anti-cd3 mab Search Results


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  • 96
    ATCC anti cd3 mab
    Anti Cd3 Mab, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 116 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher anti cd3
    Anti Cd3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12407 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson anti cd3 mab
    17-DMAG induces Hsp70 expression. Analysis of Hsp70 in PBMCs stimulated with 1 μg/ml plate-bound <t>anti-CD3</t> antibody in absence and presence of different concentrations of 17-DMAG for 24 hours. Hsp70 protein expression was analyzed in cell lysates of these cell cultures by immunoblotting. Protein concentration was expressed relative to the β-actin level using densitometry measurements. The results are presented as mean values ± SEM of n = 3 healthy blood donors. * P
    Anti Cd3 Mab, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 1111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher anti cd3 mab
    17-DMAG induces Hsp70 expression. Analysis of Hsp70 in PBMCs stimulated with 1 μg/ml plate-bound <t>anti-CD3</t> antibody in absence and presence of different concentrations of 17-DMAG for 24 hours. Hsp70 protein expression was analyzed in cell lysates of these cell cultures by immunoblotting. Protein concentration was expressed relative to the β-actin level using densitometry measurements. The results are presented as mean values ± SEM of n = 3 healthy blood donors. * P
    Anti Cd3 Mab, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 604 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher cd3 monoclonal antibody
    17-DMAG induces Hsp70 expression. Analysis of Hsp70 in PBMCs stimulated with 1 μg/ml plate-bound <t>anti-CD3</t> antibody in absence and presence of different concentrations of 17-DMAG for 24 hours. Hsp70 protein expression was analyzed in cell lysates of these cell cultures by immunoblotting. Protein concentration was expressed relative to the β-actin level using densitometry measurements. The results are presented as mean values ± SEM of n = 3 healthy blood donors. * P
    Cd3 Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 705 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pharmingen anti cd3 mab
    Diagram of Rap1 regulation by CD28/Lck. Rap1 is regulated by the balance between the actions of Rap1 GEFs and Rap1 GAPs. During <t>TCR-CD3</t> engagement, the activation of Rap1 limits signals generated by activated Ras. Costimulation of CD28 recruits Lck to its C terminus, where it can activate a Rap1 GAP. This activity functions to reverse the Rap1-dependent antagonism of Ras signaling to strongly potentiate Ras-dependent signals to ERKs.
    Anti Cd3 Mab, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 210 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Becton Dickinson plate bound anti cd3 mab
    TLR2 but not TLR1 mRNA expression is upregulated in activated CD4 + T cells. CD4 + T cells (2 × 10 6 ) were either left unstimulated or stimulated overnight with <t>anti-CD3</t> and anti-CD28 MAbs. Total cellular RNA was isolated, and TLR1
    Plate Bound Anti Cd3 Mab, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 206 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher anti cd3 cd28 antibodies
    TLR2 but not TLR1 mRNA expression is upregulated in activated CD4 + T cells. CD4 + T cells (2 × 10 6 ) were either left unstimulated or stimulated overnight with <t>anti-CD3</t> and anti-CD28 MAbs. Total cellular RNA was isolated, and TLR1
    Anti Cd3 Cd28 Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 240 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher cd3 functional grade monoclonal antibody
    Involvement T cells in the VPDT-mediated immune response. ( a ) T-cell infiltration. Tumor sections obtained at different times after VPDT were stained with antibodies against CD4 (red), CD8 (green), and Hoechst 33342 (blue). Arrows indicate the sites of infiltration. Bar = 120 μm. ( b ) Splenocyte cytotoxicity. On Day 40 post-tumor re-challenge, the tumor-free mice were sacrificed. Their splenocytes were used as the effector with CT26 cells as the target. The two cell types were mixed at a ratio of 50:1 and incubated for 4 h. The cytotoxicity of the splenocytes was evaluated using flow cytometry, n = 3. ( c ) T-cell-dependent immunological memory. Mice that had been cured by VPDT and were resistant to tumor re-challenge ( n = 4) were depleted of T cells by intraperitoneal injections of an <t>anti-CD3</t> antibody. These mice were then re-challenged with 1 × 10 6 CT26 cells. The tumor sizes were recorded on Day 12 post-inoculation. The line represents the mean value in the vertical scatter plot. * p
    Cd3 Functional Grade Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 234 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cd3e monoclonal antibody
    Involvement T cells in the VPDT-mediated immune response. ( a ) T-cell infiltration. Tumor sections obtained at different times after VPDT were stained with antibodies against CD4 (red), CD8 (green), and Hoechst 33342 (blue). Arrows indicate the sites of infiltration. Bar = 120 μm. ( b ) Splenocyte cytotoxicity. On Day 40 post-tumor re-challenge, the tumor-free mice were sacrificed. Their splenocytes were used as the effector with CT26 cells as the target. The two cell types were mixed at a ratio of 50:1 and incubated for 4 h. The cytotoxicity of the splenocytes was evaluated using flow cytometry, n = 3. ( c ) T-cell-dependent immunological memory. Mice that had been cured by VPDT and were resistant to tumor re-challenge ( n = 4) were depleted of T cells by intraperitoneal injections of an <t>anti-CD3</t> antibody. These mice were then re-challenged with 1 × 10 6 CT26 cells. The tumor sizes were recorded on Day 12 post-inoculation. The line represents the mean value in the vertical scatter plot. * p
    Cd3e Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 589 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cilag AG anti cd3 mab
    CD95 promotes upregulation of activation markers and ERK activation. Purified human CD4 + T cells were incubated in X-VIVO medium with immobilized <t>anti-CD3</t> mAb or PHA for the indicated time periods in the presence or absence of plate-bound anti-CD95 mAb anti-APO-1 (5 μ g/ml) ( a – c , e and f ) or CD95L-ST-Fc (2,5 μ g/ml) ( e ) as well as high doses of CD95L-ST-Fc ( d ) or CD95LFc ( f ) (both 20 μ g/ml). The expression of CD69 ( a and b ) and other activation markers ( b ) was determined between 1–8 h ( a ), and at d2 ( b ) of incubation by flow cytometry using FITC/PE-labeled anti-CD69, anti-OX40, anti-CTLA-4, anti-CD25, anti-IL-2R β and anti-CD95L mAb, respectively. Expression levels are given as MFI and are representative of three independent experiments with different donors. ( c–d ) After incubation at 37°C for the indicated time points, T cells were lysed and aliquots of 20 μ g of the whole-cell lysate were analyzed by western blot using antibodies against phospho-ERK and ERK as a loading control. ( e ) Cells were treated in the presence or absence of the ERK1/2 inhibitor PD 98059 (50 μ M) or DMSO for 3 days before MTS assay. The experiment was performed in triplicates. Error bars represent S.Ds. from the mean. Statistical differences were calculated by student's t -test: ** P
    Anti Cd3 Mab, supplied by Cilag AG, used in various techniques. Bioz Stars score: 92/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti cd3 mab
    CD95 promotes upregulation of activation markers and ERK activation. Purified human CD4 + T cells were incubated in X-VIVO medium with immobilized <t>anti-CD3</t> mAb or PHA for the indicated time periods in the presence or absence of plate-bound anti-CD95 mAb anti-APO-1 (5 μ g/ml) ( a – c , e and f ) or CD95L-ST-Fc (2,5 μ g/ml) ( e ) as well as high doses of CD95L-ST-Fc ( d ) or CD95LFc ( f ) (both 20 μ g/ml). The expression of CD69 ( a and b ) and other activation markers ( b ) was determined between 1–8 h ( a ), and at d2 ( b ) of incubation by flow cytometry using FITC/PE-labeled anti-CD69, anti-OX40, anti-CTLA-4, anti-CD25, anti-IL-2R β and anti-CD95L mAb, respectively. Expression levels are given as MFI and are representative of three independent experiments with different donors. ( c–d ) After incubation at 37°C for the indicated time points, T cells were lysed and aliquots of 20 μ g of the whole-cell lysate were analyzed by western blot using antibodies against phospho-ERK and ERK as a loading control. ( e ) Cells were treated in the presence or absence of the ERK1/2 inhibitor PD 98059 (50 μ M) or DMSO for 3 days before MTS assay. The experiment was performed in triplicates. Error bars represent S.Ds. from the mean. Statistical differences were calculated by student's t -test: ** P
    Anti Cd3 Mab, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies anti cd3 mab
    Comparsion of different lysosomal markers for the identification of degranulating NK cells. ( A ) PBMC were surface stained with fluorochrome-conjugated <t>anti-CD3</t> and anti-CD56 mAbs, followed by intracellular staining with fluorochrome-conjugated anti-CD63, anti-CD107a, anti-CD107b, anti-perforin, or anti-granzyme A mAbs, as indicated. Profiles show CD56 versus intracellular lysosomal protein staining, as indicated on CD3 − CD56 + NK cells. PBMC were incubated either alone ( B ), or with K562 cells ( C ) for 2 hours at 37°C, surface stained with fluorochrome-conjugated anti-CD3 and anti-CD56 mAbs, in addition to anti-CD63, anti-CD107a, or anti-CD107b mAbs, as indicated. Profiles show CD56 versus surface staining of lysosomal proteins, as indicated on CD3 − CD56 + NK cells.
    Anti Cd3 Mab, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher anti human cd3
    tTreg cells treated with miR-142b-3p antagomir show enhanced autophagy status, FoxP3 expression, with suppressive function. ( n = 3). PB CD4+CD25+CD127− tTregs were sort purified by MACS, expanded in vitro for 14 days, treated with or without agomir/antagomir for 2 more days. Immunofluorescence confocal microscopy test for tTreg autophagy status after treatment. a Representative cellular autophagy level by the laser scanning. b The ratio of punctate autophagic vacuoles was measured by ImageJ software. c LC3B western blot results to detect the autophagy status after antagomir or agomir treatment. d ) Representative example of Foxp3 vs CD127 staining on tTreg treated with antagomir or agomir (gated on CD4+ cells). Summary of overall e level of Foxp3 expression and f representative example of Ki67 histogram on tTreg treated with antagomir or agomir (gated on CD4+ cells). Summary of overall e level of Ki67 expression after antagomir/agomir treatment. g Percent suppression of in vitro, <t>anti-CD3–mediated</t> CD8+ T cell proliferation at ratios from 1:2 to 1:32 (tTreg: PBMC) as determined by CFSE dye dilution. Values indicate mean ± SEM of these experiments (* P
    Anti Human Cd3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 570 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rabbit monoclonal anti cd3
    tTreg cells treated with miR-142b-3p antagomir show enhanced autophagy status, FoxP3 expression, with suppressive function. ( n = 3). PB CD4+CD25+CD127− tTregs were sort purified by MACS, expanded in vitro for 14 days, treated with or without agomir/antagomir for 2 more days. Immunofluorescence confocal microscopy test for tTreg autophagy status after treatment. a Representative cellular autophagy level by the laser scanning. b The ratio of punctate autophagic vacuoles was measured by ImageJ software. c LC3B western blot results to detect the autophagy status after antagomir or agomir treatment. d ) Representative example of Foxp3 vs CD127 staining on tTreg treated with antagomir or agomir (gated on CD4+ cells). Summary of overall e level of Foxp3 expression and f representative example of Ki67 histogram on tTreg treated with antagomir or agomir (gated on CD4+ cells). Summary of overall e level of Ki67 expression after antagomir/agomir treatment. g Percent suppression of in vitro, <t>anti-CD3–mediated</t> CD8+ T cell proliferation at ratios from 1:2 to 1:32 (tTreg: PBMC) as determined by CFSE dye dilution. Values indicate mean ± SEM of these experiments (* P
    Rabbit Monoclonal Anti Cd3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Agilent technologies mouse monoclonal anti cd3
    Example of the processing of the image analysis. a ) Part of a <t>CD3</t> stained section. b ) CD3+ lymphocytes labelled with the red color are counted by the software
    Mouse Monoclonal Anti Cd3, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 90/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher anti cd3 28
    Th1 immune response induced by BCG in vivo is affected in Sam68 -ko mice. a Flow cytometry analysis of IFN-γ-producing CD4 + T cells in splenocytes from wt and Sam68 -ko mice treated with PBS or infected with BCG and, 28 days later, restimulated with <t>anti-CD3-28,</t> PMA and Ionomycin, or PPD for 5 h in the presence of Brefeldin-A. b Bar graphs show data from seven independent experiments performed as in a (mean and s.e.m). c ELISA of IFN-γ in supernatants of splenocytes wt and Sam68 -ko mice treated with PBS or infected with BCG and, 28 days later, restimulated for 24 h with PMA and Ionomycin, or PPD. Data are the mean and s.e.m. of 11 independent experiments. d BCG burden in lungs of wt and Sam68 -ko mice 28 days after intravenous infection of BCG. Data are expressed as CFU/g of tissue (mean and s.e.m. of eight independent experiments); * p
    Anti Cd3 28, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Janssen anti cd3 mab
    ChA6 mAb treatment prolongs human islet allograft survival in hu-PBL-NOD/SCID recipient mice. (A) Diabetic NOD/SCID mice were transplanted under the kidney capsule with human islets. Mice were injected intraperitoneally with 50 × 10 6 allogeneic human PBMCs. Normal NOD/SCID mice (*, n = 8) were used as control of human islets function. Mice were treated with vehicle (•, n = 12); with the Edmonton protocol (♦, n = 4); chA6 mAb at days 0, 3, and 5 after transplantation (▪, n = 14); or sirolimus (▴, n = 3). Glycemia levels monitored graft survival. Asterisks indicate statistical analysis in which treated mice were compared with control mice (*** ≤ 0.0001). (B) Kidney bearing the human-islet graft from control, chA6 mAb–treated, or normal NOD/SCID mice at 100 d after transplantation were snap-frozen, and 5-μm-thick sections were stained with hematoxylin and eosin (HE). Alternatively, sections were stained for the expression of <t>CD3</t> and insulin.
    Anti Cd3 Mab, supplied by Janssen, used in various techniques. Bioz Stars score: 92/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biomeda corporation anti cd3 mab
    Kv1.3 high IKCa1 low phenotype: an exclusive functional marker for activated effector memory T cells. ( a ) Fluorescent immunostained images of patch-clamped CD4 T cell subsets from control subjects showing naive CCR7 + CD45RA + (left), T CM CCR7 + CD45RA – cells (middle), and T EM CCR7 + CD45RA + cells (right, counterstained with antihuman CD4 mAb). Representative Kv1.3 currents from each cell type are shown below the respective images. ( b ) Kv1.3 channel number/cell in naive (yellow squares), T CM (MBP-stimulated, green squares; TT-stimulated, green triangles) and T EM (MBP-stimulated, orange squares; TT-stimulated, orange triangles) T cells. Activated naive CD4 + cells were identified in the peripheral blood of controls 48 hours following stimulation with <t>anti-CD3</t> mAb. Activated T CM and T EM cells were identified in the same preparations of control TCLs (stimulated ten times with either MBP or TT) 48 hours after stimulation with the appropriate antigen. ( c ) Kv1.3 versus IKCa1 channel number per cell in the three populations before and after activation. Each data point is the mean ± SEM channel number in 20–50 cells.
    Anti Cd3 Mab, supplied by Biomeda corporation, used in various techniques. Bioz Stars score: 92/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Agilent technologies monoclonal mouse anti human cd3
    Inhibiting fatty acid synthesis or accelerating pyruvate generation corrects the tissue-invasive and arthritogenic behavior of RA T cells CD4 + CD45RO − PBMC were isolated from RA patients with active disease and adoptively transferred into human synovium-NSG chimeric mice. Mice were randomly assigned to one of three treatment arms ( n = 14 synovial grafts/treatment arm): Vehicle arm (vehicle injection); RA treated with C75 (5 mg/kg, i.p. every other day) or RA treated with ML265 (10 mg/kg, i.p. daily). Synovial tissues were harvested, tissue sections were stained with anti-human <t>CD3</t> (brown) and anti-RANKL (pink) antibodies and frequencies of RANKL positive cells were quantified in randomly selected high-powered fields. ( a ) Representative tissue stains showing human CD3 + and RANKL + T cells infiltrating into synovial tissue, where they form cellular clusters. Scale bars, 20 μm. ( b ) Frequencies of CD3 + RANKL + T cells in the tissue as percent of total cells. ( c ) RT-PCR-based quantification of T cell receptor ( TRB ) transcripts. ( d ) Gene expression of IFNG and IL17 . ( e ) Gene expression of transcription factors, key cytokines and TKS5 determined by RT-PCR. ( f , g ) Co-immunofluorescence staining of tissue sections for IFN-γ and CD3. Frequencies of tissue-residing IFN-γ + CD3 + T cells in the different treatment arms. Scale bars, 20 μm. All data are mean ± s.e.m. * P
    Monoclonal Mouse Anti Human Cd3, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    17-DMAG induces Hsp70 expression. Analysis of Hsp70 in PBMCs stimulated with 1 μg/ml plate-bound anti-CD3 antibody in absence and presence of different concentrations of 17-DMAG for 24 hours. Hsp70 protein expression was analyzed in cell lysates of these cell cultures by immunoblotting. Protein concentration was expressed relative to the β-actin level using densitometry measurements. The results are presented as mean values ± SEM of n = 3 healthy blood donors. * P

    Journal: Journal of Inflammation (London, England)

    Article Title: Inhibitory effects of heat shock protein 90 blockade on proinflammatory human Th1 and Th17 cell subpopulations

    doi: 10.1186/1476-9255-11-10

    Figure Lengend Snippet: 17-DMAG induces Hsp70 expression. Analysis of Hsp70 in PBMCs stimulated with 1 μg/ml plate-bound anti-CD3 antibody in absence and presence of different concentrations of 17-DMAG for 24 hours. Hsp70 protein expression was analyzed in cell lysates of these cell cultures by immunoblotting. Protein concentration was expressed relative to the β-actin level using densitometry measurements. The results are presented as mean values ± SEM of n = 3 healthy blood donors. * P

    Article Snippet: Cells were cultured in the presence of 1 μg/ml immobilized anti-CD3 mAb (BD Bioscience) in 24-well or 96-well culture plates in 5% CO2 at 37°C without or with different concentration of 17-DMAG (InVitrogen) for 24 hours, 72 hours, or 7 days.

    Techniques: Expressing, Protein Concentration

    17-DMAG reduces frequencies of proinflammatory Th1 and Th17 cell subpopulations. PBMCs were stimulated with 1 μg/ml of plate-bound anti-CD3 antibody in absence or presence of 17-DMAG (0.1 μM) for 72 hours. GolgiStop™, PMA, and ionomycin were added in the last 5 hours of culture before permeabilization. Cells were washed, stained with anti-CD4, anti-IFN-γ, and anti-IL-17 antibodies, and analyzed by flow cytometry. The numbers in upper right quadrants of the representative dot blots show the percentage of positive cells of each CD4 + T cell subpopulation. The results are presented as mean values ± SEM of n = 4 healthy blood donors. * P

    Journal: Journal of Inflammation (London, England)

    Article Title: Inhibitory effects of heat shock protein 90 blockade on proinflammatory human Th1 and Th17 cell subpopulations

    doi: 10.1186/1476-9255-11-10

    Figure Lengend Snippet: 17-DMAG reduces frequencies of proinflammatory Th1 and Th17 cell subpopulations. PBMCs were stimulated with 1 μg/ml of plate-bound anti-CD3 antibody in absence or presence of 17-DMAG (0.1 μM) for 72 hours. GolgiStop™, PMA, and ionomycin were added in the last 5 hours of culture before permeabilization. Cells were washed, stained with anti-CD4, anti-IFN-γ, and anti-IL-17 antibodies, and analyzed by flow cytometry. The numbers in upper right quadrants of the representative dot blots show the percentage of positive cells of each CD4 + T cell subpopulation. The results are presented as mean values ± SEM of n = 4 healthy blood donors. * P

    Article Snippet: Cells were cultured in the presence of 1 μg/ml immobilized anti-CD3 mAb (BD Bioscience) in 24-well or 96-well culture plates in 5% CO2 at 37°C without or with different concentration of 17-DMAG (InVitrogen) for 24 hours, 72 hours, or 7 days.

    Techniques: Staining, Flow Cytometry, Cytometry

    17-DMAG arrests secretion of proinflammatory cytokines. ELISA-based evaluation of the cytokines IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17A, IFN-γ, TNF-α, G-CSF, and TGF-β1 secreted into the culture medium by PBMCs which were stimulated with 1 μg/ml plate-bound anti-CD3 antibody and treated without or with 17-DMAG (0.1 μM) for 72 hours. The results are presented as mean values ± SEM of n = 3 healthy blood donors. The dotted line represents the mean detection limit of the assay. * P

    Journal: Journal of Inflammation (London, England)

    Article Title: Inhibitory effects of heat shock protein 90 blockade on proinflammatory human Th1 and Th17 cell subpopulations

    doi: 10.1186/1476-9255-11-10

    Figure Lengend Snippet: 17-DMAG arrests secretion of proinflammatory cytokines. ELISA-based evaluation of the cytokines IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17A, IFN-γ, TNF-α, G-CSF, and TGF-β1 secreted into the culture medium by PBMCs which were stimulated with 1 μg/ml plate-bound anti-CD3 antibody and treated without or with 17-DMAG (0.1 μM) for 72 hours. The results are presented as mean values ± SEM of n = 3 healthy blood donors. The dotted line represents the mean detection limit of the assay. * P

    Article Snippet: Cells were cultured in the presence of 1 μg/ml immobilized anti-CD3 mAb (BD Bioscience) in 24-well or 96-well culture plates in 5% CO2 at 37°C without or with different concentration of 17-DMAG (InVitrogen) for 24 hours, 72 hours, or 7 days.

    Techniques: Enzyme-linked Immunosorbent Assay

    17-DMAG blunts NF κ B p65 activity. Analysis of NFκB p65 in PBMCs stimulated with 1 μg/ml plate-bound anti-CD3 antibody in absence and presence of different concentrations of 17-DMAG for 24 hours. NFκB p65 activity and protein expression was analyzed in lysates of these cell cultures by ELISA and immunoblotting, respectively. Protein concentration was expressed relative to the β-actin level using densitometry measurements. The results are presented as mean values ± SEM of n = 3 healthy blood donors. * P

    Journal: Journal of Inflammation (London, England)

    Article Title: Inhibitory effects of heat shock protein 90 blockade on proinflammatory human Th1 and Th17 cell subpopulations

    doi: 10.1186/1476-9255-11-10

    Figure Lengend Snippet: 17-DMAG blunts NF κ B p65 activity. Analysis of NFκB p65 in PBMCs stimulated with 1 μg/ml plate-bound anti-CD3 antibody in absence and presence of different concentrations of 17-DMAG for 24 hours. NFκB p65 activity and protein expression was analyzed in lysates of these cell cultures by ELISA and immunoblotting, respectively. Protein concentration was expressed relative to the β-actin level using densitometry measurements. The results are presented as mean values ± SEM of n = 3 healthy blood donors. * P

    Article Snippet: Cells were cultured in the presence of 1 μg/ml immobilized anti-CD3 mAb (BD Bioscience) in 24-well or 96-well culture plates in 5% CO2 at 37°C without or with different concentration of 17-DMAG (InVitrogen) for 24 hours, 72 hours, or 7 days.

    Techniques: Activity Assay, Expressing, Enzyme-linked Immunosorbent Assay, Protein Concentration

    17-DMAG disrupts Lck activation. Analysis of Lck in PBMCs stimulated with 1 μg/ml plate-bound anti-CD3 antibody in absence and presence of different concentrations of 17-DMAG for 24 hours. Lck phosphorylation at Tyr 394 position was analyzed in lysates of these cell cultures by immunoblotting. Protein concentration was expressed relative to the β-actin level using densitometry measurements. The results are presented as mean values ± SEM of n = 3 healthy blood donors. * P

    Journal: Journal of Inflammation (London, England)

    Article Title: Inhibitory effects of heat shock protein 90 blockade on proinflammatory human Th1 and Th17 cell subpopulations

    doi: 10.1186/1476-9255-11-10

    Figure Lengend Snippet: 17-DMAG disrupts Lck activation. Analysis of Lck in PBMCs stimulated with 1 μg/ml plate-bound anti-CD3 antibody in absence and presence of different concentrations of 17-DMAG for 24 hours. Lck phosphorylation at Tyr 394 position was analyzed in lysates of these cell cultures by immunoblotting. Protein concentration was expressed relative to the β-actin level using densitometry measurements. The results are presented as mean values ± SEM of n = 3 healthy blood donors. * P

    Article Snippet: Cells were cultured in the presence of 1 μg/ml immobilized anti-CD3 mAb (BD Bioscience) in 24-well or 96-well culture plates in 5% CO2 at 37°C without or with different concentration of 17-DMAG (InVitrogen) for 24 hours, 72 hours, or 7 days.

    Techniques: Activation Assay, Protein Concentration

    17-DMAG inhibits proliferation of T cells. A Proliferation response of human PBMCs stimulated by plate-bound anti-CD3 antibody (1 μg/ml) in absence and presence of 17-DMAG. Cell cultures were exposed to different concentrations of 17-DMAG and proliferation was assayed by ELISA after BrdU incorporation on the 6th day of treatment followed by 24 hours incubation. B LDH-based cytotoxicity ELISA measurement in culture medium after 24-hour incubation of PBMCs with different concentrations of 17-DMAG. LDH release from cells lysed with 1% Triton X-100 was regarded as 100%. The results are presented as mean values ± SEM of n = 4 healthy blood donors. * P

    Journal: Journal of Inflammation (London, England)

    Article Title: Inhibitory effects of heat shock protein 90 blockade on proinflammatory human Th1 and Th17 cell subpopulations

    doi: 10.1186/1476-9255-11-10

    Figure Lengend Snippet: 17-DMAG inhibits proliferation of T cells. A Proliferation response of human PBMCs stimulated by plate-bound anti-CD3 antibody (1 μg/ml) in absence and presence of 17-DMAG. Cell cultures were exposed to different concentrations of 17-DMAG and proliferation was assayed by ELISA after BrdU incorporation on the 6th day of treatment followed by 24 hours incubation. B LDH-based cytotoxicity ELISA measurement in culture medium after 24-hour incubation of PBMCs with different concentrations of 17-DMAG. LDH release from cells lysed with 1% Triton X-100 was regarded as 100%. The results are presented as mean values ± SEM of n = 4 healthy blood donors. * P

    Article Snippet: Cells were cultured in the presence of 1 μg/ml immobilized anti-CD3 mAb (BD Bioscience) in 24-well or 96-well culture plates in 5% CO2 at 37°C without or with different concentration of 17-DMAG (InVitrogen) for 24 hours, 72 hours, or 7 days.

    Techniques: Enzyme-linked Immunosorbent Assay, BrdU Incorporation Assay, Incubation

    AM80 is a potent inhibitor for Th17 cell differentiation in vitro. Whole splenocytes were stimulated with soluble anti-CD3 with retinoids added at a range of concentrations. A: Cells were cultured in the presence of IL-23 (broken line), TGF-β and IL-6 (solid line), or without the addition of cytokines (closed triangle) for 3 days and IL-17 production assessed by enzyme-linked immunosorbent assay (ELISA). B: Intracellular cytokine staining of IL-17 and IFN-γ among TcR + CD4 + cells with or without 10 nmol/L retinoid treatment assessed after 3 days of culture. Data depicted in A and B are representative of three independent experiments. In C , CD4 + CD44 − CD25 − CD62L high naïve T cells were stimulated under Th17-priming conditions with retinoids at a range of concentrations for 96 hours. Cells were rested for 48 hours before restimulation and IL-17 production was measured in culture supernatants after further 96 hours of stimulation. * P

    Journal: The American Journal of Pathology

    Article Title: Synthetic Retinoid AM80 Inhibits Th17 Cells and Ameliorates Experimental Autoimmune Encephalomyelitis

    doi: 10.2353/ajpath.2009.081084

    Figure Lengend Snippet: AM80 is a potent inhibitor for Th17 cell differentiation in vitro. Whole splenocytes were stimulated with soluble anti-CD3 with retinoids added at a range of concentrations. A: Cells were cultured in the presence of IL-23 (broken line), TGF-β and IL-6 (solid line), or without the addition of cytokines (closed triangle) for 3 days and IL-17 production assessed by enzyme-linked immunosorbent assay (ELISA). B: Intracellular cytokine staining of IL-17 and IFN-γ among TcR + CD4 + cells with or without 10 nmol/L retinoid treatment assessed after 3 days of culture. Data depicted in A and B are representative of three independent experiments. In C , CD4 + CD44 − CD25 − CD62L high naïve T cells were stimulated under Th17-priming conditions with retinoids at a range of concentrations for 96 hours. Cells were rested for 48 hours before restimulation and IL-17 production was measured in culture supernatants after further 96 hours of stimulation. * P

    Article Snippet: Cells were activated with immobilized anti-CD3 monoclonal antibody (mAb) (2C-11; 2 μg/ml) and soluble anti-CD28 mAb (BD PharMingen) or, when measuring recall responses of secondary lymphoid tissue 10 days after immunization, with MOG35–55 peptide (0–100 μmol/L).

    Techniques: Cell Differentiation, In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining

    Continuous AM80 treatment impairs IL-10 production by T cells, which are identified as RORγt + /FOXP3 + population. A: Naïve T cells were cultured under neutral (no priming) or Th17-priming conditions in the presence of retinoids (100 nmol/L). After 96 hours, expression of IL-10 mRNA was assessed by quantitative RT-PCR. For further analysis, cells were rested for 48 and restimulated with anti-CD3 antibody for another 96 hours and examined for expression of IL-10 transcript. B: Naive T cells stimulated under Th17 priming conditions without RAR agonists were then restimulated as described above. RAR agonists (100 nmol/L) were added during the secondary stimulation and assessed for their expression of IL-10 mRNA by quantitative RT-PCR. Results are representative of two independent experiments. C: CNS-infiltrating T cells were isolated from EAE animals (score 3) and restimulated with immobilized anti-CD3 mAb in the presence of AM80 or ATRA (100 nmol/L) for 72 hours. The amount of IL-10 in culture supernatants was examined by ELISA. D: Whole splenocytes were cultured for 96 hours with TGF-β and IL-6 and RAR agonists (10 nmol/L) and CBA analysis were performed for analyzing the levels of IL-10 production in the culture supernatants. Cells were then subjected to intracellular cytokine staining by fluorescence-activated cell sorting. The histogram gated on CD45 high TCR + CD4 + lymphocytes displays the comparative population of IL-10 + cells by Foxp3 + (black line) and Foxp3 − (gray line) cell populations. Broken line represents the data stained with isotype control antibody. E: CD45 high TCR + CD4 + lymphocytes derived from D were analyzed for their expression of RORγt and Foxp3. Comparative dot plots were shown with or without AM80 treatment (10 nmol/L). Addition of AM80 decreased the number of RORγt + cells, including the RORγt + FOXP3 + cells. F: The levels of IL-10 producing cells from the quadrants labeled in E are shown.

    Journal: The American Journal of Pathology

    Article Title: Synthetic Retinoid AM80 Inhibits Th17 Cells and Ameliorates Experimental Autoimmune Encephalomyelitis

    doi: 10.2353/ajpath.2009.081084

    Figure Lengend Snippet: Continuous AM80 treatment impairs IL-10 production by T cells, which are identified as RORγt + /FOXP3 + population. A: Naïve T cells were cultured under neutral (no priming) or Th17-priming conditions in the presence of retinoids (100 nmol/L). After 96 hours, expression of IL-10 mRNA was assessed by quantitative RT-PCR. For further analysis, cells were rested for 48 and restimulated with anti-CD3 antibody for another 96 hours and examined for expression of IL-10 transcript. B: Naive T cells stimulated under Th17 priming conditions without RAR agonists were then restimulated as described above. RAR agonists (100 nmol/L) were added during the secondary stimulation and assessed for their expression of IL-10 mRNA by quantitative RT-PCR. Results are representative of two independent experiments. C: CNS-infiltrating T cells were isolated from EAE animals (score 3) and restimulated with immobilized anti-CD3 mAb in the presence of AM80 or ATRA (100 nmol/L) for 72 hours. The amount of IL-10 in culture supernatants was examined by ELISA. D: Whole splenocytes were cultured for 96 hours with TGF-β and IL-6 and RAR agonists (10 nmol/L) and CBA analysis were performed for analyzing the levels of IL-10 production in the culture supernatants. Cells were then subjected to intracellular cytokine staining by fluorescence-activated cell sorting. The histogram gated on CD45 high TCR + CD4 + lymphocytes displays the comparative population of IL-10 + cells by Foxp3 + (black line) and Foxp3 − (gray line) cell populations. Broken line represents the data stained with isotype control antibody. E: CD45 high TCR + CD4 + lymphocytes derived from D were analyzed for their expression of RORγt and Foxp3. Comparative dot plots were shown with or without AM80 treatment (10 nmol/L). Addition of AM80 decreased the number of RORγt + cells, including the RORγt + FOXP3 + cells. F: The levels of IL-10 producing cells from the quadrants labeled in E are shown.

    Article Snippet: Cells were activated with immobilized anti-CD3 monoclonal antibody (mAb) (2C-11; 2 μg/ml) and soluble anti-CD28 mAb (BD PharMingen) or, when measuring recall responses of secondary lymphoid tissue 10 days after immunization, with MOG35–55 peptide (0–100 μmol/L).

    Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Isolation, Enzyme-linked Immunosorbent Assay, Crocin Bleaching Assay, Staining, Fluorescence, FACS, Derivative Assay, Labeling

    AM80 suppresses IL-17 production by differentiated Th17 cells and ameliorates EAE in therapeutic intervention. A: CD4 + CD44 + CD25 − CD62L low memory T cells were stimulated by plate-bound anti-CD3 antibody for 4 days in the presence of IL-23. Dose-dependent decrease of IL-17 production by RAR agonists was shown in left panel. Intracellular cytokine staining of TCR + CD4 + cells shows decreased percentage of IL-17 + memory T cells after treatment with 100 nmol/L AM80 ( B ). Graphs are representative of two independent experiments. C: CNS-infiltrating T cells from mice with severe EAE (score 3–4) were isolated and restimulated with immobilized anti-CD3 in the presence of RAR agonists (100 nmol/L) for 3 days. Supernatants were analyzed for IL-17 production by ELISA and cells were subjected to quantitative RT-PCR for RORγt expression. Data are a representative of four similar experiments. D: Clinical EAE scores of animals treated daily from day 13 on ( arrow ) either with vehicle (CMC) or AM80. Displayed scores are representative of two experiments with n = 6.

    Journal: The American Journal of Pathology

    Article Title: Synthetic Retinoid AM80 Inhibits Th17 Cells and Ameliorates Experimental Autoimmune Encephalomyelitis

    doi: 10.2353/ajpath.2009.081084

    Figure Lengend Snippet: AM80 suppresses IL-17 production by differentiated Th17 cells and ameliorates EAE in therapeutic intervention. A: CD4 + CD44 + CD25 − CD62L low memory T cells were stimulated by plate-bound anti-CD3 antibody for 4 days in the presence of IL-23. Dose-dependent decrease of IL-17 production by RAR agonists was shown in left panel. Intracellular cytokine staining of TCR + CD4 + cells shows decreased percentage of IL-17 + memory T cells after treatment with 100 nmol/L AM80 ( B ). Graphs are representative of two independent experiments. C: CNS-infiltrating T cells from mice with severe EAE (score 3–4) were isolated and restimulated with immobilized anti-CD3 in the presence of RAR agonists (100 nmol/L) for 3 days. Supernatants were analyzed for IL-17 production by ELISA and cells were subjected to quantitative RT-PCR for RORγt expression. Data are a representative of four similar experiments. D: Clinical EAE scores of animals treated daily from day 13 on ( arrow ) either with vehicle (CMC) or AM80. Displayed scores are representative of two experiments with n = 6.

    Article Snippet: Cells were activated with immobilized anti-CD3 monoclonal antibody (mAb) (2C-11; 2 μg/ml) and soluble anti-CD28 mAb (BD PharMingen) or, when measuring recall responses of secondary lymphoid tissue 10 days after immunization, with MOG35–55 peptide (0–100 μmol/L).

    Techniques: Staining, Mouse Assay, Isolation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing

    Continuous AM80 treatment is less effective on EAE suppression with a differentially modulated cytokine profile. A: The H E-stained sections and the Luxol fast blue-stained sections in EAE mice treated with or without AM80 are shown. The control animal (score 2) and AM80-treated animal (score 2) at day 25 were subjected to histological examination. Scale bar = 200 μm. B: Total leukocyte numbers and isolated T cell numbers from spinal cords were evaluated in animals that had received CMC or AM80 at day 25 after immunization. The upper row of C depicts purified T cells from B that were subjected to quantitative RT-PCR. Error bars represent duplicated PCR of the same samples. In the lower row of C , CNS-infiltrating T cells were restimulated with immobilized anti-CD3 mAb and cytokine or chemokine production in culture supernatants were assessed by ELISA or CBA after 72 hours. Error bars represent measurements from duplicate wells (* P

    Journal: The American Journal of Pathology

    Article Title: Synthetic Retinoid AM80 Inhibits Th17 Cells and Ameliorates Experimental Autoimmune Encephalomyelitis

    doi: 10.2353/ajpath.2009.081084

    Figure Lengend Snippet: Continuous AM80 treatment is less effective on EAE suppression with a differentially modulated cytokine profile. A: The H E-stained sections and the Luxol fast blue-stained sections in EAE mice treated with or without AM80 are shown. The control animal (score 2) and AM80-treated animal (score 2) at day 25 were subjected to histological examination. Scale bar = 200 μm. B: Total leukocyte numbers and isolated T cell numbers from spinal cords were evaluated in animals that had received CMC or AM80 at day 25 after immunization. The upper row of C depicts purified T cells from B that were subjected to quantitative RT-PCR. Error bars represent duplicated PCR of the same samples. In the lower row of C , CNS-infiltrating T cells were restimulated with immobilized anti-CD3 mAb and cytokine or chemokine production in culture supernatants were assessed by ELISA or CBA after 72 hours. Error bars represent measurements from duplicate wells (* P

    Article Snippet: Cells were activated with immobilized anti-CD3 monoclonal antibody (mAb) (2C-11; 2 μg/ml) and soluble anti-CD28 mAb (BD PharMingen) or, when measuring recall responses of secondary lymphoid tissue 10 days after immunization, with MOG35–55 peptide (0–100 μmol/L).

    Techniques: Staining, Mouse Assay, Isolation, Purification, Quantitative RT-PCR, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Crocin Bleaching Assay

    Diagram of Rap1 regulation by CD28/Lck. Rap1 is regulated by the balance between the actions of Rap1 GEFs and Rap1 GAPs. During TCR-CD3 engagement, the activation of Rap1 limits signals generated by activated Ras. Costimulation of CD28 recruits Lck to its C terminus, where it can activate a Rap1 GAP. This activity functions to reverse the Rap1-dependent antagonism of Ras signaling to strongly potentiate Ras-dependent signals to ERKs.

    Journal: Molecular and Cellular Biology

    Article Title: CD28 and the Tyrosine Kinase Lck Stimulate Mitogen-Activated Protein Kinase Activity in T Cells via Inhibition of the Small G Protein Rap1

    doi:

    Figure Lengend Snippet: Diagram of Rap1 regulation by CD28/Lck. Rap1 is regulated by the balance between the actions of Rap1 GEFs and Rap1 GAPs. During TCR-CD3 engagement, the activation of Rap1 limits signals generated by activated Ras. Costimulation of CD28 recruits Lck to its C terminus, where it can activate a Rap1 GAP. This activity functions to reverse the Rap1-dependent antagonism of Ras signaling to strongly potentiate Ras-dependent signals to ERKs.

    Article Snippet: Ca2+ fluctuations, before and after the addition of anti-CD3 MAb (Pharmingen) at 10 μg/ml, were monitored using a fluorescence-activated cell sorter (FACS) Vantage flow cytometer by gating on the GFP-positive cells.

    Techniques: Activation Assay, Generated, Activity Assay

    Interfering with Rap1 GAP blocks CD28 enhancement of ERKs. Jurkat cells were transfected with FLAG-ERK2 along with the vector and dn.Rap1GAP1 or the vector alone as indicated. Cells were treated with anti-CD3 antibody (α-CD3) and/or anti-CD28 antibody (α-CD28) or left untreated (lanes 0), as indicated. In the upper blot, the activation of FLAG-ERK2 was monitored using FLAG immunoprecipitation followed by pERK Western blotting. The position of pFlag-ERK2 is shown. A representative gel is shown ( n = 4). In the lower blot, lysates prepared as described above were subjected to a GST-RalGDS pull-down assay and Rap1 Western blotting. The position of Rap1 is shown.

    Journal: Molecular and Cellular Biology

    Article Title: CD28 and the Tyrosine Kinase Lck Stimulate Mitogen-Activated Protein Kinase Activity in T Cells via Inhibition of the Small G Protein Rap1

    doi:

    Figure Lengend Snippet: Interfering with Rap1 GAP blocks CD28 enhancement of ERKs. Jurkat cells were transfected with FLAG-ERK2 along with the vector and dn.Rap1GAP1 or the vector alone as indicated. Cells were treated with anti-CD3 antibody (α-CD3) and/or anti-CD28 antibody (α-CD28) or left untreated (lanes 0), as indicated. In the upper blot, the activation of FLAG-ERK2 was monitored using FLAG immunoprecipitation followed by pERK Western blotting. The position of pFlag-ERK2 is shown. A representative gel is shown ( n = 4). In the lower blot, lysates prepared as described above were subjected to a GST-RalGDS pull-down assay and Rap1 Western blotting. The position of Rap1 is shown.

    Article Snippet: Ca2+ fluctuations, before and after the addition of anti-CD3 MAb (Pharmingen) at 10 μg/ml, were monitored using a fluorescence-activated cell sorter (FACS) Vantage flow cytometer by gating on the GFP-positive cells.

    Techniques: Transfection, Plasmid Preparation, Activation Assay, Immunoprecipitation, Western Blot, Pull Down Assay

    CD28's inhibition of Rap 1 and enhancement of ERKs map to the 16 carboxy-terminal residues of CD28. (A) Requirement of the carboxy terminus of CD28 in the inhibition of Rap1 by CD28. Jurkat cells and Jurkat cells stably expressing wild-type mCD28 [Jurkat (mCD28-WT)] or mCD28 with its 16 carboxy-terminal amino acids deleted [Jurkat (mCD28-CΔ16)] were incubated with anti-human CD3 antibody (α-hCD3) and/or anti-mCD28 antibody (α-mCD28) for 5 min or treated with secondary antibody alone (untreated). In all experiments, T-cell lysates were prepared and assayed for Rap1 activation using GST-RalGDS and Western blotting was performed using polyclonal Rap1 antiserum. The position of Rap1 in control lysates (A and D) and following isolation of glutathione-bound proteins is shown. Representative Western blots are shown ( n = 3). (B) Ras activation does not require the carboxy terminus of CD28. Jurkat mCD28-WT cells and mCD28-CΔ16 cells were incubated with anti-human CD3 antibody (α-CD3), anti-human CD28 antibody (α-CD28), and/or anti-mCD28 antibody (α-mCD28) for 5 min or left untreated as indicated. Lysates were prepared and assayed for Ras activation using GST–Raf-1–RBD, and Western blotting was performed using Ras antiserum. The position of Ras in control lysates and following isolation of glutathione-bound proteins is shown. (C) The 16 carboxy-terminal amino acids residues of CD28 that are required for inhibiting Rap1 are required for stimulating ERK activity. Jurkat mCD28-WT cells and mCD28-CΔ16 cells were incubated with anti-human CD3 antibody (α-CD3), anti-human CD28 antibody (α-CD28), and/or anti-mCD28 antibody (α-mCD28) for 5 min or left untreated as indicated. Lysates were prepared and assayed for ERK activation using phospho-specific ERK (pERK) antibodies. Representative Western blots with the positions of pERK1 and pERK2 indicated are shown ( n = 3).

    Journal: Molecular and Cellular Biology

    Article Title: CD28 and the Tyrosine Kinase Lck Stimulate Mitogen-Activated Protein Kinase Activity in T Cells via Inhibition of the Small G Protein Rap1

    doi:

    Figure Lengend Snippet: CD28's inhibition of Rap 1 and enhancement of ERKs map to the 16 carboxy-terminal residues of CD28. (A) Requirement of the carboxy terminus of CD28 in the inhibition of Rap1 by CD28. Jurkat cells and Jurkat cells stably expressing wild-type mCD28 [Jurkat (mCD28-WT)] or mCD28 with its 16 carboxy-terminal amino acids deleted [Jurkat (mCD28-CΔ16)] were incubated with anti-human CD3 antibody (α-hCD3) and/or anti-mCD28 antibody (α-mCD28) for 5 min or treated with secondary antibody alone (untreated). In all experiments, T-cell lysates were prepared and assayed for Rap1 activation using GST-RalGDS and Western blotting was performed using polyclonal Rap1 antiserum. The position of Rap1 in control lysates (A and D) and following isolation of glutathione-bound proteins is shown. Representative Western blots are shown ( n = 3). (B) Ras activation does not require the carboxy terminus of CD28. Jurkat mCD28-WT cells and mCD28-CΔ16 cells were incubated with anti-human CD3 antibody (α-CD3), anti-human CD28 antibody (α-CD28), and/or anti-mCD28 antibody (α-mCD28) for 5 min or left untreated as indicated. Lysates were prepared and assayed for Ras activation using GST–Raf-1–RBD, and Western blotting was performed using Ras antiserum. The position of Ras in control lysates and following isolation of glutathione-bound proteins is shown. (C) The 16 carboxy-terminal amino acids residues of CD28 that are required for inhibiting Rap1 are required for stimulating ERK activity. Jurkat mCD28-WT cells and mCD28-CΔ16 cells were incubated with anti-human CD3 antibody (α-CD3), anti-human CD28 antibody (α-CD28), and/or anti-mCD28 antibody (α-mCD28) for 5 min or left untreated as indicated. Lysates were prepared and assayed for ERK activation using phospho-specific ERK (pERK) antibodies. Representative Western blots with the positions of pERK1 and pERK2 indicated are shown ( n = 3).

    Article Snippet: Ca2+ fluctuations, before and after the addition of anti-CD3 MAb (Pharmingen) at 10 μg/ml, were monitored using a fluorescence-activated cell sorter (FACS) Vantage flow cytometer by gating on the GFP-positive cells.

    Techniques: Inhibition, Stable Transfection, Expressing, Incubation, Activation Assay, Western Blot, Isolation, Activity Assay

    ) as indicated and incubated with anti-human CD3 and anti-human CD28 antibodies for 6 h in the absence and presence of the MEK inhibitor PD98059 or UO126, as indicated. For all luciferase assays, lysates were prepared and assayed for luciferase activity. The data reflect the fold activation above basal luciferase activity (lane 1), with standard errors ( n = 3).

    Journal: Molecular and Cellular Biology

    Article Title: CD28 and the Tyrosine Kinase Lck Stimulate Mitogen-Activated Protein Kinase Activity in T Cells via Inhibition of the Small G Protein Rap1

    doi:

    Figure Lengend Snippet: ) as indicated and incubated with anti-human CD3 and anti-human CD28 antibodies for 6 h in the absence and presence of the MEK inhibitor PD98059 or UO126, as indicated. For all luciferase assays, lysates were prepared and assayed for luciferase activity. The data reflect the fold activation above basal luciferase activity (lane 1), with standard errors ( n = 3).

    Article Snippet: Ca2+ fluctuations, before and after the addition of anti-CD3 MAb (Pharmingen) at 10 μg/ml, were monitored using a fluorescence-activated cell sorter (FACS) Vantage flow cytometer by gating on the GFP-positive cells.

    Techniques: Incubation, Luciferase, Activity Assay, Activation Assay

    Lck is required for CD28's inhibition of Rap1 and augmentation of ERK. (A) dn.Lck inhibits mobilization of intracellular calcium. Jurkat T cells were transfected with the vector (black line), dn.Lck (dark-gray line), or dn.Fyn (light-gray line) and preloaded with Indo-1. Cells were then stimulated with anti-CD3 MAb as indicated. Changes in the mobilization of intracellular free calcium, presented as ratios of 405 nm to 480 nm (representing Ca 2+ -associated Indo-1 [405 nm] and free Indo-1 [480 nm]), are shown as a function of time. Expression of dn.Fyn had no effect on Ca 2+ flux. However, expression of dn.Lck blocked Ca 2+ in these cells. (Inset) Successful loading of Jurkat cells, expressing dn.Lck, with Indo-1 was confirmed by treating cells with anti-CD3 MAb and ionomycin (+ iono), which resulted in a Ca 2+ flux, or treating them with anti-CD3 antibody alone (− iono), which resulted in a block in Ca 2+ flux. Results of a representative experiment are shown ( n = 3). (B) Jurkat cells were transfected with LckR273 (dn.Lck) or the vector alone and incubated with anti-human CD3 antibody (α-CD3) and/or anti-human CD28 antibody (α-CD28) for 2 min or left untreated as indicated. T-cell lysates were prepared and assayed for Rap1 activation using GST-RalGDS, and Western blotting was performed using Rap1 antiserum. The position of Rap1 in control lysates and following isolation of glutathione-bound proteins is shown. Representative Western blots are shown ( n = 3). (C) Jurkat cells were transfected with dn.lck or the vector along with FLAG-ERK2 and treated with antibodies to CD3 and/or CD28 for 5 min as indicated. The phosphorylation of FLAG-ERK2 was monitored by pERK Western blotting. The position of pFlag-ERK2 is shown. A representative Western blot is shown ( n = 3). (D) JCaM1.6 cells stably expressing the vector (JCaM/vector), wild-type Lck (JCaM/LckWT) or LckW97A (JCaM/LckW97A) were incubated with anti-human CD3 antibody and/or anti-human CD28 antibody for 5 min or left untreated as indicated. Cell lysates were prepared and assayed for Rap1 activation using GST-RalGDS, and Western blotting was performed using Rap1 antiserum (upper blot). A representative western blot with the position of Rap1 is shown following a GST-RalGDS pull-down assay ( n = 3). The lower Western blot shows the relative levels of expression of Rap1 in these cell lines. (E) The data in panel D are presented as the averages of results of three independent experiments with standard errors. Untr., untreated cells.

    Journal: Molecular and Cellular Biology

    Article Title: CD28 and the Tyrosine Kinase Lck Stimulate Mitogen-Activated Protein Kinase Activity in T Cells via Inhibition of the Small G Protein Rap1

    doi:

    Figure Lengend Snippet: Lck is required for CD28's inhibition of Rap1 and augmentation of ERK. (A) dn.Lck inhibits mobilization of intracellular calcium. Jurkat T cells were transfected with the vector (black line), dn.Lck (dark-gray line), or dn.Fyn (light-gray line) and preloaded with Indo-1. Cells were then stimulated with anti-CD3 MAb as indicated. Changes in the mobilization of intracellular free calcium, presented as ratios of 405 nm to 480 nm (representing Ca 2+ -associated Indo-1 [405 nm] and free Indo-1 [480 nm]), are shown as a function of time. Expression of dn.Fyn had no effect on Ca 2+ flux. However, expression of dn.Lck blocked Ca 2+ in these cells. (Inset) Successful loading of Jurkat cells, expressing dn.Lck, with Indo-1 was confirmed by treating cells with anti-CD3 MAb and ionomycin (+ iono), which resulted in a Ca 2+ flux, or treating them with anti-CD3 antibody alone (− iono), which resulted in a block in Ca 2+ flux. Results of a representative experiment are shown ( n = 3). (B) Jurkat cells were transfected with LckR273 (dn.Lck) or the vector alone and incubated with anti-human CD3 antibody (α-CD3) and/or anti-human CD28 antibody (α-CD28) for 2 min or left untreated as indicated. T-cell lysates were prepared and assayed for Rap1 activation using GST-RalGDS, and Western blotting was performed using Rap1 antiserum. The position of Rap1 in control lysates and following isolation of glutathione-bound proteins is shown. Representative Western blots are shown ( n = 3). (C) Jurkat cells were transfected with dn.lck or the vector along with FLAG-ERK2 and treated with antibodies to CD3 and/or CD28 for 5 min as indicated. The phosphorylation of FLAG-ERK2 was monitored by pERK Western blotting. The position of pFlag-ERK2 is shown. A representative Western blot is shown ( n = 3). (D) JCaM1.6 cells stably expressing the vector (JCaM/vector), wild-type Lck (JCaM/LckWT) or LckW97A (JCaM/LckW97A) were incubated with anti-human CD3 antibody and/or anti-human CD28 antibody for 5 min or left untreated as indicated. Cell lysates were prepared and assayed for Rap1 activation using GST-RalGDS, and Western blotting was performed using Rap1 antiserum (upper blot). A representative western blot with the position of Rap1 is shown following a GST-RalGDS pull-down assay ( n = 3). The lower Western blot shows the relative levels of expression of Rap1 in these cell lines. (E) The data in panel D are presented as the averages of results of three independent experiments with standard errors. Untr., untreated cells.

    Article Snippet: Ca2+ fluctuations, before and after the addition of anti-CD3 MAb (Pharmingen) at 10 μg/ml, were monitored using a fluorescence-activated cell sorter (FACS) Vantage flow cytometer by gating on the GFP-positive cells.

    Techniques: Inhibition, Transfection, Plasmid Preparation, Expressing, Blocking Assay, Incubation, Activation Assay, Western Blot, Isolation, Stable Transfection, Pull Down Assay

    Involvement of Lck in CD28's enhancement of Rap1 GTPase activity. (A) The 16 carboxy-terminal amino acid residues of CD28 are required for stimulating Rap1GAP activity. Jurkat cells stably expressing full-length wild-type mCD28 (mCD28-WT) or Jurkat cells stably expressing mCD28-CΔ16 were treated with anti-human CD3 antibody (α-hCD3) and/or anti-mCD28 antibody (α-mCD28) or left untreated (Untr.) as indicated, and GTPase assays were performed as described in Materials and Methods. Percentages of release of [γ- 32 P]GTP are shown with standard errors ( n = 3). (B) Lck, but not Fyn, stimulates Rap1GAP activity. Jurkat cells were transfected with the vector alone, ca.Lck, or ca.Fyn and incubated with recombinant [γ- 32 P]GTP-loaded Rap1 (left) and either the vector alone or ca.Lck and incubated with recombinant [γ- 32 P]GTP-loaded Ras (right) as indicated. Percentages of release of [γ- 32 P]GTP are shown with standard errors ( n = 5). (C) JCaM/vector, JCaM/LckWT, or JCaM/LckW97A cells were incubated with anti-human CD3 antibody (α-CD3) and/or anti-human CD28 antibody (α-CD28) for 5 min or left untreated as indicated. Cell lysates were prepared and assayed for Rap1 GAP activity as described for panel B. Percentages of release of [γ- 32 P]GTP are shown with standard errors ( n = 3).

    Journal: Molecular and Cellular Biology

    Article Title: CD28 and the Tyrosine Kinase Lck Stimulate Mitogen-Activated Protein Kinase Activity in T Cells via Inhibition of the Small G Protein Rap1

    doi:

    Figure Lengend Snippet: Involvement of Lck in CD28's enhancement of Rap1 GTPase activity. (A) The 16 carboxy-terminal amino acid residues of CD28 are required for stimulating Rap1GAP activity. Jurkat cells stably expressing full-length wild-type mCD28 (mCD28-WT) or Jurkat cells stably expressing mCD28-CΔ16 were treated with anti-human CD3 antibody (α-hCD3) and/or anti-mCD28 antibody (α-mCD28) or left untreated (Untr.) as indicated, and GTPase assays were performed as described in Materials and Methods. Percentages of release of [γ- 32 P]GTP are shown with standard errors ( n = 3). (B) Lck, but not Fyn, stimulates Rap1GAP activity. Jurkat cells were transfected with the vector alone, ca.Lck, or ca.Fyn and incubated with recombinant [γ- 32 P]GTP-loaded Rap1 (left) and either the vector alone or ca.Lck and incubated with recombinant [γ- 32 P]GTP-loaded Ras (right) as indicated. Percentages of release of [γ- 32 P]GTP are shown with standard errors ( n = 5). (C) JCaM/vector, JCaM/LckWT, or JCaM/LckW97A cells were incubated with anti-human CD3 antibody (α-CD3) and/or anti-human CD28 antibody (α-CD28) for 5 min or left untreated as indicated. Cell lysates were prepared and assayed for Rap1 GAP activity as described for panel B. Percentages of release of [γ- 32 P]GTP are shown with standard errors ( n = 3).

    Article Snippet: Ca2+ fluctuations, before and after the addition of anti-CD3 MAb (Pharmingen) at 10 μg/ml, were monitored using a fluorescence-activated cell sorter (FACS) Vantage flow cytometer by gating on the GFP-positive cells.

    Techniques: Activity Assay, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Incubation, Recombinant

    ). (B) Constitutively active Rap1 blocks ERK activation by CD28 costimulation in Jurkat cells. Jurkat cells were transfected with the vector, Rap1E63, or RasN17 as indicated. Cells were treated with anti-CD3 antibody (α-CD3) and/or anti-CD28 antibody (α-CD28) for 5 min or not treated (lanes 0) as indicated. Phosphorylation of ERK1 and -2 as monitored by Western blotting with pERK is shown. The positions of pERK1 and -2 are shown. In the lower gel, control shows equivalent levels of protein loading. (C) Jurkat cells were transfected with 10 μg each of cDNAs encoding the vector or Rap1GAP1 as indicated. All cells received 10 μg of FLAG-ERK2 cDNA. Subsequently, cells were incubated with anti-TCR-CD3 antibody for 30 min on ice or left untreated. Cells were then activated by incubation at 37°C for 10 min. Cells were lysed, and FLAG-ERK2 was immunoprecipitated. Activation of immunoprecipitated ERK2 was measured by in vitro kinase assay. Samples were subjected to SDS-PAGE and analyzed with a PhosphorImager. A representative gel with the position of the substrate MBP indicated is presented. (D) Ras activation of Raf-1 is limited by endogenous Rap1. Jurkat cells were transfected with either FLAG-Rap1 or FLAG-Ras and treated with α-CD3 or left untreated as indicated. The associated endogenous Raf-1 was measured following FLAG immunoprecipitation and anti-Raf-1 antibody Western blotting. For FLAG-Ras, cells were also transfected with the vector or Rap1GAP1 as indicated. A representative gel with the position of Raf-1 indicated is shown ( n = 3).

    Journal: Molecular and Cellular Biology

    Article Title: CD28 and the Tyrosine Kinase Lck Stimulate Mitogen-Activated Protein Kinase Activity in T Cells via Inhibition of the Small G Protein Rap1

    doi:

    Figure Lengend Snippet: ). (B) Constitutively active Rap1 blocks ERK activation by CD28 costimulation in Jurkat cells. Jurkat cells were transfected with the vector, Rap1E63, or RasN17 as indicated. Cells were treated with anti-CD3 antibody (α-CD3) and/or anti-CD28 antibody (α-CD28) for 5 min or not treated (lanes 0) as indicated. Phosphorylation of ERK1 and -2 as monitored by Western blotting with pERK is shown. The positions of pERK1 and -2 are shown. In the lower gel, control shows equivalent levels of protein loading. (C) Jurkat cells were transfected with 10 μg each of cDNAs encoding the vector or Rap1GAP1 as indicated. All cells received 10 μg of FLAG-ERK2 cDNA. Subsequently, cells were incubated with anti-TCR-CD3 antibody for 30 min on ice or left untreated. Cells were then activated by incubation at 37°C for 10 min. Cells were lysed, and FLAG-ERK2 was immunoprecipitated. Activation of immunoprecipitated ERK2 was measured by in vitro kinase assay. Samples were subjected to SDS-PAGE and analyzed with a PhosphorImager. A representative gel with the position of the substrate MBP indicated is presented. (D) Ras activation of Raf-1 is limited by endogenous Rap1. Jurkat cells were transfected with either FLAG-Rap1 or FLAG-Ras and treated with α-CD3 or left untreated as indicated. The associated endogenous Raf-1 was measured following FLAG immunoprecipitation and anti-Raf-1 antibody Western blotting. For FLAG-Ras, cells were also transfected with the vector or Rap1GAP1 as indicated. A representative gel with the position of Raf-1 indicated is shown ( n = 3).

    Article Snippet: Ca2+ fluctuations, before and after the addition of anti-CD3 MAb (Pharmingen) at 10 μg/ml, were monitored using a fluorescence-activated cell sorter (FACS) Vantage flow cytometer by gating on the GFP-positive cells.

    Techniques: Activation Assay, Transfection, Plasmid Preparation, Western Blot, Incubation, Immunoprecipitation, In Vitro, Kinase Assay, SDS Page

    Lack of regulation of Rap1 GEF activity by CD28. Jurkat cells were incubated with anti-human CD3 antibody (α-CD3) and/or anti-human CD28 antibody (α-CD28) for 5 min or left untreated (Untr.) as indicated. Lysates were prepared and incubated for 10 min at 30°C with recombinant [ 3 H]GDP-loaded Rap1 protein (left panel) or Ras protein (right panel) bound to agarose beads. The percentages of [ 3 H]GDP released from the beads are indicated. Standard errors are shown ( n = 3).

    Journal: Molecular and Cellular Biology

    Article Title: CD28 and the Tyrosine Kinase Lck Stimulate Mitogen-Activated Protein Kinase Activity in T Cells via Inhibition of the Small G Protein Rap1

    doi:

    Figure Lengend Snippet: Lack of regulation of Rap1 GEF activity by CD28. Jurkat cells were incubated with anti-human CD3 antibody (α-CD3) and/or anti-human CD28 antibody (α-CD28) for 5 min or left untreated (Untr.) as indicated. Lysates were prepared and incubated for 10 min at 30°C with recombinant [ 3 H]GDP-loaded Rap1 protein (left panel) or Ras protein (right panel) bound to agarose beads. The percentages of [ 3 H]GDP released from the beads are indicated. Standard errors are shown ( n = 3).

    Article Snippet: Ca2+ fluctuations, before and after the addition of anti-CD3 MAb (Pharmingen) at 10 μg/ml, were monitored using a fluorescence-activated cell sorter (FACS) Vantage flow cytometer by gating on the GFP-positive cells.

    Techniques: Activity Assay, Incubation, Recombinant

    Enhancement of Rap1 GTPase activity by CD28. (A) Specificity of Rap1GAP1 for Ras and selected Rap1 mutants in vitro. Human Rap1GAP1 was expressed in Cos7 cells and incubated with recombinant Rap1 mutant proteins loaded with [γ- 32 P]GTP. The percentages of hydrolysis of [γ- 32 P]GTP from wild-type Rap1(Rap1 WT), RapV12/E63 (V12/E63), Ras, and buffer alone are indicated by the percentage of γ- 32 P released from Rap or Ras loaded in vitro. (B) CD28 stimulation of Rap1 GAP activity and introduction of E63 into RapV12, which blocks GTPase activity. Jurkat cells were incubated with anti-human CD3 antibody (α-CD3) and/or anti-human CD28 antibody (α-CD28) for 5 min or left untreated (Untr.) as indicated. Lysates were prepared and incubated for 20 min with recombinant FLAG-tagged Rap1 (left graph) or RapV12/E63 (right graph) loaded in vitro with [γ- 32 P]GTP, as indicated. The percentages of release of [γ- 32 P]GTP are shown with standard errors. ( n = 3). (C) Cells were treated as described for panel B and were assayed with [γ- 32 P]GTP. Release of α- 32 P was monitored as described in Materials and Methods, and the percentages of release of [γ- 32 P]GTPase is shown with standard errors ( n = 6).

    Journal: Molecular and Cellular Biology

    Article Title: CD28 and the Tyrosine Kinase Lck Stimulate Mitogen-Activated Protein Kinase Activity in T Cells via Inhibition of the Small G Protein Rap1

    doi:

    Figure Lengend Snippet: Enhancement of Rap1 GTPase activity by CD28. (A) Specificity of Rap1GAP1 for Ras and selected Rap1 mutants in vitro. Human Rap1GAP1 was expressed in Cos7 cells and incubated with recombinant Rap1 mutant proteins loaded with [γ- 32 P]GTP. The percentages of hydrolysis of [γ- 32 P]GTP from wild-type Rap1(Rap1 WT), RapV12/E63 (V12/E63), Ras, and buffer alone are indicated by the percentage of γ- 32 P released from Rap or Ras loaded in vitro. (B) CD28 stimulation of Rap1 GAP activity and introduction of E63 into RapV12, which blocks GTPase activity. Jurkat cells were incubated with anti-human CD3 antibody (α-CD3) and/or anti-human CD28 antibody (α-CD28) for 5 min or left untreated (Untr.) as indicated. Lysates were prepared and incubated for 20 min with recombinant FLAG-tagged Rap1 (left graph) or RapV12/E63 (right graph) loaded in vitro with [γ- 32 P]GTP, as indicated. The percentages of release of [γ- 32 P]GTP are shown with standard errors. ( n = 3). (C) Cells were treated as described for panel B and were assayed with [γ- 32 P]GTP. Release of α- 32 P was monitored as described in Materials and Methods, and the percentages of release of [γ- 32 P]GTPase is shown with standard errors ( n = 6).

    Article Snippet: Ca2+ fluctuations, before and after the addition of anti-CD3 MAb (Pharmingen) at 10 μg/ml, were monitored using a fluorescence-activated cell sorter (FACS) Vantage flow cytometer by gating on the GFP-positive cells.

    Techniques: Activity Assay, In Vitro, Incubation, Recombinant, Mutagenesis

    Inhibition of Rap1 by CD28 costimulation in primary splenic T cells and in human Jurkat cells. (A) Activation of Rap1 in primary splenic T cells. Primary splenic T cells were harvested and incubated with anti-mCD3 antibody (α-CD3) and/or anti-mCD28 antibody (α-CD28) for 5 min or left untreated as indicated. (B) Activation of Rap1 in human Jurkat T cells. Jurkat cells were incubated with anti-human CD3 antibody (α-CD3) and/or anti-human CD28 antibody (α-CD28) for 5 min or left untreated as indicated. (C) Inhibition of Rap1 by CD28 following transfection of human Jurkat T cells. Wild-type Jurkat cells were transfected with CrkL/C3G or the vector alone and incubated with α-CD28 as indicated. In all experiments, T-cell lysates were prepared and assayed for Rap1 activation using GST-RalGDS and Western blotting was performed using Rap1 antiserum. The position of Rap1 in control lysates and following isolation of glutathione-bound proteins is shown. Representative Western blots are shown ( n = 3).

    Journal: Molecular and Cellular Biology

    Article Title: CD28 and the Tyrosine Kinase Lck Stimulate Mitogen-Activated Protein Kinase Activity in T Cells via Inhibition of the Small G Protein Rap1

    doi:

    Figure Lengend Snippet: Inhibition of Rap1 by CD28 costimulation in primary splenic T cells and in human Jurkat cells. (A) Activation of Rap1 in primary splenic T cells. Primary splenic T cells were harvested and incubated with anti-mCD3 antibody (α-CD3) and/or anti-mCD28 antibody (α-CD28) for 5 min or left untreated as indicated. (B) Activation of Rap1 in human Jurkat T cells. Jurkat cells were incubated with anti-human CD3 antibody (α-CD3) and/or anti-human CD28 antibody (α-CD28) for 5 min or left untreated as indicated. (C) Inhibition of Rap1 by CD28 following transfection of human Jurkat T cells. Wild-type Jurkat cells were transfected with CrkL/C3G or the vector alone and incubated with α-CD28 as indicated. In all experiments, T-cell lysates were prepared and assayed for Rap1 activation using GST-RalGDS and Western blotting was performed using Rap1 antiserum. The position of Rap1 in control lysates and following isolation of glutathione-bound proteins is shown. Representative Western blots are shown ( n = 3).

    Article Snippet: Ca2+ fluctuations, before and after the addition of anti-CD3 MAb (Pharmingen) at 10 μg/ml, were monitored using a fluorescence-activated cell sorter (FACS) Vantage flow cytometer by gating on the GFP-positive cells.

    Techniques: Inhibition, Activation Assay, Incubation, Transfection, Plasmid Preparation, Western Blot, Isolation

    Lck is sufficient to inhibit TCR stimulation of Rap1. (A) Jurkat cells were transfected with ca.Lck or the vector alone and incubated with anti-human CD3 antibody (α-CD3) for 2 min or left untreated as indicated. (B) Jurkat cells were transfected with LckF505 (ca.Lck) and/or FynF531 (ca.Fyn) or the vector alone. In both experiments, T-cell lysates were prepared and assayed for Rap1 activation using GST-RalGDS and Western blotting was performed using Rap1 antiserum. The position of Rap1 in control lysates and following isolation of glutathione-bound proteins is shown. Representative Western blots are shown ( n = 3).

    Journal: Molecular and Cellular Biology

    Article Title: CD28 and the Tyrosine Kinase Lck Stimulate Mitogen-Activated Protein Kinase Activity in T Cells via Inhibition of the Small G Protein Rap1

    doi:

    Figure Lengend Snippet: Lck is sufficient to inhibit TCR stimulation of Rap1. (A) Jurkat cells were transfected with ca.Lck or the vector alone and incubated with anti-human CD3 antibody (α-CD3) for 2 min or left untreated as indicated. (B) Jurkat cells were transfected with LckF505 (ca.Lck) and/or FynF531 (ca.Fyn) or the vector alone. In both experiments, T-cell lysates were prepared and assayed for Rap1 activation using GST-RalGDS and Western blotting was performed using Rap1 antiserum. The position of Rap1 in control lysates and following isolation of glutathione-bound proteins is shown. Representative Western blots are shown ( n = 3).

    Article Snippet: Ca2+ fluctuations, before and after the addition of anti-CD3 MAb (Pharmingen) at 10 μg/ml, were monitored using a fluorescence-activated cell sorter (FACS) Vantage flow cytometer by gating on the GFP-positive cells.

    Techniques: Transfection, Plasmid Preparation, Incubation, Activation Assay, Western Blot, Isolation

    TLR2 but not TLR1 mRNA expression is upregulated in activated CD4 + T cells. CD4 + T cells (2 × 10 6 ) were either left unstimulated or stimulated overnight with anti-CD3 and anti-CD28 MAbs. Total cellular RNA was isolated, and TLR1

    Journal: Infection and Immunity

    Article Title: Mycobacterium tuberculosis Lipoproteins Directly Regulate Human Memory CD4+ T Cell Activation via Toll-Like Receptors 1 and 2 ▿

    doi: 10.1128/IAI.00806-10

    Figure Lengend Snippet: TLR2 but not TLR1 mRNA expression is upregulated in activated CD4 + T cells. CD4 + T cells (2 × 10 6 ) were either left unstimulated or stimulated overnight with anti-CD3 and anti-CD28 MAbs. Total cellular RNA was isolated, and TLR1

    Article Snippet: CD4+ T cells (105 /well) were cultured in the presence of medium alone or 10 μg of plate-bound anti-CD3 MAb (clone HIT3a; BD Bioscience)/ml with or without 1 μg of soluble anti-CD28 MAb (clone CD28.2; BD Bioscience)/ml and the following: M. tuberculosis fractions (0.5 μg/ml), nonacylated recombinant (NA-rLprG) or acylated recombinant LprG (rLprG) (1 μg/ml), native H37Rv LpqH (nLpqH, 1 μg/ml) provided by the Tuberculosis Vaccine Testing and Research Materials Contract at Colorado State University (NIAID HHSN266200400091C), or N -palmitoyl- S -[2,3-bis(palmitoyloxy)-(2 RS )-propyl]-[ R ]-cysteinyl-[ S ]-seryl-[ S ]-lysyl-[ S ]-lysyl-[ S ]-lysyl-[ S ]-lysine × 3HCl (Pam3 CSK4 ; EMC Microcollections GmbH, Tubingen, Germany) at 0.5 μg/ml.

    Techniques: Expressing, Isolation

    Activation of human CD4 + T cells increases the expression of TLR2. TLR2 surface expression was measured on CD4 + T cells by FACS either at rest (A, D, and G) or after overnight stimulation with anti-CD3 and anti-CD28 (B, E, and H), or anti-CD3

    Journal: Infection and Immunity

    Article Title: Mycobacterium tuberculosis Lipoproteins Directly Regulate Human Memory CD4+ T Cell Activation via Toll-Like Receptors 1 and 2 ▿

    doi: 10.1128/IAI.00806-10

    Figure Lengend Snippet: Activation of human CD4 + T cells increases the expression of TLR2. TLR2 surface expression was measured on CD4 + T cells by FACS either at rest (A, D, and G) or after overnight stimulation with anti-CD3 and anti-CD28 (B, E, and H), or anti-CD3

    Article Snippet: CD4+ T cells (105 /well) were cultured in the presence of medium alone or 10 μg of plate-bound anti-CD3 MAb (clone HIT3a; BD Bioscience)/ml with or without 1 μg of soluble anti-CD28 MAb (clone CD28.2; BD Bioscience)/ml and the following: M. tuberculosis fractions (0.5 μg/ml), nonacylated recombinant (NA-rLprG) or acylated recombinant LprG (rLprG) (1 μg/ml), native H37Rv LpqH (nLpqH, 1 μg/ml) provided by the Tuberculosis Vaccine Testing and Research Materials Contract at Colorado State University (NIAID HHSN266200400091C), or N -palmitoyl- S -[2,3-bis(palmitoyloxy)-(2 RS )-propyl]-[ R ]-cysteinyl-[ S ]-seryl-[ S ]-lysyl-[ S ]-lysyl-[ S ]-lysyl-[ S ]-lysine × 3HCl (Pam3 CSK4 ; EMC Microcollections GmbH, Tubingen, Germany) at 0.5 μg/ml.

    Techniques: Activation Assay, Expressing, FACS

    Acylation is required for lipoprotein-induced costimulation of human CD4 + T cells. CD4 + T cells (10 5 cells/well) were stimulated with anti-CD3 and anti-CD28 plus medium (None), synthetic lipopeptide Pam 3 CSK 4 (P3C), M. tuberculosis fractions

    Journal: Infection and Immunity

    Article Title: Mycobacterium tuberculosis Lipoproteins Directly Regulate Human Memory CD4+ T Cell Activation via Toll-Like Receptors 1 and 2 ▿

    doi: 10.1128/IAI.00806-10

    Figure Lengend Snippet: Acylation is required for lipoprotein-induced costimulation of human CD4 + T cells. CD4 + T cells (10 5 cells/well) were stimulated with anti-CD3 and anti-CD28 plus medium (None), synthetic lipopeptide Pam 3 CSK 4 (P3C), M. tuberculosis fractions

    Article Snippet: CD4+ T cells (105 /well) were cultured in the presence of medium alone or 10 μg of plate-bound anti-CD3 MAb (clone HIT3a; BD Bioscience)/ml with or without 1 μg of soluble anti-CD28 MAb (clone CD28.2; BD Bioscience)/ml and the following: M. tuberculosis fractions (0.5 μg/ml), nonacylated recombinant (NA-rLprG) or acylated recombinant LprG (rLprG) (1 μg/ml), native H37Rv LpqH (nLpqH, 1 μg/ml) provided by the Tuberculosis Vaccine Testing and Research Materials Contract at Colorado State University (NIAID HHSN266200400091C), or N -palmitoyl- S -[2,3-bis(palmitoyloxy)-(2 RS )-propyl]-[ R ]-cysteinyl-[ S ]-seryl-[ S ]-lysyl-[ S ]-lysyl-[ S ]-lysyl-[ S ]-lysine × 3HCl (Pam3 CSK4 ; EMC Microcollections GmbH, Tubingen, Germany) at 0.5 μg/ml.

    Techniques:

    M. tuberculosis lipoprotein LprG induces NF-κB phosphorylation in the absence of TCR signaling. CD4 + T cells (10 6 cells/well) were left unstimulated (Resting [A]) or stimulated overnight with anti-CD3 and anti-CD28 (Prestimulated [B]).

    Journal: Infection and Immunity

    Article Title: Mycobacterium tuberculosis Lipoproteins Directly Regulate Human Memory CD4+ T Cell Activation via Toll-Like Receptors 1 and 2 ▿

    doi: 10.1128/IAI.00806-10

    Figure Lengend Snippet: M. tuberculosis lipoprotein LprG induces NF-κB phosphorylation in the absence of TCR signaling. CD4 + T cells (10 6 cells/well) were left unstimulated (Resting [A]) or stimulated overnight with anti-CD3 and anti-CD28 (Prestimulated [B]).

    Article Snippet: CD4+ T cells (105 /well) were cultured in the presence of medium alone or 10 μg of plate-bound anti-CD3 MAb (clone HIT3a; BD Bioscience)/ml with or without 1 μg of soluble anti-CD28 MAb (clone CD28.2; BD Bioscience)/ml and the following: M. tuberculosis fractions (0.5 μg/ml), nonacylated recombinant (NA-rLprG) or acylated recombinant LprG (rLprG) (1 μg/ml), native H37Rv LpqH (nLpqH, 1 μg/ml) provided by the Tuberculosis Vaccine Testing and Research Materials Contract at Colorado State University (NIAID HHSN266200400091C), or N -palmitoyl- S -[2,3-bis(palmitoyloxy)-(2 RS )-propyl]-[ R ]-cysteinyl-[ S ]-seryl-[ S ]-lysyl-[ S ]-lysyl-[ S ]-lysyl-[ S ]-lysine × 3HCl (Pam3 CSK4 ; EMC Microcollections GmbH, Tubingen, Germany) at 0.5 μg/ml.

    Techniques:

    LprG induces CD4 + T cell costimulation via TLR1 and TLR2. CD4 + T cells (10 5 cells/well) were stimulated overnight with anti-CD3 and anti-CD28 MAbs. Cells were then treated with control immunoglobulin (Ig), anti-TLR1, anti-TLR2 or a combination

    Journal: Infection and Immunity

    Article Title: Mycobacterium tuberculosis Lipoproteins Directly Regulate Human Memory CD4+ T Cell Activation via Toll-Like Receptors 1 and 2 ▿

    doi: 10.1128/IAI.00806-10

    Figure Lengend Snippet: LprG induces CD4 + T cell costimulation via TLR1 and TLR2. CD4 + T cells (10 5 cells/well) were stimulated overnight with anti-CD3 and anti-CD28 MAbs. Cells were then treated with control immunoglobulin (Ig), anti-TLR1, anti-TLR2 or a combination

    Article Snippet: CD4+ T cells (105 /well) were cultured in the presence of medium alone or 10 μg of plate-bound anti-CD3 MAb (clone HIT3a; BD Bioscience)/ml with or without 1 μg of soluble anti-CD28 MAb (clone CD28.2; BD Bioscience)/ml and the following: M. tuberculosis fractions (0.5 μg/ml), nonacylated recombinant (NA-rLprG) or acylated recombinant LprG (rLprG) (1 μg/ml), native H37Rv LpqH (nLpqH, 1 μg/ml) provided by the Tuberculosis Vaccine Testing and Research Materials Contract at Colorado State University (NIAID HHSN266200400091C), or N -palmitoyl- S -[2,3-bis(palmitoyloxy)-(2 RS )-propyl]-[ R ]-cysteinyl-[ S ]-seryl-[ S ]-lysyl-[ S ]-lysyl-[ S ]-lysyl-[ S ]-lysine × 3HCl (Pam3 CSK4 ; EMC Microcollections GmbH, Tubingen, Germany) at 0.5 μg/ml.

    Techniques:

    Lipoproteins LprG and LpqH are major CD4 + T cell costimulatory ligands in M. tuberculosis lysate. CD4 + T cells (10 5 cells/well) were stimulated with plate-bound anti-CD3 (10 μg/ml) and soluble anti-CD28 (1 μg/ml) in presence

    Journal: Infection and Immunity

    Article Title: Mycobacterium tuberculosis Lipoproteins Directly Regulate Human Memory CD4+ T Cell Activation via Toll-Like Receptors 1 and 2 ▿

    doi: 10.1128/IAI.00806-10

    Figure Lengend Snippet: Lipoproteins LprG and LpqH are major CD4 + T cell costimulatory ligands in M. tuberculosis lysate. CD4 + T cells (10 5 cells/well) were stimulated with plate-bound anti-CD3 (10 μg/ml) and soluble anti-CD28 (1 μg/ml) in presence

    Article Snippet: CD4+ T cells (105 /well) were cultured in the presence of medium alone or 10 μg of plate-bound anti-CD3 MAb (clone HIT3a; BD Bioscience)/ml with or without 1 μg of soluble anti-CD28 MAb (clone CD28.2; BD Bioscience)/ml and the following: M. tuberculosis fractions (0.5 μg/ml), nonacylated recombinant (NA-rLprG) or acylated recombinant LprG (rLprG) (1 μg/ml), native H37Rv LpqH (nLpqH, 1 μg/ml) provided by the Tuberculosis Vaccine Testing and Research Materials Contract at Colorado State University (NIAID HHSN266200400091C), or N -palmitoyl- S -[2,3-bis(palmitoyloxy)-(2 RS )-propyl]-[ R ]-cysteinyl-[ S ]-seryl-[ S ]-lysyl-[ S ]-lysyl-[ S ]-lysyl-[ S ]-lysine × 3HCl (Pam3 CSK4 ; EMC Microcollections GmbH, Tubingen, Germany) at 0.5 μg/ml.

    Techniques:

    M. tuberculosis lipoproteins trigger a second signal for CD4 + T cell activation that results in cytokine secretion and proliferation. CD4 + T cells (10 5 cells/well) were stimulated with anti-CD3 and either medium alone (none), anti-CD28MAb,

    Journal: Infection and Immunity

    Article Title: Mycobacterium tuberculosis Lipoproteins Directly Regulate Human Memory CD4+ T Cell Activation via Toll-Like Receptors 1 and 2 ▿

    doi: 10.1128/IAI.00806-10

    Figure Lengend Snippet: M. tuberculosis lipoproteins trigger a second signal for CD4 + T cell activation that results in cytokine secretion and proliferation. CD4 + T cells (10 5 cells/well) were stimulated with anti-CD3 and either medium alone (none), anti-CD28MAb,

    Article Snippet: CD4+ T cells (105 /well) were cultured in the presence of medium alone or 10 μg of plate-bound anti-CD3 MAb (clone HIT3a; BD Bioscience)/ml with or without 1 μg of soluble anti-CD28 MAb (clone CD28.2; BD Bioscience)/ml and the following: M. tuberculosis fractions (0.5 μg/ml), nonacylated recombinant (NA-rLprG) or acylated recombinant LprG (rLprG) (1 μg/ml), native H37Rv LpqH (nLpqH, 1 μg/ml) provided by the Tuberculosis Vaccine Testing and Research Materials Contract at Colorado State University (NIAID HHSN266200400091C), or N -palmitoyl- S -[2,3-bis(palmitoyloxy)-(2 RS )-propyl]-[ R ]-cysteinyl-[ S ]-seryl-[ S ]-lysyl-[ S ]-lysyl-[ S ]-lysyl-[ S ]-lysine × 3HCl (Pam3 CSK4 ; EMC Microcollections GmbH, Tubingen, Germany) at 0.5 μg/ml.

    Techniques: Activation Assay

    Involvement T cells in the VPDT-mediated immune response. ( a ) T-cell infiltration. Tumor sections obtained at different times after VPDT were stained with antibodies against CD4 (red), CD8 (green), and Hoechst 33342 (blue). Arrows indicate the sites of infiltration. Bar = 120 μm. ( b ) Splenocyte cytotoxicity. On Day 40 post-tumor re-challenge, the tumor-free mice were sacrificed. Their splenocytes were used as the effector with CT26 cells as the target. The two cell types were mixed at a ratio of 50:1 and incubated for 4 h. The cytotoxicity of the splenocytes was evaluated using flow cytometry, n = 3. ( c ) T-cell-dependent immunological memory. Mice that had been cured by VPDT and were resistant to tumor re-challenge ( n = 4) were depleted of T cells by intraperitoneal injections of an anti-CD3 antibody. These mice were then re-challenged with 1 × 10 6 CT26 cells. The tumor sizes were recorded on Day 12 post-inoculation. The line represents the mean value in the vertical scatter plot. * p

    Journal: Cellular and Molecular Immunology

    Article Title: Anti-tumor immunity of BAM-SiPc-mediated vascular photodynamic therapy in a BALB/c mouse model

    doi: 10.1038/cmi.2015.84

    Figure Lengend Snippet: Involvement T cells in the VPDT-mediated immune response. ( a ) T-cell infiltration. Tumor sections obtained at different times after VPDT were stained with antibodies against CD4 (red), CD8 (green), and Hoechst 33342 (blue). Arrows indicate the sites of infiltration. Bar = 120 μm. ( b ) Splenocyte cytotoxicity. On Day 40 post-tumor re-challenge, the tumor-free mice were sacrificed. Their splenocytes were used as the effector with CT26 cells as the target. The two cell types were mixed at a ratio of 50:1 and incubated for 4 h. The cytotoxicity of the splenocytes was evaluated using flow cytometry, n = 3. ( c ) T-cell-dependent immunological memory. Mice that had been cured by VPDT and were resistant to tumor re-challenge ( n = 4) were depleted of T cells by intraperitoneal injections of an anti-CD3 antibody. These mice were then re-challenged with 1 × 10 6 CT26 cells. The tumor sizes were recorded on Day 12 post-inoculation. The line represents the mean value in the vertical scatter plot. * p

    Article Snippet: BALB/c mice that had resisted tumor growth in the re-challenge test, after either VPDT or serum treatment, were given an intraperitoneal injection of 3 µg of anti-CD3 antibody (16003285, eBioscience, San Diego, CA, US) on Days −1, 0, 2, and 4.

    Techniques: Staining, Mouse Assay, Incubation, Flow Cytometry, Cytometry

    CD95 promotes upregulation of activation markers and ERK activation. Purified human CD4 + T cells were incubated in X-VIVO medium with immobilized anti-CD3 mAb or PHA for the indicated time periods in the presence or absence of plate-bound anti-CD95 mAb anti-APO-1 (5 μ g/ml) ( a – c , e and f ) or CD95L-ST-Fc (2,5 μ g/ml) ( e ) as well as high doses of CD95L-ST-Fc ( d ) or CD95LFc ( f ) (both 20 μ g/ml). The expression of CD69 ( a and b ) and other activation markers ( b ) was determined between 1–8 h ( a ), and at d2 ( b ) of incubation by flow cytometry using FITC/PE-labeled anti-CD69, anti-OX40, anti-CTLA-4, anti-CD25, anti-IL-2R β and anti-CD95L mAb, respectively. Expression levels are given as MFI and are representative of three independent experiments with different donors. ( c–d ) After incubation at 37°C for the indicated time points, T cells were lysed and aliquots of 20 μ g of the whole-cell lysate were analyzed by western blot using antibodies against phospho-ERK and ERK as a loading control. ( e ) Cells were treated in the presence or absence of the ERK1/2 inhibitor PD 98059 (50 μ M) or DMSO for 3 days before MTS assay. The experiment was performed in triplicates. Error bars represent S.Ds. from the mean. Statistical differences were calculated by student's t -test: ** P

    Journal: Cell Death and Differentiation

    Article Title: Modulation of CD4+ T-cell activation by CD95 co-stimulation

    doi: 10.1038/cdd.2010.134

    Figure Lengend Snippet: CD95 promotes upregulation of activation markers and ERK activation. Purified human CD4 + T cells were incubated in X-VIVO medium with immobilized anti-CD3 mAb or PHA for the indicated time periods in the presence or absence of plate-bound anti-CD95 mAb anti-APO-1 (5 μ g/ml) ( a – c , e and f ) or CD95L-ST-Fc (2,5 μ g/ml) ( e ) as well as high doses of CD95L-ST-Fc ( d ) or CD95LFc ( f ) (both 20 μ g/ml). The expression of CD69 ( a and b ) and other activation markers ( b ) was determined between 1–8 h ( a ), and at d2 ( b ) of incubation by flow cytometry using FITC/PE-labeled anti-CD69, anti-OX40, anti-CTLA-4, anti-CD25, anti-IL-2R β and anti-CD95L mAb, respectively. Expression levels are given as MFI and are representative of three independent experiments with different donors. ( c–d ) After incubation at 37°C for the indicated time points, T cells were lysed and aliquots of 20 μ g of the whole-cell lysate were analyzed by western blot using antibodies against phospho-ERK and ERK as a loading control. ( e ) Cells were treated in the presence or absence of the ERK1/2 inhibitor PD 98059 (50 μ M) or DMSO for 3 days before MTS assay. The experiment was performed in triplicates. Error bars represent S.Ds. from the mean. Statistical differences were calculated by student's t -test: ** P

    Article Snippet: TCR stimulation was performed with immobilized anti-CD3 mAb (OKT3, 2 μ g/ml, IgG2a, Cilag, Sulzbach, Germany) and anti-CD28 mAb (CD28.2, 5 μ g/ml, IgG1, BD Biosciences, San Diego, CA, USA).

    Techniques: Activation Assay, Purification, Incubation, Expressing, Flow Cytometry, Cytometry, Labeling, Western Blot, MTS Assay

    TCR and CD95 internalization are enhanced in TCR-triggered cells co-treated through low-dose CD95. Freshly isolated CD4 + T cells were incubated with plate-bound anti-CD3/CD28 mAb ( a and b ) or PHA ( c – e ) in the presence or absence of immobilized anti-APO-1 (5 μ g/ml). ( a and b ) TCR internalization was analyzed using a FITC-labeled TCRα/β-specific mAb between 2 and 8 h ( a ) and after 48 h of stimulation ( b ). The experiments were performed in triplicates. Error bars represent S.Ds. from the mean. Statistical differences were calculated by student's t -test: * P

    Journal: Cell Death and Differentiation

    Article Title: Modulation of CD4+ T-cell activation by CD95 co-stimulation

    doi: 10.1038/cdd.2010.134

    Figure Lengend Snippet: TCR and CD95 internalization are enhanced in TCR-triggered cells co-treated through low-dose CD95. Freshly isolated CD4 + T cells were incubated with plate-bound anti-CD3/CD28 mAb ( a and b ) or PHA ( c – e ) in the presence or absence of immobilized anti-APO-1 (5 μ g/ml). ( a and b ) TCR internalization was analyzed using a FITC-labeled TCRα/β-specific mAb between 2 and 8 h ( a ) and after 48 h of stimulation ( b ). The experiments were performed in triplicates. Error bars represent S.Ds. from the mean. Statistical differences were calculated by student's t -test: * P

    Article Snippet: TCR stimulation was performed with immobilized anti-CD3 mAb (OKT3, 2 μ g/ml, IgG2a, Cilag, Sulzbach, Germany) and anti-CD28 mAb (CD28.2, 5 μ g/ml, IgG1, BD Biosciences, San Diego, CA, USA).

    Techniques: Isolation, Incubation, Labeling

    CD95 ligands affect T-cell activation in a dose-dependent manner. MACS-purified human CD4 + T cells from three different donors were cultured overnight in X-VIVO medium in 96-well plates coated with or without anti-CD3 mAb and anti-CD28 mAb in the presence or absence of graded doses of plate-bound CD95LFc, CD95L-ST-Fc, LZ-CD95L or anti-CD95 mAb anti-APO-1 as indicated. The expression levels of CD25 and CD69 were monitored by flow cytometry. The percentage of CD25/CD69 double-positive cells is indicated in individual dot plots

    Journal: Cell Death and Differentiation

    Article Title: Modulation of CD4+ T-cell activation by CD95 co-stimulation

    doi: 10.1038/cdd.2010.134

    Figure Lengend Snippet: CD95 ligands affect T-cell activation in a dose-dependent manner. MACS-purified human CD4 + T cells from three different donors were cultured overnight in X-VIVO medium in 96-well plates coated with or without anti-CD3 mAb and anti-CD28 mAb in the presence or absence of graded doses of plate-bound CD95LFc, CD95L-ST-Fc, LZ-CD95L or anti-CD95 mAb anti-APO-1 as indicated. The expression levels of CD25 and CD69 were monitored by flow cytometry. The percentage of CD25/CD69 double-positive cells is indicated in individual dot plots

    Article Snippet: TCR stimulation was performed with immobilized anti-CD3 mAb (OKT3, 2 μ g/ml, IgG2a, Cilag, Sulzbach, Germany) and anti-CD28 mAb (CD28.2, 5 μ g/ml, IgG1, BD Biosciences, San Diego, CA, USA).

    Techniques: Activation Assay, Magnetic Cell Separation, Purification, Cell Culture, Expressing, Flow Cytometry, Cytometry

    CD95 co-stimulation induces upregulation of cell cycle-regulating proteins and promotes cell-cycle progression. Purified human CD4 + T cells were stimulated with plate-bound anti-CD3 mAb alone or in combination with anti-CD28 mAb. Anti-APO-1 (5 μ g/ml) was co-immobilized where indicated. ( a ) At d3 of incubation, cell-cycle analysis was performed by PI-staining and determination of the DNA content by flow cytometry. Individual phases of the cell cycle, including hypodiploid apoptotic nuclei are indicated. Values denote the percentage of cells in each region. ( b ) At d3 of culture, the cells were prepared for western blot and analyzed for the expression or phosphorylation of several cell-cycle-regulating proteins using antibodies specific for phospho-Rb (Ser795), phospho-Rb (Ser780), phospho-Rb (Ser807/811), CDKs 1, 2 and 6, cyclins D1, D2, E and B1, PCNA and ERK as a loading control

    Journal: Cell Death and Differentiation

    Article Title: Modulation of CD4+ T-cell activation by CD95 co-stimulation

    doi: 10.1038/cdd.2010.134

    Figure Lengend Snippet: CD95 co-stimulation induces upregulation of cell cycle-regulating proteins and promotes cell-cycle progression. Purified human CD4 + T cells were stimulated with plate-bound anti-CD3 mAb alone or in combination with anti-CD28 mAb. Anti-APO-1 (5 μ g/ml) was co-immobilized where indicated. ( a ) At d3 of incubation, cell-cycle analysis was performed by PI-staining and determination of the DNA content by flow cytometry. Individual phases of the cell cycle, including hypodiploid apoptotic nuclei are indicated. Values denote the percentage of cells in each region. ( b ) At d3 of culture, the cells were prepared for western blot and analyzed for the expression or phosphorylation of several cell-cycle-regulating proteins using antibodies specific for phospho-Rb (Ser795), phospho-Rb (Ser780), phospho-Rb (Ser807/811), CDKs 1, 2 and 6, cyclins D1, D2, E and B1, PCNA and ERK as a loading control

    Article Snippet: TCR stimulation was performed with immobilized anti-CD3 mAb (OKT3, 2 μ g/ml, IgG2a, Cilag, Sulzbach, Germany) and anti-CD28 mAb (CD28.2, 5 μ g/ml, IgG1, BD Biosciences, San Diego, CA, USA).

    Techniques: Purification, Incubation, Cell Cycle Assay, Staining, Flow Cytometry, Cytometry, Western Blot, Expressing

    The low-dose co-stimulatory effect of CD95 is associated with IL-2 production and potentially skews a Th1 response. Freshly isolated CD4 + T cells were left untreated or treated with immobilized anti-CD3 mAb plus/minus anti-CD28 mAb in the absence or presence of 5 μ g/ml anti-APO-1. ( a ) Secreted IL-2 was determined in supernatants by ELISA after 24, 48 and 72 h. Experiments were performed in triplicates with error bars indicating S.Ds., and are representative for three experiments carried out with different donors. ( b ) Exogenous rIL-2 was added where indicated. At day 3 of culture, CD25 expression was analyzed by flow cytometry using a PE-labeled anti-CD25 mAb. The percentages of CD25-positive cells are indicated in individual dot plots. ( c ) IFN γ , IL-4 and TNF α were measured by intracellular staining at d3. The experiment was performed in triplicates. Cytokine production is given as mean fluorescence intensities (MFI) for one representative experiment out of five with different donors. Error bars indicate S.Ds. from the MFI. Statistical differences were calculated by a standard t -test: * P

    Journal: Cell Death and Differentiation

    Article Title: Modulation of CD4+ T-cell activation by CD95 co-stimulation

    doi: 10.1038/cdd.2010.134

    Figure Lengend Snippet: The low-dose co-stimulatory effect of CD95 is associated with IL-2 production and potentially skews a Th1 response. Freshly isolated CD4 + T cells were left untreated or treated with immobilized anti-CD3 mAb plus/minus anti-CD28 mAb in the absence or presence of 5 μ g/ml anti-APO-1. ( a ) Secreted IL-2 was determined in supernatants by ELISA after 24, 48 and 72 h. Experiments were performed in triplicates with error bars indicating S.Ds., and are representative for three experiments carried out with different donors. ( b ) Exogenous rIL-2 was added where indicated. At day 3 of culture, CD25 expression was analyzed by flow cytometry using a PE-labeled anti-CD25 mAb. The percentages of CD25-positive cells are indicated in individual dot plots. ( c ) IFN γ , IL-4 and TNF α were measured by intracellular staining at d3. The experiment was performed in triplicates. Cytokine production is given as mean fluorescence intensities (MFI) for one representative experiment out of five with different donors. Error bars indicate S.Ds. from the MFI. Statistical differences were calculated by a standard t -test: * P

    Article Snippet: TCR stimulation was performed with immobilized anti-CD3 mAb (OKT3, 2 μ g/ml, IgG2a, Cilag, Sulzbach, Germany) and anti-CD28 mAb (CD28.2, 5 μ g/ml, IgG1, BD Biosciences, San Diego, CA, USA).

    Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Cytometry, Labeling, Staining, Fluorescence

    CD95 stimulation affects activation of primary T cells without induction of cell death. ( a ) Freshly isolated human PBMCs were stimulated for 3 days on 96-well plates coated with anti-CD3 or anti-CD3/anti-CD28 in the presence or absence of anti-CD95 mAb (here 7C11, 2 μ g/ml), 20 μ g/ml CD95LFc or huIgGFc as a control. Proliferation was determined by adding [ 3 H] TdR for 16 h before harvesting. This experiment was performed in triplicates. Error bars indicate the S.D. of the mean values. ( b – e ) Purified CD4 + T cells were cultured in X-VIVO medium for 3 days in 24-well plates coated with anti-CD3/anti-CD28 in the presence or absence of anti-CD95 mAb (here anti-APO-1, 5 μ g/ml), CD95LFc or Fc control protein (both 20 μ g/ml). ( b ) Microphotographs document the increase in cell size and cluster formation as observed by inverse light microscopy. ( c ) Blastogenesis on the basis of cell size (forward scatter) and granularity (side scatter) was determined by FACS analysis. ( d ) Freshly isolated human CD4 + T cells were labeled with CFSE and stimulated as indicated. CFSE profiles were analyzed by flow cytometry. ( e ) Cell death of stimulated CD4 + T cells was investigated by PI-staining and FACS analysis. All experiments were performed with freshly isolated cells from different donors, and are representative of at least three independent experiments performed

    Journal: Cell Death and Differentiation

    Article Title: Modulation of CD4+ T-cell activation by CD95 co-stimulation

    doi: 10.1038/cdd.2010.134

    Figure Lengend Snippet: CD95 stimulation affects activation of primary T cells without induction of cell death. ( a ) Freshly isolated human PBMCs were stimulated for 3 days on 96-well plates coated with anti-CD3 or anti-CD3/anti-CD28 in the presence or absence of anti-CD95 mAb (here 7C11, 2 μ g/ml), 20 μ g/ml CD95LFc or huIgGFc as a control. Proliferation was determined by adding [ 3 H] TdR for 16 h before harvesting. This experiment was performed in triplicates. Error bars indicate the S.D. of the mean values. ( b – e ) Purified CD4 + T cells were cultured in X-VIVO medium for 3 days in 24-well plates coated with anti-CD3/anti-CD28 in the presence or absence of anti-CD95 mAb (here anti-APO-1, 5 μ g/ml), CD95LFc or Fc control protein (both 20 μ g/ml). ( b ) Microphotographs document the increase in cell size and cluster formation as observed by inverse light microscopy. ( c ) Blastogenesis on the basis of cell size (forward scatter) and granularity (side scatter) was determined by FACS analysis. ( d ) Freshly isolated human CD4 + T cells were labeled with CFSE and stimulated as indicated. CFSE profiles were analyzed by flow cytometry. ( e ) Cell death of stimulated CD4 + T cells was investigated by PI-staining and FACS analysis. All experiments were performed with freshly isolated cells from different donors, and are representative of at least three independent experiments performed

    Article Snippet: TCR stimulation was performed with immobilized anti-CD3 mAb (OKT3, 2 μ g/ml, IgG2a, Cilag, Sulzbach, Germany) and anti-CD28 mAb (CD28.2, 5 μ g/ml, IgG1, BD Biosciences, San Diego, CA, USA).

    Techniques: Activation Assay, Isolation, Purification, Cell Culture, Light Microscopy, FACS, Labeling, Flow Cytometry, Cytometry, Staining

    Low-dose CD95 co-engagement induces caspase activation, expression of antiapoptotic proteins and promotes apoptosis resistance. Caspase-3/-8 activity ( a ) and processing ( b and c ) as well as cleavage of caspase substrates ( c ) were determined after incubation of primary human TCR-stimulated cells in the presence or absence of the indicated CD95 agonists at different concentrations. ( a ) Cleavage of the caspase-3/-8 specific substrates Ac-DEVD-AMC/Ac-IETD-AMC, followed by the release of the fluorogenic AMC, was used to detect activity of caspase-3/-8 in lysates from T cells incubated for 3 days with or without anti-CD3 +/− anti-CD28 mAb in the presence or absence of anti-CD95 mAb (anti-APO-1, 5 μ g/ml (C3)) or CD95LFc (2.5 (C2) and 40 μ g/ml (C1)). An AMC fluorescence reference standard was used to calibrate the AMC-based caspase substrates in order to quantify caspase activities ( a , lower panel). In this study, we included the caspase activity of Jurkat T cells incubated with 5 μ g/ml of soluble CD95LFc for 2 h to induce apoptosis. ( b ) Resting T cells were stimulated as indicated in the presence or absence of anti-APO-1 (5 μ g/ml). Cleavage of caspase-3 was analyzed by western blot at day 1–3. ( c ) After 2 days of stimulation, T-cell lysates were analyzed by western blot for processed caspase-3 and -8, and the cleavage of PLC γ and PARP. ERK was used as a loading control. ( d ) Similarly, levels of cFLIP, Bcl-X L , I κ B and phospho-I κ B were determined by western blot. ( e ) To determine apoptosis resistance, CD4 + T cells were incubated with immobilized anti-CD3 alone or in combination with anti-APO-1 or left untreated. At d2, the cells were collected, washed and exposed to γ -irradiation or mistletoe lectin and incubated overnight. Untreated control cells were left on coated plates used for the initial stimulation. Induction of cell death was determined by PI-staining

    Journal: Cell Death and Differentiation

    Article Title: Modulation of CD4+ T-cell activation by CD95 co-stimulation

    doi: 10.1038/cdd.2010.134

    Figure Lengend Snippet: Low-dose CD95 co-engagement induces caspase activation, expression of antiapoptotic proteins and promotes apoptosis resistance. Caspase-3/-8 activity ( a ) and processing ( b and c ) as well as cleavage of caspase substrates ( c ) were determined after incubation of primary human TCR-stimulated cells in the presence or absence of the indicated CD95 agonists at different concentrations. ( a ) Cleavage of the caspase-3/-8 specific substrates Ac-DEVD-AMC/Ac-IETD-AMC, followed by the release of the fluorogenic AMC, was used to detect activity of caspase-3/-8 in lysates from T cells incubated for 3 days with or without anti-CD3 +/− anti-CD28 mAb in the presence or absence of anti-CD95 mAb (anti-APO-1, 5 μ g/ml (C3)) or CD95LFc (2.5 (C2) and 40 μ g/ml (C1)). An AMC fluorescence reference standard was used to calibrate the AMC-based caspase substrates in order to quantify caspase activities ( a , lower panel). In this study, we included the caspase activity of Jurkat T cells incubated with 5 μ g/ml of soluble CD95LFc for 2 h to induce apoptosis. ( b ) Resting T cells were stimulated as indicated in the presence or absence of anti-APO-1 (5 μ g/ml). Cleavage of caspase-3 was analyzed by western blot at day 1–3. ( c ) After 2 days of stimulation, T-cell lysates were analyzed by western blot for processed caspase-3 and -8, and the cleavage of PLC γ and PARP. ERK was used as a loading control. ( d ) Similarly, levels of cFLIP, Bcl-X L , I κ B and phospho-I κ B were determined by western blot. ( e ) To determine apoptosis resistance, CD4 + T cells were incubated with immobilized anti-CD3 alone or in combination with anti-APO-1 or left untreated. At d2, the cells were collected, washed and exposed to γ -irradiation or mistletoe lectin and incubated overnight. Untreated control cells were left on coated plates used for the initial stimulation. Induction of cell death was determined by PI-staining

    Article Snippet: TCR stimulation was performed with immobilized anti-CD3 mAb (OKT3, 2 μ g/ml, IgG2a, Cilag, Sulzbach, Germany) and anti-CD28 mAb (CD28.2, 5 μ g/ml, IgG1, BD Biosciences, San Diego, CA, USA).

    Techniques: Activation Assay, Expressing, Activity Assay, Incubation, Fluorescence, Western Blot, Planar Chromatography, Irradiation, Staining

    Comparsion of different lysosomal markers for the identification of degranulating NK cells. ( A ) PBMC were surface stained with fluorochrome-conjugated anti-CD3 and anti-CD56 mAbs, followed by intracellular staining with fluorochrome-conjugated anti-CD63, anti-CD107a, anti-CD107b, anti-perforin, or anti-granzyme A mAbs, as indicated. Profiles show CD56 versus intracellular lysosomal protein staining, as indicated on CD3 − CD56 + NK cells. PBMC were incubated either alone ( B ), or with K562 cells ( C ) for 2 hours at 37°C, surface stained with fluorochrome-conjugated anti-CD3 and anti-CD56 mAbs, in addition to anti-CD63, anti-CD107a, or anti-CD107b mAbs, as indicated. Profiles show CD56 versus surface staining of lysosomal proteins, as indicated on CD3 − CD56 + NK cells.

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: Functional Analysis of Human NK cells by Flow Cytometry

    doi: 10.1007/978-1-60761-362-6_23

    Figure Lengend Snippet: Comparsion of different lysosomal markers for the identification of degranulating NK cells. ( A ) PBMC were surface stained with fluorochrome-conjugated anti-CD3 and anti-CD56 mAbs, followed by intracellular staining with fluorochrome-conjugated anti-CD63, anti-CD107a, anti-CD107b, anti-perforin, or anti-granzyme A mAbs, as indicated. Profiles show CD56 versus intracellular lysosomal protein staining, as indicated on CD3 − CD56 + NK cells. PBMC were incubated either alone ( B ), or with K562 cells ( C ) for 2 hours at 37°C, surface stained with fluorochrome-conjugated anti-CD3 and anti-CD56 mAbs, in addition to anti-CD63, anti-CD107a, or anti-CD107b mAbs, as indicated. Profiles show CD56 versus surface staining of lysosomal proteins, as indicated on CD3 − CD56 + NK cells.

    Article Snippet: Anti-CD3 mAb (clone UCHT1) Cascade Yellow (Dako, Glostrup, Denmark).

    Techniques: Staining, Incubation

    Analysis of multiple human NK cell responses by flow cytometric analysis. PBMC were incubated with target cells for 6 hours at 37°C, surface stained with fluorochrome-conjugated anti-CD3, anti-CD14, anti-CD19, anti-CD56, and anti-CD107a mAbs, followed by intracellular staining with fluorochrome-conjugated anti-IFN-γ, anti-MIP-1β, and anti-TNF-α mAbs. ( A ) Gating strategy for identification of CD3 − CD56 dim NK cells. ( B ) Profiles show staining for multiple responses on CD3 − CD56 dim NK cells from one representative donor after incubation with target cells as indicated for 6 hours at 37°C. One representative donor is shown. ( C ) Pies represent the distribution of cells responding with different numbers of distinct responses, as indicated. Surrounding the pies, lines indicate the nature of individual responses.

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: Functional Analysis of Human NK cells by Flow Cytometry

    doi: 10.1007/978-1-60761-362-6_23

    Figure Lengend Snippet: Analysis of multiple human NK cell responses by flow cytometric analysis. PBMC were incubated with target cells for 6 hours at 37°C, surface stained with fluorochrome-conjugated anti-CD3, anti-CD14, anti-CD19, anti-CD56, and anti-CD107a mAbs, followed by intracellular staining with fluorochrome-conjugated anti-IFN-γ, anti-MIP-1β, and anti-TNF-α mAbs. ( A ) Gating strategy for identification of CD3 − CD56 dim NK cells. ( B ) Profiles show staining for multiple responses on CD3 − CD56 dim NK cells from one representative donor after incubation with target cells as indicated for 6 hours at 37°C. One representative donor is shown. ( C ) Pies represent the distribution of cells responding with different numbers of distinct responses, as indicated. Surrounding the pies, lines indicate the nature of individual responses.

    Article Snippet: Anti-CD3 mAb (clone UCHT1) Cascade Yellow (Dako, Glostrup, Denmark).

    Techniques: Flow Cytometry, Incubation, Staining

    Donor variation in NK cell responses. NK cells were stimulated with ( A ) K562 cells or ( B ) P815 cells supplemented with anti-CD16 mAb and incubated for 6 hours at 37°C. Profiles show CD107a versus IFN-γ staining on CD3 − CD56 dim NK cells from two different donor.

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: Functional Analysis of Human NK cells by Flow Cytometry

    doi: 10.1007/978-1-60761-362-6_23

    Figure Lengend Snippet: Donor variation in NK cell responses. NK cells were stimulated with ( A ) K562 cells or ( B ) P815 cells supplemented with anti-CD16 mAb and incubated for 6 hours at 37°C. Profiles show CD107a versus IFN-γ staining on CD3 − CD56 dim NK cells from two different donor.

    Article Snippet: Anti-CD3 mAb (clone UCHT1) Cascade Yellow (Dako, Glostrup, Denmark).

    Techniques: Incubation, Staining

    Analysis of human NK cell degranulation by flow cytometric analysis. PBMC were incubated with target cells for 2 hours at 37°C, surface stained with fluorochrome-conjugated anti-CD3, anti-CD56, and anti-CD107a mAbs. ( A ) Profiles demonstrate the gating strategy [side scatter (SSC) vs. forward scatter (FCS) and CD56 vs. CD3] for identification of CD3 − CD56 + NK cells with FlowJo software. ( B ) Profiles show CD56 versus CD107a staining on CD3 − CD56 + NK cells for one representative donor after incubation of PBMC with indicated target cells.

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: Functional Analysis of Human NK cells by Flow Cytometry

    doi: 10.1007/978-1-60761-362-6_23

    Figure Lengend Snippet: Analysis of human NK cell degranulation by flow cytometric analysis. PBMC were incubated with target cells for 2 hours at 37°C, surface stained with fluorochrome-conjugated anti-CD3, anti-CD56, and anti-CD107a mAbs. ( A ) Profiles demonstrate the gating strategy [side scatter (SSC) vs. forward scatter (FCS) and CD56 vs. CD3] for identification of CD3 − CD56 + NK cells with FlowJo software. ( B ) Profiles show CD56 versus CD107a staining on CD3 − CD56 + NK cells for one representative donor after incubation of PBMC with indicated target cells.

    Article Snippet: Anti-CD3 mAb (clone UCHT1) Cascade Yellow (Dako, Glostrup, Denmark).

    Techniques: Flow Cytometry, Incubation, Staining, Software

    tTreg cells treated with miR-142b-3p antagomir show enhanced autophagy status, FoxP3 expression, with suppressive function. ( n = 3). PB CD4+CD25+CD127− tTregs were sort purified by MACS, expanded in vitro for 14 days, treated with or without agomir/antagomir for 2 more days. Immunofluorescence confocal microscopy test for tTreg autophagy status after treatment. a Representative cellular autophagy level by the laser scanning. b The ratio of punctate autophagic vacuoles was measured by ImageJ software. c LC3B western blot results to detect the autophagy status after antagomir or agomir treatment. d ) Representative example of Foxp3 vs CD127 staining on tTreg treated with antagomir or agomir (gated on CD4+ cells). Summary of overall e level of Foxp3 expression and f representative example of Ki67 histogram on tTreg treated with antagomir or agomir (gated on CD4+ cells). Summary of overall e level of Ki67 expression after antagomir/agomir treatment. g Percent suppression of in vitro, anti-CD3–mediated CD8+ T cell proliferation at ratios from 1:2 to 1:32 (tTreg: PBMC) as determined by CFSE dye dilution. Values indicate mean ± SEM of these experiments (* P

    Journal: Cell Death & Disease

    Article Title: miR-142-3p regulates autophagy by targeting ATG16L1 in thymic-derived regulatory T cell (tTreg)

    doi: 10.1038/s41419-018-0298-2

    Figure Lengend Snippet: tTreg cells treated with miR-142b-3p antagomir show enhanced autophagy status, FoxP3 expression, with suppressive function. ( n = 3). PB CD4+CD25+CD127− tTregs were sort purified by MACS, expanded in vitro for 14 days, treated with or without agomir/antagomir for 2 more days. Immunofluorescence confocal microscopy test for tTreg autophagy status after treatment. a Representative cellular autophagy level by the laser scanning. b The ratio of punctate autophagic vacuoles was measured by ImageJ software. c LC3B western blot results to detect the autophagy status after antagomir or agomir treatment. d ) Representative example of Foxp3 vs CD127 staining on tTreg treated with antagomir or agomir (gated on CD4+ cells). Summary of overall e level of Foxp3 expression and f representative example of Ki67 histogram on tTreg treated with antagomir or agomir (gated on CD4+ cells). Summary of overall e level of Ki67 expression after antagomir/agomir treatment. g Percent suppression of in vitro, anti-CD3–mediated CD8+ T cell proliferation at ratios from 1:2 to 1:32 (tTreg: PBMC) as determined by CFSE dye dilution. Values indicate mean ± SEM of these experiments (* P

    Article Snippet: Briefly, PBMNCs were purified, labeled with CFSE (Invitrogen), and stimulated with anti-CD3 mAb-coated beads (Dynal) ± cultured tTreg (1:2–1:32 tTregs/PBMNCs).

    Techniques: Expressing, Purification, Magnetic Cell Separation, In Vitro, Immunofluorescence, Confocal Microscopy, Software, Western Blot, Staining

    Attenuated tTreg autophagy status and apoptosis-related gene changes during ex vivo tTreg expansion. ( n = 3) . CD4+CD25+CD127− tTreg cells were sort-purified by MACS and stimulated with anti-CD3/28 beads in the presence of IL-2 as indicated in methods part. a Cell number and b Relative expansion folds were recorded every 2–3 days until day 21. c Immunofluorescence confocal microscopy test for tTreg autophagy status: cells were transfected with mRFP-LC3-GFP lentivirus for 48 h, representative cellular autophagy level by the laser scanning from day 3 to 21, respectively. d The ratio of punctate autophagic vacuoles was measured by ImageJ software. e LC3B western blot results to show the autophagy status in tTreg culture process. f ATG3/5/12/16L1 expression by western blot in tTreg culture process. g RNA was purified and qRT-PCR used to determine the ATG16L1 mRNA expression Values indicate mean ± SEM of these experiments. (* P

    Journal: Cell Death & Disease

    Article Title: miR-142-3p regulates autophagy by targeting ATG16L1 in thymic-derived regulatory T cell (tTreg)

    doi: 10.1038/s41419-018-0298-2

    Figure Lengend Snippet: Attenuated tTreg autophagy status and apoptosis-related gene changes during ex vivo tTreg expansion. ( n = 3) . CD4+CD25+CD127− tTreg cells were sort-purified by MACS and stimulated with anti-CD3/28 beads in the presence of IL-2 as indicated in methods part. a Cell number and b Relative expansion folds were recorded every 2–3 days until day 21. c Immunofluorescence confocal microscopy test for tTreg autophagy status: cells were transfected with mRFP-LC3-GFP lentivirus for 48 h, representative cellular autophagy level by the laser scanning from day 3 to 21, respectively. d The ratio of punctate autophagic vacuoles was measured by ImageJ software. e LC3B western blot results to show the autophagy status in tTreg culture process. f ATG3/5/12/16L1 expression by western blot in tTreg culture process. g RNA was purified and qRT-PCR used to determine the ATG16L1 mRNA expression Values indicate mean ± SEM of these experiments. (* P

    Article Snippet: Briefly, PBMNCs were purified, labeled with CFSE (Invitrogen), and stimulated with anti-CD3 mAb-coated beads (Dynal) ± cultured tTreg (1:2–1:32 tTregs/PBMNCs).

    Techniques: Ex Vivo, Purification, Magnetic Cell Separation, Immunofluorescence, Confocal Microscopy, Transfection, Software, Western Blot, Expressing, Quantitative RT-PCR

    Example of the processing of the image analysis. a ) Part of a CD3 stained section. b ) CD3+ lymphocytes labelled with the red color are counted by the software

    Journal: Diagnostic Pathology

    Article Title: Computer-assisted stereology and automated image analysis for quantification of tumor infiltrating lymphocytes in colon cancer

    doi: 10.1186/s13000-017-0653-0

    Figure Lengend Snippet: Example of the processing of the image analysis. a ) Part of a CD3 stained section. b ) CD3+ lymphocytes labelled with the red color are counted by the software

    Article Snippet: The primary antibodies were mouse monoclonal anti-CD3 (code M7254, DAKO) diluted 1:600, and anti-CD8 (code M7103, DAKO) diluted 1:300.

    Techniques: Staining, Software

    Bland Altman plots showing the differences in densities of CD3+ and CD8+ tumor infiltrating lymphocytes for the central and invasive area measured by image analysis. The horizontal red line corresponds to zero difference, the blue dashed line shows mean and the dashed red lines show ±1.96 standard deviation. a and b ) Differences for densities of CD3+ and CD8+ tumor infiltrating lymphocytes (TILs) for all sections ( n = 129). c and d ) Differences for densities of CD3+ and CD8+ TILs for “the deepest invasive sections” ( n = 43). e and f ) Differences for densities of CD3+ and CD8+ TILs per tumor, where density is calculated as an average of the densities obtained from each of the three sections from each tumor ( n = 43)

    Journal: Diagnostic Pathology

    Article Title: Computer-assisted stereology and automated image analysis for quantification of tumor infiltrating lymphocytes in colon cancer

    doi: 10.1186/s13000-017-0653-0

    Figure Lengend Snippet: Bland Altman plots showing the differences in densities of CD3+ and CD8+ tumor infiltrating lymphocytes for the central and invasive area measured by image analysis. The horizontal red line corresponds to zero difference, the blue dashed line shows mean and the dashed red lines show ±1.96 standard deviation. a and b ) Differences for densities of CD3+ and CD8+ tumor infiltrating lymphocytes (TILs) for all sections ( n = 129). c and d ) Differences for densities of CD3+ and CD8+ TILs for “the deepest invasive sections” ( n = 43). e and f ) Differences for densities of CD3+ and CD8+ TILs per tumor, where density is calculated as an average of the densities obtained from each of the three sections from each tumor ( n = 43)

    Article Snippet: The primary antibodies were mouse monoclonal anti-CD3 (code M7254, DAKO) diluted 1:600, and anti-CD8 (code M7103, DAKO) diluted 1:300.

    Techniques: Standard Deviation

    Correlation between cell counts as obtained by stereology and image analysis. a ) Correlation for CD3+ tumor infiltrating lymphocytes (TILs) numerical density in the central area of the tumor. b ) Correlation for CD3+ TILs numerical density in the invasive tumor front. c ) Correlation for CD8+ TILs numerical density in the central area of the tumor. d ) Correlation for CD8+ TILs numerical density in the invasive tumor front

    Journal: Diagnostic Pathology

    Article Title: Computer-assisted stereology and automated image analysis for quantification of tumor infiltrating lymphocytes in colon cancer

    doi: 10.1186/s13000-017-0653-0

    Figure Lengend Snippet: Correlation between cell counts as obtained by stereology and image analysis. a ) Correlation for CD3+ tumor infiltrating lymphocytes (TILs) numerical density in the central area of the tumor. b ) Correlation for CD3+ TILs numerical density in the invasive tumor front. c ) Correlation for CD8+ TILs numerical density in the central area of the tumor. d ) Correlation for CD8+ TILs numerical density in the invasive tumor front

    Article Snippet: The primary antibodies were mouse monoclonal anti-CD3 (code M7254, DAKO) diluted 1:600, and anti-CD8 (code M7103, DAKO) diluted 1:300.

    Techniques:

    Correlation between area fractions as estimated by stereology and image analysis. a ) Correlation for CD3+ tumor infiltrating lymphocytes (TILs) area fraction in the central tumor area. b ) Correlation for CD3+ TILs area fraction in the invasive tumor front. c ) Correlation for CD8+ TILs area fraction in the central tumor area. d ) Correlation for CD8+ TILs area fraction in the invasive tumor front

    Journal: Diagnostic Pathology

    Article Title: Computer-assisted stereology and automated image analysis for quantification of tumor infiltrating lymphocytes in colon cancer

    doi: 10.1186/s13000-017-0653-0

    Figure Lengend Snippet: Correlation between area fractions as estimated by stereology and image analysis. a ) Correlation for CD3+ tumor infiltrating lymphocytes (TILs) area fraction in the central tumor area. b ) Correlation for CD3+ TILs area fraction in the invasive tumor front. c ) Correlation for CD8+ TILs area fraction in the central tumor area. d ) Correlation for CD8+ TILs area fraction in the invasive tumor front

    Article Snippet: The primary antibodies were mouse monoclonal anti-CD3 (code M7254, DAKO) diluted 1:600, and anti-CD8 (code M7103, DAKO) diluted 1:300.

    Techniques:

    Field of vision in a CD3-stained section magnified ×40. The density estimation was performed using the 2D unbiased counting frame with left and bottom edges, and their extensions, serving as exclusion lines (red), and with the upper and right edges of the frame as inclusion lines (green). Cell profiles were counted when completely inside the counting frame or partly inside the frame, provided that they did not touch the exclusion lines or their extensions. Thus, three cell profiles were counted (red crosses). The area fraction estimation was performed using the point grid. Points hitting CD3 positive cells = 2 (red ring) and points hitting tumor = 30, giving an area fraction of 0.07 in this field of vision

    Journal: Diagnostic Pathology

    Article Title: Computer-assisted stereology and automated image analysis for quantification of tumor infiltrating lymphocytes in colon cancer

    doi: 10.1186/s13000-017-0653-0

    Figure Lengend Snippet: Field of vision in a CD3-stained section magnified ×40. The density estimation was performed using the 2D unbiased counting frame with left and bottom edges, and their extensions, serving as exclusion lines (red), and with the upper and right edges of the frame as inclusion lines (green). Cell profiles were counted when completely inside the counting frame or partly inside the frame, provided that they did not touch the exclusion lines or their extensions. Thus, three cell profiles were counted (red crosses). The area fraction estimation was performed using the point grid. Points hitting CD3 positive cells = 2 (red ring) and points hitting tumor = 30, giving an area fraction of 0.07 in this field of vision

    Article Snippet: The primary antibodies were mouse monoclonal anti-CD3 (code M7254, DAKO) diluted 1:600, and anti-CD8 (code M7103, DAKO) diluted 1:300.

    Techniques: Staining

    Th1 immune response induced by BCG in vivo is affected in Sam68 -ko mice. a Flow cytometry analysis of IFN-γ-producing CD4 + T cells in splenocytes from wt and Sam68 -ko mice treated with PBS or infected with BCG and, 28 days later, restimulated with anti-CD3-28, PMA and Ionomycin, or PPD for 5 h in the presence of Brefeldin-A. b Bar graphs show data from seven independent experiments performed as in a (mean and s.e.m). c ELISA of IFN-γ in supernatants of splenocytes wt and Sam68 -ko mice treated with PBS or infected with BCG and, 28 days later, restimulated for 24 h with PMA and Ionomycin, or PPD. Data are the mean and s.e.m. of 11 independent experiments. d BCG burden in lungs of wt and Sam68 -ko mice 28 days after intravenous infection of BCG. Data are expressed as CFU/g of tissue (mean and s.e.m. of eight independent experiments); * p

    Journal: Cell Death and Differentiation

    Article Title: The RNA binding protein Sam68 controls T helper 1 differentiation and anti-mycobacterial response through modulation of miR-29

    doi: 10.1038/s41418-018-0201-9

    Figure Lengend Snippet: Th1 immune response induced by BCG in vivo is affected in Sam68 -ko mice. a Flow cytometry analysis of IFN-γ-producing CD4 + T cells in splenocytes from wt and Sam68 -ko mice treated with PBS or infected with BCG and, 28 days later, restimulated with anti-CD3-28, PMA and Ionomycin, or PPD for 5 h in the presence of Brefeldin-A. b Bar graphs show data from seven independent experiments performed as in a (mean and s.e.m). c ELISA of IFN-γ in supernatants of splenocytes wt and Sam68 -ko mice treated with PBS or infected with BCG and, 28 days later, restimulated for 24 h with PMA and Ionomycin, or PPD. Data are the mean and s.e.m. of 11 independent experiments. d BCG burden in lungs of wt and Sam68 -ko mice 28 days after intravenous infection of BCG. Data are expressed as CFU/g of tissue (mean and s.e.m. of eight independent experiments); * p

    Article Snippet: For analysis of Th1 response in vivo by ELISA, splenocytes or total CD4 T cells sorted by high sped cell sorter MoFlo (Coulter) were restimulated for 5 h with PMA and ionomycin (both 200 ng/ml) (both from Sigma), or anti-CD3/28 (ratio 2 beads/1 cell) (Life Technologies) for 24 h. Splenocytes were also restimulated with PPD (300 IU/ml) (Life Technologies).

    Techniques: In Vivo, Mouse Assay, Flow Cytometry, Infection, Enzyme-linked Immunosorbent Assay

    ChA6 mAb treatment prolongs human islet allograft survival in hu-PBL-NOD/SCID recipient mice. (A) Diabetic NOD/SCID mice were transplanted under the kidney capsule with human islets. Mice were injected intraperitoneally with 50 × 10 6 allogeneic human PBMCs. Normal NOD/SCID mice (*, n = 8) were used as control of human islets function. Mice were treated with vehicle (•, n = 12); with the Edmonton protocol (♦, n = 4); chA6 mAb at days 0, 3, and 5 after transplantation (▪, n = 14); or sirolimus (▴, n = 3). Glycemia levels monitored graft survival. Asterisks indicate statistical analysis in which treated mice were compared with control mice (*** ≤ 0.0001). (B) Kidney bearing the human-islet graft from control, chA6 mAb–treated, or normal NOD/SCID mice at 100 d after transplantation were snap-frozen, and 5-μm-thick sections were stained with hematoxylin and eosin (HE). Alternatively, sections were stained for the expression of CD3 and insulin.

    Journal: The Journal of Experimental Medicine

    Article Title: An anti-CD45RO/RB monoclonal antibody modulates T cell responses via induction of apoptosis and generation of regulatory T cells

    doi: 10.1084/jem.20040912

    Figure Lengend Snippet: ChA6 mAb treatment prolongs human islet allograft survival in hu-PBL-NOD/SCID recipient mice. (A) Diabetic NOD/SCID mice were transplanted under the kidney capsule with human islets. Mice were injected intraperitoneally with 50 × 10 6 allogeneic human PBMCs. Normal NOD/SCID mice (*, n = 8) were used as control of human islets function. Mice were treated with vehicle (•, n = 12); with the Edmonton protocol (♦, n = 4); chA6 mAb at days 0, 3, and 5 after transplantation (▪, n = 14); or sirolimus (▴, n = 3). Glycemia levels monitored graft survival. Asterisks indicate statistical analysis in which treated mice were compared with control mice (*** ≤ 0.0001). (B) Kidney bearing the human-islet graft from control, chA6 mAb–treated, or normal NOD/SCID mice at 100 d after transplantation were snap-frozen, and 5-μm-thick sections were stained with hematoxylin and eosin (HE). Alternatively, sections were stained for the expression of CD3 and insulin.

    Article Snippet: To evaluate secondary responses, primary cultures were carried on for 10 d; then cells were collected, washed, and plated in 96-well plates with newly prepared stimulator cells with or without of chA6 mAb (10 μg/ml) for 2 d. To analyze proliferation in response to polyclonal activation, CD4+ T cells (105 /well) were cultured in 96-well plates precoated with anti-CD3 mAb (OKT3, Janssen Cilag, Ltd.) for 3 d in the presence or absence of chA6 or chimeric isotype control mAbs (10 μg/ml) with or without soluble anti-CD28 mAb (1 μg/ml) (BD Biosciences).

    Techniques: Mouse Assay, Injection, Transplantation Assay, Staining, Expressing

    ChA6 mAb inhibits allogeneic and TT-specific proliferation of CD4 + T cells. (A) Primary MLR: CD4 + T cells were stimulated with allogeneic CD3-depleted cells with or without chA6 mAb (10 μg/ml). Results from one representative donor out of 14 are shown. Secondary MLR: CD4 + T cells were primed with allogeneic CD3-depleted cells for 10 d. Cells were collected and washed, and proliferative responses to the same allogenic cells used in the primary stimulation in the presence or absence of chA6 mAb (10 μg/ml) were tested. Results from one representative donor out of four are shown. Dose-dependent inhibition of primary MLR: CD4 + T cells were stimulated with allogeneic CD3-depleted cells in the presence of the indicated concentrations of chA6 or chimeric isotype control mAbs. Results from one representative donor out of five are shown. R, responder; S, stimulator. (B) TT response: total PBMCs were stimulated with TT with or without chA6 mAb (10 μg/ml). Results from one representative donor out of 16 are shown. The percentages of inhibition of proliferation in the presence of chA6 mAb relative to control are indicated.

    Journal: The Journal of Experimental Medicine

    Article Title: An anti-CD45RO/RB monoclonal antibody modulates T cell responses via induction of apoptosis and generation of regulatory T cells

    doi: 10.1084/jem.20040912

    Figure Lengend Snippet: ChA6 mAb inhibits allogeneic and TT-specific proliferation of CD4 + T cells. (A) Primary MLR: CD4 + T cells were stimulated with allogeneic CD3-depleted cells with or without chA6 mAb (10 μg/ml). Results from one representative donor out of 14 are shown. Secondary MLR: CD4 + T cells were primed with allogeneic CD3-depleted cells for 10 d. Cells were collected and washed, and proliferative responses to the same allogenic cells used in the primary stimulation in the presence or absence of chA6 mAb (10 μg/ml) were tested. Results from one representative donor out of four are shown. Dose-dependent inhibition of primary MLR: CD4 + T cells were stimulated with allogeneic CD3-depleted cells in the presence of the indicated concentrations of chA6 or chimeric isotype control mAbs. Results from one representative donor out of five are shown. R, responder; S, stimulator. (B) TT response: total PBMCs were stimulated with TT with or without chA6 mAb (10 μg/ml). Results from one representative donor out of 16 are shown. The percentages of inhibition of proliferation in the presence of chA6 mAb relative to control are indicated.

    Article Snippet: To evaluate secondary responses, primary cultures were carried on for 10 d; then cells were collected, washed, and plated in 96-well plates with newly prepared stimulator cells with or without of chA6 mAb (10 μg/ml) for 2 d. To analyze proliferation in response to polyclonal activation, CD4+ T cells (105 /well) were cultured in 96-well plates precoated with anti-CD3 mAb (OKT3, Janssen Cilag, Ltd.) for 3 d in the presence or absence of chA6 or chimeric isotype control mAbs (10 μg/ml) with or without soluble anti-CD28 mAb (1 μg/ml) (BD Biosciences).

    Techniques: Inhibition

    Dose-dependent apoptosis induced in CD4 + A6 bright T cells by chA6 mAb. (A) CD4 + T cells were incubated with the indicated concentrations of chA6 or isotype control mAbs. Cells were cultured overnight without (unstimulated) or with (stimulated) coated anti-CD3 (0.1 μg/ml) and soluble anti-CD28 (1 μg/ml) mAb and were analyzed for apoptosis. The percentages of early apoptotic (annexin V + /PI − ) cells and mean ± SE are shown. P values were calculated by t test: * P , effect of chA6 mAb on total CD4 + T cells; $ P , effect on annexin V–depleted CD4 + T cells (* P or $ P ≤ 0.05; ** P ≤ 0.005). (B) PBMCs of three healthy donors were cultured overnight in the presence of the indicated concentrations of chA6 mAb (filled symbols) or an IgG1 isotype control antibody (open symbols) and analyzed for apoptosis. Curve fitting and ED 50 value calculations were performed. (C) Total or annexin V–depleted CD4 + T cells were incubated with the indicated concentrations of chA6 mAb. Cells were cultured overnight without or with (stimulated) coated anti-CD3 (0.1 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs and were stained with annexin V FITC mAb and chA6 mAb followed by anti-human IgG1PE mAb and analyzed by flow cytometry. Percentages of positive cells, set according to the isotype-matched controls (not shown), are shown in the top corner of the quadrant. Results from one representative out of 10 different donors tested are shown. (D) CD4 + T cells were incubated with the indicated concentrations of anti-CD45RO (UCLH-1), anti-CD45RA (HI-100), chA6, or isotype control mAbs. Cells were stimulated with coated anti-CD3 (0.1 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs, cultured overnight, and analyzed for apoptosis. The percentages of early-apoptotic (annexinV + /PI − ) cells are shown. (E) Total or annexin V–depleted CD4 + T cells were stimulated with allogeneic CD3-depleted cells with or without chA6 mAb (10 μg/ml). Results from one representative donor out of four are shown. The percentages of inhibition of proliferation in the presence of chA6 mAb relative to control are presented.

    Journal: The Journal of Experimental Medicine

    Article Title: An anti-CD45RO/RB monoclonal antibody modulates T cell responses via induction of apoptosis and generation of regulatory T cells

    doi: 10.1084/jem.20040912

    Figure Lengend Snippet: Dose-dependent apoptosis induced in CD4 + A6 bright T cells by chA6 mAb. (A) CD4 + T cells were incubated with the indicated concentrations of chA6 or isotype control mAbs. Cells were cultured overnight without (unstimulated) or with (stimulated) coated anti-CD3 (0.1 μg/ml) and soluble anti-CD28 (1 μg/ml) mAb and were analyzed for apoptosis. The percentages of early apoptotic (annexin V + /PI − ) cells and mean ± SE are shown. P values were calculated by t test: * P , effect of chA6 mAb on total CD4 + T cells; $ P , effect on annexin V–depleted CD4 + T cells (* P or $ P ≤ 0.05; ** P ≤ 0.005). (B) PBMCs of three healthy donors were cultured overnight in the presence of the indicated concentrations of chA6 mAb (filled symbols) or an IgG1 isotype control antibody (open symbols) and analyzed for apoptosis. Curve fitting and ED 50 value calculations were performed. (C) Total or annexin V–depleted CD4 + T cells were incubated with the indicated concentrations of chA6 mAb. Cells were cultured overnight without or with (stimulated) coated anti-CD3 (0.1 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs and were stained with annexin V FITC mAb and chA6 mAb followed by anti-human IgG1PE mAb and analyzed by flow cytometry. Percentages of positive cells, set according to the isotype-matched controls (not shown), are shown in the top corner of the quadrant. Results from one representative out of 10 different donors tested are shown. (D) CD4 + T cells were incubated with the indicated concentrations of anti-CD45RO (UCLH-1), anti-CD45RA (HI-100), chA6, or isotype control mAbs. Cells were stimulated with coated anti-CD3 (0.1 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs, cultured overnight, and analyzed for apoptosis. The percentages of early-apoptotic (annexinV + /PI − ) cells are shown. (E) Total or annexin V–depleted CD4 + T cells were stimulated with allogeneic CD3-depleted cells with or without chA6 mAb (10 μg/ml). Results from one representative donor out of four are shown. The percentages of inhibition of proliferation in the presence of chA6 mAb relative to control are presented.

    Article Snippet: To evaluate secondary responses, primary cultures were carried on for 10 d; then cells were collected, washed, and plated in 96-well plates with newly prepared stimulator cells with or without of chA6 mAb (10 μg/ml) for 2 d. To analyze proliferation in response to polyclonal activation, CD4+ T cells (105 /well) were cultured in 96-well plates precoated with anti-CD3 mAb (OKT3, Janssen Cilag, Ltd.) for 3 d in the presence or absence of chA6 or chimeric isotype control mAbs (10 μg/ml) with or without soluble anti-CD28 mAb (1 μg/ml) (BD Biosciences).

    Techniques: Incubation, Cell Culture, Staining, Flow Cytometry, Cytometry, Inhibition

    ChA6 mAb induce apoptosis in CD4 + T cells through the activation of the intrinsic pathway. (A) CD4 + T cells were incubated with the indicated concentrations of chA6 mAb. Cells were cultured overnight with or without coated anti-CD3 (0.1 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs. Western blot tests with anti-caspase-3, anti-caspase-8, and anti-caspase-9 mAb were performed. As positive control, Hela cells were treated with staurosporine, 5 μM. Amounts of loaded proteins have been controlled for homogeneity by probing membranes with an anti- β-actin mAb. Weak expression of the cleavage products of caspase-9 in CD4 + T cells cultured in medium is considered background apoptosis. Results from one representative donor out of three are shown. (B) CD4 + T cells were incubated with chA6 mAb (5 μg/ml) in the presence of 15 μg/ml of cross-linking goat anti–human IgG (F(ab′) 2 ). Mitochondrial-based death pathways were assessed using 3,3′-dihexyloxacarbocyanine iodide (DiOC 6 (3)) accumulation that reflects changes in Δψm in the mitochondria. Results from one representative donor out of three are shown.

    Journal: The Journal of Experimental Medicine

    Article Title: An anti-CD45RO/RB monoclonal antibody modulates T cell responses via induction of apoptosis and generation of regulatory T cells

    doi: 10.1084/jem.20040912

    Figure Lengend Snippet: ChA6 mAb induce apoptosis in CD4 + T cells through the activation of the intrinsic pathway. (A) CD4 + T cells were incubated with the indicated concentrations of chA6 mAb. Cells were cultured overnight with or without coated anti-CD3 (0.1 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs. Western blot tests with anti-caspase-3, anti-caspase-8, and anti-caspase-9 mAb were performed. As positive control, Hela cells were treated with staurosporine, 5 μM. Amounts of loaded proteins have been controlled for homogeneity by probing membranes with an anti- β-actin mAb. Weak expression of the cleavage products of caspase-9 in CD4 + T cells cultured in medium is considered background apoptosis. Results from one representative donor out of three are shown. (B) CD4 + T cells were incubated with chA6 mAb (5 μg/ml) in the presence of 15 μg/ml of cross-linking goat anti–human IgG (F(ab′) 2 ). Mitochondrial-based death pathways were assessed using 3,3′-dihexyloxacarbocyanine iodide (DiOC 6 (3)) accumulation that reflects changes in Δψm in the mitochondria. Results from one representative donor out of three are shown.

    Article Snippet: To evaluate secondary responses, primary cultures were carried on for 10 d; then cells were collected, washed, and plated in 96-well plates with newly prepared stimulator cells with or without of chA6 mAb (10 μg/ml) for 2 d. To analyze proliferation in response to polyclonal activation, CD4+ T cells (105 /well) were cultured in 96-well plates precoated with anti-CD3 mAb (OKT3, Janssen Cilag, Ltd.) for 3 d in the presence or absence of chA6 or chimeric isotype control mAbs (10 μg/ml) with or without soluble anti-CD28 mAb (1 μg/ml) (BD Biosciences).

    Techniques: Activation Assay, Incubation, Cell Culture, Western Blot, Positive Control, Expressing

    ChA6 mAb inhibits polyclonal proliferation of CD4 + T cells. (A) CD4 + T cells were stimulated with the indicated concentrations of coated anti-CD3 mAb in the presence or absence of chA6 or chimeric isotype control mAb (10 μg/ml) with or without soluble anti-CD28 mAb (1 μg/ml). Results from one representative donor out of four are shown. (B) Dose-dependent inhibition of proliferative polyclonal T cell response by chA6 mAb. CD4 + T cells were stimulated with 0.1 and 0.01 μg/ml of coated anti-CD3 mAb in the presence of the indicated concentrations of soluble chA6 mAb. Results from one representative donor out of three (0.1 μg/ml of anti-CD3 mAb) and out of two (0.01 μg/ml of anti-CD3 mAb) are shown. The percentages of inhibition of proliferation in the presence of chA6 mAb relative to control are indicated.

    Journal: The Journal of Experimental Medicine

    Article Title: An anti-CD45RO/RB monoclonal antibody modulates T cell responses via induction of apoptosis and generation of regulatory T cells

    doi: 10.1084/jem.20040912

    Figure Lengend Snippet: ChA6 mAb inhibits polyclonal proliferation of CD4 + T cells. (A) CD4 + T cells were stimulated with the indicated concentrations of coated anti-CD3 mAb in the presence or absence of chA6 or chimeric isotype control mAb (10 μg/ml) with or without soluble anti-CD28 mAb (1 μg/ml). Results from one representative donor out of four are shown. (B) Dose-dependent inhibition of proliferative polyclonal T cell response by chA6 mAb. CD4 + T cells were stimulated with 0.1 and 0.01 μg/ml of coated anti-CD3 mAb in the presence of the indicated concentrations of soluble chA6 mAb. Results from one representative donor out of three (0.1 μg/ml of anti-CD3 mAb) and out of two (0.01 μg/ml of anti-CD3 mAb) are shown. The percentages of inhibition of proliferation in the presence of chA6 mAb relative to control are indicated.

    Article Snippet: To evaluate secondary responses, primary cultures were carried on for 10 d; then cells were collected, washed, and plated in 96-well plates with newly prepared stimulator cells with or without of chA6 mAb (10 μg/ml) for 2 d. To analyze proliferation in response to polyclonal activation, CD4+ T cells (105 /well) were cultured in 96-well plates precoated with anti-CD3 mAb (OKT3, Janssen Cilag, Ltd.) for 3 d in the presence or absence of chA6 or chimeric isotype control mAbs (10 μg/ml) with or without soluble anti-CD28 mAb (1 μg/ml) (BD Biosciences).

    Techniques: Inhibition

    Kv1.3 high IKCa1 low phenotype: an exclusive functional marker for activated effector memory T cells. ( a ) Fluorescent immunostained images of patch-clamped CD4 T cell subsets from control subjects showing naive CCR7 + CD45RA + (left), T CM CCR7 + CD45RA – cells (middle), and T EM CCR7 + CD45RA + cells (right, counterstained with antihuman CD4 mAb). Representative Kv1.3 currents from each cell type are shown below the respective images. ( b ) Kv1.3 channel number/cell in naive (yellow squares), T CM (MBP-stimulated, green squares; TT-stimulated, green triangles) and T EM (MBP-stimulated, orange squares; TT-stimulated, orange triangles) T cells. Activated naive CD4 + cells were identified in the peripheral blood of controls 48 hours following stimulation with anti-CD3 mAb. Activated T CM and T EM cells were identified in the same preparations of control TCLs (stimulated ten times with either MBP or TT) 48 hours after stimulation with the appropriate antigen. ( c ) Kv1.3 versus IKCa1 channel number per cell in the three populations before and after activation. Each data point is the mean ± SEM channel number in 20–50 cells.

    Journal: Journal of Clinical Investigation

    Article Title: The voltage-gated Kv1.3 K+ channel in effector memory T cells as new target for MS

    doi: 10.1172/JCI200316921

    Figure Lengend Snippet: Kv1.3 high IKCa1 low phenotype: an exclusive functional marker for activated effector memory T cells. ( a ) Fluorescent immunostained images of patch-clamped CD4 T cell subsets from control subjects showing naive CCR7 + CD45RA + (left), T CM CCR7 + CD45RA – cells (middle), and T EM CCR7 + CD45RA + cells (right, counterstained with antihuman CD4 mAb). Representative Kv1.3 currents from each cell type are shown below the respective images. ( b ) Kv1.3 channel number/cell in naive (yellow squares), T CM (MBP-stimulated, green squares; TT-stimulated, green triangles) and T EM (MBP-stimulated, orange squares; TT-stimulated, orange triangles) T cells. Activated naive CD4 + cells were identified in the peripheral blood of controls 48 hours following stimulation with anti-CD3 mAb. Activated T CM and T EM cells were identified in the same preparations of control TCLs (stimulated ten times with either MBP or TT) 48 hours after stimulation with the appropriate antigen. ( c ) Kv1.3 versus IKCa1 channel number per cell in the three populations before and after activation. Each data point is the mean ± SEM channel number in 20–50 cells.

    Article Snippet: Background counts were below 2,000 cpm for cells that were not reactivated with anti-CD3 mAb, and maximal counts in anti-CD3 mAb–reactivated cells were about 30,000 cpm.

    Techniques: Functional Assay, Marker, Activation Assay

    Kv1.3 blockers selectively and persistently suppress MBP-specific T EM cells. ( a ) ShK potently blocked Kv1.3 in Kv1.3 high IKCa1 low MBP-specific T EM cells K d , 9 ± 1 pM). Each data point represents the mean ± SD of three cells. ( b ) ShK suppressed anti-CD3 mAb–stimulated [ 3 H]TdR incorporation by a Kv1.3 high IKCa1 low MBP-specific MS patient TCL stimulated three times with MBP (left) and a control TCL stimulated 12 times with MBP (right). ( c ) ShK (10 nM) partially suppressed anti-CD3 mAb–stimulated [ 3 H]TdR incorporation by normal peripheral blood T lymphocytes (left) but was unable to suppress proliferation when these cells were rechallenged with anti-CD3 mAb. One representative experiment from a total of three experiments is shown. Each experiment was done in triplicate. ( d ) IKCa1 expression is enhanced in activated naive and T CM cells following anti-CD3 mAb stimulation, despite the presence of a suppressive dose of ShK. Open circles, naive resting; filled circles, naive T cells activated by anti-CD3 mAb in the absence or presence of ShK; open triangles, T CM resting; filled triangles, T CM cells activated by anti-CD3 mAb in the absence or presence of ShK.

    Journal: Journal of Clinical Investigation

    Article Title: The voltage-gated Kv1.3 K+ channel in effector memory T cells as new target for MS

    doi: 10.1172/JCI200316921

    Figure Lengend Snippet: Kv1.3 blockers selectively and persistently suppress MBP-specific T EM cells. ( a ) ShK potently blocked Kv1.3 in Kv1.3 high IKCa1 low MBP-specific T EM cells K d , 9 ± 1 pM). Each data point represents the mean ± SD of three cells. ( b ) ShK suppressed anti-CD3 mAb–stimulated [ 3 H]TdR incorporation by a Kv1.3 high IKCa1 low MBP-specific MS patient TCL stimulated three times with MBP (left) and a control TCL stimulated 12 times with MBP (right). ( c ) ShK (10 nM) partially suppressed anti-CD3 mAb–stimulated [ 3 H]TdR incorporation by normal peripheral blood T lymphocytes (left) but was unable to suppress proliferation when these cells were rechallenged with anti-CD3 mAb. One representative experiment from a total of three experiments is shown. Each experiment was done in triplicate. ( d ) IKCa1 expression is enhanced in activated naive and T CM cells following anti-CD3 mAb stimulation, despite the presence of a suppressive dose of ShK. Open circles, naive resting; filled circles, naive T cells activated by anti-CD3 mAb in the absence or presence of ShK; open triangles, T CM resting; filled triangles, T CM cells activated by anti-CD3 mAb in the absence or presence of ShK.

    Article Snippet: Background counts were below 2,000 cpm for cells that were not reactivated with anti-CD3 mAb, and maximal counts in anti-CD3 mAb–reactivated cells were about 30,000 cpm.

    Techniques: Mass Spectrometry, Expressing

    Myelin antigen–activated T cells from MS patients express the distinctive Kv1.3 high IKCa1 low phenotype. ( a ) Kv1.3 and IKCa1 currents in TCLs from MS patients and controls measured 48 hours after activation with MBP. The numbers 1, 2, and 3 in the Kv1.3 traces represent traces after the first, second, and third depolarizing 200-ms pulse; the pulse interval is 1 second. ( b ) Kv1.3 channel number/cell in myelin antigen–activated TCLs (left) or mitogen-activated T cells (right) from MS patients or controls. Each data point constitutes the mean ± SEM of 20–30 cells from two to seven independent TCLs. Myelin antigens used were MBP (filled and open squares), MOG (filled and open triangles), and PLP (filled diamonds). Filled symbols, MSpatients; open symbols, control subjects. Mitogens used were soluble anti-CD3 mAb (50 ng/ml) and PMA (10 nM) plus ionomycin (175 nM) (data for both mitogens are grouped together). Filled circles, MS patients; open circles, controls. ( c ) Kv1.3 channel number/cell in MS patient T cells activated for 48 hours with the control antigens GAD65, insulin peptide, and ovalbumin. Due to the paucity of cells in these TCLs, IKCa1 expression was measured in only two to three cells per TCL.

    Journal: Journal of Clinical Investigation

    Article Title: The voltage-gated Kv1.3 K+ channel in effector memory T cells as new target for MS

    doi: 10.1172/JCI200316921

    Figure Lengend Snippet: Myelin antigen–activated T cells from MS patients express the distinctive Kv1.3 high IKCa1 low phenotype. ( a ) Kv1.3 and IKCa1 currents in TCLs from MS patients and controls measured 48 hours after activation with MBP. The numbers 1, 2, and 3 in the Kv1.3 traces represent traces after the first, second, and third depolarizing 200-ms pulse; the pulse interval is 1 second. ( b ) Kv1.3 channel number/cell in myelin antigen–activated TCLs (left) or mitogen-activated T cells (right) from MS patients or controls. Each data point constitutes the mean ± SEM of 20–30 cells from two to seven independent TCLs. Myelin antigens used were MBP (filled and open squares), MOG (filled and open triangles), and PLP (filled diamonds). Filled symbols, MSpatients; open symbols, control subjects. Mitogens used were soluble anti-CD3 mAb (50 ng/ml) and PMA (10 nM) plus ionomycin (175 nM) (data for both mitogens are grouped together). Filled circles, MS patients; open circles, controls. ( c ) Kv1.3 channel number/cell in MS patient T cells activated for 48 hours with the control antigens GAD65, insulin peptide, and ovalbumin. Due to the paucity of cells in these TCLs, IKCa1 expression was measured in only two to three cells per TCL.

    Article Snippet: Background counts were below 2,000 cpm for cells that were not reactivated with anti-CD3 mAb, and maximal counts in anti-CD3 mAb–reactivated cells were about 30,000 cpm.

    Techniques: Mass Spectrometry, Activation Assay, Plasmid Purification, Expressing

    Inhibiting fatty acid synthesis or accelerating pyruvate generation corrects the tissue-invasive and arthritogenic behavior of RA T cells CD4 + CD45RO − PBMC were isolated from RA patients with active disease and adoptively transferred into human synovium-NSG chimeric mice. Mice were randomly assigned to one of three treatment arms ( n = 14 synovial grafts/treatment arm): Vehicle arm (vehicle injection); RA treated with C75 (5 mg/kg, i.p. every other day) or RA treated with ML265 (10 mg/kg, i.p. daily). Synovial tissues were harvested, tissue sections were stained with anti-human CD3 (brown) and anti-RANKL (pink) antibodies and frequencies of RANKL positive cells were quantified in randomly selected high-powered fields. ( a ) Representative tissue stains showing human CD3 + and RANKL + T cells infiltrating into synovial tissue, where they form cellular clusters. Scale bars, 20 μm. ( b ) Frequencies of CD3 + RANKL + T cells in the tissue as percent of total cells. ( c ) RT-PCR-based quantification of T cell receptor ( TRB ) transcripts. ( d ) Gene expression of IFNG and IL17 . ( e ) Gene expression of transcription factors, key cytokines and TKS5 determined by RT-PCR. ( f , g ) Co-immunofluorescence staining of tissue sections for IFN-γ and CD3. Frequencies of tissue-residing IFN-γ + CD3 + T cells in the different treatment arms. Scale bars, 20 μm. All data are mean ± s.e.m. * P

    Journal: Nature immunology

    Article Title: Metabolic control of the scaffold protein TKS5 in tissue-invasive, pro-inflammatory T cells

    doi: 10.1038/ni.3808

    Figure Lengend Snippet: Inhibiting fatty acid synthesis or accelerating pyruvate generation corrects the tissue-invasive and arthritogenic behavior of RA T cells CD4 + CD45RO − PBMC were isolated from RA patients with active disease and adoptively transferred into human synovium-NSG chimeric mice. Mice were randomly assigned to one of three treatment arms ( n = 14 synovial grafts/treatment arm): Vehicle arm (vehicle injection); RA treated with C75 (5 mg/kg, i.p. every other day) or RA treated with ML265 (10 mg/kg, i.p. daily). Synovial tissues were harvested, tissue sections were stained with anti-human CD3 (brown) and anti-RANKL (pink) antibodies and frequencies of RANKL positive cells were quantified in randomly selected high-powered fields. ( a ) Representative tissue stains showing human CD3 + and RANKL + T cells infiltrating into synovial tissue, where they form cellular clusters. Scale bars, 20 μm. ( b ) Frequencies of CD3 + RANKL + T cells in the tissue as percent of total cells. ( c ) RT-PCR-based quantification of T cell receptor ( TRB ) transcripts. ( d ) Gene expression of IFNG and IL17 . ( e ) Gene expression of transcription factors, key cytokines and TKS5 determined by RT-PCR. ( f , g ) Co-immunofluorescence staining of tissue sections for IFN-γ and CD3. Frequencies of tissue-residing IFN-γ + CD3 + T cells in the different treatment arms. Scale bars, 20 μm. All data are mean ± s.e.m. * P

    Article Snippet: Antibodies Primary antibodies used: APC/Cy7 anti-human CD3 (Clone SK7, BioLegend), FITC anti-human CD4 (Clone RPA-T4, BioLegend), anti-human CD8a PE-Cy5 (Clone RPA-T8, eBioscience), PE-Cy™7 Mouse Anti-Human CD45RA (Clone HI100, BD Biosciences), APC anti-human CD45RA (Clone HI100, BioLegend), APC Mouse Anti-Human CD45RO (Clone UCHL1, BD Biosciences), Brilliant Violet 711™ anti-human CD45 (HI30, BioLegend), cortactin antibody (PA5-29799) (ThermoFisher Scientific), FAS mouse monoclonal antibody (M01) (Clone 3F2-1F3, Abnova), Acetyl-CoA Carboxylase (C83B10) Rabbit mAb (Cell Signaling Technology), SCD1 (C12H5) Rabbit mAb (Cell Signaling Technology), RANKL/CD254 Antibody (12A668) (ThermoFisher Scientific), Monoclonal Mouse Anti-Human CD3 (Clone F7.2.38, Dako), Polyclonal Rabbit Anti-Human CD3 (#A045201-2, Dako), β-Actin (8H10D10) Mouse mAb (#3700, Cell Signaling), Anti-rabbit IgG, HRP-linked Antibody (#7074, Cell Signaling), WesternSure HRP Goat anti-Mouse IgG (H+L) (LI-COR), Anti-Interferon gamma antibody (ab25101, Abcam), Anti-PPAR gamma antibody (ab178860, Abcam), PPARγ (81B8) Rabbit mAb (Cell Signaling), Monoclonal anti-flag (HRP) antibody (A8592-.2MG, Sigma-Aldrich).

    Techniques: Isolation, Mouse Assay, Injection, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunofluorescence

    CD4 T cells from RA patients accumulate cytoplasmic lipid droplets CD4 + CD45RA + T cells from RA patients and age-matched healthy individuals were stimulated for 96 h and stained with BODIPY (493/503) to quantify intracellular lipids. ( a ) Confocal microscopy imaging of intracellular lipid droplets (green) in RA CD4 + T cells. Scale bar, 20 μm. ( b ) Fluorescent Bodipy quantification in activated CD4 + CD45RA + T cells from 10 RA patients, 6 PsA patients and 10 healthy individuals. ( c ) Intracellular neutral lipids labeled with Bodipy in naive and memory populations of CD4 + T cells from healthy controls, RA patients and PsA patients. Representative histograms from 3 experiments. ( d ) Frozen sections from RA synovial tissues were stained with Bodipy (493/503) and analyzed by fluorescence microscopy. Representative images show accumulation of lipid droplets (green) in tissue-residing CD3 + T cells (red). Scale bar, 20 μm. ( e ) Palmitic acid measured in activated CD4 + CD45RA + T cells from 8 RA patients, 8 healthy donors and 4 PsA patients. ( f ) Expression levels of genes involved in lipid droplet formation from 6 RA patients, 5 PsA patients and 6 controls. All data are mean ± s.e.m. * P

    Journal: Nature immunology

    Article Title: Metabolic control of the scaffold protein TKS5 in tissue-invasive, pro-inflammatory T cells

    doi: 10.1038/ni.3808

    Figure Lengend Snippet: CD4 T cells from RA patients accumulate cytoplasmic lipid droplets CD4 + CD45RA + T cells from RA patients and age-matched healthy individuals were stimulated for 96 h and stained with BODIPY (493/503) to quantify intracellular lipids. ( a ) Confocal microscopy imaging of intracellular lipid droplets (green) in RA CD4 + T cells. Scale bar, 20 μm. ( b ) Fluorescent Bodipy quantification in activated CD4 + CD45RA + T cells from 10 RA patients, 6 PsA patients and 10 healthy individuals. ( c ) Intracellular neutral lipids labeled with Bodipy in naive and memory populations of CD4 + T cells from healthy controls, RA patients and PsA patients. Representative histograms from 3 experiments. ( d ) Frozen sections from RA synovial tissues were stained with Bodipy (493/503) and analyzed by fluorescence microscopy. Representative images show accumulation of lipid droplets (green) in tissue-residing CD3 + T cells (red). Scale bar, 20 μm. ( e ) Palmitic acid measured in activated CD4 + CD45RA + T cells from 8 RA patients, 8 healthy donors and 4 PsA patients. ( f ) Expression levels of genes involved in lipid droplet formation from 6 RA patients, 5 PsA patients and 6 controls. All data are mean ± s.e.m. * P

    Article Snippet: Antibodies Primary antibodies used: APC/Cy7 anti-human CD3 (Clone SK7, BioLegend), FITC anti-human CD4 (Clone RPA-T4, BioLegend), anti-human CD8a PE-Cy5 (Clone RPA-T8, eBioscience), PE-Cy™7 Mouse Anti-Human CD45RA (Clone HI100, BD Biosciences), APC anti-human CD45RA (Clone HI100, BioLegend), APC Mouse Anti-Human CD45RO (Clone UCHL1, BD Biosciences), Brilliant Violet 711™ anti-human CD45 (HI30, BioLegend), cortactin antibody (PA5-29799) (ThermoFisher Scientific), FAS mouse monoclonal antibody (M01) (Clone 3F2-1F3, Abnova), Acetyl-CoA Carboxylase (C83B10) Rabbit mAb (Cell Signaling Technology), SCD1 (C12H5) Rabbit mAb (Cell Signaling Technology), RANKL/CD254 Antibody (12A668) (ThermoFisher Scientific), Monoclonal Mouse Anti-Human CD3 (Clone F7.2.38, Dako), Polyclonal Rabbit Anti-Human CD3 (#A045201-2, Dako), β-Actin (8H10D10) Mouse mAb (#3700, Cell Signaling), Anti-rabbit IgG, HRP-linked Antibody (#7074, Cell Signaling), WesternSure HRP Goat anti-Mouse IgG (H+L) (LI-COR), Anti-Interferon gamma antibody (ab25101, Abcam), Anti-PPAR gamma antibody (ab178860, Abcam), PPARγ (81B8) Rabbit mAb (Cell Signaling), Monoclonal anti-flag (HRP) antibody (A8592-.2MG, Sigma-Aldrich).

    Techniques: Staining, Confocal Microscopy, Imaging, Labeling, Fluorescence, Microscopy, Expressing

    Reducing glycolytic flux renders CD4 + T cells tissue-invasive and pro-inflammatory CD4 + CD45RO − PBMC were isolated from healthy individuals. Glycolytic activity was inhibited by transfecting the cells with PFKFB3 siRNA ( a–f ) or treating with the PFKFB3 inhibitor 3PO (200 nM) ( g–l ). Cells were adoptively transferred into human synovium-engrafted NSG mice. Seven days later, synovial tissue grafts were harvested and processed for immuno-histochemical analysis or for RT-PCR analysis of cytokine and transcription factor gene expression. Data indicate mean ± s.e.m. from 20 different synovial tissues. ( a , g ) Immunohistochemical staining for tissue-residing CD3 + T cells. Scale bars, 20 μm. ( b , h ) Quantification of T cell receptor ( TRB ) transcripts and of tissue-invading CD3 + T cells, expressed as percent of total cells. ( c , i ) Gene expression of IFNG and IL17 . ( d , j ) Co-immunofluorescence staining of tissue sections for IFN-γ and CD3 + T cells. Scale bars, 20 μm. ( e , k ) Frequencies of IFN-γ + CD3 + T cells in the tissue. ( f , l ) Gene expression of lineage-determining transcription factors, key inflammatory markers and TKS5 were determined by RT-PCR. Scale bars, 20 μm. All data are mean ± s.e.m. * P

    Journal: Nature immunology

    Article Title: Metabolic control of the scaffold protein TKS5 in tissue-invasive, pro-inflammatory T cells

    doi: 10.1038/ni.3808

    Figure Lengend Snippet: Reducing glycolytic flux renders CD4 + T cells tissue-invasive and pro-inflammatory CD4 + CD45RO − PBMC were isolated from healthy individuals. Glycolytic activity was inhibited by transfecting the cells with PFKFB3 siRNA ( a–f ) or treating with the PFKFB3 inhibitor 3PO (200 nM) ( g–l ). Cells were adoptively transferred into human synovium-engrafted NSG mice. Seven days later, synovial tissue grafts were harvested and processed for immuno-histochemical analysis or for RT-PCR analysis of cytokine and transcription factor gene expression. Data indicate mean ± s.e.m. from 20 different synovial tissues. ( a , g ) Immunohistochemical staining for tissue-residing CD3 + T cells. Scale bars, 20 μm. ( b , h ) Quantification of T cell receptor ( TRB ) transcripts and of tissue-invading CD3 + T cells, expressed as percent of total cells. ( c , i ) Gene expression of IFNG and IL17 . ( d , j ) Co-immunofluorescence staining of tissue sections for IFN-γ and CD3 + T cells. Scale bars, 20 μm. ( e , k ) Frequencies of IFN-γ + CD3 + T cells in the tissue. ( f , l ) Gene expression of lineage-determining transcription factors, key inflammatory markers and TKS5 were determined by RT-PCR. Scale bars, 20 μm. All data are mean ± s.e.m. * P

    Article Snippet: Antibodies Primary antibodies used: APC/Cy7 anti-human CD3 (Clone SK7, BioLegend), FITC anti-human CD4 (Clone RPA-T4, BioLegend), anti-human CD8a PE-Cy5 (Clone RPA-T8, eBioscience), PE-Cy™7 Mouse Anti-Human CD45RA (Clone HI100, BD Biosciences), APC anti-human CD45RA (Clone HI100, BioLegend), APC Mouse Anti-Human CD45RO (Clone UCHL1, BD Biosciences), Brilliant Violet 711™ anti-human CD45 (HI30, BioLegend), cortactin antibody (PA5-29799) (ThermoFisher Scientific), FAS mouse monoclonal antibody (M01) (Clone 3F2-1F3, Abnova), Acetyl-CoA Carboxylase (C83B10) Rabbit mAb (Cell Signaling Technology), SCD1 (C12H5) Rabbit mAb (Cell Signaling Technology), RANKL/CD254 Antibody (12A668) (ThermoFisher Scientific), Monoclonal Mouse Anti-Human CD3 (Clone F7.2.38, Dako), Polyclonal Rabbit Anti-Human CD3 (#A045201-2, Dako), β-Actin (8H10D10) Mouse mAb (#3700, Cell Signaling), Anti-rabbit IgG, HRP-linked Antibody (#7074, Cell Signaling), WesternSure HRP Goat anti-Mouse IgG (H+L) (LI-COR), Anti-Interferon gamma antibody (ab25101, Abcam), Anti-PPAR gamma antibody (ab178860, Abcam), PPARγ (81B8) Rabbit mAb (Cell Signaling), Monoclonal anti-flag (HRP) antibody (A8592-.2MG, Sigma-Aldrich).

    Techniques: Isolation, Activity Assay, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunohistochemistry, Staining, Immunofluorescence

    Hypermotility and tissue-invasiveness of RA T cells ( a ) Migration of activated CD4 + CD45RA + T cells in transwells without chemokines for 48 h ( n =10). ( b–d ) CD4 + T cells invasiveness in 3D-collagen matrices measured by confocal imaging DAPI-stained nuclei. ( b ) Representative confocal images at indicated depths after 48 h. Scale bar, 200 μm. ( c ) DAPI indices (signal at defined depth/signal at surface) (5 control-RA pairs). ( d ) Maximum invasion distances ( n =5). ( e, f ) Cytoskeletal organization and membrane protrusions analyzed by confocal microscopy. Membrane ruffles and podosomes identified through F-actin/cortactin co-localization. ( e ) Representative micrographs showing actin mesh co-localizing with cortactin in lamellopodia and membrane ruffles. Scale bars, 20 μm. ( f ) Quantification of F-actin, cortactin and F-actin + cortactin + membrane protrusions (5 control-RA pairs). ( g ) Gene expression profiling (RT-PCR) of genes involved in actin nucleation and cell motility. Heat map presentation from 6 RA-control pairs. Scaled z-score: red indicates high and blue low transcript levels. ( h–i ) Representative histograms and bar graphs of T cell TKS5 protein expression (11 control-RA pairs). ( j–l ) Human synovium-NSG mice ( n =14) were reconstituted with CD4 + CD45RO − PBMC from healthy individuals or RA patients. ( j ) Immunohistochemistry of synovial CD3 + T cells, scale bars, 20 μm. ( k ) Tissue-invading T cells as percentage of total cells. ( l ) T cell receptor ( TRB ) transcript concentrations. ( m–o ) Human synovium-NSG chimeras ( n =14) were reconstituted with RA CD4 + CD45RO − PBMC transfected with SH3PXD2A or control siRNA. ( m ) Immunohistochemistry of synovial CD3 + T cells. ( n ) Number of tissue-invading T cells. ( o ) TRB transcript concentrations. ( p–r ) Human synovium-NSG chimeras ( n =14) were reconstituted with healthy CD4 + CD45RO − PBMC transfected with SH3PXD2A or control plasmids. ( p, q ) Immunohistochemistry of synovial CD3 + T cells. ( r ) T cell receptor ( TRB ) transcript concentrations. ( s ) RA disease activity assessed by the Clinical Disease Activity Index (CDAI) correlated with TKS5 transcripts in activated CD4 + CD45RA + T cells. Data from 29 patients. All data are mean ± s.e.m. * P

    Journal: Nature immunology

    Article Title: Metabolic control of the scaffold protein TKS5 in tissue-invasive, pro-inflammatory T cells

    doi: 10.1038/ni.3808

    Figure Lengend Snippet: Hypermotility and tissue-invasiveness of RA T cells ( a ) Migration of activated CD4 + CD45RA + T cells in transwells without chemokines for 48 h ( n =10). ( b–d ) CD4 + T cells invasiveness in 3D-collagen matrices measured by confocal imaging DAPI-stained nuclei. ( b ) Representative confocal images at indicated depths after 48 h. Scale bar, 200 μm. ( c ) DAPI indices (signal at defined depth/signal at surface) (5 control-RA pairs). ( d ) Maximum invasion distances ( n =5). ( e, f ) Cytoskeletal organization and membrane protrusions analyzed by confocal microscopy. Membrane ruffles and podosomes identified through F-actin/cortactin co-localization. ( e ) Representative micrographs showing actin mesh co-localizing with cortactin in lamellopodia and membrane ruffles. Scale bars, 20 μm. ( f ) Quantification of F-actin, cortactin and F-actin + cortactin + membrane protrusions (5 control-RA pairs). ( g ) Gene expression profiling (RT-PCR) of genes involved in actin nucleation and cell motility. Heat map presentation from 6 RA-control pairs. Scaled z-score: red indicates high and blue low transcript levels. ( h–i ) Representative histograms and bar graphs of T cell TKS5 protein expression (11 control-RA pairs). ( j–l ) Human synovium-NSG mice ( n =14) were reconstituted with CD4 + CD45RO − PBMC from healthy individuals or RA patients. ( j ) Immunohistochemistry of synovial CD3 + T cells, scale bars, 20 μm. ( k ) Tissue-invading T cells as percentage of total cells. ( l ) T cell receptor ( TRB ) transcript concentrations. ( m–o ) Human synovium-NSG chimeras ( n =14) were reconstituted with RA CD4 + CD45RO − PBMC transfected with SH3PXD2A or control siRNA. ( m ) Immunohistochemistry of synovial CD3 + T cells. ( n ) Number of tissue-invading T cells. ( o ) TRB transcript concentrations. ( p–r ) Human synovium-NSG chimeras ( n =14) were reconstituted with healthy CD4 + CD45RO − PBMC transfected with SH3PXD2A or control plasmids. ( p, q ) Immunohistochemistry of synovial CD3 + T cells. ( r ) T cell receptor ( TRB ) transcript concentrations. ( s ) RA disease activity assessed by the Clinical Disease Activity Index (CDAI) correlated with TKS5 transcripts in activated CD4 + CD45RA + T cells. Data from 29 patients. All data are mean ± s.e.m. * P

    Article Snippet: Antibodies Primary antibodies used: APC/Cy7 anti-human CD3 (Clone SK7, BioLegend), FITC anti-human CD4 (Clone RPA-T4, BioLegend), anti-human CD8a PE-Cy5 (Clone RPA-T8, eBioscience), PE-Cy™7 Mouse Anti-Human CD45RA (Clone HI100, BD Biosciences), APC anti-human CD45RA (Clone HI100, BioLegend), APC Mouse Anti-Human CD45RO (Clone UCHL1, BD Biosciences), Brilliant Violet 711™ anti-human CD45 (HI30, BioLegend), cortactin antibody (PA5-29799) (ThermoFisher Scientific), FAS mouse monoclonal antibody (M01) (Clone 3F2-1F3, Abnova), Acetyl-CoA Carboxylase (C83B10) Rabbit mAb (Cell Signaling Technology), SCD1 (C12H5) Rabbit mAb (Cell Signaling Technology), RANKL/CD254 Antibody (12A668) (ThermoFisher Scientific), Monoclonal Mouse Anti-Human CD3 (Clone F7.2.38, Dako), Polyclonal Rabbit Anti-Human CD3 (#A045201-2, Dako), β-Actin (8H10D10) Mouse mAb (#3700, Cell Signaling), Anti-rabbit IgG, HRP-linked Antibody (#7074, Cell Signaling), WesternSure HRP Goat anti-Mouse IgG (H+L) (LI-COR), Anti-Interferon gamma antibody (ab25101, Abcam), Anti-PPAR gamma antibody (ab178860, Abcam), PPARγ (81B8) Rabbit mAb (Cell Signaling), Monoclonal anti-flag (HRP) antibody (A8592-.2MG, Sigma-Aldrich).

    Techniques: Migration, Imaging, Staining, Confocal Microscopy, Expressing, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Immunohistochemistry, Transfection, Activity Assay