograms. The numbers indicate percentages of T cells that express each activation marker. The IL-2 levels were determined by intracellular staining of unstimulated (0 h) or T cells that have been stimulated with anti-CD3 plus anti-CD28 antibodies for (6 h), followed by flow cytometry. The numbers indicate mean fluorescent intensities.

    Techniques Used: Activation Assay, Isolation, Purification, Staining, Flow Cytometry, Cytometry, Expressing, Marker

    Analysis of T cell division kinetics and cell cycle progression profiles. (A) Purified T cells were labeled with CFSE, stimulated with anti-CD3 plus anti-CD28 antibodies, analyzed by flow cytometry at the indicated times and cell division visualized using flow cytometric histograms. The numbers present on each histogram indicate the percentage of cells that had undergone specific cell divisions. (B) Purified T cells were stimulated with anti-CD3 plus anti-CD28 antibodies and at the indicated times cells stained with PI and analyzed by flow cytometry. Relative DNA content (PI) at each time point is presented in histograms, and the percentages of T cells in each cell cycle stage are indicated.
    Figure Legend Snippet: Analysis of T cell division kinetics and cell cycle progression profiles. (A) Purified T cells were labeled with CFSE, stimulated with anti-CD3 plus anti-CD28 antibodies, analyzed by flow cytometry at the indicated times and cell division visualized using flow cytometric histograms. The numbers present on each histogram indicate the percentage of cells that had undergone specific cell divisions. (B) Purified T cells were stimulated with anti-CD3 plus anti-CD28 antibodies and at the indicated times cells stained with PI and analyzed by flow cytometry. Relative DNA content (PI) at each time point is presented in histograms, and the percentages of T cells in each cell cycle stage are indicated.

    Techniques Used: Purification, Labeling, Flow Cytometry, Cytometry, Staining

    Western blot analyses of cell cycle proteins in Nit1 −/− T cells. (A) Expression of Cyc D1 in total splenocytes and freshly isolated T cells (0 h) and purified T cells stimulated with anti-CD3 plus anti-CD28 antibodies for 28 h. Wild-type mouse embryonic fibroblasts (MEFs) were used as positive controls. (B) Purified T cells were stimulated with anti-CD3 a plus anti-CD28 antibodies, and at the indicated times cells were harvested, proteins extracted and western blotting performed with the indicated antibodies.
    Figure Legend Snippet: Western blot analyses of cell cycle proteins in Nit1 −/− T cells. (A) Expression of Cyc D1 in total splenocytes and freshly isolated T cells (0 h) and purified T cells stimulated with anti-CD3 plus anti-CD28 antibodies for 28 h. Wild-type mouse embryonic fibroblasts (MEFs) were used as positive controls. (B) Purified T cells were stimulated with anti-CD3 a plus anti-CD28 antibodies, and at the indicated times cells were harvested, proteins extracted and western blotting performed with the indicated antibodies.

    Techniques Used: Western Blot, Expressing, Isolation, Purification

    10) Product Images from "Pharmacological Activation of Pyruvate Kinase M2 Inhibits CD4+ T Cell Pathogenicity and Suppresses Autoimmunity"

    Article Title: Pharmacological Activation of Pyruvate Kinase M2 Inhibits CD4+ T Cell Pathogenicity and Suppresses Autoimmunity

    Journal: Cell Metabolism

    doi: 10.1016/j.cmet.2019.10.015

    TEPP-46 Limits Th17 Cell Polarization Murine CD4 + CD62 + T cells were activated in vitro for 3 days with CD3/CD28 antibodies under Th17-polarizing conditions in the presence of DMSO (CTRL condition), TEPP-46 50 μM, or 100 μM. (A) Flow cytometry evaluation of IL-17A and TNF-α production by Th17 cells after intracellular cytokine staining. Left, representative plots showing reduced IL-17A and TNF-α production by TEPP-46 treated cells. Right, quantification of the percentage of IL-17A/TNF-α-producing cells and of IL-17A/TNF-α MFI in CTRL versus TEPP-46-treated cells (n = 6–9 from 3 independent experiments). (B and C) Quantification of the mRNA levels of Th17 signature cytokines (B) and transcription factors (C) by qRT-PCR (n = 6–8 from 3–4 independent experiments). For all panels, data are the mean ± SD. ∗ p
    Figure Legend Snippet: TEPP-46 Limits Th17 Cell Polarization Murine CD4 + CD62 + T cells were activated in vitro for 3 days with CD3/CD28 antibodies under Th17-polarizing conditions in the presence of DMSO (CTRL condition), TEPP-46 50 μM, or 100 μM. (A) Flow cytometry evaluation of IL-17A and TNF-α production by Th17 cells after intracellular cytokine staining. Left, representative plots showing reduced IL-17A and TNF-α production by TEPP-46 treated cells. Right, quantification of the percentage of IL-17A/TNF-α-producing cells and of IL-17A/TNF-α MFI in CTRL versus TEPP-46-treated cells (n = 6–9 from 3 independent experiments). (B and C) Quantification of the mRNA levels of Th17 signature cytokines (B) and transcription factors (C) by qRT-PCR (n = 6–8 from 3–4 independent experiments). For all panels, data are the mean ± SD. ∗ p

    Techniques Used: In Vitro, Flow Cytometry, Staining, Quantitative RT-PCR

    TEPP-46 Constrains Th1 Cell Development Murine CD4 + CD62 + T cells were activated in vitro for 3 days with CD3/CD28 antibodies under Th1-polarizing conditions in the presence of DMSO (CTRL condition) or TEPP-46 50 μM or 100 μM. (A) Flow cytometry evaluation of IFN-γ and TNF-α production by Th1 cells after intracellular cytokine staining. Left, representative plots showing reduced IFN-γ and TNF-α production by TEPP-46 treated cells. Right, quantification of the percentage of IFN-γ/TNF-α-producing cells and of IFN-γ/TNF-α MFI in CTRL versus TEPP-46-treated cells (n = 5–6 from 4 independent experiments). (B and C) Quantification of Tnfa / Ifng (B) and Tbx21/Eomes (C) mRNA levels by qPCR (n = 5–6 from 3 independent experiments). For all panels, data are the mean ± SD. ∗∗∗ p
    Figure Legend Snippet: TEPP-46 Constrains Th1 Cell Development Murine CD4 + CD62 + T cells were activated in vitro for 3 days with CD3/CD28 antibodies under Th1-polarizing conditions in the presence of DMSO (CTRL condition) or TEPP-46 50 μM or 100 μM. (A) Flow cytometry evaluation of IFN-γ and TNF-α production by Th1 cells after intracellular cytokine staining. Left, representative plots showing reduced IFN-γ and TNF-α production by TEPP-46 treated cells. Right, quantification of the percentage of IFN-γ/TNF-α-producing cells and of IFN-γ/TNF-α MFI in CTRL versus TEPP-46-treated cells (n = 5–6 from 4 independent experiments). (B and C) Quantification of Tnfa / Ifng (B) and Tbx21/Eomes (C) mRNA levels by qPCR (n = 5–6 from 3 independent experiments). For all panels, data are the mean ± SD. ∗∗∗ p

    Techniques Used: In Vitro, Flow Cytometry, Staining, Real-time Polymerase Chain Reaction

    PKM2 Tetramerization Blocks T Cell Activation In Vitro Murine CD4 + CD62 + T cells were stimulated in vitro with CD3/CD28 antibodies in the presence of DMSO (CTRL), TEPP-46 50 μM, or 100 μM. (A and B) Cells were collected after 24 h of stimulation. (A) Quantification of Il2 mRNA in activated T cells by qRT-PCR (n = 9 from three independent experiments). (B) Cells were re-stimulated in vitro with PMA and ionomycin in the presence of brefeldin A. IL-2 production was then evaluated by flow cytometry after intracellular cytokine staining. Left, representative plot showing reduced IL-2 production by TEPP-46-treated cells. Right, quantification of the percentage of IL-2-producing cells and IL-2 mean fluorescence intensity (MFI) in CTRL versus TEPP-46-treated cells (n = 8 from 3 independent experiments). (C–F) Cells were collected after 3 days of stimulation. (C) Top, representative flow cytometry plot displaying T cell proliferation assessed as CellTrace violet dilution. Bottom, a division index was calculated with FlowJo software to quantify T cell proliferation (n = 5 from four independent experiments). (D) Expression of surface CD62L, CD44, and CD25 was evaluated by flow cytometry. The percentage of expressing cells and the MFI are shown (n = 3 from 2 independent experiments). (E) Quantification of Tnfa mRNA levels in activated T cells by qRT-PCR (n = 6 from 6 independent experiments). (F) Cells were re-stimulated in vitro with PMA and ionomycin in the presence of brefeldin A. TNF-α production was then evaluated by flow cytometry after intracellular cytokine staining. Left, representative plot showing reduced TNF-α production by TEPP-46 treated cells. Right, quantification of the percentage of TNF-α-producing cells and TNF-α MFI in CTRL versus TEPP-46-treated cells (n = 5 from 2 independent experiments). For all panels, data are the mean ± SD. ∗ p
    Figure Legend Snippet: PKM2 Tetramerization Blocks T Cell Activation In Vitro Murine CD4 + CD62 + T cells were stimulated in vitro with CD3/CD28 antibodies in the presence of DMSO (CTRL), TEPP-46 50 μM, or 100 μM. (A and B) Cells were collected after 24 h of stimulation. (A) Quantification of Il2 mRNA in activated T cells by qRT-PCR (n = 9 from three independent experiments). (B) Cells were re-stimulated in vitro with PMA and ionomycin in the presence of brefeldin A. IL-2 production was then evaluated by flow cytometry after intracellular cytokine staining. Left, representative plot showing reduced IL-2 production by TEPP-46-treated cells. Right, quantification of the percentage of IL-2-producing cells and IL-2 mean fluorescence intensity (MFI) in CTRL versus TEPP-46-treated cells (n = 8 from 3 independent experiments). (C–F) Cells were collected after 3 days of stimulation. (C) Top, representative flow cytometry plot displaying T cell proliferation assessed as CellTrace violet dilution. Bottom, a division index was calculated with FlowJo software to quantify T cell proliferation (n = 5 from four independent experiments). (D) Expression of surface CD62L, CD44, and CD25 was evaluated by flow cytometry. The percentage of expressing cells and the MFI are shown (n = 3 from 2 independent experiments). (E) Quantification of Tnfa mRNA levels in activated T cells by qRT-PCR (n = 6 from 6 independent experiments). (F) Cells were re-stimulated in vitro with PMA and ionomycin in the presence of brefeldin A. TNF-α production was then evaluated by flow cytometry after intracellular cytokine staining. Left, representative plot showing reduced TNF-α production by TEPP-46 treated cells. Right, quantification of the percentage of TNF-α-producing cells and TNF-α MFI in CTRL versus TEPP-46-treated cells (n = 5 from 2 independent experiments). For all panels, data are the mean ± SD. ∗ p

    Techniques Used: Activation Assay, In Vitro, Quantitative RT-PCR, Flow Cytometry, Staining, Fluorescence, Software, Expressing

    TCR Stimulation Induces PKM2 Upregulation and Nuclear Translocation in Human T Cells, and TEPP-46 Limits Their Activation Human naive CD4 + T cells were stimulated in vitro for 4 days with CD3/CD28 antibodies and collected at different time points of activation. (A) Quantification of PKM2 mRNA in resting versus activated human CD4 + T cells by qRT-PCR (n = 5–6 from three independent experiments). (B) Left, western blot showing upregulation of PKM2 protein in human CD4 + cells following activation. Right, quantification of PKM2 expression by densitometry analysis (n = 3 from 2 independent experiments). (C) Western blots showing time-dependent increase in PKM2 phosphorylation on Tyr105 in activated human T cells. One representative experiment out of two is shown. (D) Cells were collected at different time points of activation, crosslinked with DSS, and analyzed for PKM2 expression by western blot. A representative blot showing the upregulation of monomeric/dimeric and tetrameric PKM2 in activated human CD4 + T cells is presented. (E) Western blots showing time-dependent increase in PKM2 phosphorylation on Ser37 in activated human CD4 + T cells. (F) Cells were collected at different time points of activation. Nuclear and cytoplasmic fractions were isolated by cell fractionation and analyzed for PKM2 expression by western blot. A representative blot showing accumulation of PKM2 in the nucleus of activated human CD4 + T cells is presented. For (D–F), one representative experiment out of three is shown. (G–K) Human naïve CD4 + T cells were stimulated in vitro for 48 h (G and H) or 4 days (I–K) with CD3/CD28 antibodies, in the presence of DMSO (CTRL condition) or TEPP-46 100 μM. (G) Quantification of IL2 mRNA in activated human cells by qRT-PCR (n = 9 from five independent experiments). (H) IL-2 production evaluated by flow cytometry after intracellular cytokine staining. Left, representative plot showing reduced IL-2 production by TEPP-46 treated cells. Right, quantification of the percentage of IL-2-producing cells and IL-2 MFI in CTRL versus TEPP-46-treated cells (n = 5 from 3 independent experiments). (I) Human CD4 + T cells proliferation evaluated as in Figure 2 C (n = 5 from 2 independent experiments). (J) Percentage of CD71 + cells (left) and CD71 MFI (right) in CTRL and TEPP-46-treated activated human T cells (n = 7 from three independent experiments). (K) Percentage of FOXP3 + CD25 high CD127 neg cells in CTRL and TEPP-46-treated activated CD4 + human T cells (n = 8 from 3 independent experiments). For all panels, data are the mean ± SD. ∗ p
    Figure Legend Snippet: TCR Stimulation Induces PKM2 Upregulation and Nuclear Translocation in Human T Cells, and TEPP-46 Limits Their Activation Human naive CD4 + T cells were stimulated in vitro for 4 days with CD3/CD28 antibodies and collected at different time points of activation. (A) Quantification of PKM2 mRNA in resting versus activated human CD4 + T cells by qRT-PCR (n = 5–6 from three independent experiments). (B) Left, western blot showing upregulation of PKM2 protein in human CD4 + cells following activation. Right, quantification of PKM2 expression by densitometry analysis (n = 3 from 2 independent experiments). (C) Western blots showing time-dependent increase in PKM2 phosphorylation on Tyr105 in activated human T cells. One representative experiment out of two is shown. (D) Cells were collected at different time points of activation, crosslinked with DSS, and analyzed for PKM2 expression by western blot. A representative blot showing the upregulation of monomeric/dimeric and tetrameric PKM2 in activated human CD4 + T cells is presented. (E) Western blots showing time-dependent increase in PKM2 phosphorylation on Ser37 in activated human CD4 + T cells. (F) Cells were collected at different time points of activation. Nuclear and cytoplasmic fractions were isolated by cell fractionation and analyzed for PKM2 expression by western blot. A representative blot showing accumulation of PKM2 in the nucleus of activated human CD4 + T cells is presented. For (D–F), one representative experiment out of three is shown. (G–K) Human naïve CD4 + T cells were stimulated in vitro for 48 h (G and H) or 4 days (I–K) with CD3/CD28 antibodies, in the presence of DMSO (CTRL condition) or TEPP-46 100 μM. (G) Quantification of IL2 mRNA in activated human cells by qRT-PCR (n = 9 from five independent experiments). (H) IL-2 production evaluated by flow cytometry after intracellular cytokine staining. Left, representative plot showing reduced IL-2 production by TEPP-46 treated cells. Right, quantification of the percentage of IL-2-producing cells and IL-2 MFI in CTRL versus TEPP-46-treated cells (n = 5 from 3 independent experiments). (I) Human CD4 + T cells proliferation evaluated as in Figure 2 C (n = 5 from 2 independent experiments). (J) Percentage of CD71 + cells (left) and CD71 MFI (right) in CTRL and TEPP-46-treated activated human T cells (n = 7 from three independent experiments). (K) Percentage of FOXP3 + CD25 high CD127 neg cells in CTRL and TEPP-46-treated activated CD4 + human T cells (n = 8 from 3 independent experiments). For all panels, data are the mean ± SD. ∗ p

    Techniques Used: Translocation Assay, Activation Assay, In Vitro, Quantitative RT-PCR, Western Blot, Expressing, Isolation, Cell Fractionation, Flow Cytometry, Staining

    PKM2 Tetramerization Limits HIF1-α, Myc, and mTORC1 Signaling and Engagement of Glycolysis in CD4 + T Cells Murine CD4 + T cells were collected after 72 h of in vitro activation with CD3/CD28 antibodies in the presence of DMSO (CTRL condition) or TEPP-46 100 μM. (A) Results of unbiased Ingenuity Pathway Analysis predicting downregulation of Myc-, Hif-1-α-, and mTOR-regulated pathways by TEPP-46. (B) Heatmaps showing expression of Myc-, Hif-1-α-, and mTOR-regulated genes in resting T cells and T cells activated in the presence of DMSO (Th0 Ctrl) or TEPP-46 (Th0 TEPP). (C) Heatmap showing expression of glycolytic genes in resting, Th0 Ctrl, and Th0 TEPP-46 cells. (D and E) Cells were tested for their glycolytic capacity and oxygen consumption rate (OCR) on a Seahorse XFe96 Analyzer. (D) Quantitative analysis of glycolytic rate and glycolytic capacity of CTRL and TEPP-46-treated cells (n = 4 from 2 independent experiments). (E) Quantitative analysis of basal OCR, maximum respiration and spare respiratory capacity of CTRL and TEPP-46-treated cells (n = 7 from three independent experiments). For (D) and (E), data are the mean ± SD. ∗∗ p
    Figure Legend Snippet: PKM2 Tetramerization Limits HIF1-α, Myc, and mTORC1 Signaling and Engagement of Glycolysis in CD4 + T Cells Murine CD4 + T cells were collected after 72 h of in vitro activation with CD3/CD28 antibodies in the presence of DMSO (CTRL condition) or TEPP-46 100 μM. (A) Results of unbiased Ingenuity Pathway Analysis predicting downregulation of Myc-, Hif-1-α-, and mTOR-regulated pathways by TEPP-46. (B) Heatmaps showing expression of Myc-, Hif-1-α-, and mTOR-regulated genes in resting T cells and T cells activated in the presence of DMSO (Th0 Ctrl) or TEPP-46 (Th0 TEPP). (C) Heatmap showing expression of glycolytic genes in resting, Th0 Ctrl, and Th0 TEPP-46 cells. (D and E) Cells were tested for their glycolytic capacity and oxygen consumption rate (OCR) on a Seahorse XFe96 Analyzer. (D) Quantitative analysis of glycolytic rate and glycolytic capacity of CTRL and TEPP-46-treated cells (n = 4 from 2 independent experiments). (E) Quantitative analysis of basal OCR, maximum respiration and spare respiratory capacity of CTRL and TEPP-46-treated cells (n = 7 from three independent experiments). For (D) and (E), data are the mean ± SD. ∗∗ p

    Techniques Used: In Vitro, Activation Assay, Expressing

    CD3/CD28 Activation Induces PKM2 Expression and Nuclear Accumulation in Murine CD4 + CD62L + T Cells Murine CD4 + CD62 + T cells were stimulated in vitro for 3 days with CD3/CD28 antibodies and collected at different time points of activation. (A) Quantification of Pkm2 mRNA in resting versus activated murine CD4 + CD62L + T cells by qRT-PCR (n = 5–6 from 4 independent experiments). ∗ p
    Figure Legend Snippet: CD3/CD28 Activation Induces PKM2 Expression and Nuclear Accumulation in Murine CD4 + CD62L + T Cells Murine CD4 + CD62 + T cells were stimulated in vitro for 3 days with CD3/CD28 antibodies and collected at different time points of activation. (A) Quantification of Pkm2 mRNA in resting versus activated murine CD4 + CD62L + T cells by qRT-PCR (n = 5–6 from 4 independent experiments). ∗ p

    Techniques Used: Activation Assay, Expressing, In Vitro, Quantitative RT-PCR

    11) Product Images from "The enhanced expression of IL-17-secreting T cells during the early progression of atherosclerosis in ApoE-deficient mice fed on a western-type diet"

    Article Title: The enhanced expression of IL-17-secreting T cells during the early progression of atherosclerosis in ApoE-deficient mice fed on a western-type diet

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/emm.2015.19

    The systemic expression of T-cell-derived cytokines in ApoE −/− and control mice. ( a ) The proportion of IL-17-, IFN-γ- and IL-4-positive CD4 T cells in splenocytes. ( b ) The production of IL-17 and IFN-γ from splenocytes at 12 weeks after anti-CD3/CD28 stimulation. * P
    Figure Legend Snippet: The systemic expression of T-cell-derived cytokines in ApoE −/− and control mice. ( a ) The proportion of IL-17-, IFN-γ- and IL-4-positive CD4 T cells in splenocytes. ( b ) The production of IL-17 and IFN-γ from splenocytes at 12 weeks after anti-CD3/CD28 stimulation. * P

    Techniques Used: Expressing, Derivative Assay, Mouse Assay

    12) Product Images from "MicroRNA-155 controls CD8+ T cell responses by regulating interferon signaling"

    Article Title: MicroRNA-155 controls CD8+ T cell responses by regulating interferon signaling

    Journal: Nature immunology

    doi: 10.1038/ni.2576

    miR-155 is expressed in CD8 + T cells. (a) miR-155 is highly upregulated with in vitro activation of CD8 + T cells. Sorted splenic CD8 + T cells from wild-type C57BL/6 mice were stimulated in vitro with anti-CD3, anti-CD28 antibodies for 1, 3 and 5 days and miR-155 expression was quantified by RT-PCR. Graph shows fold increase of miR-155 expression over sorted unstimulated CD8 + T cells. Bars represent mean and standard errors of 5 animals per group tested in 3 independent experiments. ( b) miR-155 is expressed in vivo in primary effector and effector memory CD8 + T cells. Donor day 10 lung effector CD44 + CD62L- CD8 + T cells and donor day 60 splenic effector memory CD44 + CD62L - or central memory CD44 + CD62L + CD8 + T cells were sorted from congenic animals that had received OT-I adoptive transfers and been infected with WSN-OVA influenza virus. Naive CD44-CD62L + CD8 + T cells were sorted from spleens of naive OT-I mice. MiR-155 expression was quantified by RT-PCR. Graph depicts fold increase of miR-155 over naive CD8 + T cells. Bars represent mean and SEM from 3-5 mice/groups and 2 independent experiments. All values were normalized to 18S rRNA expression.
    Figure Legend Snippet: miR-155 is expressed in CD8 + T cells. (a) miR-155 is highly upregulated with in vitro activation of CD8 + T cells. Sorted splenic CD8 + T cells from wild-type C57BL/6 mice were stimulated in vitro with anti-CD3, anti-CD28 antibodies for 1, 3 and 5 days and miR-155 expression was quantified by RT-PCR. Graph shows fold increase of miR-155 expression over sorted unstimulated CD8 + T cells. Bars represent mean and standard errors of 5 animals per group tested in 3 independent experiments. ( b) miR-155 is expressed in vivo in primary effector and effector memory CD8 + T cells. Donor day 10 lung effector CD44 + CD62L- CD8 + T cells and donor day 60 splenic effector memory CD44 + CD62L - or central memory CD44 + CD62L + CD8 + T cells were sorted from congenic animals that had received OT-I adoptive transfers and been infected with WSN-OVA influenza virus. Naive CD44-CD62L + CD8 + T cells were sorted from spleens of naive OT-I mice. MiR-155 expression was quantified by RT-PCR. Graph depicts fold increase of miR-155 over naive CD8 + T cells. Bars represent mean and SEM from 3-5 mice/groups and 2 independent experiments. All values were normalized to 18S rRNA expression.

    Techniques Used: In Vitro, Activation Assay, Mouse Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, In Vivo, Infection

    13) Product Images from "IFN-? Inhibits the Increased Expression of IL-9 during Experimental Autoimmune Uveoretinitis"

    Article Title: IFN-? Inhibits the Increased Expression of IL-9 during Experimental Autoimmune Uveoretinitis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0048566

    Effect of IFN-β treatment on IL-9 production. Splenocytes (A) and DLN cells (B) from the immunized mice following IFN-β or PBS treatment were activated with IRBP 161–180 (20 µg/ml) or anti-CD3/CD28 (1 µg/ml) for 3 days. IL-9 was analyzed by ELISA. Data are representative of three independent experiments with at least five mice per group. ND: not detected.
    Figure Legend Snippet: Effect of IFN-β treatment on IL-9 production. Splenocytes (A) and DLN cells (B) from the immunized mice following IFN-β or PBS treatment were activated with IRBP 161–180 (20 µg/ml) or anti-CD3/CD28 (1 µg/ml) for 3 days. IL-9 was analyzed by ELISA. Data are representative of three independent experiments with at least five mice per group. ND: not detected.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    14) Product Images from "Targeting T cell oxidative metabolism to improve influenza survival in a mouse model of obesity"

    Article Title: Targeting T cell oxidative metabolism to improve influenza survival in a mouse model of obesity

    Journal: International journal of obesity (2005)

    doi: 10.1038/s41366-020-00692-3

    Obesity is associated with changes in CD4 + T cell metabolism. CD4 + T cells were isolated from the spleens of lean (control) versus diet induced obese (DIO) mice. Cells were rested or activated by plate-bound anti-CD3 and anti-CD28 for 48hr. Cells were then plated at 250,000 cells per well, and extracellular flux analysis was performed using the Seahorse XFe96 Flux analyzer in response to the mitochondrial stress test with injections of 1 μM oligomycin, 0.5 μM flurorcarbonyl cynade phenylhydrazone (FCCP) and 0.75 μM rotenone with 1.5μM antimycin A. OCR and ECAR were measured for rested (a) and activated (b) CD4 cells. Data were pooled from two independent experiments ( n=5 ). T-test with Welch’s correction was used to compare groups. *p
    Figure Legend Snippet: Obesity is associated with changes in CD4 + T cell metabolism. CD4 + T cells were isolated from the spleens of lean (control) versus diet induced obese (DIO) mice. Cells were rested or activated by plate-bound anti-CD3 and anti-CD28 for 48hr. Cells were then plated at 250,000 cells per well, and extracellular flux analysis was performed using the Seahorse XFe96 Flux analyzer in response to the mitochondrial stress test with injections of 1 μM oligomycin, 0.5 μM flurorcarbonyl cynade phenylhydrazone (FCCP) and 0.75 μM rotenone with 1.5μM antimycin A. OCR and ECAR were measured for rested (a) and activated (b) CD4 cells. Data were pooled from two independent experiments ( n=5 ). T-test with Welch’s correction was used to compare groups. *p

    Techniques Used: Isolation, Mouse Assay

    Obesity is associated with changes in CD4 + T cell nutrient uptake. CD4 + T cells were isolated from lean (control) and diet induced obese (DIO) mice. Cells were rested or activated by plate-bound anti-CD3 and anti-CD28 for 48hr after which were stained for 2NBDG (a, c) and BODIPY (b, d) and analyzed by flow cytometry. Data were pooled from two independent experiments except for BODIPY uptake were the results represent data from one of the experiments both showed same trend and statistical significance ( n=5 ). T-test with Welch’s correction was used to compare groups. **p
    Figure Legend Snippet: Obesity is associated with changes in CD4 + T cell nutrient uptake. CD4 + T cells were isolated from lean (control) and diet induced obese (DIO) mice. Cells were rested or activated by plate-bound anti-CD3 and anti-CD28 for 48hr after which were stained for 2NBDG (a, c) and BODIPY (b, d) and analyzed by flow cytometry. Data were pooled from two independent experiments except for BODIPY uptake were the results represent data from one of the experiments both showed same trend and statistical significance ( n=5 ). T-test with Welch’s correction was used to compare groups. **p

    Techniques Used: Isolation, Mouse Assay, Staining, Flow Cytometry

    The antidiabetic drug metformin reverses T cell metabolic dysfunction in CD4 + T cells and enhances memory markers. CD4 + T cells were isolated from C57BL/6J male mice and activated for 24 hours by plate bound anti-CD3 and anti-CD28 antibodies then treated with 2 mM metformin for an additional 24 hours. The cells were assayed for extracellular metabolic flux (a) , and stained for activation marker expression (b-d) . Splenocytes were pooled from 3 mice and data represents results from two independent experiments. T-test with Welch’s correction was used to compare groups. *p
    Figure Legend Snippet: The antidiabetic drug metformin reverses T cell metabolic dysfunction in CD4 + T cells and enhances memory markers. CD4 + T cells were isolated from C57BL/6J male mice and activated for 24 hours by plate bound anti-CD3 and anti-CD28 antibodies then treated with 2 mM metformin for an additional 24 hours. The cells were assayed for extracellular metabolic flux (a) , and stained for activation marker expression (b-d) . Splenocytes were pooled from 3 mice and data represents results from two independent experiments. T-test with Welch’s correction was used to compare groups. *p

    Techniques Used: Isolation, Mouse Assay, Staining, Activation Assay, Marker, Expressing

    15) Product Images from "The DHODH Inhibitor PTC299 Arrests SARS-CoV-2 Replication and Suppresses Induction of Inflammatory Cytokines"

    Article Title: The DHODH Inhibitor PTC299 Arrests SARS-CoV-2 Replication and Suppresses Induction of Inflammatory Cytokines

    Journal: Virus Research

    doi: 10.1016/j.virusres.2020.198246

    PTC299 inhibits IL-17A and IL-17 F production in Th17 cells. (A-D) PMBCs were stimulated with T-cell activator CD3/CD28 Dynabeads and a combination of cytokines and antibodies to promote T-cell differentiation while blocking Th1 and Th2 differentiation. Cells were incubated with increasing concentrations of PTC299 and levels of (A) IL-17A and (B) IL-17 F in the medium were measured by ELISA. Following PMBC stimulation and incubation in the presence of 1 µM PTC299 and100 µM uridine, levels of (C) IL-17A and (D) IL-17 F in the medium were measured by ELISA. Vehicle (Medium plus 0.5%DMSO) was used as a negative control Secreted IL-17 F was reported as both pg/mL in the medium and as (pg/mL) normalized for cell number. Bars indicate standard deviation. Abbreviations: IL, interleukin; PBMC, peripheral blood mononuclear cell.
    Figure Legend Snippet: PTC299 inhibits IL-17A and IL-17 F production in Th17 cells. (A-D) PMBCs were stimulated with T-cell activator CD3/CD28 Dynabeads and a combination of cytokines and antibodies to promote T-cell differentiation while blocking Th1 and Th2 differentiation. Cells were incubated with increasing concentrations of PTC299 and levels of (A) IL-17A and (B) IL-17 F in the medium were measured by ELISA. Following PMBC stimulation and incubation in the presence of 1 µM PTC299 and100 µM uridine, levels of (C) IL-17A and (D) IL-17 F in the medium were measured by ELISA. Vehicle (Medium plus 0.5%DMSO) was used as a negative control Secreted IL-17 F was reported as both pg/mL in the medium and as (pg/mL) normalized for cell number. Bars indicate standard deviation. Abbreviations: IL, interleukin; PBMC, peripheral blood mononuclear cell.

    Techniques Used: Cell Differentiation, Blocking Assay, Incubation, Enzyme-linked Immunosorbent Assay, Negative Control, Standard Deviation

    16) Product Images from "Downregulation of the AU-Rich RNA-Binding Protein ZFP36 in Chronic HBV Patients: Implications for Anti-Inflammatory Therapy"

    Article Title: Downregulation of the AU-Rich RNA-Binding Protein ZFP36 in Chronic HBV Patients: Implications for Anti-Inflammatory Therapy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033356

    Effect of siRNA-mediated silencing of ZFP36 on T cell activation, proliferation, and survival. PBMCs from healthy donors were transfected by nucleofection with non-specific siRNA (NC siRNA) or ZFP36-specific siRNA (ZFP36 siRNA). A. Expression of CD25 and CD69 in CD4 + and CD8 + T cells was measured 24 h after stimulation with anti-CD3/CD28. Dotted lines: un-stimulated cells; solid lines: stimulated cells; grey lines: NC siRNA-transfected cells; black lines: ZFP36 siRNA-transfected cells. B. CFSE dilution of labeled CD4 + and CD8 + T lymphocytes. Dotted lines: cells transfected with NC siRNA; solid lines: cells transfected with ZFP36 siRNA. C. Cell cycle status of ZFP36 siRNA-transfected T cells. Cells were stained with PI. Solid lines: cells transfected with NC siRNA; dotted lines: cells transfected with ZFP36 siRNA. D. Apoptotic rates of ZFP36 siRNA-transfected T cells as determined by Annexin V staining. Percentages represent the frequency of Annexin V + cells within the CD4 + and CD8 + T cell populations of PBMCs transfected with NC siRNA or ZFP36 siRNA.
    Figure Legend Snippet: Effect of siRNA-mediated silencing of ZFP36 on T cell activation, proliferation, and survival. PBMCs from healthy donors were transfected by nucleofection with non-specific siRNA (NC siRNA) or ZFP36-specific siRNA (ZFP36 siRNA). A. Expression of CD25 and CD69 in CD4 + and CD8 + T cells was measured 24 h after stimulation with anti-CD3/CD28. Dotted lines: un-stimulated cells; solid lines: stimulated cells; grey lines: NC siRNA-transfected cells; black lines: ZFP36 siRNA-transfected cells. B. CFSE dilution of labeled CD4 + and CD8 + T lymphocytes. Dotted lines: cells transfected with NC siRNA; solid lines: cells transfected with ZFP36 siRNA. C. Cell cycle status of ZFP36 siRNA-transfected T cells. Cells were stained with PI. Solid lines: cells transfected with NC siRNA; dotted lines: cells transfected with ZFP36 siRNA. D. Apoptotic rates of ZFP36 siRNA-transfected T cells as determined by Annexin V staining. Percentages represent the frequency of Annexin V + cells within the CD4 + and CD8 + T cell populations of PBMCs transfected with NC siRNA or ZFP36 siRNA.

    Techniques Used: Activation Assay, Transfection, Expressing, Labeling, Staining

    Related Articles

    Expressing:

    Article Title: Decreased sensitivity to 1,25-dihydroxyvitamin D3 in T cells from the rheumatoid joint
    Article Snippet: For experiments using isolated CD45RA + CD4+ naïve T cells, CD45RO + CD4+ memory T cells and CD14 + monocytes, cells were enriched by negative selection using cell separation reagents (StemCell Technologies and Biolegend). .. For 24 h post-stimulation analysis of gene expression, T cells were stimulated with anti-CD3/CD28 dynabeads (Life Technologies) at a ratio of 1 bead: 2 T cells in medium supplemented with 5% human AB serum (TCS Biosciences, Buckingham UK). ..

    Article Title: CD160-Associated CD8 T-Cell Functional Impairment Is Independent of PD-1 Expression
    Article Snippet: Frequencies of proliferating CFSElow CD8 T cells were assessed by flow cytometry. .. Evaluation of the kinetic of CD160 and PD-1 expression on CD8 T cells after activation and expansion Cryopreserved blood mononuclear cells (106 in 1 ml of complete medium) were stained with 0.25 µM 5,6-carboxyfluorescein succinimidyl ester (CFSE, Molecular Probes, USA) as previously described , and stimulated with anti-CD3/anti-CD28 magnetic beads (Dynabeads, Invitrogen). .. The expression of CD160 and PD-1 on CD8 T cells was evaluated after 0, 24, 48, 72 and 120 hours of stimulation by anti-CD3/anti-CD28 magnetic beads.

    Transfection:

    Article Title: The miR-27a-calreticulin axis affects drug-induced immunogenic cell death in human colorectal cancer cells
    Article Snippet: CD4+ T-cell isolation, proliferation and IFN-γ production After Ficoll hypaque (GE-Healthcare, Pittsburgh, PA, USA) gradient centrifugation, CD4+ T cells were isolated from PBMC by negative selection using human CD4+ CD25+ T cell kit (Thermo Fisher) (cell purity > 98% by FACS analysis) and cultured (1 × 106 cells/ml) in 96-well plates (BD Biosciences) with RPMI 1640 medium supplemented with 100 UI/ml penicillin, 100 μ g/ml streptomycin (Thermo Fisher) and supplemented with 5% FCS (Thermo Fisher). .. Cells were stimulated for 3 days with an anti-CD3/CD28 Dynabeads (0.1 bead/cell) (Thermo Fisher) in the presence or absence of the CM obtained either from co-cultures, as reported above, or from HCT116 or RKO cells transfected with the miR-27a S, AS or C RNA and treated with MTX or OXP. .. On the last day, cells were exposed to [3 H] thymidine (0.5 μ Ci/well) (GE Healthcare) and harvested 12 h later.

    Selection:

    Article Title: Ly6C defines a subset of memory-like CD27+ γδ T cells with inducible cancer-killing function
    Article Snippet: In brief, CD3+ T cells were enriched by negative selection on a LD column and QuadroMACS separator (Miltenyi Biotec), before positive selection of γδ T cells using MS columns with an OctoMacs separator (Miltenyi Biotec). .. To yield a purer γδ T cell population, positive selection was performed twice. γδ T cells were cultured in IMDM medium supplemented with 10% FCS, 100U/mL penicillin/streptomycin, 2mM glutamine, 50μM 2-Mercaptoethanol (β-ME), 10ng/mL murine interleukin-15 (IL-15) (PeproTech) and CD3/CD28 coated Dynabeads (Thermo Fisher Scientific) at a 1:1 ratio. .. Cells were kept in 96 U-well plates (Greiner Bio-One) in normoxic incubators at 37°C and propagated at a density of 4×104 cells per well.

    Cell Culture:

    Article Title: Ly6C defines a subset of memory-like CD27+ γδ T cells with inducible cancer-killing function
    Article Snippet: In brief, CD3+ T cells were enriched by negative selection on a LD column and QuadroMACS separator (Miltenyi Biotec), before positive selection of γδ T cells using MS columns with an OctoMacs separator (Miltenyi Biotec). .. To yield a purer γδ T cell population, positive selection was performed twice. γδ T cells were cultured in IMDM medium supplemented with 10% FCS, 100U/mL penicillin/streptomycin, 2mM glutamine, 50μM 2-Mercaptoethanol (β-ME), 10ng/mL murine interleukin-15 (IL-15) (PeproTech) and CD3/CD28 coated Dynabeads (Thermo Fisher Scientific) at a 1:1 ratio. .. Cells were kept in 96 U-well plates (Greiner Bio-One) in normoxic incubators at 37°C and propagated at a density of 4×104 cells per well.

    Activation Assay:

    Article Title: CD160-Associated CD8 T-Cell Functional Impairment Is Independent of PD-1 Expression
    Article Snippet: Frequencies of proliferating CFSElow CD8 T cells were assessed by flow cytometry. .. Evaluation of the kinetic of CD160 and PD-1 expression on CD8 T cells after activation and expansion Cryopreserved blood mononuclear cells (106 in 1 ml of complete medium) were stained with 0.25 µM 5,6-carboxyfluorescein succinimidyl ester (CFSE, Molecular Probes, USA) as previously described , and stimulated with anti-CD3/anti-CD28 magnetic beads (Dynabeads, Invitrogen). .. The expression of CD160 and PD-1 on CD8 T cells was evaluated after 0, 24, 48, 72 and 120 hours of stimulation by anti-CD3/anti-CD28 magnetic beads.

    Staining:

    Article Title: CD160-Associated CD8 T-Cell Functional Impairment Is Independent of PD-1 Expression
    Article Snippet: Frequencies of proliferating CFSElow CD8 T cells were assessed by flow cytometry. .. Evaluation of the kinetic of CD160 and PD-1 expression on CD8 T cells after activation and expansion Cryopreserved blood mononuclear cells (106 in 1 ml of complete medium) were stained with 0.25 µM 5,6-carboxyfluorescein succinimidyl ester (CFSE, Molecular Probes, USA) as previously described , and stimulated with anti-CD3/anti-CD28 magnetic beads (Dynabeads, Invitrogen). .. The expression of CD160 and PD-1 on CD8 T cells was evaluated after 0, 24, 48, 72 and 120 hours of stimulation by anti-CD3/anti-CD28 magnetic beads.

    Magnetic Beads:

    Article Title: CD160-Associated CD8 T-Cell Functional Impairment Is Independent of PD-1 Expression
    Article Snippet: Frequencies of proliferating CFSElow CD8 T cells were assessed by flow cytometry. .. Evaluation of the kinetic of CD160 and PD-1 expression on CD8 T cells after activation and expansion Cryopreserved blood mononuclear cells (106 in 1 ml of complete medium) were stained with 0.25 µM 5,6-carboxyfluorescein succinimidyl ester (CFSE, Molecular Probes, USA) as previously described , and stimulated with anti-CD3/anti-CD28 magnetic beads (Dynabeads, Invitrogen). .. The expression of CD160 and PD-1 on CD8 T cells was evaluated after 0, 24, 48, 72 and 120 hours of stimulation by anti-CD3/anti-CD28 magnetic beads.

    Positive Control:

    Article Title: Targeting DNGR-1 (CLEC9A) with antibody/MUC1 peptide conjugates as a vaccine for carcinomas
    Article Snippet: After a 4-hour incubation, the production of IFN-γ was measured by intracellular cytokine staining as previously described (Correa et al. [26]). .. As a positive control for the assay, an aliquot of T cells was incubated with PBMC:Dynabeads® human T-Expander CD3/CD28 (Life Technologies) at a 1:1 ratio (cell:bead) instead of cancer cells. ..

    Incubation:

    Article Title: Targeting DNGR-1 (CLEC9A) with antibody/MUC1 peptide conjugates as a vaccine for carcinomas
    Article Snippet: After a 4-hour incubation, the production of IFN-γ was measured by intracellular cytokine staining as previously described (Correa et al. [26]). .. As a positive control for the assay, an aliquot of T cells was incubated with PBMC:Dynabeads® human T-Expander CD3/CD28 (Life Technologies) at a 1:1 ratio (cell:bead) instead of cancer cells. ..

    Functional Assay:

    Article Title: TIGIT Marks Exhausted T Cells, Correlates with Disease Progression, and Serves as a Target for Immune Restoration in HIV and SIV Infection
    Article Snippet: .. Functional assays In the intracellular cytokine stimulation assay studies, thawed cryopreserved PBMCs were stimulated for 12 hours in an incubator at 37°C with 5% CO2 with 5 μg/ml brefeldin A and 5 μg/ml monensin (Sigma-Aldrich) culture media, DMSO alone, pooled HIV-1 Gag peptides, or anti-CD3/CD28 dynabeads (Life Technologies) in the presence or absence of purified isotype IgG control, anti-TIGIT and/or anti-PD-L1 mAbs. .. After stimulation, the cells were washed and stained for viability with AARD and cultured with surface phenotype panel against CD8, TIGIT or an isotype control antibody, followed by intracellular staining of CD3 and IFN-γ and acquisition on the flow cytometer as above.

    Purification:

    Article Title: TIGIT Marks Exhausted T Cells, Correlates with Disease Progression, and Serves as a Target for Immune Restoration in HIV and SIV Infection
    Article Snippet: .. Functional assays In the intracellular cytokine stimulation assay studies, thawed cryopreserved PBMCs were stimulated for 12 hours in an incubator at 37°C with 5% CO2 with 5 μg/ml brefeldin A and 5 μg/ml monensin (Sigma-Aldrich) culture media, DMSO alone, pooled HIV-1 Gag peptides, or anti-CD3/CD28 dynabeads (Life Technologies) in the presence or absence of purified isotype IgG control, anti-TIGIT and/or anti-PD-L1 mAbs. .. After stimulation, the cells were washed and stained for viability with AARD and cultured with surface phenotype panel against CD8, TIGIT or an isotype control antibody, followed by intracellular staining of CD3 and IFN-γ and acquisition on the flow cytometer as above.

    Concentration Assay:

    Article Title: Differential Reliance on Lipid Metabolism as a Salvage Pathway Underlies Functional Differences of T Cell Subsets in Poor Nutrient Environments
    Article Snippet: The medium was supplemented with 1× (500 μL), 2× (1,000 μL), or 3× (1,500 μL), when indicated, of a chemically defined lipid mixture (ThermoFisher Scientific, 11905031; individual lipid concentrations available online) and 8 mM L-glutamine. .. Cells were then activated at 1 million cells/mL using Dynabeads Human T-Expander CD3/CD28 (ThermoFisher Scientific, 11131D) at a concentration of 3 beads per cell. .. Cells were treated with TOFA (5 or 10 μM; Sigma Aldrich, T6575), or etomoxir (200 or 400 μM; Sigma Aldrich, E1905). cDNA encoding LC3B was synthesized (IDT) and transferred into pTRPE, a lentiviral transfer vector ( ).

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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