anti cd28  (BioLegend)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    Name:
    Purified anti human CD28
    Description:
    Purified anti human CD28 CD28 2 Isotype Mouse IgG1 κ Reactivity Human Cross Reactivity Chimpanzee Baboon Cynomolgus Rhesus Pigtailed Macaque Capuchin Monkey Sooty Mangabey Squirrel Monkey Apps FC IP IHC Size 25 μg
    Catalog Number:
    302901
    Price:
    20
    Category:
    Human Immunology
    Source:
    Mouse
    Applications:
    FC, IP, IHC
    Conjugate:
    PURE
    Size:
    25 μg
    Quantity:
    1
    Preparation:
    The antibody was purified by affinity chromatography
    Buy from Supplier


    Structured Review

    BioLegend anti cd28
    Purified anti human CD28
    Purified anti human CD28 CD28 2 Isotype Mouse IgG1 κ Reactivity Human Cross Reactivity Chimpanzee Baboon Cynomolgus Rhesus Pigtailed Macaque Capuchin Monkey Sooty Mangabey Squirrel Monkey Apps FC IP IHC Size 25 μg
    https://www.bioz.com/result/anti cd28/product/BioLegend
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti cd28 - by Bioz Stars, 2021-05
    97/100 stars

    Images

    1) Product Images from "Allosteric activation of MALT1 by its ubiquitin-binding Ig3 domain"

    Article Title: Allosteric activation of MALT1 by its ubiquitin-binding Ig3 domain

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

    doi: 10.1073/pnas.1912681117

    The Ig3-ubiquitin interaction is required for monoubiquitination-dependent MALT1 activation and T cell function. ( A and B ) Assessment of MALT1-dependent FRET reporter cleavage ( A ) or RelB cleavage ( B ) in 293T cells. The reporter or RelB were coexpressed with oncogenic CARMA1(G116S) and WT MALT1 or the indicated MALT1 mutants of E696 and D697 (ED/AA or ED/KK). FRET reporter cleavage was assessed by flow cytometry ( A ) and protein expression, and RelB cleavage was assessed by Western blot as indicated ( A and B ). Positions of uncleaved (black arrowhead) and cleaved (open arrowhead) RelB are indicated. ( C ). ( D ) MALT1-deficient Jurkat T cells were reconstituted with the indicated MALT1 constructs and incubated for 1 h with the MALT1 active site inhibitor z-LVSR-fmk before stimulation with PMA and ionomycin for 1 h. MALT1 monoubiquitination, phosphorylation of IκBα, and phosphorylation of JNK were analyzed by Western blot. ( E ) MALT1-deficient Jurkat T cells reconstituted with the indicated empty vector (mock) or MALT1 constructs were stimulated with PMA and ionomycin for 0, 5, or 30 min. Substrate cleavage (CYLD and Roquin-1) and phosphorylation of IκBα were analyzed by Western blot. Tubulin was used as a loading control, and positions of molecular weight markers (in kDa) are indicated ( A – E ). ( F ) IL-2 secretion of MALT1-deficient Jurkat T cells reconstituted with the indicated MALT1 constructs, stimulated for 16 h with PMA and ionomycin or solvent alone. ( G and H ) Analysis of intracellular IL-2 in isolated primary CD4 + T cells from four healthy donors, lentivirally transduced with mock, MALT1 WT, or MALT1(ED/KK) constructs and stimulated with anti-CD3, anti-CD28, and a cross-linking antibody in the presence of brefeldin A. The percentage of IL-2 + cells among infected GFP + cells was determined by flow cytometry. ( G ) Gating strategy for one donor for MALT1 WT and MALT1(ED/KK) transduced cells. ( H ) Combined analysis of four donors. Bars represent means ± SD ( A and F ) or means ± SEM ( H ); * P
    Figure Legend Snippet: The Ig3-ubiquitin interaction is required for monoubiquitination-dependent MALT1 activation and T cell function. ( A and B ) Assessment of MALT1-dependent FRET reporter cleavage ( A ) or RelB cleavage ( B ) in 293T cells. The reporter or RelB were coexpressed with oncogenic CARMA1(G116S) and WT MALT1 or the indicated MALT1 mutants of E696 and D697 (ED/AA or ED/KK). FRET reporter cleavage was assessed by flow cytometry ( A ) and protein expression, and RelB cleavage was assessed by Western blot as indicated ( A and B ). Positions of uncleaved (black arrowhead) and cleaved (open arrowhead) RelB are indicated. ( C ). ( D ) MALT1-deficient Jurkat T cells were reconstituted with the indicated MALT1 constructs and incubated for 1 h with the MALT1 active site inhibitor z-LVSR-fmk before stimulation with PMA and ionomycin for 1 h. MALT1 monoubiquitination, phosphorylation of IκBα, and phosphorylation of JNK were analyzed by Western blot. ( E ) MALT1-deficient Jurkat T cells reconstituted with the indicated empty vector (mock) or MALT1 constructs were stimulated with PMA and ionomycin for 0, 5, or 30 min. Substrate cleavage (CYLD and Roquin-1) and phosphorylation of IκBα were analyzed by Western blot. Tubulin was used as a loading control, and positions of molecular weight markers (in kDa) are indicated ( A – E ). ( F ) IL-2 secretion of MALT1-deficient Jurkat T cells reconstituted with the indicated MALT1 constructs, stimulated for 16 h with PMA and ionomycin or solvent alone. ( G and H ) Analysis of intracellular IL-2 in isolated primary CD4 + T cells from four healthy donors, lentivirally transduced with mock, MALT1 WT, or MALT1(ED/KK) constructs and stimulated with anti-CD3, anti-CD28, and a cross-linking antibody in the presence of brefeldin A. The percentage of IL-2 + cells among infected GFP + cells was determined by flow cytometry. ( G ) Gating strategy for one donor for MALT1 WT and MALT1(ED/KK) transduced cells. ( H ) Combined analysis of four donors. Bars represent means ± SD ( A and F ) or means ± SEM ( H ); * P

    Techniques Used: Activation Assay, Cell Function Assay, Flow Cytometry, Expressing, Western Blot, Construct, Incubation, Plasmid Preparation, Molecular Weight, Isolation, Transduction, Infection

    2) Product Images from "Rotenone Treatment Reveals a Role for Electron Transport Complex I in the Subcellular Localization of Key Transcriptional Regulators During T Helper Cell Differentiation"

    Article Title: Rotenone Treatment Reveals a Role for Electron Transport Complex I in the Subcellular Localization of Key Transcriptional Regulators During T Helper Cell Differentiation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01284

    T helper (Th)17 cells lose expression of nuclear RORγt following rotenone treatment. CD4 T cells were left untreated or treated with 20 µM rotenone for 2 h and then stimulated with plate-bound anti-CD3ε plus anti-CD28 for 24, 48, 72, and 96 h in the presence of specific Th17- or induced regulatory T cell (iTreg)-specific polarization conditions. For each master transcriptional regulator, we determined the total, nuclear, and non-nuclear median fluorescence intensity (MFI), using an AMNIS Imaging Flow Cytometer. nuclear localization scores were determined by an algorithm that calculates the probability of the protein of interest resides within the nucleus. The higher the nuclear localization score, the greater the probability of the protein of interest is in the nucleus. Scores above “1” are considered to be positive for nuclear expression. Nuclear and non-nuclear MFI were calculated using AMNIS IDEAS Software and applying the nuclear mask. Percent total nuclear and non-nuclear transcription factor proteins were calculated for cells differentiated in the absence or presence of rotenone as follows: [nuclear MFI]/[Total MFI] × 100, or [non-nuclear MFI]/[Total MFI] × 100, respectively. For cells differentiated without or with rotenone, (A) percent distribution of total RORγt protein and RORγt nuclear localization score in Th17-polarized cells. (B) Representative images showing nuclear localization of RORγt. (C) Percent distribution of total Foxp3 protein and Foxp3 nuclear localization score in iTreg-polarized cells. (D) Representative images for nuclear localization of Foxp3 in iTreg cells. For cells differentiated without or with rotenone. (E) Percent of nuclear Notch1-expressing Th17 cells and their representative corresponding Nuclear Localization Scores and representative images of Notch1 nuclear localization in Th17 cells after 72 h of differentiation without or with rotenone in Th17 cells 72 h after differentiation, and (F) percent of nuclear Notch1-expressing iTreg cells and their representative corresponding Nuclear Localization Scores and representative images of Notch1 nuclear localization in iTreg cells after 48 h of differentiation without or with rotenone. Data represent the mean ± SEM of three independent experiments. * p
    Figure Legend Snippet: T helper (Th)17 cells lose expression of nuclear RORγt following rotenone treatment. CD4 T cells were left untreated or treated with 20 µM rotenone for 2 h and then stimulated with plate-bound anti-CD3ε plus anti-CD28 for 24, 48, 72, and 96 h in the presence of specific Th17- or induced regulatory T cell (iTreg)-specific polarization conditions. For each master transcriptional regulator, we determined the total, nuclear, and non-nuclear median fluorescence intensity (MFI), using an AMNIS Imaging Flow Cytometer. nuclear localization scores were determined by an algorithm that calculates the probability of the protein of interest resides within the nucleus. The higher the nuclear localization score, the greater the probability of the protein of interest is in the nucleus. Scores above “1” are considered to be positive for nuclear expression. Nuclear and non-nuclear MFI were calculated using AMNIS IDEAS Software and applying the nuclear mask. Percent total nuclear and non-nuclear transcription factor proteins were calculated for cells differentiated in the absence or presence of rotenone as follows: [nuclear MFI]/[Total MFI] × 100, or [non-nuclear MFI]/[Total MFI] × 100, respectively. For cells differentiated without or with rotenone, (A) percent distribution of total RORγt protein and RORγt nuclear localization score in Th17-polarized cells. (B) Representative images showing nuclear localization of RORγt. (C) Percent distribution of total Foxp3 protein and Foxp3 nuclear localization score in iTreg-polarized cells. (D) Representative images for nuclear localization of Foxp3 in iTreg cells. For cells differentiated without or with rotenone. (E) Percent of nuclear Notch1-expressing Th17 cells and their representative corresponding Nuclear Localization Scores and representative images of Notch1 nuclear localization in Th17 cells after 72 h of differentiation without or with rotenone in Th17 cells 72 h after differentiation, and (F) percent of nuclear Notch1-expressing iTreg cells and their representative corresponding Nuclear Localization Scores and representative images of Notch1 nuclear localization in iTreg cells after 48 h of differentiation without or with rotenone. Data represent the mean ± SEM of three independent experiments. * p

    Techniques Used: Expressing, Fluorescence, Imaging, Flow Cytometry, Cytometry, Software

    Rotenone treatment alters the kinetics of Th17 and induced regulatory T cell (iTreg) cell differentiation. Mouse splenic CD4 T cells and CD4 + CD25 − T cells were used for T helper (Th) cell and iTreg differentiation, respectively. Cells were left untreated or treated with 20 µM rotenone for 2 h and then stimulated with plate-bound anti-CD3ε plus anti-CD28 for 24, 48, 72, and 96 h in the presence of specific Th cell polarization conditions. At the indicated time points, cell supernatants were collected, and signature cytokines were quantified using standard enzyme-linked immunosorbent assay techniques for (A) IFNγ by Th1-polarized cells, (B) IL-4 by Th2-polarized cells, (C) IL-17A by Th17-polarized cells, and (D) IL-10 by iTreg-polarized cells. Data represent the mean ± SEM of three independent experiments. * p
    Figure Legend Snippet: Rotenone treatment alters the kinetics of Th17 and induced regulatory T cell (iTreg) cell differentiation. Mouse splenic CD4 T cells and CD4 + CD25 − T cells were used for T helper (Th) cell and iTreg differentiation, respectively. Cells were left untreated or treated with 20 µM rotenone for 2 h and then stimulated with plate-bound anti-CD3ε plus anti-CD28 for 24, 48, 72, and 96 h in the presence of specific Th cell polarization conditions. At the indicated time points, cell supernatants were collected, and signature cytokines were quantified using standard enzyme-linked immunosorbent assay techniques for (A) IFNγ by Th1-polarized cells, (B) IL-4 by Th2-polarized cells, (C) IL-17A by Th17-polarized cells, and (D) IL-10 by iTreg-polarized cells. Data represent the mean ± SEM of three independent experiments. * p

    Techniques Used: Cell Differentiation, Enzyme-linked Immunosorbent Assay

    Notch1 expression and cellular localization in T helper (Th) cells is differentially affected by rotenone treatment. Notch1 levels were measured in Th cells, using flow cytometric approaches. Cells were left untreated or treated with 20 µM rotenone for 2 h and then stimulated with plate-bound anti-CD3ε plus anti-CD28 for 24, 48, 72, and 96 h in the presence of specific Th cell polarization conditions. At the indicated timepoints, cells were harvested to determine the (A) percent Notch1-positive and (B) Notch1 median fluorescence intensity (MFI) in Th17-polarized cells, and the (C) percent Notch1-positive, and (D) Notch1 MFI in induced regulatory T cell (iTreg)-polarized cells. We visualized mitochondria within the cells using Mitotracker Red CMXRos and imaging flow cytometry. Mitochondrial mass was determined based on the MFI of Red CMXRos for DMSO control and rotenone-treated cells under (E) Th17-polarizing conditions 72 h after stimulation and (F) iTreg-polarizing conditions 48 h after stimulation. (G) We calculated the percent of Th17 (left panel) and iTreg (right panel) cells which showed mitochondrial Notch1 localization. Representative cell images showing Notch1 and mitochondrial colocalization in (H) Th17 and (I) iTreg cells, differentiated in the presence and absence of rotenone. (J) We used the AMNIS Colocalization Wizard to calculate Th17 and iTreg cell frequency histograms that show Notch1 colocalized with mitochondria, together with their corresponding colocalization scores. Data represent the mean ± SEM of three independent experiments. * p
    Figure Legend Snippet: Notch1 expression and cellular localization in T helper (Th) cells is differentially affected by rotenone treatment. Notch1 levels were measured in Th cells, using flow cytometric approaches. Cells were left untreated or treated with 20 µM rotenone for 2 h and then stimulated with plate-bound anti-CD3ε plus anti-CD28 for 24, 48, 72, and 96 h in the presence of specific Th cell polarization conditions. At the indicated timepoints, cells were harvested to determine the (A) percent Notch1-positive and (B) Notch1 median fluorescence intensity (MFI) in Th17-polarized cells, and the (C) percent Notch1-positive, and (D) Notch1 MFI in induced regulatory T cell (iTreg)-polarized cells. We visualized mitochondria within the cells using Mitotracker Red CMXRos and imaging flow cytometry. Mitochondrial mass was determined based on the MFI of Red CMXRos for DMSO control and rotenone-treated cells under (E) Th17-polarizing conditions 72 h after stimulation and (F) iTreg-polarizing conditions 48 h after stimulation. (G) We calculated the percent of Th17 (left panel) and iTreg (right panel) cells which showed mitochondrial Notch1 localization. Representative cell images showing Notch1 and mitochondrial colocalization in (H) Th17 and (I) iTreg cells, differentiated in the presence and absence of rotenone. (J) We used the AMNIS Colocalization Wizard to calculate Th17 and iTreg cell frequency histograms that show Notch1 colocalized with mitochondria, together with their corresponding colocalization scores. Data represent the mean ± SEM of three independent experiments. * p

    Techniques Used: Expressing, Flow Cytometry, Fluorescence, Imaging, Cytometry

    Rotenone treatment does not affect the expression of T helper (Th) cell master transcriptional regulators. CD4 T cells were left untreated or treated with 20 µM rotenone for 2 h and then stimulated with plate-bound anti-CD3ε plus anti-CD28 for 24, 48, 72, and 96 h in the presence of specific Th17- or induced regulatory T cell (iTreg)-specific polarization conditions. At the indicated timepoints, cells were harvested, and we determined the (A) percent RORγt-positive and (B) median fluorescence intensity (MFI) of RORγt expressed in Th17-polarized cells, and the (C) percent Foxp3-positive and (D) MFI of Foxp3 expressed in iTreg-polarized cells. (E) Representative dot plot and (F) collated data showing percentages of CD4 + CD25 + Foxp3 + iTregs following differentiation in the absence or presence of rotenone. Data represent the mean ± SEM of three independent experiments. * p
    Figure Legend Snippet: Rotenone treatment does not affect the expression of T helper (Th) cell master transcriptional regulators. CD4 T cells were left untreated or treated with 20 µM rotenone for 2 h and then stimulated with plate-bound anti-CD3ε plus anti-CD28 for 24, 48, 72, and 96 h in the presence of specific Th17- or induced regulatory T cell (iTreg)-specific polarization conditions. At the indicated timepoints, cells were harvested, and we determined the (A) percent RORγt-positive and (B) median fluorescence intensity (MFI) of RORγt expressed in Th17-polarized cells, and the (C) percent Foxp3-positive and (D) MFI of Foxp3 expressed in iTreg-polarized cells. (E) Representative dot plot and (F) collated data showing percentages of CD4 + CD25 + Foxp3 + iTregs following differentiation in the absence or presence of rotenone. Data represent the mean ± SEM of three independent experiments. * p

    Techniques Used: Expressing, Fluorescence

    Rotenone treatment inhibits mitochondrial localization of pyruvate dehydrogenase kinase 1 (PDHK1) and promotes RORγt and Notch1 colocalization with its phosphorylated substrate, pPDH-E1α, in Th17-polarized cells. CD4 T cells were treated either with 20 µM rotenone for 2 h or 1 mM dichloroacetate (DCA) (left in the cell suspension throughout) and then stimulated with plate-bound anti-CD3ε plus anti-CD28 for 72 h in the presence of Th17 polarization conditions. After 72 h, samples were split and half of the cells were stained for PDHK1, total PDH-E1α, pPDH-E1α (Ser232), RORγt, and Notch1 to determine their localization in cytosol (Tubulin AF647 staining) and mitochondria (Mitotracker CMXRos). Data were acquired via AMNIS ImageStream X Mark Imaging Flow Cytometer. Quantification of percent of cytosolic or mitochondrial (A) PDHK1-localizing cells and (B) total PDH-E1α-localizing cells along with corresponding representative images are shown. For colocalization, percent of (C) Notch1 + pPDH-E1α (Ser232)-colocalizing and (D) RORγt + pPDH-E1α (Ser232) Th17-polarized cells for each treatment condition and representative images are shown. Total RNA was collected from the other half of treated cells and qPCR was performed for measuring (E) foxp3 and rorgt expressions upon rotenone treatment (DCA treatment was used as a control which triggers foxp3 expression). Data represent the mean ± SEM of three independent experiments. * p
    Figure Legend Snippet: Rotenone treatment inhibits mitochondrial localization of pyruvate dehydrogenase kinase 1 (PDHK1) and promotes RORγt and Notch1 colocalization with its phosphorylated substrate, pPDH-E1α, in Th17-polarized cells. CD4 T cells were treated either with 20 µM rotenone for 2 h or 1 mM dichloroacetate (DCA) (left in the cell suspension throughout) and then stimulated with plate-bound anti-CD3ε plus anti-CD28 for 72 h in the presence of Th17 polarization conditions. After 72 h, samples were split and half of the cells were stained for PDHK1, total PDH-E1α, pPDH-E1α (Ser232), RORγt, and Notch1 to determine their localization in cytosol (Tubulin AF647 staining) and mitochondria (Mitotracker CMXRos). Data were acquired via AMNIS ImageStream X Mark Imaging Flow Cytometer. Quantification of percent of cytosolic or mitochondrial (A) PDHK1-localizing cells and (B) total PDH-E1α-localizing cells along with corresponding representative images are shown. For colocalization, percent of (C) Notch1 + pPDH-E1α (Ser232)-colocalizing and (D) RORγt + pPDH-E1α (Ser232) Th17-polarized cells for each treatment condition and representative images are shown. Total RNA was collected from the other half of treated cells and qPCR was performed for measuring (E) foxp3 and rorgt expressions upon rotenone treatment (DCA treatment was used as a control which triggers foxp3 expression). Data represent the mean ± SEM of three independent experiments. * p

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

    Rotenone treatment reduces T cell activation upon anti-CD3ε plus anti-CD28 stimulation. Mouse splenic CD4 T cells were left untreated or treated with 20 µM rotenone for 2 h, then stimulated with plate-bound anti-CD3ε plus anti-CD28 for 24 h. CD25 and Notch1 levels were measured as read-outs of T cell activation via flow cytometry. (A) Representative contour plot showing CD25-expressing cells left unstimulated or stimulated in the presence or absence of rotenone for 24 h. The (B) percent CD25-positive, and (C) median fluorescence intensity (MFI) of CD25, as well as the (D) percent Notch1-positive (E) MFI of Notch1 on CD4 T cells, cultured as described above. At the end of 24 h of stimulation, culture supernatants were collected, and standard enzyme-linked immunosorbent assay techniques were used to quantify secreted (F) IFNγ and (G) IL-2. Data represent the mean ± SEM of three independent experiments. * p
    Figure Legend Snippet: Rotenone treatment reduces T cell activation upon anti-CD3ε plus anti-CD28 stimulation. Mouse splenic CD4 T cells were left untreated or treated with 20 µM rotenone for 2 h, then stimulated with plate-bound anti-CD3ε plus anti-CD28 for 24 h. CD25 and Notch1 levels were measured as read-outs of T cell activation via flow cytometry. (A) Representative contour plot showing CD25-expressing cells left unstimulated or stimulated in the presence or absence of rotenone for 24 h. The (B) percent CD25-positive, and (C) median fluorescence intensity (MFI) of CD25, as well as the (D) percent Notch1-positive (E) MFI of Notch1 on CD4 T cells, cultured as described above. At the end of 24 h of stimulation, culture supernatants were collected, and standard enzyme-linked immunosorbent assay techniques were used to quantify secreted (F) IFNγ and (G) IL-2. Data represent the mean ± SEM of three independent experiments. * p

    Techniques Used: Activation Assay, Flow Cytometry, Cytometry, Expressing, Fluorescence, Cell Culture, Enzyme-linked Immunosorbent Assay

    3) Product Images from "The Human CD8? M-4 Isoform Dominant in Effector Memory T Cells Has Distinct Cytoplasmic Motifs That Confer Unique Properties"

    Article Title: The Human CD8? M-4 Isoform Dominant in Effector Memory T Cells Has Distinct Cytoplasmic Motifs That Confer Unique Properties

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0059374

    CD4 + T cells transduced with the M-4 CD8β isoform showed increased frequency of cells producing MIP-1β after stimulation. Peripheral blood CD4 + T cells were stimulated with anti-CD3 and anti-CD28 antibodies for 24 hours, and then co-transduced with a lentivirus expressing a NY-ESO-1 TCR and another lentivirus expressing CD8α and one of the CD8β isoforms. Cells were stimulated and analyzed for cytokine/chemokine production after day 10–12. ( A ) Schematic representation of lentiviral vectors used for co-transduction of primary CD4 + T cells is followed by histograms for surface expression of CD8αβ, CD8α and TCR and dot plots showing cell population co-expressing NY-ESO-1 TCR and CD8α. Data were collected by flow cytometry using antibodies against CD8 and an MHC tetramer specific to NY-ESO-1 (NY-ESO- tetramer). Live CD3 + lymphocytes were gated using side vs. forward scatter, anti-CD3 antibody and live/dead cell dye. ( B ) Frequency of transduced CD4 + T cells producing MIP-1β (top panel) after stimulation with K562 target cells expressing the NY-ESO-1 antigen. T cells without targets served as negative control and cells stimulated with PMA and Ionomycin (bottom panel) were used as positive control. One representative of three independent experiments is shown. Values that are statistically different from the wild type M-1 protein as determined by two-population Student’s paired t-test are indicated as one star (*) for p
    Figure Legend Snippet: CD4 + T cells transduced with the M-4 CD8β isoform showed increased frequency of cells producing MIP-1β after stimulation. Peripheral blood CD4 + T cells were stimulated with anti-CD3 and anti-CD28 antibodies for 24 hours, and then co-transduced with a lentivirus expressing a NY-ESO-1 TCR and another lentivirus expressing CD8α and one of the CD8β isoforms. Cells were stimulated and analyzed for cytokine/chemokine production after day 10–12. ( A ) Schematic representation of lentiviral vectors used for co-transduction of primary CD4 + T cells is followed by histograms for surface expression of CD8αβ, CD8α and TCR and dot plots showing cell population co-expressing NY-ESO-1 TCR and CD8α. Data were collected by flow cytometry using antibodies against CD8 and an MHC tetramer specific to NY-ESO-1 (NY-ESO- tetramer). Live CD3 + lymphocytes were gated using side vs. forward scatter, anti-CD3 antibody and live/dead cell dye. ( B ) Frequency of transduced CD4 + T cells producing MIP-1β (top panel) after stimulation with K562 target cells expressing the NY-ESO-1 antigen. T cells without targets served as negative control and cells stimulated with PMA and Ionomycin (bottom panel) were used as positive control. One representative of three independent experiments is shown. Values that are statistically different from the wild type M-1 protein as determined by two-population Student’s paired t-test are indicated as one star (*) for p

    Techniques Used: Transduction, Expressing, Flow Cytometry, Cytometry, Negative Control, Positive Control

    4) Product Images from "CRISPR-mediated deletion of the Protein tyrosine phosphatase, non-receptor type 22 (PTPN22) improves human T cell function for adoptive T cell therapy"

    Article Title: CRISPR-mediated deletion of the Protein tyrosine phosphatase, non-receptor type 22 (PTPN22) improves human T cell function for adoptive T cell therapy

    Journal: bioRxiv

    doi: 10.1101/2020.12.03.410043

    Surface marker expression on PTPN22 KO and control T cells Expression of CD98, CD71, CD27, and CD28 was evaluated by cell surface staining and flow cytometry. Data of 2 different healthy donors are shown.
    Figure Legend Snippet: Surface marker expression on PTPN22 KO and control T cells Expression of CD98, CD71, CD27, and CD28 was evaluated by cell surface staining and flow cytometry. Data of 2 different healthy donors are shown.

    Techniques Used: Marker, Expressing, Staining, Flow Cytometry

    EBV/LMP2-specific PTPN22 KO and control T cells have similar CD8/CD4 frequencies and expression of surface markers (A-B) TCR expression was determined before (A) and after (B) magnetic enrichment using antibodies against mouse TCRβ chain. Bar charts below show data summarized from 7 donors. (C) Representative dot plots and bar charts of data pooled from 7 donors show the frequencies of CD8 and CD4 T cells in control and PTPN22 KO samples. (D) Expression of CD98, CD71, CD27, and CD28 was evaluated by cell surface staining and flow cytometry. Data are shown from one representative experiment from 3 different donors (two other donors are shown in Figure S1). Significance was determined using two-way ANOVA with Tukey’s post test for multiple comparisons. ns = not significant
    Figure Legend Snippet: EBV/LMP2-specific PTPN22 KO and control T cells have similar CD8/CD4 frequencies and expression of surface markers (A-B) TCR expression was determined before (A) and after (B) magnetic enrichment using antibodies against mouse TCRβ chain. Bar charts below show data summarized from 7 donors. (C) Representative dot plots and bar charts of data pooled from 7 donors show the frequencies of CD8 and CD4 T cells in control and PTPN22 KO samples. (D) Expression of CD98, CD71, CD27, and CD28 was evaluated by cell surface staining and flow cytometry. Data are shown from one representative experiment from 3 different donors (two other donors are shown in Figure S1). Significance was determined using two-way ANOVA with Tukey’s post test for multiple comparisons. ns = not significant

    Techniques Used: Expressing, Staining, Flow Cytometry

    5) Product Images from "Intestinal microbiota-derived short-chain fatty acids regulation of immune cell IL-22 production and gut immunity"

    Article Title: Intestinal microbiota-derived short-chain fatty acids regulation of immune cell IL-22 production and gut immunity

    Journal: Nature Communications

    doi: 10.1038/s41467-020-18262-6

    Butyrate induces human CD4 + T cell IL-22 production. a – e Peripheral blood CD4 + T cells were isolated from healthy controls (HC, n = 8 biologically independent samples), patients with active Crohn’s colitis (CD, n = 10 biologically independent samples) and ulcerative colitis (UC, n = 7 biologically independent samples), and activated with anti-CD3/CD28 mAbs with or without butyrate (0.5 mM). Il22 expression was assessed at day 3 by qRT-PCR ( a ), IL-22 + cells were measured by flow cytometry at day 5 ( b ), and IL-22 production in supernatants was measured at day 3 by ELISA ( c ). Hif1a ( d ) and Ahr ( e ) expression in CD4 + T cells were analyzed by qRT-PCR at day 3. f Peripheral blood CD4 + T cells from healthy controls, CD, and UC patients were treated with or without butyrate (0.5 mM) ± YC-1 (20 µM) or CH-223191 (5 µM) for 5 days ( n = 3/group). IL-22 production was analyzed by flow cytometry. One representative of three independent experiments was shown. Scale bar, 300 µm. Data were expressed as mean ± SD. Statistical significance was tested by two-tailed paired Student t -test ( a – e ), or two-tailed one-way ANOVA ( f ). a ** p = 0.0024, *** p = 0.0008 (CD), and 0.0003 (UC); b *** p = 0.0001, **** p
    Figure Legend Snippet: Butyrate induces human CD4 + T cell IL-22 production. a – e Peripheral blood CD4 + T cells were isolated from healthy controls (HC, n = 8 biologically independent samples), patients with active Crohn’s colitis (CD, n = 10 biologically independent samples) and ulcerative colitis (UC, n = 7 biologically independent samples), and activated with anti-CD3/CD28 mAbs with or without butyrate (0.5 mM). Il22 expression was assessed at day 3 by qRT-PCR ( a ), IL-22 + cells were measured by flow cytometry at day 5 ( b ), and IL-22 production in supernatants was measured at day 3 by ELISA ( c ). Hif1a ( d ) and Ahr ( e ) expression in CD4 + T cells were analyzed by qRT-PCR at day 3. f Peripheral blood CD4 + T cells from healthy controls, CD, and UC patients were treated with or without butyrate (0.5 mM) ± YC-1 (20 µM) or CH-223191 (5 µM) for 5 days ( n = 3/group). IL-22 production was analyzed by flow cytometry. One representative of three independent experiments was shown. Scale bar, 300 µm. Data were expressed as mean ± SD. Statistical significance was tested by two-tailed paired Student t -test ( a – e ), or two-tailed one-way ANOVA ( f ). a ** p = 0.0024, *** p = 0.0008 (CD), and 0.0003 (UC); b *** p = 0.0001, **** p

    Techniques Used: Isolation, Expressing, Quantitative RT-PCR, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Stat3 and mTOR regulate IL-22 production by CD4 + T cells. a–d WT CD4 + T cells were activated with anti-CD3/CD28 mAbs under Th1 conditions with or without butyrate (0.5 mM) ( n = 3/group). Phosphorylated Stat3 (6 h) ( a , b ) and phosphorylated mTOR (24 h) ( c , d ) were assessed by western blot and flow cytometry. Phosphorylated S6K was analyzed by flow cytometry ( e ). f – i CBir1 Tg CD4 + T cells were activated with APCs and CBir1 peptide under Th1 conditions with butyrate (0.5 mM) ± rapamycin (1 µM) or HJC0152 (1 µM). IL-22 mRNA ( f ) and protein ( g ) were assessed by qRT-PCR and ELISA at 60 h ( n = 3/group). Expression of Hif1a ( h ) and Ahr ( i ) was analyzed at 48 h by qRT-PCR. j WT and Stat3 −/− CD4 + T cells were treated with or without butyrate (0.5 mM) for 5 days ( n = 3/group). IL-22 production was measured by flow cytometry. One representative of three independent experiments was shown. Data were expressed as mean ± SD. Statistical significance was tested by two-tailed unpaired Student t -test ( a – e , j ) or two-tailed one-way ANOVA ( f – i ). a * p = 0.0134; b *** p = 0.0002; c ** p = 0.0059; d *** p = 0.0002; e ** p = 0.0010; f , **** p
    Figure Legend Snippet: Stat3 and mTOR regulate IL-22 production by CD4 + T cells. a–d WT CD4 + T cells were activated with anti-CD3/CD28 mAbs under Th1 conditions with or without butyrate (0.5 mM) ( n = 3/group). Phosphorylated Stat3 (6 h) ( a , b ) and phosphorylated mTOR (24 h) ( c , d ) were assessed by western blot and flow cytometry. Phosphorylated S6K was analyzed by flow cytometry ( e ). f – i CBir1 Tg CD4 + T cells were activated with APCs and CBir1 peptide under Th1 conditions with butyrate (0.5 mM) ± rapamycin (1 µM) or HJC0152 (1 µM). IL-22 mRNA ( f ) and protein ( g ) were assessed by qRT-PCR and ELISA at 60 h ( n = 3/group). Expression of Hif1a ( h ) and Ahr ( i ) was analyzed at 48 h by qRT-PCR. j WT and Stat3 −/− CD4 + T cells were treated with or without butyrate (0.5 mM) for 5 days ( n = 3/group). IL-22 production was measured by flow cytometry. One representative of three independent experiments was shown. Data were expressed as mean ± SD. Statistical significance was tested by two-tailed unpaired Student t -test ( a – e , j ) or two-tailed one-way ANOVA ( f – i ). a * p = 0.0134; b *** p = 0.0002; c ** p = 0.0059; d *** p = 0.0002; e ** p = 0.0010; f , **** p

    Techniques Used: Western Blot, Flow Cytometry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Two Tailed Test

    Butyrate promotes HIF1α binding to Il22 promoter in CD4 + T cells. a Schematic diagram of HIF1α binding to Il22 promoter. b WT CD4 + T cells were activated with anti-CD3/CD28 mAbs under Th1 conditions for 2 days ( n = 3/group). HIF1α binding to Il22 promoter was analyzed by CHIP assay. c – e WT CD4 + T cells were cultured under Th1 conditions with or without butyrate (0.5 mM) for 2 days ( n = 3/group). HIF1α binding to Il22 promoter was analyzed by CHIP assay ( c ). The H3K9 acetylation ( d ) and trimethylation ( e ) levels in HIF1α-binding site on Il22 promoter were assessed by CHIP assay. One representative of three independent experiments ( b , c ), or two independent experiments ( d , e ) was shown. Data were expressed as mean ± SD. Statistical significance was tested by two-tailed unpaired Student t -test. b ** p = 0.0047; c * p = 0.0278; d * p = 0.0105; e ** p = 0.0094.
    Figure Legend Snippet: Butyrate promotes HIF1α binding to Il22 promoter in CD4 + T cells. a Schematic diagram of HIF1α binding to Il22 promoter. b WT CD4 + T cells were activated with anti-CD3/CD28 mAbs under Th1 conditions for 2 days ( n = 3/group). HIF1α binding to Il22 promoter was analyzed by CHIP assay. c – e WT CD4 + T cells were cultured under Th1 conditions with or without butyrate (0.5 mM) for 2 days ( n = 3/group). HIF1α binding to Il22 promoter was analyzed by CHIP assay ( c ). The H3K9 acetylation ( d ) and trimethylation ( e ) levels in HIF1α-binding site on Il22 promoter were assessed by CHIP assay. One representative of three independent experiments ( b , c ), or two independent experiments ( d , e ) was shown. Data were expressed as mean ± SD. Statistical significance was tested by two-tailed unpaired Student t -test. b ** p = 0.0047; c * p = 0.0278; d * p = 0.0105; e ** p = 0.0094.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

    SCFAs promote IL-22 production in CD4 + T cells and ILCs in vitro. a WT splenic CD4 + T cells were activated with anti-CD3/CD28 mAbs ± butyrate (0.5 mM) for 2 days ( n = 3 biologically independent samples per group). RNA sequencing was performed. Il10 , Ifng , and Il22 expressions were shown in heatmap. b , c CBir1 Tg CD4 + T cells were cultured with APCs and CBir1 peptide ± acetate (10 mM), propionate (0.5 mM), or butyrate (0.5 mM) for 2 days ( n = 3/group). Il22 expression was analyzed by qRT-PCR ( b ), and IL-22 in supernatants was assessed by ELISA ( c ). d CBir1 Tg CD4 + T cells were cultured with APCs and CBir1 peptide ± butyrate (0.5 mM) for 2 days ( n = 3/group) under neutral, Th1, Th17, or Treg conditions. Il22 was analyzed by qRT-PCR. e CD4 + T cells were activated with anti-CD3/CD28 mAbs ± butyrate (0.5 mM) for 2 days ( n = 3 biologically independent samples per group) under Th1 conditions. RNA sequencing was performed. Expression of Il10 , Ifng , and Il22 was shown in heatmap. f–h CBir1 Tg CD4 + T cells were activated with APCs and CBir1 peptide ± butyrate (0.5 mM) under Th1 conditions ( n = 3/group). IL-22 was analyzed by qRT-PCR at different time point ( f ), and ELISA at 60 h ( g ), and IL-22 and IL-17 were measured flow cytometry on day 5 ( h ). i CD4 + T cell-depleted splenic cells were treated with IL-23 (20 ng/ml) ± acetate (10 mM), propionate (0.5 mM), or butyrate (0.5 mM) for 16 h ( n = 3/groups). IL-22 and IL-17 production in ILCs were analyzed by flow cytometry. One representative of three independent experiments was shown ( b – d , f–i ). Data were expressed as mean ± SD. Statistical significance was tested by two-tailed one-way ANOVA ( b , c , i ) or two-tailed unpaired Student t -test ( d , g , h ). b ** p = 0.0014 (acetate vs control) and 0.0028 (propionate vs control), **** p
    Figure Legend Snippet: SCFAs promote IL-22 production in CD4 + T cells and ILCs in vitro. a WT splenic CD4 + T cells were activated with anti-CD3/CD28 mAbs ± butyrate (0.5 mM) for 2 days ( n = 3 biologically independent samples per group). RNA sequencing was performed. Il10 , Ifng , and Il22 expressions were shown in heatmap. b , c CBir1 Tg CD4 + T cells were cultured with APCs and CBir1 peptide ± acetate (10 mM), propionate (0.5 mM), or butyrate (0.5 mM) for 2 days ( n = 3/group). Il22 expression was analyzed by qRT-PCR ( b ), and IL-22 in supernatants was assessed by ELISA ( c ). d CBir1 Tg CD4 + T cells were cultured with APCs and CBir1 peptide ± butyrate (0.5 mM) for 2 days ( n = 3/group) under neutral, Th1, Th17, or Treg conditions. Il22 was analyzed by qRT-PCR. e CD4 + T cells were activated with anti-CD3/CD28 mAbs ± butyrate (0.5 mM) for 2 days ( n = 3 biologically independent samples per group) under Th1 conditions. RNA sequencing was performed. Expression of Il10 , Ifng , and Il22 was shown in heatmap. f–h CBir1 Tg CD4 + T cells were activated with APCs and CBir1 peptide ± butyrate (0.5 mM) under Th1 conditions ( n = 3/group). IL-22 was analyzed by qRT-PCR at different time point ( f ), and ELISA at 60 h ( g ), and IL-22 and IL-17 were measured flow cytometry on day 5 ( h ). i CD4 + T cell-depleted splenic cells were treated with IL-23 (20 ng/ml) ± acetate (10 mM), propionate (0.5 mM), or butyrate (0.5 mM) for 16 h ( n = 3/groups). IL-22 and IL-17 production in ILCs were analyzed by flow cytometry. One representative of three independent experiments was shown ( b – d , f–i ). Data were expressed as mean ± SD. Statistical significance was tested by two-tailed one-way ANOVA ( b , c , i ) or two-tailed unpaired Student t -test ( d , g , h ). b ** p = 0.0014 (acetate vs control) and 0.0028 (propionate vs control), **** p

    Techniques Used: In Vitro, RNA Sequencing Assay, Cell Culture, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Two Tailed Test

    HIF1α and AhR mediate butyrate induction of IL-22 in CD4 + T cells. a WT CD4 + T cells were activated with anti-CD3/CD28 mAbs under Th1 conditions ± butyrate (0.5 mM) for 2 days ( n = 3 biologically independent samples per group). RNA sequencing was performed. Hif1α , Ahr , and Prdm1 were shown in heatmap. b – f CD4 + T cells were activated with anti-CD3/CD28 mAbs ± butyrate (0.5 mM) under Th1 conditions ( n = 3/group). Hif1a ( b ) and Ahr ( c ) were analyzed by qRT-PCR. HIF1α ( d ) and AhR ( e ) protein was analyzed by western blot on day 2. HIF1α activity was measured using HIF1α Transcription Factor Assay Kit ( f ). g Raw 264.7 cells were transduced with XRE/AhR Luciferase Reporter Gene Lentivirus, and treated ± butyrate (0.5 mM) 3 days post transduction. AhR activity was assessed by luciferase. h–j Cbir1 Tg CD4 + T cells were activated with APCs and Cbir1 peptide under Th1 conditions with butyrate (0.5 mM) ± YC-1 (5 µM) or/and CH-223191 (3 µM) for 60 h ( n = 3/group). IL-22 mRNA ( h ) and protein ( i ) were measured by qRT-PCR and ELISA. j IL-22 was measured by flow cytometry on day 5. k WT and HIF1α −/− CD4 + T cells were activated with anti-CD3/CD28 mAbs ± butyrate (0.5 mM) for 5 days ( n = 3/group). IL-22 was assessed by flow cytometry. l , m CD4 + T cells were activated with anti-CD3/CD28 mAbs under Th1 conditions with or without butyrate (0.5 mM), AR420626 (5 µM), or TSA (10 nM) for 60 h ( n = 3/group). Hif1a ( l ) and Ahr ( m ) were measured by qRT-PCR. One representative of three independent experiments was shown ( b – m ). Data were expressed as mean ± SD. Statistical significance was tested by two-tailed unpaired Student t -test ( b – g ) or two-tailed one-way ANOVA ( h – m ). b ** p = 0.0033 (24 h), *** p = 0.0002 (36 h), ** p = 0.0032 (48 h), * p = 0.0310 (60 h); c * p = 0.0338 (24 h), ** p = 0.0054 (36 h), *** p = 0.0003 (48 h), *** p = 0.0007 (60 h); d * p = 0.0178; e * p = 0.0325; f * p = 0.0273; g * p = 0.0435; h **** p
    Figure Legend Snippet: HIF1α and AhR mediate butyrate induction of IL-22 in CD4 + T cells. a WT CD4 + T cells were activated with anti-CD3/CD28 mAbs under Th1 conditions ± butyrate (0.5 mM) for 2 days ( n = 3 biologically independent samples per group). RNA sequencing was performed. Hif1α , Ahr , and Prdm1 were shown in heatmap. b – f CD4 + T cells were activated with anti-CD3/CD28 mAbs ± butyrate (0.5 mM) under Th1 conditions ( n = 3/group). Hif1a ( b ) and Ahr ( c ) were analyzed by qRT-PCR. HIF1α ( d ) and AhR ( e ) protein was analyzed by western blot on day 2. HIF1α activity was measured using HIF1α Transcription Factor Assay Kit ( f ). g Raw 264.7 cells were transduced with XRE/AhR Luciferase Reporter Gene Lentivirus, and treated ± butyrate (0.5 mM) 3 days post transduction. AhR activity was assessed by luciferase. h–j Cbir1 Tg CD4 + T cells were activated with APCs and Cbir1 peptide under Th1 conditions with butyrate (0.5 mM) ± YC-1 (5 µM) or/and CH-223191 (3 µM) for 60 h ( n = 3/group). IL-22 mRNA ( h ) and protein ( i ) were measured by qRT-PCR and ELISA. j IL-22 was measured by flow cytometry on day 5. k WT and HIF1α −/− CD4 + T cells were activated with anti-CD3/CD28 mAbs ± butyrate (0.5 mM) for 5 days ( n = 3/group). IL-22 was assessed by flow cytometry. l , m CD4 + T cells were activated with anti-CD3/CD28 mAbs under Th1 conditions with or without butyrate (0.5 mM), AR420626 (5 µM), or TSA (10 nM) for 60 h ( n = 3/group). Hif1a ( l ) and Ahr ( m ) were measured by qRT-PCR. One representative of three independent experiments was shown ( b – m ). Data were expressed as mean ± SD. Statistical significance was tested by two-tailed unpaired Student t -test ( b – g ) or two-tailed one-way ANOVA ( h – m ). b ** p = 0.0033 (24 h), *** p = 0.0002 (36 h), ** p = 0.0032 (48 h), * p = 0.0310 (60 h); c * p = 0.0338 (24 h), ** p = 0.0054 (36 h), *** p = 0.0003 (48 h), *** p = 0.0007 (60 h); d * p = 0.0178; e * p = 0.0325; f * p = 0.0273; g * p = 0.0435; h **** p

    Techniques Used: RNA Sequencing Assay, Quantitative RT-PCR, Western Blot, Activity Assay, Transcription Factor Assay, Transduction, Luciferase, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Two Tailed Test

    Butyrate promotes IL-22 production through GPR41 and HDAC inhibition. a , b CBir1 Tg CD4 + T cells were cultured with APCs and Cbir1 peptide with or without butyrate (0.5 mM) ± AR420626 (5 µM) or/and TSA (10 mM) under Th1 conditions ( n = 3/group). IL-22 mRNA ( a ) and protein ( b ) were measured by qRT-PCR and ELISA at 60 h. IL-22 production was measured by flow cytometry on day 5 ( c ). d CD4 + T cells were cultured with anti-CD3/CD28 mAbs under Th1 conditions with or without butyrate (0.5 mM) or TSA (10 mM) ( n = 3/group). Cells were collected at 24 h for analysis of HDAC activity at fluorescence intensity at excitation/emission (490/525 nm) by using the HDAC Activity Assay Kit. One representative of three independent experiments was shown. Data were expressed as mean ± SD. Statistical significance was tested by two-tailed one-way ANOVA. a **** p
    Figure Legend Snippet: Butyrate promotes IL-22 production through GPR41 and HDAC inhibition. a , b CBir1 Tg CD4 + T cells were cultured with APCs and Cbir1 peptide with or without butyrate (0.5 mM) ± AR420626 (5 µM) or/and TSA (10 mM) under Th1 conditions ( n = 3/group). IL-22 mRNA ( a ) and protein ( b ) were measured by qRT-PCR and ELISA at 60 h. IL-22 production was measured by flow cytometry on day 5 ( c ). d CD4 + T cells were cultured with anti-CD3/CD28 mAbs under Th1 conditions with or without butyrate (0.5 mM) or TSA (10 mM) ( n = 3/group). Cells were collected at 24 h for analysis of HDAC activity at fluorescence intensity at excitation/emission (490/525 nm) by using the HDAC Activity Assay Kit. One representative of three independent experiments was shown. Data were expressed as mean ± SD. Statistical significance was tested by two-tailed one-way ANOVA. a **** p

    Techniques Used: Inhibition, Cell Culture, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Activity Assay, Fluorescence, HDAC Activity Assay, Two Tailed Test

    6) Product Images from "Effect of Gemcitabine based chemotherapy on the immunogenicity of pancreatic tumour cells and T-cells"

    Article Title: Effect of Gemcitabine based chemotherapy on the immunogenicity of pancreatic tumour cells and T-cells

    Journal: Clinical & Translational Oncology

    doi: 10.1007/s12094-020-02429-0

    Pomalidomide modulates activation of CD8 + T-cells pre-incubated with anti-PD-1 antibody. T-cells were incubated with anti-PD-1 (10 μg/ml) ± GEM (1–100 nM) or POM (1–100 nM) or combinations of GEM (10 nM) and POM (10 nM) for 48 h prior to activation with anti-CD3 and anti-CD28 antibodies for a further 48 h ( a ). Effect of Anti-PD-1 pre-stimulation ± POM or GEM on IFN-γ expression ( b ), or Annexin V expression ( c ) from CD8+ T-cells. N = 4
    Figure Legend Snippet: Pomalidomide modulates activation of CD8 + T-cells pre-incubated with anti-PD-1 antibody. T-cells were incubated with anti-PD-1 (10 μg/ml) ± GEM (1–100 nM) or POM (1–100 nM) or combinations of GEM (10 nM) and POM (10 nM) for 48 h prior to activation with anti-CD3 and anti-CD28 antibodies for a further 48 h ( a ). Effect of Anti-PD-1 pre-stimulation ± POM or GEM on IFN-γ expression ( b ), or Annexin V expression ( c ) from CD8+ T-cells. N = 4

    Techniques Used: Activation Assay, Incubation, Expressing

    7) Product Images from "CD3-T Cell Receptor Co-stimulation through SLAMF3 and SLAMF6 Receptors Enhances ROR?t Recruitment to the IL17A Promoter in Human T Lymphocytes *"

    Article Title: CD3-T Cell Receptor Co-stimulation through SLAMF3 and SLAMF6 Receptors Enhances ROR?t Recruitment to the IL17A Promoter in Human T Lymphocytes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.415067

    IL17A promoter activity in response to CD28, SLAMF3, or SLAMF6 co-stimulation requires intact NFAT binding sites. A , the IL17A promoter harbors multiple putative transcription factor binding sites, including three consensus binding sequences for RORγt
    Figure Legend Snippet: IL17A promoter activity in response to CD28, SLAMF3, or SLAMF6 co-stimulation requires intact NFAT binding sites. A , the IL17A promoter harbors multiple putative transcription factor binding sites, including three consensus binding sequences for RORγt

    Techniques Used: Activity Assay, Binding Assay

    IL-17A expression depends on RORγt. The effects of RORγt signaling on IL-17A expression by T lymphocytes in response to stimulation with anti-CD3 plus anti-CD28, anti-SLAMF3, or anti-SLAMF6 antibodies were investigated, using the RORγt
    Figure Legend Snippet: IL-17A expression depends on RORγt. The effects of RORγt signaling on IL-17A expression by T lymphocytes in response to stimulation with anti-CD3 plus anti-CD28, anti-SLAMF3, or anti-SLAMF6 antibodies were investigated, using the RORγt

    Techniques Used: Expressing

    Differences in the IL-17A response to co-stimulation with anti-CD28 versus anti-SLAMF3 or anti-SLAMF6 antibodies are mediated by RORγt but not NFAT. Differences in the nuclear abundance of NFAT ( A ) and RORγt ( B ) in response to co-stimulation
    Figure Legend Snippet: Differences in the IL-17A response to co-stimulation with anti-CD28 versus anti-SLAMF3 or anti-SLAMF6 antibodies are mediated by RORγt but not NFAT. Differences in the nuclear abundance of NFAT ( A ) and RORγt ( B ) in response to co-stimulation

    Techniques Used:

    IL17A promoter activity in response to co-stimulation with anti-SLAMF3 and anti-SLAMF6 antibodies requires an intact RORγt binding site. The effects of T lymphocyte stimulation with anti-CD3 plus anti-CD28, anti-SLAMF3 ( A and B ), or anti-SLAMF6
    Figure Legend Snippet: IL17A promoter activity in response to co-stimulation with anti-SLAMF3 and anti-SLAMF6 antibodies requires an intact RORγt binding site. The effects of T lymphocyte stimulation with anti-CD3 plus anti-CD28, anti-SLAMF3 ( A and B ), or anti-SLAMF6

    Techniques Used: Activity Assay, Binding Assay

    Model of the effects of canonical co-stimulation through CD28 versus co-stimulation with SLAMF3 or SLAMF6 on IL-17 expression in T lymphocytes. Co-stimulation of T lymphocytes through the canonical CD28 pathway results in massively increased NFAT abundance
    Figure Legend Snippet: Model of the effects of canonical co-stimulation through CD28 versus co-stimulation with SLAMF3 or SLAMF6 on IL-17 expression in T lymphocytes. Co-stimulation of T lymphocytes through the canonical CD28 pathway results in massively increased NFAT abundance

    Techniques Used: Expressing

    Differences in the IL-17A response to co-stimulation with anti-CD28 versus anti-SLAMF3 or anti-SLAMF6 antibodies are mediated by increased binding of RORγt but not NFAT to the IL-17A promoter. Differences in the recruitment of NFAT ( A ) and RORγt
    Figure Legend Snippet: Differences in the IL-17A response to co-stimulation with anti-CD28 versus anti-SLAMF3 or anti-SLAMF6 antibodies are mediated by increased binding of RORγt but not NFAT to the IL-17A promoter. Differences in the recruitment of NFAT ( A ) and RORγt

    Techniques Used: Binding Assay

    IL-17A expression depends on NFAT. A , the effects of NFAT signaling on IL-17A expression by T lymphocytes in response to stimulation with anti-CD3 plus anti-CD28, anti-SLAMF3 or anti-SLAMF6 antibodies were investigated, using the NFAT inhibitor FKK506/tacrolimus
    Figure Legend Snippet: IL-17A expression depends on NFAT. A , the effects of NFAT signaling on IL-17A expression by T lymphocytes in response to stimulation with anti-CD3 plus anti-CD28, anti-SLAMF3 or anti-SLAMF6 antibodies were investigated, using the NFAT inhibitor FKK506/tacrolimus

    Techniques Used: Expressing

    8) Product Images from "Decreased B and T lymphocyte attenuator in Behcet’s disease may trigger abnormal Th17 and Th1 immune responses"

    Article Title: Decreased B and T lymphocyte attenuator in Behcet’s disease may trigger abnormal Th17 and Th1 immune responses

    Journal: Scientific Reports

    doi: 10.1038/srep20401

    Decreased expression of BTLA is associated with increased Th17 and Th1 cell responses. Purified CD4 + T cells from ocular BD patients ( n = 8 ) and normal controls ( n = 8 ) were stimulated with anti-CD3/CD28 for 3 days. The cells were analyzed for intracellular expression of IL-17 and IFN-gamma by flow cytometry. ( A ) A scatter diagram of a representative subject for each group is presented. ( B ) Frequency of IL-17- and IFN-gamma-producing CD4 + T cells in patients and controls. Anti-CD3/CD28 stimulated CD4 + BTLA lo cells and CD4 + BTLA hi cells from patients ( n = 8 ) and controls ( n = 8 ) were analyzed for the expression of IL-17 and IFN-gamma by flow cytometry. ( C ) Scatter diagrams of a representative subject for each group are shown. ( D ) Frequency of IL-17- and IFN-gamma-producing in CD4 + BTLA hi cells and CD4 + BTLA lo cells. *p
    Figure Legend Snippet: Decreased expression of BTLA is associated with increased Th17 and Th1 cell responses. Purified CD4 + T cells from ocular BD patients ( n = 8 ) and normal controls ( n = 8 ) were stimulated with anti-CD3/CD28 for 3 days. The cells were analyzed for intracellular expression of IL-17 and IFN-gamma by flow cytometry. ( A ) A scatter diagram of a representative subject for each group is presented. ( B ) Frequency of IL-17- and IFN-gamma-producing CD4 + T cells in patients and controls. Anti-CD3/CD28 stimulated CD4 + BTLA lo cells and CD4 + BTLA hi cells from patients ( n = 8 ) and controls ( n = 8 ) were analyzed for the expression of IL-17 and IFN-gamma by flow cytometry. ( C ) Scatter diagrams of a representative subject for each group are shown. ( D ) Frequency of IL-17- and IFN-gamma-producing in CD4 + BTLA hi cells and CD4 + BTLA lo cells. *p

    Techniques Used: Expressing, Purification, Flow Cytometry, Cytometry

    Agonistic anti-BTLA antibody inhibits overreacted Th17 and Th1 cell responses. Purified CD4 + T cells from ocular BD patients ( n = 8 ) and normal controls ( n = 8 ) were stimulated with anti-CD3/CD28 in the presence or absence of 1μg/ml agonistic anti-BTLA antibody for 3 days. ( A,B ) The cells were stimulated with PMA/ionomycin and analyzed for intracellular expression of IL-17 and IFN-gamma by flow cytometry. Scatter diagrams of a representative subject for each group are shown. ( C,D ) Frequency of IL-17- and IFN-gamma-producing CD4 + T cells. ( E ) The production of IL-17, IL-22 and IFN-gamma in the supernatants was determined by ELISA. *p
    Figure Legend Snippet: Agonistic anti-BTLA antibody inhibits overreacted Th17 and Th1 cell responses. Purified CD4 + T cells from ocular BD patients ( n = 8 ) and normal controls ( n = 8 ) were stimulated with anti-CD3/CD28 in the presence or absence of 1μg/ml agonistic anti-BTLA antibody for 3 days. ( A,B ) The cells were stimulated with PMA/ionomycin and analyzed for intracellular expression of IL-17 and IFN-gamma by flow cytometry. Scatter diagrams of a representative subject for each group are shown. ( C,D ) Frequency of IL-17- and IFN-gamma-producing CD4 + T cells. ( E ) The production of IL-17, IL-22 and IFN-gamma in the supernatants was determined by ELISA. *p

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

    9) Product Images from "IRAP-dependent endosomal T cell receptor signalling is essential for T cell responses"

    Article Title: IRAP-dependent endosomal T cell receptor signalling is essential for T cell responses

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16471-7

    IRAP-deficient mice have low numbers of peripheral T cells. a TCRb expression on the cell surface of effector OT1 or IRO T cells (culture day 7), measured by flow cytometry. Each symbol represents an individual experiment. Values represent mean ± SEM of three independent experiments, * p = 0.0468. b Immunoblot analysis of effector OT1 or IRO T cells after activation for the indicated time points by anti-CD3ε/CD28 antibody. Values on quantification graphs represent mean ± SEM of pool of three independent experiments, * p = 0.0352, ** p = 0.0031. c CD8 + T-cell absolute number quantification in OT1 or IRO mouse lymph nodes by flow cytometry. Values represent mean ± SEM of five mice from two independent experiments, * p = 0.0172. CD8 + cells are gated on CD45 + TCRb + live cells. d Quantification of number of OT1 and IRO T cells after activation by DC2.4 loaded with N4 or Q4 peptides. Values represent mean ± SEM of three or four independent experiments, Q4 day 3 * p = 0.017, Q4 day 7 * p = 0.011. Representative proliferation graphs at day 3 are shown. e CD8 + and CD4 + T-cell absolute number quantification in wt (IRAP loxlox ) or IRAP Tcellko mouse lymph nodes or spleen by flow cytometry. CD8 + and CD4 + cells are gated on CD45 + TCRb + live cells. Values represent mean ± SEM of six mice from two independent experiments, LN CD8 + *** p = 0.0003, CD4 + ** p = 0.0017; spleen CD8 + ** p = 0.0037, CD4 + ** p = 0.0096. All p -values ( a , b , c , d , e ) were calculated with two-tailed unpaired t tests. For additional information, see Supplementary Figs. 4, 5, 6 and 7.
    Figure Legend Snippet: IRAP-deficient mice have low numbers of peripheral T cells. a TCRb expression on the cell surface of effector OT1 or IRO T cells (culture day 7), measured by flow cytometry. Each symbol represents an individual experiment. Values represent mean ± SEM of three independent experiments, * p = 0.0468. b Immunoblot analysis of effector OT1 or IRO T cells after activation for the indicated time points by anti-CD3ε/CD28 antibody. Values on quantification graphs represent mean ± SEM of pool of three independent experiments, * p = 0.0352, ** p = 0.0031. c CD8 + T-cell absolute number quantification in OT1 or IRO mouse lymph nodes by flow cytometry. Values represent mean ± SEM of five mice from two independent experiments, * p = 0.0172. CD8 + cells are gated on CD45 + TCRb + live cells. d Quantification of number of OT1 and IRO T cells after activation by DC2.4 loaded with N4 or Q4 peptides. Values represent mean ± SEM of three or four independent experiments, Q4 day 3 * p = 0.017, Q4 day 7 * p = 0.011. Representative proliferation graphs at day 3 are shown. e CD8 + and CD4 + T-cell absolute number quantification in wt (IRAP loxlox ) or IRAP Tcellko mouse lymph nodes or spleen by flow cytometry. CD8 + and CD4 + cells are gated on CD45 + TCRb + live cells. Values represent mean ± SEM of six mice from two independent experiments, LN CD8 + *** p = 0.0003, CD4 + ** p = 0.0017; spleen CD8 + ** p = 0.0037, CD4 + ** p = 0.0096. All p -values ( a , b , c , d , e ) were calculated with two-tailed unpaired t tests. For additional information, see Supplementary Figs. 4, 5, 6 and 7.

    Techniques Used: Mouse Assay, Expressing, Flow Cytometry, Activation Assay, Two Tailed Test

    IRAP is required for proper TCR signalling and activation of Jurkat T cells. a Immunoblot analysis of TCR signalling molecules in wt and IRAP ko Jurkat T cells after activation by anti-CD3ε/CD28 for various time points. Values represent mean ± SEM of pool of three independent experiments for all signalling components excepting pPLCγ ( n = 4) and pZAP70 ( n = 2). pLAT * p = 0.0429, pCD3ζ * p = 0.0231, pLck * p = 0.0107, pPLCγ * p 1.5 min = 0.0262, * p 5 min = 0.0199. b , c IL-2 response of Jurkat T cells measured by ELISA: b wt or IRAP ko Jurkat T cells were incubated for 6 h with Raji B cells presenting the SEE superantigen, c wt or IRAP ko Jurkat T cells expressing the MART1 TCR were incubated overnight with Daju-A2 cells presenting the MART1 peptide. Values represent mean ± SEM of three replicates of one representative experiment of three independent experiments. SEE **** p
    Figure Legend Snippet: IRAP is required for proper TCR signalling and activation of Jurkat T cells. a Immunoblot analysis of TCR signalling molecules in wt and IRAP ko Jurkat T cells after activation by anti-CD3ε/CD28 for various time points. Values represent mean ± SEM of pool of three independent experiments for all signalling components excepting pPLCγ ( n = 4) and pZAP70 ( n = 2). pLAT * p = 0.0429, pCD3ζ * p = 0.0231, pLck * p = 0.0107, pPLCγ * p 1.5 min = 0.0262, * p 5 min = 0.0199. b , c IL-2 response of Jurkat T cells measured by ELISA: b wt or IRAP ko Jurkat T cells were incubated for 6 h with Raji B cells presenting the SEE superantigen, c wt or IRAP ko Jurkat T cells expressing the MART1 TCR were incubated overnight with Daju-A2 cells presenting the MART1 peptide. Values represent mean ± SEM of three replicates of one representative experiment of three independent experiments. SEE **** p

    Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Expressing

    IRAP S-acylation is important for TCR activation. a Intracellular IRAP expression in wt, IRAP ko and IRAP ko cells reconstituted with IRAP E465A or IRAP 3CA, measured by flow cytometry ( n = 3 experiments, ** p wt = 0.0034, ** p E465A = 0.0071, *** p = 0.0002). Values represent mean ± SEM of three independent experiments. b Representative images of Jurkat T cells expressing endogenous IRAP (wt) and IRAP ko cells expressing IRAP E465A or IRAP 3CA via lentiviral transductions. The cells were activated on CD3ε/CD28-coated slides for 10 min, fixed and stained with antibodies specific for IRAP and Stx6. Bars represent 5-μm scale. c CD3ε expression on the cell surface of wt, IRAP ko Jurkat T cells and IRAP ko reconstituted with IRAP E465A and IRAP 3CA, measured by flow cytometry ( n = 3 experiments, *** p = 0.0006, * p = 0.0398). Values represent mean ± SEM of three independent experiments. d Representative images of Jurkat T cells expressing endogenous IRAP (wt) and IRAP ko cells expressing IRAP E465A or IRAP 3CA via lentiviral transductions. The cells were activated on CD3ε/CD28-coated slides for 10 min, fixed and stained with antibodies specific for IRAP and phospho-CD3ζ. Bars represent 5-μm scale. e wt, IRAP ko Jurkat T cells and IRAP ko transduced with lentiviruses expressing IRAP E465A or IRAP 3CA were incubated with SEE-pulsed Raji cells. Values represent mean ± SEM of three replicates of one representative experiment of three independent experiments. Wt: * p = 0.0116, ** p = 0.0010, **** p
    Figure Legend Snippet: IRAP S-acylation is important for TCR activation. a Intracellular IRAP expression in wt, IRAP ko and IRAP ko cells reconstituted with IRAP E465A or IRAP 3CA, measured by flow cytometry ( n = 3 experiments, ** p wt = 0.0034, ** p E465A = 0.0071, *** p = 0.0002). Values represent mean ± SEM of three independent experiments. b Representative images of Jurkat T cells expressing endogenous IRAP (wt) and IRAP ko cells expressing IRAP E465A or IRAP 3CA via lentiviral transductions. The cells were activated on CD3ε/CD28-coated slides for 10 min, fixed and stained with antibodies specific for IRAP and Stx6. Bars represent 5-μm scale. c CD3ε expression on the cell surface of wt, IRAP ko Jurkat T cells and IRAP ko reconstituted with IRAP E465A and IRAP 3CA, measured by flow cytometry ( n = 3 experiments, *** p = 0.0006, * p = 0.0398). Values represent mean ± SEM of three independent experiments. d Representative images of Jurkat T cells expressing endogenous IRAP (wt) and IRAP ko cells expressing IRAP E465A or IRAP 3CA via lentiviral transductions. The cells were activated on CD3ε/CD28-coated slides for 10 min, fixed and stained with antibodies specific for IRAP and phospho-CD3ζ. Bars represent 5-μm scale. e wt, IRAP ko Jurkat T cells and IRAP ko transduced with lentiviruses expressing IRAP E465A or IRAP 3CA were incubated with SEE-pulsed Raji cells. Values represent mean ± SEM of three replicates of one representative experiment of three independent experiments. Wt: * p = 0.0116, ** p = 0.0010, **** p

    Techniques Used: Activation Assay, Expressing, Flow Cytometry, Staining, Transduction, Incubation

    The TCR signals from IRAP/Stx6+ intracellular compartments. a CD3ζ reporter composed of a CD3ζ chain followed by a GFP molecule, an mCherry molecule and an SH2 domain of ZAP-70. At the activated state, Lck phosphorylates the CD3ζ ITAMs, which then recruit the SH2 domain of ZAP-70. Then GFP and mCherry come to a distance where FRET can occur. b Representative images and quantification of GFP average lifetime in CD3ε/CD28-activated Jurkat T cells. Jurkat T cells expressing the CD3ζ reporter and transduced with either shNT or shIRAP lentivirus were activated on CD3ε/CD28-coated slides for 10 min, fixed and GFP average lifetime was measured by FRET–FLIM. Green symbols represent quantification at the plasma membrane (mb), and red symbols quantification in endosomes (endo). Each dot represents an individual cell (GFP n = 46; GFP-mCherry n = 38; wt mb n = 43; ko mb n = 39; wt endo n = 28; ko endo n = 30, ** p = 0.0064, **** p
    Figure Legend Snippet: The TCR signals from IRAP/Stx6+ intracellular compartments. a CD3ζ reporter composed of a CD3ζ chain followed by a GFP molecule, an mCherry molecule and an SH2 domain of ZAP-70. At the activated state, Lck phosphorylates the CD3ζ ITAMs, which then recruit the SH2 domain of ZAP-70. Then GFP and mCherry come to a distance where FRET can occur. b Representative images and quantification of GFP average lifetime in CD3ε/CD28-activated Jurkat T cells. Jurkat T cells expressing the CD3ζ reporter and transduced with either shNT or shIRAP lentivirus were activated on CD3ε/CD28-coated slides for 10 min, fixed and GFP average lifetime was measured by FRET–FLIM. Green symbols represent quantification at the plasma membrane (mb), and red symbols quantification in endosomes (endo). Each dot represents an individual cell (GFP n = 46; GFP-mCherry n = 38; wt mb n = 43; ko mb n = 39; wt endo n = 28; ko endo n = 30, ** p = 0.0064, **** p

    Techniques Used: Expressing, Transduction

    10) Product Images from "Multi-Parameter Analysis of Biobanked Human Bone Marrow Stromal Cells Shows Little Influence for Donor Age and Mild Comorbidities on Phenotypic and Functional Properties"

    Article Title: Multi-Parameter Analysis of Biobanked Human Bone Marrow Stromal Cells Shows Little Influence for Donor Age and Mild Comorbidities on Phenotypic and Functional Properties

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02474

    Immunomodulatory and paracrine activity of BMSCs. (A) Immunomodulatory activity of BMSCs ( n = 9 adult vs. n = 12 elderly and n = 14 non-diabetic vs. n = 7 diabetic donors, passage 3) to suppress anti-CD3/CD28- or phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cell (PBMC)-proliferation. The PBMCs were labeled with the cell proliferation-tracker dye CFSE, activated with either of the two different stimuli, and cocultured for 5 days with BMSCs from adult or elderly donors, in order to assess their capacity to inhibit the proliferation of CD4+ and CD8+ T-cells with flow cytometry, with representative histograms shown to the right. The quantitative assessment of T-cell proliferation is expressed as the percentage proliferation of CD4+ and CD8+ T cells relative to the positive control without BMSCs. Both adult vs. elderly and non-diabetic vs. diabetic donor-derived MSCs are equally potent in inhibiting CD4 and CD8 T-cell proliferation. (B,C) Paracrine and proangiogenic activity of BMSCs ( n = 9 adult vs. n = 12 elderly and n = 14 non-diabetic vs. n = 7 diabetic donors) with and without cytokine stimulation (10 ng/mL of TNF-alpha and IFN-gamma for 24 h): (B) secretion of IL-6 and VEGF (pg/mL) in BMSC-conditioned culture media was assessed with ELISA, detecting elevated levels of IL-6 secretion by BMSCs obtained from elderly or diabetic donors, and (C) proangiogenic activity of BMSC-conditioned media compared to blank group (medium only) in an endothelial tube formation assay, with a representative image for the quantification of endothelial network formation by quantification of the total master segment length (TMSL/field, with 3–5 images assessed per test condition); TMSL/field was slightly increased for adult and non-diabetic donors. Results are given as box plot ± min-max whiskers. Statistical analysis was performed using either a Student's t -test or ANOVA followed by post-tests (* p
    Figure Legend Snippet: Immunomodulatory and paracrine activity of BMSCs. (A) Immunomodulatory activity of BMSCs ( n = 9 adult vs. n = 12 elderly and n = 14 non-diabetic vs. n = 7 diabetic donors, passage 3) to suppress anti-CD3/CD28- or phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cell (PBMC)-proliferation. The PBMCs were labeled with the cell proliferation-tracker dye CFSE, activated with either of the two different stimuli, and cocultured for 5 days with BMSCs from adult or elderly donors, in order to assess their capacity to inhibit the proliferation of CD4+ and CD8+ T-cells with flow cytometry, with representative histograms shown to the right. The quantitative assessment of T-cell proliferation is expressed as the percentage proliferation of CD4+ and CD8+ T cells relative to the positive control without BMSCs. Both adult vs. elderly and non-diabetic vs. diabetic donor-derived MSCs are equally potent in inhibiting CD4 and CD8 T-cell proliferation. (B,C) Paracrine and proangiogenic activity of BMSCs ( n = 9 adult vs. n = 12 elderly and n = 14 non-diabetic vs. n = 7 diabetic donors) with and without cytokine stimulation (10 ng/mL of TNF-alpha and IFN-gamma for 24 h): (B) secretion of IL-6 and VEGF (pg/mL) in BMSC-conditioned culture media was assessed with ELISA, detecting elevated levels of IL-6 secretion by BMSCs obtained from elderly or diabetic donors, and (C) proangiogenic activity of BMSC-conditioned media compared to blank group (medium only) in an endothelial tube formation assay, with a representative image for the quantification of endothelial network formation by quantification of the total master segment length (TMSL/field, with 3–5 images assessed per test condition); TMSL/field was slightly increased for adult and non-diabetic donors. Results are given as box plot ± min-max whiskers. Statistical analysis was performed using either a Student's t -test or ANOVA followed by post-tests (* p

    Techniques Used: Activity Assay, Labeling, Flow Cytometry, Cytometry, Positive Control, Derivative Assay, Enzyme-linked Immunosorbent Assay, Endothelial Tube Formation Assay

    11) Product Images from "1,25-Dihydroxyvitamin D3 Ameliorates Collagen-Induced Arthritis via Suppression of Th17 Cells Through miR-124 Mediated Inhibition of IL-6 Signaling"

    Article Title: 1,25-Dihydroxyvitamin D3 Ameliorates Collagen-Induced Arthritis via Suppression of Th17 Cells Through miR-124 Mediated Inhibition of IL-6 Signaling

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00178

    VD could also function directly on T cells to shape T cell responses. CD4 + CD62L + cells from C57BL/6J or C57BL6 Foxp3 gfp reporter mice were cultured in the Th0 or Th17-polarizing conditions with immobilized anti-CD3 and soluble anti-CD28 instead of APCs, in the presence or absence of 1uM VD. (A,B) qRT-PCR results showed VDR and CYP24 mRNA relative expression on CD4 + T cells stimulated in Th0 or Th17 conditions without APCs for 24 h in CTRL (without VD) and VD groups. Data are presented as the mean ± SEM. * P
    Figure Legend Snippet: VD could also function directly on T cells to shape T cell responses. CD4 + CD62L + cells from C57BL/6J or C57BL6 Foxp3 gfp reporter mice were cultured in the Th0 or Th17-polarizing conditions with immobilized anti-CD3 and soluble anti-CD28 instead of APCs, in the presence or absence of 1uM VD. (A,B) qRT-PCR results showed VDR and CYP24 mRNA relative expression on CD4 + T cells stimulated in Th0 or Th17 conditions without APCs for 24 h in CTRL (without VD) and VD groups. Data are presented as the mean ± SEM. * P

    Techniques Used: Mouse Assay, Cell Culture, Quantitative RT-PCR, Expressing

    VD restrained Th17 cells differentiation through miR-124 mediated inhibition of IL-6 signaling. CD4 + CD62L + cells from C57BL/6J were cultured in the Th17-polarizing conditions with immobilized anti-CD3 and soluble anti-CD28 with or without VD. In some experiments, naïve CD4 + T cells were transduced with 10 nM miR-124 inhibitor for 24 h using Lipofectamine® 3000 before polarized into Th17 cells. (A) qRT-PCR showed that VD upregulates miR-124 expression in Th17 cells (72 h). (B–D) naïve CD4 + T cells were transduced with miR-124 inhibitor (i) or inhibitor control (iNC) before Th17-polarization using immobilized anti-CD3 or APCs system and flow cytometry was used to confirm IL-17 expression at protein level. Data are presented as the mean ± SEM ( n = 4). (E,F) naïve CD4 + T cells were transduced with miR-124 inhibitor (+) or inhibitor control (–) before Th17-polarization using immobilized anti-CD3. CD126 expression and (p)-STAT3 activity were checked by western blots. Data are presented as the mean ± SEM. NS means no significance, * P
    Figure Legend Snippet: VD restrained Th17 cells differentiation through miR-124 mediated inhibition of IL-6 signaling. CD4 + CD62L + cells from C57BL/6J were cultured in the Th17-polarizing conditions with immobilized anti-CD3 and soluble anti-CD28 with or without VD. In some experiments, naïve CD4 + T cells were transduced with 10 nM miR-124 inhibitor for 24 h using Lipofectamine® 3000 before polarized into Th17 cells. (A) qRT-PCR showed that VD upregulates miR-124 expression in Th17 cells (72 h). (B–D) naïve CD4 + T cells were transduced with miR-124 inhibitor (i) or inhibitor control (iNC) before Th17-polarization using immobilized anti-CD3 or APCs system and flow cytometry was used to confirm IL-17 expression at protein level. Data are presented as the mean ± SEM ( n = 4). (E,F) naïve CD4 + T cells were transduced with miR-124 inhibitor (+) or inhibitor control (–) before Th17-polarization using immobilized anti-CD3. CD126 expression and (p)-STAT3 activity were checked by western blots. Data are presented as the mean ± SEM. NS means no significance, * P

    Techniques Used: Inhibition, Cell Culture, Transduction, Quantitative RT-PCR, Expressing, Flow Cytometry, Cytometry, Activity Assay, Western Blot

    12) Product Images from "Cell-templated Supported Lipid Bilayers for T Cell Activation"

    Article Title: Cell-templated Supported Lipid Bilayers for T Cell Activation

    Journal: Advanced healthcare materials

    doi: 10.1002/adhm.201801188

    Activation particle characterization. (a) Scanning electron microscope (SEM) images of red blood cell, microsphere, and HeLa silica microparticles. (b) Fluorescent and brightfield microscope images of activation particles at 20× magnification. (c) Biotin loading on activation particles quantified by FITC-neutravidin and flow cytometry. (d) Ratiometric loading of anti-CD3ε and anti-CD28 antibodies with fluorescently labeled neutravidin and flow cytometry.
    Figure Legend Snippet: Activation particle characterization. (a) Scanning electron microscope (SEM) images of red blood cell, microsphere, and HeLa silica microparticles. (b) Fluorescent and brightfield microscope images of activation particles at 20× magnification. (c) Biotin loading on activation particles quantified by FITC-neutravidin and flow cytometry. (d) Ratiometric loading of anti-CD3ε and anti-CD28 antibodies with fluorescently labeled neutravidin and flow cytometry.

    Techniques Used: Activation Assay, Microscopy, Flow Cytometry, Cytometry, Labeling

    13) Product Images from "Severe immunosuppression and not a cytokine storm characterizes COVID-19 infections"

    Article Title: Severe immunosuppression and not a cytokine storm characterizes COVID-19 infections

    Journal: JCI Insight

    doi: 10.1172/jci.insight.140329

    IL-7 restores adaptive immune function in patients with COVID-19. Line plot demonstrating change in the number of cytokine-producing cells using ELISpot between control (anti-CD3/anti-CD28 antibody or LPS) samples and stimulation with IL-7 for IFN-ɣ ( A ) and TNF-α ( B ). Each dot represents and individual patient. Red lines represent values for patients who died. IL-7 caused a significant increase in the number of IFN-ɣ–producing T cells in COVID-19 patients; **** P
    Figure Legend Snippet: IL-7 restores adaptive immune function in patients with COVID-19. Line plot demonstrating change in the number of cytokine-producing cells using ELISpot between control (anti-CD3/anti-CD28 antibody or LPS) samples and stimulation with IL-7 for IFN-ɣ ( A ) and TNF-α ( B ). Each dot represents and individual patient. Red lines represent values for patients who died. IL-7 caused a significant increase in the number of IFN-ɣ–producing T cells in COVID-19 patients; **** P

    Techniques Used: Enzyme-linked Immunospot

    Adaptive immune suppression in COVID-19 patients. Representative ELISpot photomicrographs displaying IFN-ɣ production following overnight stimulation with anti-CD3/anti-CD28 antibodies for ( A ) healthy volunteers, ( B ) CINS patients, and ( C ) septic non–COVID-19 patients. ( D ) Three representative COVID-19–positive samples. Number of spots demonstrates the number of cytokine-producing T cells. Counts are presented as the corrected number of spots per thousand lymphocytes plated as fraction of the 2.5 × 10 4 PBMCs plated in each well. Note the reduction in IFN-ɣ production in both septic and COVID-19 patients compared with CINS patients. Note also a degree of heterogeneity in IFN-ɣ production in COVID-19 and septic patients. Each photomicrograph was captured with the same magnification, and each image is to scale. ELISpot assays were performed using the PBMC fraction from freshly drawn whole blood. Each condition was run in duplicate for control samples and triplicate for COVID-19 samples.
    Figure Legend Snippet: Adaptive immune suppression in COVID-19 patients. Representative ELISpot photomicrographs displaying IFN-ɣ production following overnight stimulation with anti-CD3/anti-CD28 antibodies for ( A ) healthy volunteers, ( B ) CINS patients, and ( C ) septic non–COVID-19 patients. ( D ) Three representative COVID-19–positive samples. Number of spots demonstrates the number of cytokine-producing T cells. Counts are presented as the corrected number of spots per thousand lymphocytes plated as fraction of the 2.5 × 10 4 PBMCs plated in each well. Note the reduction in IFN-ɣ production in both septic and COVID-19 patients compared with CINS patients. Note also a degree of heterogeneity in IFN-ɣ production in COVID-19 and septic patients. Each photomicrograph was captured with the same magnification, and each image is to scale. ELISpot assays were performed using the PBMC fraction from freshly drawn whole blood. Each condition was run in duplicate for control samples and triplicate for COVID-19 samples.

    Techniques Used: Enzyme-linked Immunospot

    Functional immune cytokine production measured by ELISpot in COVID-19, CINS, and septic patients and healthy volunteers. Comparison graphs for ex vivo cytokine production using ELISpot, comparing healthy volunteers and CINS, septic, and COVID-19 patients. ( A ) Number of spots per 1000 lymphocytes plated following overnight culture stimulated with anti-CD3/anti-CD28 for IFN-ɣ samples. ( B ) Number of spots per 1000 myeloid cells plated, stimulated with LPS for TNF-α production. Each dot represents an individual patient. Red dots represent nonsurvivors. Horizontal bars represent mean ± SEM. Healthy n = 27 for IFN-ɣ, 28 for TNF-α; CINS n = 18; septic n = 46; COVID-19 n = 25 for IFN-ɣ, 24 for TNF-α. ANOVA comparing all groups for IFN-γ production showed that there was a difference between COVID-19 and the other groups ( P = 0.003); and for TNF-α groups there was a statistically significant difference as well ( P = 0.009). ** P
    Figure Legend Snippet: Functional immune cytokine production measured by ELISpot in COVID-19, CINS, and septic patients and healthy volunteers. Comparison graphs for ex vivo cytokine production using ELISpot, comparing healthy volunteers and CINS, septic, and COVID-19 patients. ( A ) Number of spots per 1000 lymphocytes plated following overnight culture stimulated with anti-CD3/anti-CD28 for IFN-ɣ samples. ( B ) Number of spots per 1000 myeloid cells plated, stimulated with LPS for TNF-α production. Each dot represents an individual patient. Red dots represent nonsurvivors. Horizontal bars represent mean ± SEM. Healthy n = 27 for IFN-ɣ, 28 for TNF-α; CINS n = 18; septic n = 46; COVID-19 n = 25 for IFN-ɣ, 24 for TNF-α. ANOVA comparing all groups for IFN-γ production showed that there was a difference between COVID-19 and the other groups ( P = 0.003); and for TNF-α groups there was a statistically significant difference as well ( P = 0.009). ** P

    Techniques Used: Functional Assay, Enzyme-linked Immunospot, Ex Vivo

    14) Product Images from "The transcription factor Zfp281 sustains CD4+ T lymphocyte activation through directly repressing Ctla-4 transcription"

    Article Title: The transcription factor Zfp281 sustains CD4+ T lymphocyte activation through directly repressing Ctla-4 transcription

    Journal: Cellular and Molecular Immunology

    doi: 10.1038/s41423-019-0289-y

    Zfp281-deficient CD4 + T cells show decreased expression of T-cell activation-related genes. a Scatter plot of the expression of all genes in WT and Zfp281-deficient naive CD4 + T cells stimulated with the anti-CD3 mAb for 4 h in the presence of APC detected by RNA-Seq analysis. Downregulated genes are indicated in blue, upregulated genes are indicated in red. b Gene ontology (GO) analysis of the differentially expressed genes as shown in a . Blue, downregulated genes; red, upregulated genes. c Heat map comparing the indicated mRNA transcripts. d q-PCR analysis of the Ctla-4 transcript levels in naive CD4 + T cells from WT and cKO mice unstimulated or stimulated with coated anti-CD3 (5 μg/mL) and soluble anti-CD28 (3 μg/mL) for 4 h. e CTLA-4 expression in Tconv cells with or without plate-bound anti-CD3 stimulation for 8 h. f Detection of CTLA-4 in Tconv cells from mice with the OT-II background treated with or without the OVA peptide for 8 h. Data are representative of three independent experiments (mean ± s.d.). A two-tailed, unpaired t -test was used. NS not significant. * P
    Figure Legend Snippet: Zfp281-deficient CD4 + T cells show decreased expression of T-cell activation-related genes. a Scatter plot of the expression of all genes in WT and Zfp281-deficient naive CD4 + T cells stimulated with the anti-CD3 mAb for 4 h in the presence of APC detected by RNA-Seq analysis. Downregulated genes are indicated in blue, upregulated genes are indicated in red. b Gene ontology (GO) analysis of the differentially expressed genes as shown in a . Blue, downregulated genes; red, upregulated genes. c Heat map comparing the indicated mRNA transcripts. d q-PCR analysis of the Ctla-4 transcript levels in naive CD4 + T cells from WT and cKO mice unstimulated or stimulated with coated anti-CD3 (5 μg/mL) and soluble anti-CD28 (3 μg/mL) for 4 h. e CTLA-4 expression in Tconv cells with or without plate-bound anti-CD3 stimulation for 8 h. f Detection of CTLA-4 in Tconv cells from mice with the OT-II background treated with or without the OVA peptide for 8 h. Data are representative of three independent experiments (mean ± s.d.). A two-tailed, unpaired t -test was used. NS not significant. * P

    Techniques Used: Expressing, Activation Assay, RNA Sequencing Assay, Polymerase Chain Reaction, Mouse Assay, Two Tailed Test

    Zfp281 expression is induced by TCR stimulation in CD4 + T cells and does not affect T-cell development. a q-PCR detection of the Zfp281 expression levels in different T-cell subsets, including DN (Lin − CD4 − CD8 − ), DP (CD4 + CD8 + ), CD4 SP (TCRβ + CD4 + CD8 − ), CD8 SP (TCRβ + CD4 − CD8 + ) thymocytes and splenic CD4 + , CD8 + T cells ( n = 3). b Immunoblot detection of the Zfp281 protein in sorted CD4 + , CD8 + splenic T cells. q-PCR ( c ) and immunoblot analysis ( d ) of Zfp281 in purified CD4 + splenic T cells stimulated with anti-CD3 and anti-CD28 mAbs for the indicated times. The results are presented relative to Actb expression ( n = 3). N.S, non-specific band. e Flow cytometric analysis and quantification of the DN, DP, CD4 SP, and CD8 SP thymocyte subpopulations from Zfp281 fl/fl (WT) and Zfp281 fl/fl Cd4 Cre (cKO) mice ( n = 5). f Flow cytometric analysis of CD4 and CD8 expression in splenocytes from the indicated mice, and quantitation of the absolute cell numbers ( n = 5). Data are presented as the mean ± s.d. A two-tailed, unpaired t -test was used. NS not significant. * P
    Figure Legend Snippet: Zfp281 expression is induced by TCR stimulation in CD4 + T cells and does not affect T-cell development. a q-PCR detection of the Zfp281 expression levels in different T-cell subsets, including DN (Lin − CD4 − CD8 − ), DP (CD4 + CD8 + ), CD4 SP (TCRβ + CD4 + CD8 − ), CD8 SP (TCRβ + CD4 − CD8 + ) thymocytes and splenic CD4 + , CD8 + T cells ( n = 3). b Immunoblot detection of the Zfp281 protein in sorted CD4 + , CD8 + splenic T cells. q-PCR ( c ) and immunoblot analysis ( d ) of Zfp281 in purified CD4 + splenic T cells stimulated with anti-CD3 and anti-CD28 mAbs for the indicated times. The results are presented relative to Actb expression ( n = 3). N.S, non-specific band. e Flow cytometric analysis and quantification of the DN, DP, CD4 SP, and CD8 SP thymocyte subpopulations from Zfp281 fl/fl (WT) and Zfp281 fl/fl Cd4 Cre (cKO) mice ( n = 5). f Flow cytometric analysis of CD4 and CD8 expression in splenocytes from the indicated mice, and quantitation of the absolute cell numbers ( n = 5). Data are presented as the mean ± s.d. A two-tailed, unpaired t -test was used. NS not significant. * P

    Techniques Used: Expressing, Polymerase Chain Reaction, Purification, Mouse Assay, Quantitation Assay, Two Tailed Test

    TCR signaling is interrupted in Zfp281-deficient CD4 + T cells. a Immunoblot analysis of total PLC-γ1 and phosphorylated (p-) PLC-γ1 and ZAP-70 in extracts of sorted naive CD4 + T cells from WT and cKO splenocytes that were stimulated for 0, 5, 10, and 20 min with the anti-CD3 mAb and anti-CD28 mAb. b Immunoblot analysis of total and phosphorylated (p-) ERK1/2, JNK and P38 in extracts of cells stimulated as in a . c Calcium flux in naive CD4 + T cells from Zfp281 fl/fl and Zfp281 fl/fl Cd4 Cre mice after stimulation with anti-CD3 and anti-CD28 mAbs. d Immunoblot analysis of NFAT1 in nuclear extracts of naive CD4 + T cells stimulated as in a . The numbers below the lanes ( a , b , d ) indicate densitometry analysis. Data are representative of at least three independent experiments
    Figure Legend Snippet: TCR signaling is interrupted in Zfp281-deficient CD4 + T cells. a Immunoblot analysis of total PLC-γ1 and phosphorylated (p-) PLC-γ1 and ZAP-70 in extracts of sorted naive CD4 + T cells from WT and cKO splenocytes that were stimulated for 0, 5, 10, and 20 min with the anti-CD3 mAb and anti-CD28 mAb. b Immunoblot analysis of total and phosphorylated (p-) ERK1/2, JNK and P38 in extracts of cells stimulated as in a . c Calcium flux in naive CD4 + T cells from Zfp281 fl/fl and Zfp281 fl/fl Cd4 Cre mice after stimulation with anti-CD3 and anti-CD28 mAbs. d Immunoblot analysis of NFAT1 in nuclear extracts of naive CD4 + T cells stimulated as in a . The numbers below the lanes ( a , b , d ) indicate densitometry analysis. Data are representative of at least three independent experiments

    Techniques Used: Planar Chromatography, Mouse Assay

    15) Product Images from "Targeting Interleukin-2-Inducible T-Cell Kinase (ITK) Differentiates GVL and GVHD in Allo-HSCT"

    Article Title: Targeting Interleukin-2-Inducible T-Cell Kinase (ITK) Differentiates GVL and GVHD in Allo-HSCT

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.593863

    ITK deficiency results in reduced cytokine production. (A) Serum from several GVHD patients was isolated and examined for inflammatory cytokine production (IL-33, IL1α, IFN-γ and TNF-α, IL1β and IL-17A) as determined by ELISA. (B , C) 1 × 10 6 purified WT or Itk -/- CD8 + T or CD4 + T cells were separately transplanted with into irradiated BALB/c mice. At day 7 post allo-HSCT, recipient BALB/c were euthanized and serum cytokines (IL-33, IL1α, IFN-γ, and TNF-α and IL-17A) were measured by ELISA. (D) Intracellular IFN-γ and TNF-α expression by donor CD8 + and CD4 + T cells after stimulation with anti-CD3/anti-CD28 as determined by flow cytometry. (E , F) Combined data from 3 independent experiments is shown for each experiment shown in figures. (F) Flow cytometry analysis of purified WT and Itk -/- T cells that were mixed at a 1:1 ratio for transplantation into irradiated BALB/c mice. At day 7 donor T cells were gated for expression of H-2K b , CD45.1, and CD45.2 and intracellular expression of IFN-γ and TNF-α was analyzed by flow cytometry after stimulation with anti-CD3/anti-CD28. Combined data from four independent experiments is shown, and the p value for each experiment is shown. (G) Purified WT or Itk -/- donor CD8 + and CD4 + T cells were transplanted into irradiated BALB/c. At day 7 donor cells were analyzed for donor T cell proliferation by examining BrdU incorporation by flow cytometry. (H) Purified WT and Itk -/- donor T cells were mixed at a 1:1 WT: Itk -/- ratio and transplanted into irradiated BALB/c mice, at day 7 splenic donor T cells were gated for the expression of H-2K b , CD45.1, and CD45.2 and analyzed for BrDU incorporation. (I) Purified WT and Itk -/- T cells were stimulated with CD3 and CD28 overnight examined for the expression and phosphorylation of IRF4, JAK1/2 and STAT3 by western blot. For statistical analysis we used two-way ANOVA and student’s t test, p values are presented.
    Figure Legend Snippet: ITK deficiency results in reduced cytokine production. (A) Serum from several GVHD patients was isolated and examined for inflammatory cytokine production (IL-33, IL1α, IFN-γ and TNF-α, IL1β and IL-17A) as determined by ELISA. (B , C) 1 × 10 6 purified WT or Itk -/- CD8 + T or CD4 + T cells were separately transplanted with into irradiated BALB/c mice. At day 7 post allo-HSCT, recipient BALB/c were euthanized and serum cytokines (IL-33, IL1α, IFN-γ, and TNF-α and IL-17A) were measured by ELISA. (D) Intracellular IFN-γ and TNF-α expression by donor CD8 + and CD4 + T cells after stimulation with anti-CD3/anti-CD28 as determined by flow cytometry. (E , F) Combined data from 3 independent experiments is shown for each experiment shown in figures. (F) Flow cytometry analysis of purified WT and Itk -/- T cells that were mixed at a 1:1 ratio for transplantation into irradiated BALB/c mice. At day 7 donor T cells were gated for expression of H-2K b , CD45.1, and CD45.2 and intracellular expression of IFN-γ and TNF-α was analyzed by flow cytometry after stimulation with anti-CD3/anti-CD28. Combined data from four independent experiments is shown, and the p value for each experiment is shown. (G) Purified WT or Itk -/- donor CD8 + and CD4 + T cells were transplanted into irradiated BALB/c. At day 7 donor cells were analyzed for donor T cell proliferation by examining BrdU incorporation by flow cytometry. (H) Purified WT and Itk -/- donor T cells were mixed at a 1:1 WT: Itk -/- ratio and transplanted into irradiated BALB/c mice, at day 7 splenic donor T cells were gated for the expression of H-2K b , CD45.1, and CD45.2 and analyzed for BrDU incorporation. (I) Purified WT and Itk -/- T cells were stimulated with CD3 and CD28 overnight examined for the expression and phosphorylation of IRF4, JAK1/2 and STAT3 by western blot. For statistical analysis we used two-way ANOVA and student’s t test, p values are presented.

    Techniques Used: Isolation, Enzyme-linked Immunosorbent Assay, Purification, Irradiation, Mouse Assay, Expressing, Flow Cytometry, Transplantation Assay, BrdU Incorporation Assay, Western Blot

    16) Product Images from "Fine phenotypic and functional characterization of effector CD8+ T cells in patients with Primary Biliary Cirrhosis"

    Article Title: Fine phenotypic and functional characterization of effector CD8+ T cells in patients with Primary Biliary Cirrhosis

    Journal: Hepatology (Baltimore, Md.)

    doi: 10.1002/hep.24526

    Analysis of the expression of CXCR3, CCR5, CCR7, CCR9, CD28 and α4β7 on CD45RO high CD57 + CD8 high gated T lymphocytes from healthy controls (cont), all PBC patients (PBC), PBC patients at early stages (I/II) and late stages (III/IV). Bars
    Figure Legend Snippet: Analysis of the expression of CXCR3, CCR5, CCR7, CCR9, CD28 and α4β7 on CD45RO high CD57 + CD8 high gated T lymphocytes from healthy controls (cont), all PBC patients (PBC), PBC patients at early stages (I/II) and late stages (III/IV). Bars

    Techniques Used: Expressing

    17) Product Images from "Dual function of polycomb group proteins in differentiated murine T helper (CD4+) cells"

    Article Title: Dual function of polycomb group proteins in differentiated murine T helper (CD4+) cells

    Journal: Journal of Molecular Signaling

    doi: 10.1186/1750-2187-6-5

    Mel-18 is a positive regulator of cytokine genes in D5 and D10 cells . (A) ChIP experiment assessing the binding of Mel-18 at the Ifng (left) and Il4 (right) promoters in D5 and D10 cells. The cells were stimulated with antigen-presenting cells (APCs) with the appropriate peptides, cultured for 2-3 weeks, and then left unstimulated or were re-stimulated (P+I) for the indicated time points. In the panel of the Ifng promoter the result of 1 hr stimulation of D5 cells was set as 1, and in the Il4 promoter panel the result of 1 hr stimulation in D10 was set as 1. (B) Quantitative RT-PCR for the indicated mRNAs following Mel-18 knockdown in D5 (left) and D10 (right) cells. The cells were re-stimulated with APCs with the appropriate peptides, transduced with lentiviral shRNAs, cultured in the presence of puromycin for 2-3 weeks, and then re-stimulated with anti-CD3 and anti-CD28 antibodies for 2 hours. The results are presented relative to the control, defined as 1. Differences between knockdown and control with p values ≤ 0.05 are indicated with an asterisk (C) Intracellular staining of Mel-18 in the indicated transduced D5 and D10 cells. (D) Quantitative RT-PCR for Mel-18 in resting and 2-hour-restimulated cells as indicated. The expression level in resting Th1 cells was set as 1. The results in Figure 5 are the mean of three to six independent experiments +S.D., except panel 5C, which is a representative experiment.
    Figure Legend Snippet: Mel-18 is a positive regulator of cytokine genes in D5 and D10 cells . (A) ChIP experiment assessing the binding of Mel-18 at the Ifng (left) and Il4 (right) promoters in D5 and D10 cells. The cells were stimulated with antigen-presenting cells (APCs) with the appropriate peptides, cultured for 2-3 weeks, and then left unstimulated or were re-stimulated (P+I) for the indicated time points. In the panel of the Ifng promoter the result of 1 hr stimulation of D5 cells was set as 1, and in the Il4 promoter panel the result of 1 hr stimulation in D10 was set as 1. (B) Quantitative RT-PCR for the indicated mRNAs following Mel-18 knockdown in D5 (left) and D10 (right) cells. The cells were re-stimulated with APCs with the appropriate peptides, transduced with lentiviral shRNAs, cultured in the presence of puromycin for 2-3 weeks, and then re-stimulated with anti-CD3 and anti-CD28 antibodies for 2 hours. The results are presented relative to the control, defined as 1. Differences between knockdown and control with p values ≤ 0.05 are indicated with an asterisk (C) Intracellular staining of Mel-18 in the indicated transduced D5 and D10 cells. (D) Quantitative RT-PCR for Mel-18 in resting and 2-hour-restimulated cells as indicated. The expression level in resting Th1 cells was set as 1. The results in Figure 5 are the mean of three to six independent experiments +S.D., except panel 5C, which is a representative experiment.

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Cell Culture, Quantitative RT-PCR, Transduction, Staining, Expressing

    18) Product Images from "The chaperonin CCT8 controls proteostasis essential for T cell maturation, selection, and function"

    Article Title: The chaperonin CCT8 controls proteostasis essential for T cell maturation, selection, and function

    Journal: bioRxiv

    doi: 10.1101/2020.08.11.246215

    Proteomic analysis of resting and activated CD4 + T cells from CCT8 T+/+ and CCT8 T−/− mice. ( a ) T cells activated in vitro by CD3/CD28 cross linking. (i) Expansion index; (ii) normalised cell viability in the presence or absence of exogenous IL-2. ( b ) Expression of individual CCT subunits (i) and the CCT substrates actin and tubulin measured by mass spectrometry, ( c ) Nuclear actin filaments in activated CD4 + T cells in the presence of absence of CK-666, an inhibitor of Arp2/3. (i) STED images with segmentation of the nuclear actin filaments (right panels), and (ii) frequency of nuclear actin filaments detected in CCT8 T+/+ (white bars) and CCT8 T−/− T cells (grey bars). ( d ) Differentially expressed proteins in resting (0h) and activated (24h) T cells. ( e ) Go analysis of cellular components enriched in CCT8 T+/+ CD4 + cells at 0 hrs and 24 hrs (ii) after stimulation **p
    Figure Legend Snippet: Proteomic analysis of resting and activated CD4 + T cells from CCT8 T+/+ and CCT8 T−/− mice. ( a ) T cells activated in vitro by CD3/CD28 cross linking. (i) Expansion index; (ii) normalised cell viability in the presence or absence of exogenous IL-2. ( b ) Expression of individual CCT subunits (i) and the CCT substrates actin and tubulin measured by mass spectrometry, ( c ) Nuclear actin filaments in activated CD4 + T cells in the presence of absence of CK-666, an inhibitor of Arp2/3. (i) STED images with segmentation of the nuclear actin filaments (right panels), and (ii) frequency of nuclear actin filaments detected in CCT8 T+/+ (white bars) and CCT8 T−/− T cells (grey bars). ( d ) Differentially expressed proteins in resting (0h) and activated (24h) T cells. ( e ) Go analysis of cellular components enriched in CCT8 T+/+ CD4 + cells at 0 hrs and 24 hrs (ii) after stimulation **p

    Techniques Used: Mouse Assay, In Vitro, Expressing, Mass Spectrometry

    Peripheral T cell functions in the absence of CCT8 expression. Analysis of naïve CD4 + T cells from 4-6 week old CCT8 T+/+ (grey bars) and CCT8 T−/− mice (white bars). ( a) qPCR analysis of ER stress response elements in T cells activated by CD3 and CD28 crosslinking and cultured in the presence or absence of tunicamycin; expression normalised to GPDH and displayed as 2 −ΔΔCt CT values relative to values from CCT8 T+/+ T cells arbitrarily set 1. ( b ) In vitro differentiation of peripheral naïve CD4 + T cells grown for 5 days under differentiating conditions: frequency of live cells (left) and cells (right) adopting (i) Th1 polarisation; (ii) Th2 polarisation (iii); T reg differentiation; and (iv) Th17 differentiation. ( c ) (i) Uptake of fatty acids in CD4+ CD62L+CD44-(naïve) and CD62L-CD44+ (memory) cells ex vivo activated by CD3 and CD28 cross-linking for 20h; (ii) expression of long-chain fatty acid receptor CD36 on CD3/CD28-activated cells, and (iii) uptake of glucose analogue 6-NBDG in CD4+ CD62L+CD44-(naïve) and CD62L-CD44+ (memory) cells.*p
    Figure Legend Snippet: Peripheral T cell functions in the absence of CCT8 expression. Analysis of naïve CD4 + T cells from 4-6 week old CCT8 T+/+ (grey bars) and CCT8 T−/− mice (white bars). ( a) qPCR analysis of ER stress response elements in T cells activated by CD3 and CD28 crosslinking and cultured in the presence or absence of tunicamycin; expression normalised to GPDH and displayed as 2 −ΔΔCt CT values relative to values from CCT8 T+/+ T cells arbitrarily set 1. ( b ) In vitro differentiation of peripheral naïve CD4 + T cells grown for 5 days under differentiating conditions: frequency of live cells (left) and cells (right) adopting (i) Th1 polarisation; (ii) Th2 polarisation (iii); T reg differentiation; and (iv) Th17 differentiation. ( c ) (i) Uptake of fatty acids in CD4+ CD62L+CD44-(naïve) and CD62L-CD44+ (memory) cells ex vivo activated by CD3 and CD28 cross-linking for 20h; (ii) expression of long-chain fatty acid receptor CD36 on CD3/CD28-activated cells, and (iii) uptake of glucose analogue 6-NBDG in CD4+ CD62L+CD44-(naïve) and CD62L-CD44+ (memory) cells.*p

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Cell Culture, In Vitro, Ex Vivo

    19) Product Images from "Microglia Require CD4 T Cells to Complete the Fetal-to-Adult Transition"

    Article Title: Microglia Require CD4 T Cells to Complete the Fetal-to-Adult Transition

    Journal: Cell

    doi: 10.1016/j.cell.2020.06.026

    A Conserved Residency Program for CD4 T Cells and Tregs in the Healthy Mouse and Human Brain (A) Healthy perfused mouse brains were compared to blood by high-dimensional flow cytometry. n = 5. t-Distributed Stochastic Neighbor Embedding (t-SNE) of conventional T cells built on key markers (CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, and CTLA4). Colors indicate FlowSOM clusters, quantified in side panel. (B) Representative histograms for conventional T cells from wild-type mouse blood and brain. (C) t-SNE of Tregs built on key markers. Colors indicate FlowSOM clusters, quantified in side panel. (D) Representative histograms for Tregs from mouse blood and brain. (E) Unaffected human brain tissues were compared to peripheral blood mononuclear cells (PBMCs) by high-dimensional flow cytometry, n = 4. t-SNE of conventional T cells built on key markers (ICOS, CD28, CD69, Ki-67, CD95, CD31, HLA-DR, CCR2, CXCR5, CD25, PD-1, CXCR3, RORγT, CCR4, CTLA-4, CCR7, and CD45RA). Colors indicate FlowSOM clusters. (F) Dendrogram showing the relationship across the brain regions based on cross-entropy differences in t-SNE. (G) 10× single-cell sequencing was performed on sorted CD4 T cells from the human brain and PBMCs. Quality control filtering and gating based on expression markers identified 86 CD4 + T cells from the brain and 567 CD4 + T cells from the blood. t-SNE visualizing cell clusters built on the combined population of 653 CD4 + cells. Clusters are identified with different colors and labeled based on signature expression of transcriptional markers (left) or tissue origin (right). (H) Volcano plot of differential expression between brain and PBMC CD4 T cells. Indicated cut-offs are used for pathway analysis. See also Figure S2 .
    Figure Legend Snippet: A Conserved Residency Program for CD4 T Cells and Tregs in the Healthy Mouse and Human Brain (A) Healthy perfused mouse brains were compared to blood by high-dimensional flow cytometry. n = 5. t-Distributed Stochastic Neighbor Embedding (t-SNE) of conventional T cells built on key markers (CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, and CTLA4). Colors indicate FlowSOM clusters, quantified in side panel. (B) Representative histograms for conventional T cells from wild-type mouse blood and brain. (C) t-SNE of Tregs built on key markers. Colors indicate FlowSOM clusters, quantified in side panel. (D) Representative histograms for Tregs from mouse blood and brain. (E) Unaffected human brain tissues were compared to peripheral blood mononuclear cells (PBMCs) by high-dimensional flow cytometry, n = 4. t-SNE of conventional T cells built on key markers (ICOS, CD28, CD69, Ki-67, CD95, CD31, HLA-DR, CCR2, CXCR5, CD25, PD-1, CXCR3, RORγT, CCR4, CTLA-4, CCR7, and CD45RA). Colors indicate FlowSOM clusters. (F) Dendrogram showing the relationship across the brain regions based on cross-entropy differences in t-SNE. (G) 10× single-cell sequencing was performed on sorted CD4 T cells from the human brain and PBMCs. Quality control filtering and gating based on expression markers identified 86 CD4 + T cells from the brain and 567 CD4 + T cells from the blood. t-SNE visualizing cell clusters built on the combined population of 653 CD4 + cells. Clusters are identified with different colors and labeled based on signature expression of transcriptional markers (left) or tissue origin (right). (H) Volcano plot of differential expression between brain and PBMC CD4 T cells. Indicated cut-offs are used for pathway analysis. See also Figure S2 .

    Techniques Used: Flow Cytometry, Sequencing, Expressing, Labeling

    A Conserved Residency Profile for CD4 T Cells and Regulatory T Cells in the Healthy Mouse and Human Brain, Related to Figure 2 (A) Healthy perfused mouse brains were compared to blood by high-dimensional flow cytometry. Comparison of expression levels of CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4 on blood versus brain CD4 + CD3 + CD45 + CD8 - Foxp3 - T cells or (B) CD4 + CD3 + CD45 + CD8 - Foxp3 + Tregs (n = 5). (C) Transcription profile of CD4 + T cells purified from the murine brain, with analysis through the 10X single cell pipeline and filtering for known cytokines. Naive, activated and regulatory cells were defined based on tSNE clusters and the relative expression of CD44, CD62L and Foxp3 within each cluster. (D) CD4 T cells were assessed in the perfused mouse brain by high-dimensional flow cytometry. Wild-type mice were sampled across the late embryonic (day 19), post-natal development (day 5, 10, 21, 30) and during healthy aging (weeks 8, 12, 30 and 52). n = 9,3,3,5,2,8,5,6,5. Quantification of CD4 + cells per gram of brain tissue, (E) CD4 + T cells, as percentage of CD45 + cells, (F) CD4 + Foxp3 - conventional T cells and (G) CD4 + Foxp3 + regulatory T cells. (H) tSNE of CD4 + Foxp3 - T cells gated on CD4 + Foxp3 - CD3 + CD8 - CD45 + cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4. FlowSOM clusters identified in color. P values refer to cross-entropy difference between age-matched blood and brain samples. Dendrogram represents cross-entropy distance between samples. (I) Comparison of expression levels of CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4 on blood versus brain CD4 + CD3 + CD45 + CD8 - Foxp3 - T cells at different ages. (G) tSNE of CD4 + Foxp3 + T cells gated on CD4 + Foxp3 + CD3 + CD8 - CD45 + cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4. FlowSOM clusters identified in color. P values refer to cross-entropy difference between age-matched blood and brain samples. (J) Comparison of expression levels of CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4 on blood versus brain CD4 + CD3 + CD45 + CD8 - Foxp3 + T cells at different ages. (K) Brain regions were surgically dissected and resident CD4 T cells were characterized by high-dimensional flow cytometry at 10 days, 30 weeks, 60 weeks and 90 weeks of age (n = 6,4,4,5). (L) Numbers and frequencies of CD4 T cells across brain regions in pups and adult mice. (M) tSNE of CD4 + Foxp3 - T cells gated on CD4 + Foxp3 - CD3 + CD8 - CD45 + cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4, shown for blood and different brain regions in adult mice. The adjusted P value reflects the cross-entropy difference between tSNE plots in brain region versus blood. (N) Dendrogram showing the relationship in Tconv across the brain regions based on cross-entropy differences in tSNE. (O) CD69 expression in Tconv from the brain regions in pups and adult mice. (P) Heatmap showing expression of markers in brain region Tconv. (Q) tSNE of CD4 + Foxp3 + T cells gated on CD4 + Foxp3 + CD3 + CD8 - CD45 + cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4, shown for blood and different brain regions. The adjusted P value reflects the cross-entropy difference between tSNE plots in brain region versus blood. (R) CD69 expression in Treg from the brain regions in pups and adult mice. (S) Heatmap showing expression of markers in brain region Treg. (T) Unaffected human brain tissues removed during brain surgery were compared to peripheral blood mononuclear cells by high-dimensional flow cytometry (n = 4). Representative histograms for CD4 + Foxp3 - T cells from human peripheral blood mononuclear cells, white matter, gray matter and meninges for CCR2, CXCR3, PD-1 and CD69. (U) Expression of ICOS, CD28, CD69, Ki-67, CD95, CD31, HLA-DR, CCR2, CXCR5, CD25, PD-1, CXCR3, RORγT, CCR4, CTLA-4, CCR7 and CD45RA on CD4 + Foxp3 - T cells. (V) tSNE of CD4 + Foxp3 + T cells gated on CD4 + Foxp3 + CD3 + CD8 - CD14 - cells and built on ICOS, CD28, CD69, Ki-67, CD95, CD31, HLA-DR, CCR2, CXCR5, CD25, PD-1, CXCR3, RORγT, CCR4, CTLA-4, CCR7 and CD45RA. Colors indicate FlowSOM clusters. Dendrogram showing the relationship across the brain regions based on cross-entropy differences in tSNE. (W) Representative histograms for CD4 + Foxp3 + T cells from human peripheral blood mononuclear cells, white matter, gray matter and meninges for CCR2, CXCR3, PD-1 and CD69. (X) Expression of ICOS, CD28, CD69, Ki-67, CD95, CD31, HLA-DR, CCR2, CXCR5, CD25, PD-1, CXCR3, RORγT, CCR4, CTLA-4, CCR7 and CD45RA on CD4 + Foxp3 + T cells. (Y) Single cell RNaseq data from sorted CD4 + CD3 + CD8 - cells from the human brain and PBMCs. Quality control filtering and gating based on expression markers identified 86 CD4 + T cells from the brain and 567 CD4 + T cells from the blood. tSNE dimensionality reduction is used for cluster display, with lineage marker expression indicated by color for CD3D , CD4 , IL7R , IL2RA , FOXP3 , CD44 , SELL , AREG , CD69 , KLRG1 and NR4A1 . (Z) Differentially expressed genes were assessed for pathway by GSEA.
    Figure Legend Snippet: A Conserved Residency Profile for CD4 T Cells and Regulatory T Cells in the Healthy Mouse and Human Brain, Related to Figure 2 (A) Healthy perfused mouse brains were compared to blood by high-dimensional flow cytometry. Comparison of expression levels of CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4 on blood versus brain CD4 + CD3 + CD45 + CD8 - Foxp3 - T cells or (B) CD4 + CD3 + CD45 + CD8 - Foxp3 + Tregs (n = 5). (C) Transcription profile of CD4 + T cells purified from the murine brain, with analysis through the 10X single cell pipeline and filtering for known cytokines. Naive, activated and regulatory cells were defined based on tSNE clusters and the relative expression of CD44, CD62L and Foxp3 within each cluster. (D) CD4 T cells were assessed in the perfused mouse brain by high-dimensional flow cytometry. Wild-type mice were sampled across the late embryonic (day 19), post-natal development (day 5, 10, 21, 30) and during healthy aging (weeks 8, 12, 30 and 52). n = 9,3,3,5,2,8,5,6,5. Quantification of CD4 + cells per gram of brain tissue, (E) CD4 + T cells, as percentage of CD45 + cells, (F) CD4 + Foxp3 - conventional T cells and (G) CD4 + Foxp3 + regulatory T cells. (H) tSNE of CD4 + Foxp3 - T cells gated on CD4 + Foxp3 - CD3 + CD8 - CD45 + cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4. FlowSOM clusters identified in color. P values refer to cross-entropy difference between age-matched blood and brain samples. Dendrogram represents cross-entropy distance between samples. (I) Comparison of expression levels of CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4 on blood versus brain CD4 + CD3 + CD45 + CD8 - Foxp3 - T cells at different ages. (G) tSNE of CD4 + Foxp3 + T cells gated on CD4 + Foxp3 + CD3 + CD8 - CD45 + cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4. FlowSOM clusters identified in color. P values refer to cross-entropy difference between age-matched blood and brain samples. (J) Comparison of expression levels of CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4 on blood versus brain CD4 + CD3 + CD45 + CD8 - Foxp3 + T cells at different ages. (K) Brain regions were surgically dissected and resident CD4 T cells were characterized by high-dimensional flow cytometry at 10 days, 30 weeks, 60 weeks and 90 weeks of age (n = 6,4,4,5). (L) Numbers and frequencies of CD4 T cells across brain regions in pups and adult mice. (M) tSNE of CD4 + Foxp3 - T cells gated on CD4 + Foxp3 - CD3 + CD8 - CD45 + cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4, shown for blood and different brain regions in adult mice. The adjusted P value reflects the cross-entropy difference between tSNE plots in brain region versus blood. (N) Dendrogram showing the relationship in Tconv across the brain regions based on cross-entropy differences in tSNE. (O) CD69 expression in Tconv from the brain regions in pups and adult mice. (P) Heatmap showing expression of markers in brain region Tconv. (Q) tSNE of CD4 + Foxp3 + T cells gated on CD4 + Foxp3 + CD3 + CD8 - CD45 + cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4, shown for blood and different brain regions. The adjusted P value reflects the cross-entropy difference between tSNE plots in brain region versus blood. (R) CD69 expression in Treg from the brain regions in pups and adult mice. (S) Heatmap showing expression of markers in brain region Treg. (T) Unaffected human brain tissues removed during brain surgery were compared to peripheral blood mononuclear cells by high-dimensional flow cytometry (n = 4). Representative histograms for CD4 + Foxp3 - T cells from human peripheral blood mononuclear cells, white matter, gray matter and meninges for CCR2, CXCR3, PD-1 and CD69. (U) Expression of ICOS, CD28, CD69, Ki-67, CD95, CD31, HLA-DR, CCR2, CXCR5, CD25, PD-1, CXCR3, RORγT, CCR4, CTLA-4, CCR7 and CD45RA on CD4 + Foxp3 - T cells. (V) tSNE of CD4 + Foxp3 + T cells gated on CD4 + Foxp3 + CD3 + CD8 - CD14 - cells and built on ICOS, CD28, CD69, Ki-67, CD95, CD31, HLA-DR, CCR2, CXCR5, CD25, PD-1, CXCR3, RORγT, CCR4, CTLA-4, CCR7 and CD45RA. Colors indicate FlowSOM clusters. Dendrogram showing the relationship across the brain regions based on cross-entropy differences in tSNE. (W) Representative histograms for CD4 + Foxp3 + T cells from human peripheral blood mononuclear cells, white matter, gray matter and meninges for CCR2, CXCR3, PD-1 and CD69. (X) Expression of ICOS, CD28, CD69, Ki-67, CD95, CD31, HLA-DR, CCR2, CXCR5, CD25, PD-1, CXCR3, RORγT, CCR4, CTLA-4, CCR7 and CD45RA on CD4 + Foxp3 + T cells. (Y) Single cell RNaseq data from sorted CD4 + CD3 + CD8 - cells from the human brain and PBMCs. Quality control filtering and gating based on expression markers identified 86 CD4 + T cells from the brain and 567 CD4 + T cells from the blood. tSNE dimensionality reduction is used for cluster display, with lineage marker expression indicated by color for CD3D , CD4 , IL7R , IL2RA , FOXP3 , CD44 , SELL , AREG , CD69 , KLRG1 and NR4A1 . (Z) Differentially expressed genes were assessed for pathway by GSEA.

    Techniques Used: Flow Cytometry, Expressing, Purification, Mouse Assay, Marker

    20) Product Images from "PD1-CD28 Fusion Protein Enables CD4+ T Cell Help for Adoptive T Cell Therapy in Models of Pancreatic Cancer and Non-hodgkin Lymphoma"

    Article Title: PD1-CD28 Fusion Protein Enables CD4+ T Cell Help for Adoptive T Cell Therapy in Models of Pancreatic Cancer and Non-hodgkin Lymphoma

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01955

    In vitro and in vivo assessment of anti-tumor efficacy of PD1-CD28 fusion receptor (PTM receptor)-transduced CD4+ and CD8+ T cells. (A) PTM-transduced, untransduced primary murine OT-1, PTM-transduced, untransduced primary murine OT-2 T cells, or OT-1 together with OT-2 T cells were prestimulated for 24 h with anti-CD3 antibody plus recombinant PD-L1. T cells were then cocultured with Panc02-OVA or E.G7-PD-L1 cells. Interferon-γ (IFN-γ) secretion was measured by enzyme linked immunosorbent assay (ELISA). (B) Interleukin-2 (IL-2) release was measured by ELISA. (C) PTM-transduced, untransduced primary murine OT-1, PTM-transduced, untransduced primary murine OT-2 T cells, or OT-1 together with OT-2 T cells were prestimulated for 24 h with anti-CD3 antibody and recombinant PD-L1. In the meantime, Panc02-OVA or E.G7-PD-L1 cells were seeded and grown prior to the addition of T cells. LDH release measurement from lysed tumor cells was performed after 16 h of coculture. (D) Granzyme B secretion by T cells cocultured with E.G7-PD-L1 cells for 16 h measured by ELISA. (E) PTM-transduced, untransduced primary murine OT-1, PTM-transduced, untransduced primary murine OT-2 T cells or OT-1 together with OT-2 T cells were prestimulated for 24 h with anti-CD3 antibody plus recombinant PD-L1 and then cocultured with Panc02-OVA cells. T cell numbers were analyzed by flow cytometry and normalized to standardized counting beads. (F) 30 mice were subcutaneously injected with E.G7-OVA-PD-L1 tumor cells in two independent experiments. As soon as all tumors were established, the mice were randomized, assigned to five different treatment groups and treated with either PTM-transduced ( n = 6) or untransduced primary murine OT1 T cells ( n = 7) or with PTM-transduced ( n = 4) or untransduced ( n = 4) primary OT2 T cells in combination with OT1 T cells or PTM-transduced OT-1 T cells ( n = 9). Tumor growth was assessed every other day in a blinded fashion and tumor volume was calculated as indicated. Pooled data from two independent experiments is shown here. Curves are censored by the time the first mice had to be taken out of the experiment either due to tumor size or ulceration (day 10). Experiments (A–E) are representative of three independent experiments each performed in triplicates. Experiment (F) represents pooled data of two independent experiments. Bars represent SEM and P values from Student's t -test are shown. All tests are two-sided.
    Figure Legend Snippet: In vitro and in vivo assessment of anti-tumor efficacy of PD1-CD28 fusion receptor (PTM receptor)-transduced CD4+ and CD8+ T cells. (A) PTM-transduced, untransduced primary murine OT-1, PTM-transduced, untransduced primary murine OT-2 T cells, or OT-1 together with OT-2 T cells were prestimulated for 24 h with anti-CD3 antibody plus recombinant PD-L1. T cells were then cocultured with Panc02-OVA or E.G7-PD-L1 cells. Interferon-γ (IFN-γ) secretion was measured by enzyme linked immunosorbent assay (ELISA). (B) Interleukin-2 (IL-2) release was measured by ELISA. (C) PTM-transduced, untransduced primary murine OT-1, PTM-transduced, untransduced primary murine OT-2 T cells, or OT-1 together with OT-2 T cells were prestimulated for 24 h with anti-CD3 antibody and recombinant PD-L1. In the meantime, Panc02-OVA or E.G7-PD-L1 cells were seeded and grown prior to the addition of T cells. LDH release measurement from lysed tumor cells was performed after 16 h of coculture. (D) Granzyme B secretion by T cells cocultured with E.G7-PD-L1 cells for 16 h measured by ELISA. (E) PTM-transduced, untransduced primary murine OT-1, PTM-transduced, untransduced primary murine OT-2 T cells or OT-1 together with OT-2 T cells were prestimulated for 24 h with anti-CD3 antibody plus recombinant PD-L1 and then cocultured with Panc02-OVA cells. T cell numbers were analyzed by flow cytometry and normalized to standardized counting beads. (F) 30 mice were subcutaneously injected with E.G7-OVA-PD-L1 tumor cells in two independent experiments. As soon as all tumors were established, the mice were randomized, assigned to five different treatment groups and treated with either PTM-transduced ( n = 6) or untransduced primary murine OT1 T cells ( n = 7) or with PTM-transduced ( n = 4) or untransduced ( n = 4) primary OT2 T cells in combination with OT1 T cells or PTM-transduced OT-1 T cells ( n = 9). Tumor growth was assessed every other day in a blinded fashion and tumor volume was calculated as indicated. Pooled data from two independent experiments is shown here. Curves are censored by the time the first mice had to be taken out of the experiment either due to tumor size or ulceration (day 10). Experiments (A–E) are representative of three independent experiments each performed in triplicates. Experiment (F) represents pooled data of two independent experiments. Bars represent SEM and P values from Student's t -test are shown. All tests are two-sided.

    Techniques Used: In Vitro, In Vivo, Recombinant, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Mouse Assay, Injection

    In vitro characterization of PD1-CD28 fusion protein (PTM)-transduced CD4+ T cells. PTM-transduced or untransduced primary murine CD4+ T cells were either stimulated with anti-CD3 antibody, anti-CD3 antibody, and recombinant PD-L1 or anti-CD3 antibody and anti-CD28 antibody. (A) Interferon-γ (IFN-γ) secretion was measured by enzyme linked immunosorbent assay (ELISA). (B) T cell number was analyzed by flow cytometry and normalized to standardized counting beads. (C) Viability of T cells was assessed by flow cytometry. (D) After 48 h of stimulation T cells were intracellularly stained for Ki67, a mitosis marker or (E) for the differentiation marker eomesodermin (EOMES). Experiments (A–E) are representative of three independent experiments each performed in triplicates. Bars represent SEM and P values from Student's t -test are shown. All tests are two-sided.
    Figure Legend Snippet: In vitro characterization of PD1-CD28 fusion protein (PTM)-transduced CD4+ T cells. PTM-transduced or untransduced primary murine CD4+ T cells were either stimulated with anti-CD3 antibody, anti-CD3 antibody, and recombinant PD-L1 or anti-CD3 antibody and anti-CD28 antibody. (A) Interferon-γ (IFN-γ) secretion was measured by enzyme linked immunosorbent assay (ELISA). (B) T cell number was analyzed by flow cytometry and normalized to standardized counting beads. (C) Viability of T cells was assessed by flow cytometry. (D) After 48 h of stimulation T cells were intracellularly stained for Ki67, a mitosis marker or (E) for the differentiation marker eomesodermin (EOMES). Experiments (A–E) are representative of three independent experiments each performed in triplicates. Bars represent SEM and P values from Student's t -test are shown. All tests are two-sided.

    Techniques Used: In Vitro, Recombinant, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Staining, Marker

    In vitro characterization of PD1-CD28 fusion protein (PTM)-transduced CD4+ and CD8+ T cells in T cell-tumor cell cocultures at different CD4+ to CD8+ T cell ratios in the presence or absence of a neutralizing anti-IL-2-antibody. (A,B) PTM-transduced or untransduced primary murine OT-1, PTM-transduced or untransduced primary murine OT-2 T cells or combinations of these were prestimulated for 24 h with anti-CD3 antibodies plus recombinant PD-L1. Three different ratios of CD4+ to CD8+ T cells were applied (i.e., 3:1, 1:1, or 1:3 CD4+ to CD8+ T cell ratio). After prestimulation, the T cells were cocultured with Panc02-OVA or E.G7-PD-L1 cells for a further 48 h. The resulting Interferon-γ (IFN-γ) release was measured by enzyme linked immunosorbent assay (ELISA). (C,D) The concentration of interleukin-2 (IL-2) in the supernatants was measured by ELISA. (E) PTM-transduced or untransduced primary murine OT-1, PTM-transduced or untransduced primary murine OT-2 T cells or combinations of these were prestimulated for 24 h with anti-CD3 antibodies plus recombinant PD-L1. T cells were then cocultured with Panc02-OVA. In the blocking condition, a neutralizing anti-IL-2 antibody was present during the period of prestimulation and coculture. The resulting IFN-γ release was measured by ELISA. Experiments (A–E) are representative of three independent experiments each performed at least in triplicates. Bars represent SEM and P values from Student's t -test are shown. All tests are two-sided.
    Figure Legend Snippet: In vitro characterization of PD1-CD28 fusion protein (PTM)-transduced CD4+ and CD8+ T cells in T cell-tumor cell cocultures at different CD4+ to CD8+ T cell ratios in the presence or absence of a neutralizing anti-IL-2-antibody. (A,B) PTM-transduced or untransduced primary murine OT-1, PTM-transduced or untransduced primary murine OT-2 T cells or combinations of these were prestimulated for 24 h with anti-CD3 antibodies plus recombinant PD-L1. Three different ratios of CD4+ to CD8+ T cells were applied (i.e., 3:1, 1:1, or 1:3 CD4+ to CD8+ T cell ratio). After prestimulation, the T cells were cocultured with Panc02-OVA or E.G7-PD-L1 cells for a further 48 h. The resulting Interferon-γ (IFN-γ) release was measured by enzyme linked immunosorbent assay (ELISA). (C,D) The concentration of interleukin-2 (IL-2) in the supernatants was measured by ELISA. (E) PTM-transduced or untransduced primary murine OT-1, PTM-transduced or untransduced primary murine OT-2 T cells or combinations of these were prestimulated for 24 h with anti-CD3 antibodies plus recombinant PD-L1. T cells were then cocultured with Panc02-OVA. In the blocking condition, a neutralizing anti-IL-2 antibody was present during the period of prestimulation and coculture. The resulting IFN-γ release was measured by ELISA. Experiments (A–E) are representative of three independent experiments each performed at least in triplicates. Bars represent SEM and P values from Student's t -test are shown. All tests are two-sided.

    Techniques Used: In Vitro, Recombinant, Enzyme-linked Immunosorbent Assay, Concentration Assay, Blocking Assay

    In vitro characterization of the MHC I, MHC II, and PD-L1 expression on tumor cells and its effect on interferon-γ (IFN-γ) release by PD1-CD28 fusion protein (PTM)- transduced CD4+ and CD8+ T cells in T cell-tumor cell cocultures. (A) Panc02-OVA or (B) E.G7-OVA-PD-L1 cells were stimulated with increasing concentrations of recombinant murine IFN-γ (2, 20, 100 ng/ml) during a 48 h period. MHC I, MHC II, and PD-L1 expression was assessed by flow cytometry. (C–F) PTM-transduced or untransduced primary murine OT-1, PTM-transduced or untransduced primary murine OT2 T cells or OT1 plus OT-2 T cells were prestimulated for 24 h with anti-CD3 antibody plus recombinant PD-L1. T cells were then cocultured with EL4 (D) or with E.G7-OVA-PDL-1 in the absence (C) or presence of neutralizing anti-MHC. I antibody (E) or of neutralizing anti-MHCII-antibody (F) . The resulting interferon-γ (IFN-γ) release was measured by enzyme-linked immunosorbent assay (ELISA). Experiments (A–F) were performed in triplicates. Experiments (C–F) are representative of two independent experiments. Bars represent SEM and P values from Student's t -test are shown. All tests are two-sided.
    Figure Legend Snippet: In vitro characterization of the MHC I, MHC II, and PD-L1 expression on tumor cells and its effect on interferon-γ (IFN-γ) release by PD1-CD28 fusion protein (PTM)- transduced CD4+ and CD8+ T cells in T cell-tumor cell cocultures. (A) Panc02-OVA or (B) E.G7-OVA-PD-L1 cells were stimulated with increasing concentrations of recombinant murine IFN-γ (2, 20, 100 ng/ml) during a 48 h period. MHC I, MHC II, and PD-L1 expression was assessed by flow cytometry. (C–F) PTM-transduced or untransduced primary murine OT-1, PTM-transduced or untransduced primary murine OT2 T cells or OT1 plus OT-2 T cells were prestimulated for 24 h with anti-CD3 antibody plus recombinant PD-L1. T cells were then cocultured with EL4 (D) or with E.G7-OVA-PDL-1 in the absence (C) or presence of neutralizing anti-MHC. I antibody (E) or of neutralizing anti-MHCII-antibody (F) . The resulting interferon-γ (IFN-γ) release was measured by enzyme-linked immunosorbent assay (ELISA). Experiments (A–F) were performed in triplicates. Experiments (C–F) are representative of two independent experiments. Bars represent SEM and P values from Student's t -test are shown. All tests are two-sided.

    Techniques Used: In Vitro, Expressing, Recombinant, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    Related Articles

    Activation Assay:

    Article Title: Phosphorylation of the RB C-terminus regulates condensin II release from chromatin
    Article Snippet: Anti-rabbit or anti-mouse IgG goat antibody conjugated to HRP was from GE Healthcare. .. For TCR activation studies, anti-CD3ε (OKT3) and CD28 (CD28.2) antibodies were from BioLegend, and light chain-specific goat anti-mouse IgG (115-005-174) was from Jackson ImmunoResearch Labs. .. Cell culture and stimulation Suspension Jurkat cells were grown in DMEM enriched with 10% v/v FBS, L-glutamine, penicillin, and streptomycin at 37 °C in 5% CO2 .

    Centrifugation:

    Article Title: Increased Serum Levels of Brain-Derived Neurotrophic Factor Contribute to Inflammatory Responses in Patients with Rheumatoid Arthritis
    Article Snippet: After centrifugation at 250× g for 25 min, PBMCs were aspirated from the interface. .. Then PBMCs (1 × 106 /mL) were stimulated with 1 μg/mL anti-human CD3 and 1 μg/mL anti-human CD28 (BioLegend, San Diego, CA, USA) plus different concentrations of BDNF (0, 20, or 200 ng/mL) at 37 °C in 5% CO2 for 24 h. After culture, cells were pelleted by centrifugation at 300× g, and the supernatant was concomitantly collected and stored at −80 °C for the measurement of cytokines. ..

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Inclusion of Strep-Tag II in design of antigen receptors for T cell immunotherapy
    Article Snippet: .. Antibodies CD45-FITC (BD Pharmagen; Cat: 555482); CD8-PE-Cy7 (BD Pharmagen; Cat:557746); CD4-APC (BD Pharmagen; Cat: 555349); Strep-tag II Antibody (biotin) (Genscript: Cat: A10737); CD28-PE-Cy7 (BioLegend; Cat: 302925); CD27-PE (eBioscience; Cat: 12-0279); CD62L-APC-Cy7 (BioLegend; Cat: 304813);EGFR Ab, ERBITUX® (cetuximab) (Bristol-Myers Squibb; NDC: 66733094823); CD25-APC (BD Phamagen; Cat: 555434); StrepTavidin-PE(BD Pharmagen; Cat: 554061); CD28 (BioLegend; Cat: 302902); Propidium Iodine (BD Pharmagen; Cat: 556463) .. Cell lines Raji, K562, and MDA-MB-231 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

    Strep-tag:

    Article Title: Inclusion of Strep-Tag II in design of antigen receptors for T cell immunotherapy
    Article Snippet: .. Antibodies CD45-FITC (BD Pharmagen; Cat: 555482); CD8-PE-Cy7 (BD Pharmagen; Cat:557746); CD4-APC (BD Pharmagen; Cat: 555349); Strep-tag II Antibody (biotin) (Genscript: Cat: A10737); CD28-PE-Cy7 (BioLegend; Cat: 302925); CD27-PE (eBioscience; Cat: 12-0279); CD62L-APC-Cy7 (BioLegend; Cat: 304813);EGFR Ab, ERBITUX® (cetuximab) (Bristol-Myers Squibb; NDC: 66733094823); CD25-APC (BD Phamagen; Cat: 555434); StrepTavidin-PE(BD Pharmagen; Cat: 554061); CD28 (BioLegend; Cat: 302902); Propidium Iodine (BD Pharmagen; Cat: 556463) .. Cell lines Raji, K562, and MDA-MB-231 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

    Staining:

    Article Title: CD28 family of receptors on T cells in chronic HBV infection: Expression characteristics, clinical significance and correlations with PD-1 blockade
    Article Snippet: Antibodies against CD8 allophycocyanin (APC; cat. no.17-0088; 1:20), CD8 fluorescein isothiocyanate (FITC; cat. no. 11-0088; 1:20), CD4 phycoerythrin (PE; cat. no. 12-0049; 1:20), CD4 FITC (cat. no. 11-0049; 1:20), CD3 peridinin chlorophyll protein (cat. no. 45-0037; 1:20), ICOS APC (cat. no. 17-9948; 1:20), CD28 PE (cat. no. 12-0289; 1:20), IFN-γ FITC (cat. no. 11-7319; 1:20), PD-L1 (cat. no. 16-5983; 1:200) and their corresponding isotype control antibodies (ICOS; cat. no. 17-4714; 1:20, CD28; cat. no. 12-4714; 1:20, IFN-γ; cat. no. 11-4714; 1:20, PD-L1; cat. no. 16-4714; 1:200) were purchased from eBioscience, Inc. (San Diego, CA, USA). .. Antibodies against PD-1 FITC (cat. no. 329904; 1:20), CTLA-4 APC (cat. no. 349908; 1:20), BTLA PE (cat. no. 344505; 1:20), CD28 (cat. no. 302902), their isotype control antibodies (PD-1; cat. no. 400107; 1:20, CTLA-4; cat. no. 400121; 1:20, BTLA; cat. no. 400211; 1:20) and 7-AAD Viability Staining Solution [7-amino-actinomycin D (7-AAD)] were purchased from Biolegend, Inc. (San Diego, CA, USA). .. PCR primers for HLA-A2 (forward, 5′-GTGGATAGAGCAGGAGGGT-3′ and reverse, 5′-CCAAGAGCGCAGGTCCTCT-3′) were purchased from Invitrogen; Thermo Fisher Scientific, Inc. Real-time quantitative primers specific for the transcription factors, T-bet (cat. no. QT00042217), GATA-3 (cat. no. QT00095501) and β-actin (cat. no. QT01680476) were purchased from Qiagen GmbH (Hilden, Germany).

    other:

    Article Title: Methamphetamine alters T cell cycle entry and progression: role in immune dysfunction
    Article Snippet: Alexa Fluor 647 mouse anti-human CD20, brilliant violet 421 mouse anti-human CD4 and CD8, mouse anti-human CD3 and CD28 antibodies were purchased from Biolegend (San Diego, CA).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    BioLegend anti cd28
    Respiratory restriction induces an energetic crisis within the matrix compartment in early activated CD8 + T Cell. A ) Splenocytes were stimulated using anti <t>CD3/CD28</t> for 9, 12 and 24 hr. Cells were then treated with oligomycin for one hour or left untreated. Isolated CD8 + T cells were then analyzed using qRT-PCR for unprocessed mt-Rnr transcript (unpmt-Rnr) and its two processed products, mt-Rnr1 and mt-Rnr2. Figure shows ratio between the relative expression of unp mt-Rnr and mt-Rnr1 . (n = 6 biological replicates, P value ** 0.0022 ** 0.0043 ** 0.0095). ( B ) Schematic describing the way matrix ATP deficiency leads to accumulation of ubiquitinated mitochondrial matrix-localized proteins and pre-proteins. ( C-D ) Splenocytes were stimulated using anti CD3/CD28 for 9 or 12 hr (T-Early and T-Late respectively). One hour prior to CD8 + T cells isolation, cells were treated with oligomycin or left untreated. Protein extracts from isolated CD8 + T cells were then subjected to immunoprecipitation using anti-ubiquitin antibody. Sample analysis was performed by MS focusing on leader peptides. ( C ) Table shows sequences of leader peptides and their corresponding proteins identified in T-Early and T-Late in respect to untreated samples. ( D ) Comparison of the presence of leader peptides identified in T-Early and T-Late. Statistical method, non-parametric Mann-Whitney test, mean ± s.e.m.
    Anti Cd28, supplied by BioLegend, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti cd28/product/BioLegend
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti cd28 - by Bioz Stars, 2021-05
    97/100 stars
      Buy from Supplier

    97
    BioLegend anti cd3 plate coated
    Drp1 Controls the Metabolic Reprogramming of Activated T Cells (A) Mitochondria (TOM20) distribution in +/+ cre+ control and fl/fl cre+ Drp1 KO T cells stimulated with <t>anti-CD3-coated</t> beads (referred to as B, labeled with anti-CD3 antibody, red) (n = 4). (B) Fluo3-AM-loaded +/+ cre+ control and fl/fl cre+ Drp1 KO T cells were incubated with the aCD3 antibody. After acquiring Fluo3-AM baseline fluorescence, a secondary antibody was added, and fluorescence was acquired up to 6 min. The fold increase in maximum (at 2 min) and residual (at 5 min) Fluo3-AM fluorescence relative to baseline is reported in the graph on the right (n = 5 ctrl, 4 KO). (C) Expression levels of the indicated (phospho)-protein in +/+ cre+ control and fl/fl cre+ Drp1 KO T cells stimulated in vitro for the indicated time. Quantification of the KO:ctrl ratio for the indicated (phospho)-proteins is reported in the graph on the right (AMPK-mTOR, n = 5; cMyc, n = 4; S6, n = 3). cMyc levels are reported 48 hr post-stimulation (maximal upregulation), but similar results were also obtained at 5 hr. (D and E) Expression levels and relative quantifications of the indicated (phospho)-protein from +/+ cre+ control and fl/fl cre+ Drp1 KO T cells activated in vitro for 5 hr in the presence of the calcium chelators 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and EDTA (D, n = 3) or the AMPK inhibitor Compound-C (E, n = 3). (F and G) RNA sequencing (RNA-seq) analysis in 3-day in vitro -activated +/+ cre+ control and fl/fl cre+ Drp1 KO T cells. (F) Heatmap of cMyc-dependent metabolic genes in T cells (cMyc-MG) expression in +/+ cre+ control and fl/fl cre+ Drp1 KO T cells, with glycolytic genes highlighted. (G) The differential mRNA expressions (normalized association score) from enrichment gene set association analysis (GSAA) of the cMyc-MG group from (F) and additional metabolic pathways (whose heatmaps are reported in Figure S3 F). TCA, tricarboxylic acid; PPP, pentose phosphate pathway; FAS, fatty acid synthesis; FAO, fatty acid oxidation. For each group, transcriptional enrichment in KO cells compared with controls is highlighted in red, downregulation in blue and no net difference in black (n = 3). (H–J) Seahorse analysis of extracellular acidification rate (ECAR) (H) and oxygen consumption rate (OCR) (I and J) rates in 6-day in vitro -activated +/+ cre+ control and fl/fl cre+ Drp1 KO CD8+ T cells (2-DG, 2-deoxyglucose; Rot/an, rotenone and antimycin). FA oxidation was measured with BSA-palmitate with or without etomoxir (J). The following parameters were quantified: glycolysis (Glyc), maximal glycolytic capacity (MGC); basal OXPHOS (basal OX), maximum respiratory capacity (MRC), and basal (basal) and maximal (max) FA oxidation (n = 3). (K) MFI for IL7Ra (n = 17), CD44 (n = 12), KLRG1 (n = 9), IFNγ (n = 10), TNF-α (n = 6), IL-2 (n = 4), and IL-4 (n = 7) and for the Tbet:Eomes ratio (n = 8) in 6-day in vitro -activated CD8+ +/+ cre+ control and fl/fl cre+ Drp1 KO T cells under the indicated polarizing conditions. Data are represented as mean ± SEM. Scale bar, 10 μm in (A). Significance is indicated as follows: ∗ p
    Anti Cd3 Plate Coated, supplied by BioLegend, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti cd3 plate coated/product/BioLegend
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti cd3 plate coated - by Bioz Stars, 2021-05
    97/100 stars
      Buy from Supplier

    N/A
    Brilliant Violet 785 anti human CD28 CD28 2 Isotype Mouse IgG1 κ Reactivity Human Cross Reactivity Chimpanzee Baboon Cynomolgus Rhesus Pigtailed Macaque Capuchin Monkey Sooty Mangabey Squirrel Monkey Apps FC
      Buy from Supplier

    N/A
    Brilliant Violet 650 anti human CD28 CD28 2 Isotype Mouse IgG1 κ Reactivity Human Cross Reactivity Chimpanzee Baboon Cynomolgus Rhesus Pigtailed Macaque Capuchin Monkey Sooty Mangabey Squirrel Monkey Apps FC
      Buy from Supplier

    Image Search Results


    Respiratory restriction induces an energetic crisis within the matrix compartment in early activated CD8 + T Cell. A ) Splenocytes were stimulated using anti CD3/CD28 for 9, 12 and 24 hr. Cells were then treated with oligomycin for one hour or left untreated. Isolated CD8 + T cells were then analyzed using qRT-PCR for unprocessed mt-Rnr transcript (unpmt-Rnr) and its two processed products, mt-Rnr1 and mt-Rnr2. Figure shows ratio between the relative expression of unp mt-Rnr and mt-Rnr1 . (n = 6 biological replicates, P value ** 0.0022 ** 0.0043 ** 0.0095). ( B ) Schematic describing the way matrix ATP deficiency leads to accumulation of ubiquitinated mitochondrial matrix-localized proteins and pre-proteins. ( C-D ) Splenocytes were stimulated using anti CD3/CD28 for 9 or 12 hr (T-Early and T-Late respectively). One hour prior to CD8 + T cells isolation, cells were treated with oligomycin or left untreated. Protein extracts from isolated CD8 + T cells were then subjected to immunoprecipitation using anti-ubiquitin antibody. Sample analysis was performed by MS focusing on leader peptides. ( C ) Table shows sequences of leader peptides and their corresponding proteins identified in T-Early and T-Late in respect to untreated samples. ( D ) Comparison of the presence of leader peptides identified in T-Early and T-Late. Statistical method, non-parametric Mann-Whitney test, mean ± s.e.m.

    Journal: eLife

    Article Title: Systemic hypoxia inhibits T cell response by limiting mitobiogenesis via matrix substrate-level phosphorylation arrest

    doi: 10.7554/eLife.56612

    Figure Lengend Snippet: Respiratory restriction induces an energetic crisis within the matrix compartment in early activated CD8 + T Cell. A ) Splenocytes were stimulated using anti CD3/CD28 for 9, 12 and 24 hr. Cells were then treated with oligomycin for one hour or left untreated. Isolated CD8 + T cells were then analyzed using qRT-PCR for unprocessed mt-Rnr transcript (unpmt-Rnr) and its two processed products, mt-Rnr1 and mt-Rnr2. Figure shows ratio between the relative expression of unp mt-Rnr and mt-Rnr1 . (n = 6 biological replicates, P value ** 0.0022 ** 0.0043 ** 0.0095). ( B ) Schematic describing the way matrix ATP deficiency leads to accumulation of ubiquitinated mitochondrial matrix-localized proteins and pre-proteins. ( C-D ) Splenocytes were stimulated using anti CD3/CD28 for 9 or 12 hr (T-Early and T-Late respectively). One hour prior to CD8 + T cells isolation, cells were treated with oligomycin or left untreated. Protein extracts from isolated CD8 + T cells were then subjected to immunoprecipitation using anti-ubiquitin antibody. Sample analysis was performed by MS focusing on leader peptides. ( C ) Table shows sequences of leader peptides and their corresponding proteins identified in T-Early and T-Late in respect to untreated samples. ( D ) Comparison of the presence of leader peptides identified in T-Early and T-Late. Statistical method, non-parametric Mann-Whitney test, mean ± s.e.m.

    Article Snippet: Purified anti-CD3ε (OKT3) and anti-CD28 (CD28.2; both from Biolegend) were used at the appropriate concentration for human T cell activation.

    Techniques: Isolation, Quantitative RT-PCR, Expressing, Immunoprecipitation, MANN-WHITNEY

    Impaired calcium flux in Atg7-deficient T cells (A). Calcium influx in autophagy-deficient T cells upon TCR engagement. Lymph node cells from Atg7 f/f or Atg7 f/f Lck-Cre mice were loaded with Indo-1 and then stained with7-AAD, anti-CD44-FITC and either anti-CD4-PE or anti-CD8-PE antibodies. Cells were resuspended in HBSS buffer containing 1.26 mM CaCl 2 . The Indo-1 loaded cells were stimulated with biotin-anti-CD3 (5 μg/ml) and biotin-anti-CD4 (1 μg/ml) antibodies for analysis of CD4 + T cells (or biotin-anti-CD8 antibody for CD8 + T cells) for 1 min. After establishing a base line, the cells were crosslinked with 25 μg/ml streptavidin and the stimulation was indicated by the arrow in the figure (same in other calcium flux figures). Intracellular Ca 2+ kinetics were expressed as the ratio of emission at 405 nm to that at 510 nm. Graphs represent calcium influx in live naïve 7-AAD - CD44 low CD4 + or 7-AAD - CD44 low CD8 + cells. This experiment was repeated three times independently. (B). Phosphorylation of p38, ERK, and PLCγ1 in autophagy-deficient T cells upon TCR stimulation. Purified naïve CD44 low T lymphocytes were stimulated with biotin-labeled anti-CD3 (5 μg/ml), biotin-labeled anti-CD4 (1 μg/ml) and biotin-labeled anti-CD8 (1 μg/ml) for 1 min and crosslinked with streptavidin (25 μg/ml) for 1, 1.5, 3, 5, or 10 min. Phosphorylated proteins were detected with anti-phosphorylated p38, ERK, or PLCγ1 and visualized using Alexa Fluor 680- or IRDye 800-conjugated anti-species antibodies. Numbers represent the ratios of intensity of the target molecule bands to intensity of actin bands. These Western blots analysis were repeated five times using purified T cells from different pairs of wildtype and Atg7 f/f Lck-Cre mice. (C). IkBα degradation in autophagy-deficient T cells after CD3/CD28 stimulation. T cells were stimulated with anti-CD3 (5μg/ml) and anti-CD28 (2μg/ml) antibodies overnight and whole cell lysates were subjected to Western blot analysis. Numbers represent the ratios of intensity of the IkBα bands to intensity of actin bands. (D). Increased calcium store in Atg7-deficient T cells. Lymph node cells from Atg7 f/f or Atg7 f/f Lck-Cre mice were loaded with Indo-1 in Ca 2+ -free HBSS buffer. Cells were stained with anti-CD44-FITC and either anti-CD4-PE or anti-CD8-PE, and resuspended in calcium-free HBSS containing 1 mM EGTA. A total of 1×10 6 cells were incubated in calcium-free conditions with biotin-anti-CD3 and either biotin-anti-CD4 or biotin-anti-CD8 antibodies for 1 min. After establishing a base line using flow cytometry, Ca 2+ -free streptavidin was added to crosslink anti-CD3 and anti-CD4 (or anti-CD8) antibodies (upper two panels). Above Indo-1 loaded cells were also stimulated with 1μM thapsigargin (TG) in calcium-free HBSS containing 1 mM EGTA to measure the calcium store in ER (lower two panels). The kinetic changes in [Ca 2+ ] i were visualized as the ratio of emission at 405 nm to that at 510 nm over a period of 7 min. All cells were gated on CD44 low and 7-AAD negative cells. Above experiments were repeated three times independently.

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

    Article Title: Autophagy Regulates Endoplasmic Reticulum Homeostasis and Calcium Mobilization in T Lymphocytes

    doi: 10.4049/jimmunol.1001822

    Figure Lengend Snippet: Impaired calcium flux in Atg7-deficient T cells (A). Calcium influx in autophagy-deficient T cells upon TCR engagement. Lymph node cells from Atg7 f/f or Atg7 f/f Lck-Cre mice were loaded with Indo-1 and then stained with7-AAD, anti-CD44-FITC and either anti-CD4-PE or anti-CD8-PE antibodies. Cells were resuspended in HBSS buffer containing 1.26 mM CaCl 2 . The Indo-1 loaded cells were stimulated with biotin-anti-CD3 (5 μg/ml) and biotin-anti-CD4 (1 μg/ml) antibodies for analysis of CD4 + T cells (or biotin-anti-CD8 antibody for CD8 + T cells) for 1 min. After establishing a base line, the cells were crosslinked with 25 μg/ml streptavidin and the stimulation was indicated by the arrow in the figure (same in other calcium flux figures). Intracellular Ca 2+ kinetics were expressed as the ratio of emission at 405 nm to that at 510 nm. Graphs represent calcium influx in live naïve 7-AAD - CD44 low CD4 + or 7-AAD - CD44 low CD8 + cells. This experiment was repeated three times independently. (B). Phosphorylation of p38, ERK, and PLCγ1 in autophagy-deficient T cells upon TCR stimulation. Purified naïve CD44 low T lymphocytes were stimulated with biotin-labeled anti-CD3 (5 μg/ml), biotin-labeled anti-CD4 (1 μg/ml) and biotin-labeled anti-CD8 (1 μg/ml) for 1 min and crosslinked with streptavidin (25 μg/ml) for 1, 1.5, 3, 5, or 10 min. Phosphorylated proteins were detected with anti-phosphorylated p38, ERK, or PLCγ1 and visualized using Alexa Fluor 680- or IRDye 800-conjugated anti-species antibodies. Numbers represent the ratios of intensity of the target molecule bands to intensity of actin bands. These Western blots analysis were repeated five times using purified T cells from different pairs of wildtype and Atg7 f/f Lck-Cre mice. (C). IkBα degradation in autophagy-deficient T cells after CD3/CD28 stimulation. T cells were stimulated with anti-CD3 (5μg/ml) and anti-CD28 (2μg/ml) antibodies overnight and whole cell lysates were subjected to Western blot analysis. Numbers represent the ratios of intensity of the IkBα bands to intensity of actin bands. (D). Increased calcium store in Atg7-deficient T cells. Lymph node cells from Atg7 f/f or Atg7 f/f Lck-Cre mice were loaded with Indo-1 in Ca 2+ -free HBSS buffer. Cells were stained with anti-CD44-FITC and either anti-CD4-PE or anti-CD8-PE, and resuspended in calcium-free HBSS containing 1 mM EGTA. A total of 1×10 6 cells were incubated in calcium-free conditions with biotin-anti-CD3 and either biotin-anti-CD4 or biotin-anti-CD8 antibodies for 1 min. After establishing a base line using flow cytometry, Ca 2+ -free streptavidin was added to crosslink anti-CD3 and anti-CD4 (or anti-CD8) antibodies (upper two panels). Above Indo-1 loaded cells were also stimulated with 1μM thapsigargin (TG) in calcium-free HBSS containing 1 mM EGTA to measure the calcium store in ER (lower two panels). The kinetic changes in [Ca 2+ ] i were visualized as the ratio of emission at 405 nm to that at 510 nm over a period of 7 min. All cells were gated on CD44 low and 7-AAD negative cells. Above experiments were repeated three times independently.

    Article Snippet: To test whether calcium flux defect affects IL-2 production in autophagy-deficient T cells, both purified wildtype and autophagy-deficient naïve CD4+ T cells were stimulated with plate bound anti-CD3 or anti-CD3 plus anti-CD28.

    Techniques: Mouse Assay, Staining, Purification, Labeling, Western Blot, Incubation, Flow Cytometry, Cytometry

    Impaired redistribution of STIM-1 in Atg7-deficient T cells Purified T cells were added to glass coverslips coated with anti-CD3 antibody or anti-H-2K b antibody, incubated for 20 min, and fixed with 2% paraformaldehyde. Cells were surface stained with anti-CD4 and intracellularly stained with anti-STIM-1 antibody. Z-stack images were captured at 1 μm intervals using 100x oil objective. The best focused layers were used to quantitate the fluorescence intensity of STIM-1 in resting wildtype and Atg7 f/f Lck-Cre CD4 + cells. More than 30 individual cells were analysed. For the analysis of STIM-1 punctae, one layer was selected from 3D deconvolution images, and the layers on either side of the selected layer were subtracted. The size and intensity of STIM-1 punctae were measured using MetaMorph 7.6 software. Fifty wildtype or Atg7 f/f Lck-Cre CD4 + cells were randomly selected for analysis. (A). Representative images of endogenous of STIM-1 and STIM-1 punctae in wildtype and Atg7 f/f Lck-Cre CD4 + cells stimulated by anti-CD3 mAb or thapsigargin. Images were obtained with Zeiss ApoTome system using AxioVision software with a 63x oil objective. (B). Fluorescence intensity of STIM-1 in resting wildtype and Atg7 f/f Lck-Cre CD4 + cells. (C). Area of STIM-1 punctae in anti-CD3 antibody activated wildtype and Atg7 f/f Lck-Cre CD4 + cells (p=0.6). (D). Fluorescence intensity of STIM-1 punctae in anti-CD3 antibody activated CD4 cells (p=3.5×10 -15 ). (E). Area of STIM-1 punctae in wildtype and Atg7 f/f Lck-Cre CD4 + cells stimulated with thapsigargin (p=0.03). The LN cells from wildtype or Atg7 f/f Lck-Cre mice were stimulated with 100nM thapsigargin for 20min. The cells were fixed and stained intracellularly with STIM-1 antibody. The images of STIM-1 punctae were captured and quantitated using the same method as described above. (F). Fluorescence intensity of STIM-1 punctae in wildtype and Atg7 f/f Lck-Cre CD4 + cells stimulated with thapsigargin (p=0.16). (G). Protein expressions of STIM-1 and Orai1 in Atg7-deficient T cells. The Western blot was repeated three times using purified T cells from three different pairs of wildtype and Atg7 f/f Lck-Cre mice. The numbers represents the ratios of intensity of STIM-1 or Orai1 bands to the intensity of actin bands. Normalized Western blot intensities were quantified and shown in the right panel. STIM-1 (mean±SD, *p=0.0069). Orai1 (mean±SD, p=0.47). (H). IL-2 production by autophagy-deficient T cells. Purified naïve CD4 + T cells were stimulated with plate-bound anti-CD3 (5μg/ml) or anti-CD3 plus anti-CD28 (2μg/ml) over night. IL-2 in the cell culture supernatants was measured by ELISA. (*p=0.0057, **p=0.00049).

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

    Article Title: Autophagy Regulates Endoplasmic Reticulum Homeostasis and Calcium Mobilization in T Lymphocytes

    doi: 10.4049/jimmunol.1001822

    Figure Lengend Snippet: Impaired redistribution of STIM-1 in Atg7-deficient T cells Purified T cells were added to glass coverslips coated with anti-CD3 antibody or anti-H-2K b antibody, incubated for 20 min, and fixed with 2% paraformaldehyde. Cells were surface stained with anti-CD4 and intracellularly stained with anti-STIM-1 antibody. Z-stack images were captured at 1 μm intervals using 100x oil objective. The best focused layers were used to quantitate the fluorescence intensity of STIM-1 in resting wildtype and Atg7 f/f Lck-Cre CD4 + cells. More than 30 individual cells were analysed. For the analysis of STIM-1 punctae, one layer was selected from 3D deconvolution images, and the layers on either side of the selected layer were subtracted. The size and intensity of STIM-1 punctae were measured using MetaMorph 7.6 software. Fifty wildtype or Atg7 f/f Lck-Cre CD4 + cells were randomly selected for analysis. (A). Representative images of endogenous of STIM-1 and STIM-1 punctae in wildtype and Atg7 f/f Lck-Cre CD4 + cells stimulated by anti-CD3 mAb or thapsigargin. Images were obtained with Zeiss ApoTome system using AxioVision software with a 63x oil objective. (B). Fluorescence intensity of STIM-1 in resting wildtype and Atg7 f/f Lck-Cre CD4 + cells. (C). Area of STIM-1 punctae in anti-CD3 antibody activated wildtype and Atg7 f/f Lck-Cre CD4 + cells (p=0.6). (D). Fluorescence intensity of STIM-1 punctae in anti-CD3 antibody activated CD4 cells (p=3.5×10 -15 ). (E). Area of STIM-1 punctae in wildtype and Atg7 f/f Lck-Cre CD4 + cells stimulated with thapsigargin (p=0.03). The LN cells from wildtype or Atg7 f/f Lck-Cre mice were stimulated with 100nM thapsigargin for 20min. The cells were fixed and stained intracellularly with STIM-1 antibody. The images of STIM-1 punctae were captured and quantitated using the same method as described above. (F). Fluorescence intensity of STIM-1 punctae in wildtype and Atg7 f/f Lck-Cre CD4 + cells stimulated with thapsigargin (p=0.16). (G). Protein expressions of STIM-1 and Orai1 in Atg7-deficient T cells. The Western blot was repeated three times using purified T cells from three different pairs of wildtype and Atg7 f/f Lck-Cre mice. The numbers represents the ratios of intensity of STIM-1 or Orai1 bands to the intensity of actin bands. Normalized Western blot intensities were quantified and shown in the right panel. STIM-1 (mean±SD, *p=0.0069). Orai1 (mean±SD, p=0.47). (H). IL-2 production by autophagy-deficient T cells. Purified naïve CD4 + T cells were stimulated with plate-bound anti-CD3 (5μg/ml) or anti-CD3 plus anti-CD28 (2μg/ml) over night. IL-2 in the cell culture supernatants was measured by ELISA. (*p=0.0057, **p=0.00049).

    Article Snippet: To test whether calcium flux defect affects IL-2 production in autophagy-deficient T cells, both purified wildtype and autophagy-deficient naïve CD4+ T cells were stimulated with plate bound anti-CD3 or anti-CD3 plus anti-CD28.

    Techniques: Purification, Incubation, Staining, Fluorescence, Software, Mouse Assay, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

    ( A ) Immunoblot analyses of control (ctr) and IFT20 KD Jurkat cells unstimulated or activated for the indicated times with α-CD3 and α-CD28 mAb (1 μg/mL). Four different experiments are shown. ( B ) Quantification of the immunoblot

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

    Article Title: IFT20 controls LAT recruitment to the immune synapse and T-cell activation in vivo

    doi: 10.1073/pnas.1513601113

    Figure Lengend Snippet: ( A ) Immunoblot analyses of control (ctr) and IFT20 KD Jurkat cells unstimulated or activated for the indicated times with α-CD3 and α-CD28 mAb (1 μg/mL). Four different experiments are shown. ( B ) Quantification of the immunoblot

    Article Snippet: Anti-CD28 (E18) and isotype control were purchased from Biolegend.

    Techniques:

    Impaired activation of CD4 + T cells in vitro. ( A ) CD25 expression was analyzed by FACS after 20 h of culture of total CD4 + T cells isolated from spleens and LNs in the presence or absence of α-CD3 (10 μg/mL) and α-CD28 (1 μg/mL)

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

    Article Title: IFT20 controls LAT recruitment to the immune synapse and T-cell activation in vivo

    doi: 10.1073/pnas.1513601113

    Figure Lengend Snippet: Impaired activation of CD4 + T cells in vitro. ( A ) CD25 expression was analyzed by FACS after 20 h of culture of total CD4 + T cells isolated from spleens and LNs in the presence or absence of α-CD3 (10 μg/mL) and α-CD28 (1 μg/mL)

    Article Snippet: Anti-CD28 (E18) and isotype control were purchased from Biolegend.

    Techniques: Activation Assay, In Vitro, Expressing, FACS, Isolation

    Drp1 Controls the Metabolic Reprogramming of Activated T Cells (A) Mitochondria (TOM20) distribution in +/+ cre+ control and fl/fl cre+ Drp1 KO T cells stimulated with anti-CD3-coated beads (referred to as B, labeled with anti-CD3 antibody, red) (n = 4). (B) Fluo3-AM-loaded +/+ cre+ control and fl/fl cre+ Drp1 KO T cells were incubated with the aCD3 antibody. After acquiring Fluo3-AM baseline fluorescence, a secondary antibody was added, and fluorescence was acquired up to 6 min. The fold increase in maximum (at 2 min) and residual (at 5 min) Fluo3-AM fluorescence relative to baseline is reported in the graph on the right (n = 5 ctrl, 4 KO). (C) Expression levels of the indicated (phospho)-protein in +/+ cre+ control and fl/fl cre+ Drp1 KO T cells stimulated in vitro for the indicated time. Quantification of the KO:ctrl ratio for the indicated (phospho)-proteins is reported in the graph on the right (AMPK-mTOR, n = 5; cMyc, n = 4; S6, n = 3). cMyc levels are reported 48 hr post-stimulation (maximal upregulation), but similar results were also obtained at 5 hr. (D and E) Expression levels and relative quantifications of the indicated (phospho)-protein from +/+ cre+ control and fl/fl cre+ Drp1 KO T cells activated in vitro for 5 hr in the presence of the calcium chelators 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and EDTA (D, n = 3) or the AMPK inhibitor Compound-C (E, n = 3). (F and G) RNA sequencing (RNA-seq) analysis in 3-day in vitro -activated +/+ cre+ control and fl/fl cre+ Drp1 KO T cells. (F) Heatmap of cMyc-dependent metabolic genes in T cells (cMyc-MG) expression in +/+ cre+ control and fl/fl cre+ Drp1 KO T cells, with glycolytic genes highlighted. (G) The differential mRNA expressions (normalized association score) from enrichment gene set association analysis (GSAA) of the cMyc-MG group from (F) and additional metabolic pathways (whose heatmaps are reported in Figure S3 F). TCA, tricarboxylic acid; PPP, pentose phosphate pathway; FAS, fatty acid synthesis; FAO, fatty acid oxidation. For each group, transcriptional enrichment in KO cells compared with controls is highlighted in red, downregulation in blue and no net difference in black (n = 3). (H–J) Seahorse analysis of extracellular acidification rate (ECAR) (H) and oxygen consumption rate (OCR) (I and J) rates in 6-day in vitro -activated +/+ cre+ control and fl/fl cre+ Drp1 KO CD8+ T cells (2-DG, 2-deoxyglucose; Rot/an, rotenone and antimycin). FA oxidation was measured with BSA-palmitate with or without etomoxir (J). The following parameters were quantified: glycolysis (Glyc), maximal glycolytic capacity (MGC); basal OXPHOS (basal OX), maximum respiratory capacity (MRC), and basal (basal) and maximal (max) FA oxidation (n = 3). (K) MFI for IL7Ra (n = 17), CD44 (n = 12), KLRG1 (n = 9), IFNγ (n = 10), TNF-α (n = 6), IL-2 (n = 4), and IL-4 (n = 7) and for the Tbet:Eomes ratio (n = 8) in 6-day in vitro -activated CD8+ +/+ cre+ control and fl/fl cre+ Drp1 KO T cells under the indicated polarizing conditions. Data are represented as mean ± SEM. Scale bar, 10 μm in (A). Significance is indicated as follows: ∗ p

    Journal: Cell Reports

    Article Title: Drp1 Controls Effective T Cell Immune-Surveillance by Regulating T Cell Migration, Proliferation, and cMyc-Dependent Metabolic Reprogramming

    doi: 10.1016/j.celrep.2018.11.018

    Figure Lengend Snippet: Drp1 Controls the Metabolic Reprogramming of Activated T Cells (A) Mitochondria (TOM20) distribution in +/+ cre+ control and fl/fl cre+ Drp1 KO T cells stimulated with anti-CD3-coated beads (referred to as B, labeled with anti-CD3 antibody, red) (n = 4). (B) Fluo3-AM-loaded +/+ cre+ control and fl/fl cre+ Drp1 KO T cells were incubated with the aCD3 antibody. After acquiring Fluo3-AM baseline fluorescence, a secondary antibody was added, and fluorescence was acquired up to 6 min. The fold increase in maximum (at 2 min) and residual (at 5 min) Fluo3-AM fluorescence relative to baseline is reported in the graph on the right (n = 5 ctrl, 4 KO). (C) Expression levels of the indicated (phospho)-protein in +/+ cre+ control and fl/fl cre+ Drp1 KO T cells stimulated in vitro for the indicated time. Quantification of the KO:ctrl ratio for the indicated (phospho)-proteins is reported in the graph on the right (AMPK-mTOR, n = 5; cMyc, n = 4; S6, n = 3). cMyc levels are reported 48 hr post-stimulation (maximal upregulation), but similar results were also obtained at 5 hr. (D and E) Expression levels and relative quantifications of the indicated (phospho)-protein from +/+ cre+ control and fl/fl cre+ Drp1 KO T cells activated in vitro for 5 hr in the presence of the calcium chelators 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and EDTA (D, n = 3) or the AMPK inhibitor Compound-C (E, n = 3). (F and G) RNA sequencing (RNA-seq) analysis in 3-day in vitro -activated +/+ cre+ control and fl/fl cre+ Drp1 KO T cells. (F) Heatmap of cMyc-dependent metabolic genes in T cells (cMyc-MG) expression in +/+ cre+ control and fl/fl cre+ Drp1 KO T cells, with glycolytic genes highlighted. (G) The differential mRNA expressions (normalized association score) from enrichment gene set association analysis (GSAA) of the cMyc-MG group from (F) and additional metabolic pathways (whose heatmaps are reported in Figure S3 F). TCA, tricarboxylic acid; PPP, pentose phosphate pathway; FAS, fatty acid synthesis; FAO, fatty acid oxidation. For each group, transcriptional enrichment in KO cells compared with controls is highlighted in red, downregulation in blue and no net difference in black (n = 3). (H–J) Seahorse analysis of extracellular acidification rate (ECAR) (H) and oxygen consumption rate (OCR) (I and J) rates in 6-day in vitro -activated +/+ cre+ control and fl/fl cre+ Drp1 KO CD8+ T cells (2-DG, 2-deoxyglucose; Rot/an, rotenone and antimycin). FA oxidation was measured with BSA-palmitate with or without etomoxir (J). The following parameters were quantified: glycolysis (Glyc), maximal glycolytic capacity (MGC); basal OXPHOS (basal OX), maximum respiratory capacity (MRC), and basal (basal) and maximal (max) FA oxidation (n = 3). (K) MFI for IL7Ra (n = 17), CD44 (n = 12), KLRG1 (n = 9), IFNγ (n = 10), TNF-α (n = 6), IL-2 (n = 4), and IL-4 (n = 7) and for the Tbet:Eomes ratio (n = 8) in 6-day in vitro -activated CD8+ +/+ cre+ control and fl/fl cre+ Drp1 KO T cells under the indicated polarizing conditions. Data are represented as mean ± SEM. Scale bar, 10 μm in (A). Significance is indicated as follows: ∗ p

    Article Snippet: For in vitro activation, 2x 105 CD4+ or CD8+ T cells have been stimulated with 5μg/ml anti-CD3 (plate-coated) (Biolegend), 1μg/ml anti-CD28 (Biolegend) and 20ng/ml mouse IL-2 (R & D System) for 2 days.

    Techniques: Labeling, Incubation, Fluorescence, Expressing, In Vitro, RNA Sequencing Assay