anti-cd28 Search Results


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  • 99
    Thermo Fisher anti cd28
    MicroRNA‐126 (Mir‐126) deficiency altered the expression of cytokines in CD4 + T cells. CD4 + CD62L + T cells purified by magnetic‐activated cell sorting (MACS) from splenocytes in miR‐126 knock‐down (KD) mice and wild‐type (WT) mice (8–10 weeks old, n = 6) were cultured in the presence of anti‐CD3 (20 <t>μg/ml)/anti‐CD28</t> (4 ng/ml) antibody plus interleukin (IL)‐2 (10 ng/ml); 72 h later, the relative expression of cytokines including IL‐4, IL‐6, IL‐10, IL‐12, transforming growth factor (TGF)‐β, tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ were detected by real‐time polymerase chain reaction (RT–PCR) and calculated (a). (b) The expression levels of IFN‐γ, IL‐4 and IL‐17A in CD4 + T cells were analysed by fluorescence activated cell sorting (FACS) and calculated (c). One representative example of three independent experiments is shown. * P
    Anti Cd28, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5168 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore anti cd28
    RFX1 inhibits IL-17A expression in CD4 + T cells. a CD4 + T cells from healthy controls were transfected with negative control siRNA (NC), RFX1-specific siRNA#1 or siRNA#2, along with anti-CD3 and <t>-CD28</t> antibody stimulation. The RFX1 protein level was downregulated significantly in CD4 + T cells transfected with siRNA#1 or siRNA#2 compared with NC. Representative results of western blot analysis are shown in the left panel, and the quantification of three independent experiments is shown in the right panel. b mRNA expression levels of IL-17A in cells from three groups were quantified by RT-qPCR. c IL-17 protein levels in the supernatants of culture media of three groups were measured by ELISA. d CD4 + T cells from SLE patients were transfected with RFX1 expression plasmid or empty control plasmid. Representative results of western blot analysis are shown in the left panel and the quantification of three independent experiments is shown in the right panel. e mRNA expression levels of IL-17A in cells of two groups were quantified by RT-qPCR. f IL-17A protein levels in supernatants of culture media of two groups were measured by ELISA. Data are representative of three independent experiments (mean ± s.d.; n = 3). * P
    Anti Cd28, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 762 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson anti cd28
    Western blot analysis of the levels of (active) tyrosine-phosphorylated (Y536) SHP-1. (A) Purified T cells of young and elderly donors were left non-stimulated (NS) or exposed to a mixture of anti-CD3 and <t>anti-CD28</t> (5 μg/ml each) mAbs for 30 s or 5 min, as indicated. Cell lysates were sized by SDS-PAGE under reducing conditions, electrotransfered to nitrocellulose membranes and proteins revealed using an anti-Y536 SHP-1 mAb and the chemiluminescence technique. β-Actin was used as control of gel loading. The protein transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of protein had been loaded in each lane. (B) Semi-quantitative densitometric analysis of protein bands of young (filled columns) and elderly (empty columns) donors reported in arbitrary units. Data are represented as the mean ± SD and are representative of one of 20 independent experiments. (C) Flow cytometry measurements of pSHP-1-Y536 MFI in T cells of young and elderly subjects at non-stimulated (NS) and stimulated for 30 seconds (S) states as decribed in the Materials and Methods section. The non-stimulated is normalized as 100% and significant difference is shown by **, p
    Anti Cd28, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 9921 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher anti cd28 antibodies
    SIRT5 deficiency does not affect proliferation of and IL-2 production by splenocytes. SIRT5 +/+ and SIRT5 −/− splenocytes were incubated for 48 h with LPS (5 μg/ml), CpG (2 μg/ml), Pam 3 CSK 4 (5 μg/ml), TSST-1 (2 μg/ml), <t>anti-CD3/CD28</t> antibodies (1μg/ml) and PMA + ionomycin (PMA/iono, 10 ng/ml/100 ng/ml). (A) Proliferation was measured by 3 H-thymidine incorporation. (B) IL-2 concentrations in cell culture supernatants were quantified by ELISA. Data are means ± SD of one experiment performed with three mice and are representative of two experiments. P > 0.05 for all conditions.
    Anti Cd28 Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1197 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher anti cd3 cd28 beads
    Microarray analysis of CD4+ cells treated with CP655 (7‐diethylamino‐ N ‐((5‐hydroxy‐6‐methyl‐4‐oxo‐1,4‐dihydropyridin‐3‐yl)methyl)‐ N ‐methyl‐2‐oxo‐chromen‐3‐carboxamide) vs CP655OMe. Cells from five individual donors were either left unstimulated or stimulated with <t>anti‐CD3/CD28</t> beads and left untreated or treated with either CP655 or CP655OMe for 18 hours. Extracted messenger RNA was used for microarray hybridization and analysis. A, Proliferation of CD4+ T‐cell samples used for microarray. CD4+ T cells were isolated from peripheral blood mononuclear cells of healthy donors. Cells were stimulated with either anti‐CD3/CD28 beads (1:20) and left untreated or treated with either CP655 (5 µM) or the control CP655OMe (5 µM) for 18 hours. Proliferation was measured by 3 H‐thymidine incorporation. * P
    Anti Cd3 Cd28 Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1703 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher soluble anti cd28
    ILDR2 inhibits activation of human and murine T cells. ( A ) Membrane-expressed ILDR2 inhibits human T cell activation. Stimulator cells (a murine thymoma cell line engineered to express membrane-bound anti-human CD3 single-chain Ab fragments) were transfected with expression constructs encoding the long or the short splice variant of human ILDR2. Cells transfected with the empty vector served as negative control. Proliferation of human CD3 + T cells was evaluated upon coculture with stimulator cells expressing the ILDR2 short and long variants, as well as ICOSL or PD-L1, as reference. Shown is a representative experiment of four experiments carried out with a total of three donors. Data are mean ± SEM of triplicate samples. ( B ) Soluble ILDR2-hFc inhibits activation of human T cells. Naive CD4 + T cells obtained from human PBMCs were activated by soluble anti-CD3 and <t>anti-CD28</t> in the presence of irradiated autologous PBMCs. ILDR2-hFc or isotype control (human IgG1; Synagis) was added at the specified final concentrations. Cell proliferation was evaluated by [ 3 H]thymidine incorporation at 72 h. Shown are representative results obtained with three human donors. ( C . IL-2 secretion was measured by ELISA after 24 h. PD-L1–Fc (recombinant human B7-H1/Fc) was used as positive control. Shown are representative results for one of three experiments. ( D ) Bead-immobilized ILDR2-mFc reduces polyclonal murine T cell proliferation. Naive CD4 + . The beads were also coated with the indicated concentrations of ILDR2-mFc or isotype control (mIgG2a). Cells were activated with the coated beads at a 1:1 ratio, and cell proliferation was evaluated by [ 3 H]thymidine incorporation after 72 h. ( E ) Soluble ILDR2-mFc reduces Ag-specific murine T cell activation. Naive CD4 + T cells from DO11.1 mice were activated by 20 μg/ml OVA 323–339 peptide and coincubated with irradiated APCs at a 1:1 ratio in the presence of different concentrations of ILDR2-mFc or isotype control (mIgG2a). Proliferation was evaluated by [ 3 H] thymidine incorporation after 72 h. ** p
    Soluble Anti Cd28, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1310 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio X Cell anti cd28
    Different TNFRSF agonists induced a similar transcriptomic signature. RNA sequencing was performed on Tregs stimulated with <t>anti‐CD3/CD28</t> mAbs and agonists of TNFRSF for 18 h. Biological triplicates of one experiment is shown. (A) Principal component analysis. (B) FC/FC plots (expressed in log2) of differentially expressed genes (DEG) compared to controls (FDR
    Anti Cd28, supplied by Bio X Cell, used in various techniques. Bioz Stars score: 92/100, based on 1143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher anti cd3 cd28 dynabeads
    Construction of CAR-T cells secreting α-PD-1 scFv. (A) The surface expressions of CAR were determined by protein-L staining using FACS test. (B,C) The secreted α-PD-1 scFv with His tag by CAR-T cells cold bind with CAR-T cells. CAR-T cells were activated by <t>anti-CD3/CD28</t> beads for 24 h. Then the supernatants were subjected to western blot assay and scFv secretion from CAR-meso-α-PD-1 cells were confirmed (B) . CAR-meso and CAR-meso-α-PD-1 cells were further stained with AF488-labeled antibody against His tag, and checked on image flow cytometer (C) . CAR-T cells derived from 5 different donors were tested and the representative results were shown.
    Anti Cd3 Cd28 Dynabeads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 985 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pharmingen anti cd28
    Analysis of STAT5 binding to the Il4ra and Il4 loci. (a-f) ChIP-Seq analysis was performed to analyze STAT5 binding at the Il4ra ( a-c) and Il4-Il13-Il5 (d-f) loci in CD4 + T cells cultured under T H 2 conditions (anti-CD3 + <t>anti-CD28</t> + 10 ng/ml IL-4 + 10 ug/ml anti—IFN-γ) for the indicated amounts of time. Cells subjected to two rounds of T H 2 differentiation refers to cells cultured under T H 2 conditions for 3 days, expanded with IL-4 and anti—IFN-γ for 2 days, washed, re-cultured under T H 2 conditions for another 3 days and then analyzed without further cytokine stimulation. These cells were not exposed to exogenous IL-2. Unique sequence reads were first adjusted to center them on the corresponding chromatin fragments. The adjusted reads were then summed in 400 bp windows and displayed as custom tracks on the UCSC genome browser. ChIP was performed with IgG as a control for STAT5A- and STAT5B-specific antibodies. Schematics of the Il4ra ( a-c ) and Il4-Il13-Il5 ( d-f ) loci with standard conservation tracks from the UCSC genome browser indicating the areas of highest conservation among 17 vertebrate species are shown in blue at the bottom of each panel. The experiment was preformed three independent times, with similar results. ( g ) T H 2 cells polarized for 2 rounds were incubated in the presence of 10 μg/ml each of anti-IL-2 (S4B6), anti-IL-2Rα (PC61) and anti-IL-2Rβ, all from BD Bioscience, for an extra 18 h and ChIP was preformed to assess STAT5B binding to indicated gene regions. (h) IL-2-induced binding of STAT5A and STAT5B to indicated gene regions, as measured by ChIP. This is representative of two similar experiments. See Supplementary Table 10 for sequences or primers used in ChIP experiments.
    Anti Cd28, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 716 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher anti cd3 cd28 coated beads
    The Effect of Protocol A and Protocol B on the Expansion and Function of Treg Lines (A) Tregs were expanded by stimulation with <t>anti-CD3/CD28</t> beads and expanded for 36 days with IL-2 in the presence (rapa) or absence (untreated) of rapamycin. There was no difference in the fold expansion of Tregs between the different isolation strategies (protocol A and protocol B) or culture conditions. (B) The suppressive function of freshly isolated Tregs and expanded Treg lines. Suppressive ability of Tregs was measured as a decrease of proliferation of effector T cells in the presence of different concentrations of Tregs (Treg:Teff 1:1 and 1:10). Data represent the percentage Treg suppression at a Treg:Teff ratio of 1:1. Data represent mean ± SD of 3 independent experiments. Statistical analysis was performed using 1-way ANOVA and revealed no significant difference.
    Anti Cd3 Cd28 Coated Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 724 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher anti cd28 monoclonal antibody
    The frequencies and function of ICOS + Tregs in patients with AML. (A,B) Representative plots (left panel) and statistical data (right panel) showed that the frequencies of CD4 + CD25 + FoxP3 + cells and CD4 + FoxP3 + ICOS + cells in 11 healthy donors and 121 patients with AML. Unpaired t -test was used to determine the difference. (C) The CD4 + CD25 high ICOS + cells and CD4 + CD25 high ICOS − cells were sorted from bone marrow mononuclear cells of patients with AML using flow cytometry, and then incubated for 5 days with PBMCs treated with 20 μg/ml mitomycin and CFSE-labeled CD4 + CD25 − T cells, with stimulation with plate-coated anti-CD3 (1 μg/ml) and soluble <t>anti-CD28</t> (3 μg/ml) and IL-2 (20 ng/ml). The cell division was measured by levels of CFSE dilution by flow cytometry. Histograms were representatives of four independent experiments and ANOVA was used to determine the differences.
    Anti Cd28 Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson soluble anti cd28
    <t>CAR-CD28</t> heterodimers are B7-unresponsive but reduce CD28 expression. ( A ) Representative example showing CD71 upregulation in CAR T cells containing an IgG 4 -HD/CD28-TMD co-cultured for 48h with irradiated (4000Rad) CD19-wild type or deficient Raji cells with or without CTLA-4 Ig. ( B ) CD25 + CD71 + T cells were analyzed in low, intermediate (int) or high mCherry-expressing CAR-T cells using the gating strategy described in Supplementary Figure 5. Data were pooled from 4 independent experiments using T cells from 4-5 unrelated donors. ( C ) Editing strategy and homology-directed repair-mediated integration into the TRAC locus of various CD19 CARs using an AAV-6 transduction protocol. ( D ) Myc and CD28 expression in a representative example analyzed 6 days after editing and beads removal. ( E ) CD28 MFI ratio was calculated by dividing CD28 MFI of Myc + cells by Myc - cells in the same culture. Pooled data from 3-4 independent experiments across 5 unrelated donors are shown. Each dot represents one independent editing condition. Two-way ANOVA was used for statistical analysis. *p
    Soluble Anti Cd28, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 2503 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher anti cd3 cd28
    The effect of compounds of regulatory and inflammatory protein and gene expression basal levels of FOXP3 were measured by flow cytometry in unstimulated perientheseal bone (PEB) and peripheral blood mononuclear cell (PBMC) and show a clear population of Tregs in blood but not the enthesis (A). CD4+ and CD8T cells were isolated from PEB or matched peripheral blood and stimulated with <t>anti-CD3/CD28</t> for 48 hours with and without therapeutic agents indicated. qRT-PCR was used to determine the expression of FOXP3 and TGFβ1. (B–E) 2 -ΔΔCt was used to measure relative expression fold change. IL-17A and TNF protein secretion was measured by ELISA (F–G) (n=8). The effect of therapeutic agents on CD4+ and CD8+ viability following a 48-hour incubation (H), n=3. The difference in efficacy between phosphodiesterase type 4 inhibitor (PDE4i) and retinoic acid receptor-related orphan nuclear receptor gamma t inhibitor (RORγti) in attenuating cytokine secretion in enthesis (PEB) compared with blood in CD4+ populations and CD8+ populations (I). One-way t test, *p
    Anti Cd3 Cd28, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 360 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher anti mouse cd28
    Inhibition of lymphocyte proliferation with Imatinib . Imatinib inhibits proliferation of splenic lymphocytes from naïve BR.RIII- Eae27 and B10.RIII mice upon in vitro stimulation with ( a ) LPS (10 μg/ml), ( b ) anti-IgM (40 μg/ml), or ( c ) <t>anti-CD3/anti-CD28</t> (1 μg/ml and 3 μg/ml, respectively). Proliferation was measured as 3 H-thymidine incorporation. Data represent mean CPM ± SEM
    Anti Mouse Cd28, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 371 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    BioLegend anti cd28
    Butyrate induces human CD4 + T cell IL-22 production. a – e Peripheral blood CD4 + T cells were isolated from healthy controls (HC, n = 8 biologically independent samples), patients with active Crohn’s colitis (CD, n = 10 biologically independent samples) and ulcerative colitis (UC, n = 7 biologically independent samples), and activated with <t>anti-CD3/CD28</t> mAbs with or without butyrate (0.5 mM). Il22 expression was assessed at day 3 by qRT-PCR ( a ), IL-22 + cells were measured by flow cytometry at day 5 ( b ), and IL-22 production in supernatants was measured at day 3 by ELISA ( c ). Hif1a ( d ) and Ahr ( e ) expression in CD4 + T cells were analyzed by qRT-PCR at day 3. f Peripheral blood CD4 + T cells from healthy controls, CD, and UC patients were treated with or without butyrate (0.5 mM) ± YC-1 (20 µM) or CH-223191 (5 µM) for 5 days ( n = 3/group). IL-22 production was analyzed by flow cytometry. One representative of three independent experiments was shown. Scale bar, 300 µm. Data were expressed as mean ± SD. Statistical significance was tested by two-tailed paired Student t -test ( a – e ), or two-tailed one-way ANOVA ( f ). a ** p = 0.0024, *** p = 0.0008 (CD), and 0.0003 (UC); b *** p = 0.0001, **** p
    Anti Cd28, supplied by BioLegend, used in various techniques. Bioz Stars score: 92/100, based on 2363 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher anti cd28 coated beads
    RORα and IL-1β mediate the enhanced pathogenic Th17 cell differentiation of RIP2 deficient T cells. Naïve CD4 + T cells were isolated from the spleens of WT and Rip2 −/− mice and differentiated in vitro with anti-CD3 and <t>anti-CD28</t> Ab in the presence of pTh17 cell differentiation cytokines. (A-C) qPCR of Il17a (A), Rora (B) and Rorc (C) mRNA expression from pTh17 cells (n=5–6). (D) Flow cytometry analysis of IL-17A expression in in vitro derived pTh17 cells differentiated following transduction with control or Rip2 shRNA retrovirus (n=6). (E) qPCR of Rora mRNA expression in in vitro derived pTh17 cells differentiated following transduction with control or Rip2 shRNA retrovirus (n=5). (F-I) Percent of in vitro derived pTh17 cells expressing RORα (F) and IL-17A (G). Percent of in vitro derived pTh17 cells expressing IL-17A and RORα (H). MFI of RORα expression in IL-17A + pTh17 cells (I) (n=5). (J) RORα binding to CNS1 and CNS2 in the Il17a locus as measured by ChIP with anti-RORα antibody followed by qPCR (n=5). (K) IL-17A expression in in vitro derived WT and Rip2 −/− pTh17 cells differentiated in the presence of control siRNA or siRNA targeting Rora (n=5–6). (L) IL-17A expression in in vitro derived pTh17 cells differentiated in the presence of control siRNA or siRNA targeting Rora and/or Rip2 (n=7–9). (M) qPCR of Tbx21 , Ifng , Il23r , Il1r1 , Csf2 and Il10 expression in pTh17 cells (n=5). (N) qPCR or Il1r1 expression in Rip2 −/− pTh17 cells differentiated in the presence of control siRNA or siRNA targeting Rora (n=8). (O) IL-17A expression in in vitro derived Th17 cells differentiated in the presence of indicated cytokines (n=10–11). (P) qPCR of Rora expression in in vitro derived Th17 cells differentiated in the presence of indicated cytokines (n=11–12). Data are representative of two to three independent experiments (A-L, N-P) or one independent experiment (M). Statistical analyses: Student’s t -test (A-B, E-J, M, N-P), paired Student’s t -test (D-E) or one-way ANOVA followed by Tukey’s post-hoc test (K-L). * p
    Anti Cd28 Coated Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 290 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    BioLegend anti mouse cd28
    INCB057643 reduces the expression of FOXP3 in CD4 T cells and PD-L1 expression in macrophages. Immunohistochemistry for FOXP3 ( A ) or PD-L1 ( C ) in pancreas and liver of KPC mice treated with vehicle control or INCB057643 for 16 weeks. Scale bar = 30 µm. ( B ) CD4 T cells were isolated from a spleen of a wild type mouse using negative magnetic bead selection. CD4 T cells were plated with anti-CD3 (or PBS, a control for non-stimulated T cells), <t>anti-CD28,</t> IL-2 and TGF-β for 24 h prior to adding INCB057643 (100 nM) for 4 days. CD4 T cells were collected and levels of FOXP3 were determined by PCR. p
    Anti Mouse Cd28, supplied by BioLegend, used in various techniques. Bioz Stars score: 91/100, based on 155 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Syrian Hamster monoclonal CD28 antibody
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    MicroRNA‐126 (Mir‐126) deficiency altered the expression of cytokines in CD4 + T cells. CD4 + CD62L + T cells purified by magnetic‐activated cell sorting (MACS) from splenocytes in miR‐126 knock‐down (KD) mice and wild‐type (WT) mice (8–10 weeks old, n = 6) were cultured in the presence of anti‐CD3 (20 μg/ml)/anti‐CD28 (4 ng/ml) antibody plus interleukin (IL)‐2 (10 ng/ml); 72 h later, the relative expression of cytokines including IL‐4, IL‐6, IL‐10, IL‐12, transforming growth factor (TGF)‐β, tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ were detected by real‐time polymerase chain reaction (RT–PCR) and calculated (a). (b) The expression levels of IFN‐γ, IL‐4 and IL‐17A in CD4 + T cells were analysed by fluorescence activated cell sorting (FACS) and calculated (c). One representative example of three independent experiments is shown. * P

    Journal: Clinical and Experimental Immunology

    Article Title: MicroRNA‐126 deficiency enhanced the activation and function of CD4+T cells by elevating IRS‐1 pathway

    doi: 10.1111/cei.13067

    Figure Lengend Snippet: MicroRNA‐126 (Mir‐126) deficiency altered the expression of cytokines in CD4 + T cells. CD4 + CD62L + T cells purified by magnetic‐activated cell sorting (MACS) from splenocytes in miR‐126 knock‐down (KD) mice and wild‐type (WT) mice (8–10 weeks old, n = 6) were cultured in the presence of anti‐CD3 (20 μg/ml)/anti‐CD28 (4 ng/ml) antibody plus interleukin (IL)‐2 (10 ng/ml); 72 h later, the relative expression of cytokines including IL‐4, IL‐6, IL‐10, IL‐12, transforming growth factor (TGF)‐β, tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ were detected by real‐time polymerase chain reaction (RT–PCR) and calculated (a). (b) The expression levels of IFN‐γ, IL‐4 and IL‐17A in CD4 + T cells were analysed by fluorescence activated cell sorting (FACS) and calculated (c). One representative example of three independent experiments is shown. * P

    Article Snippet: Coating with 20 μg/ml anti‐CD3e (eBioscience, San Diego, CA, USA; 16‐0031‐86) for 4°C overnight in plates and cells were co‐stimulated with 4 ng/ml anti‐CD28 (eBioscience; 16‐0281‐85) antibody and 10 ng/ml plus interleukin (IL)‐2 (ProSpec, cyt‐370‐b).

    Techniques: Expressing, Purification, FACS, Magnetic Cell Separation, Mouse Assay, Cell Culture, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Fluorescence

    MicroRNA‐126 (Mir‐126) deficiency enhanced the activation and proliferation of CD4 + T cells. CD4 + CD62L + T cells purified by magnetic‐activated cell sorting (MACS) from splenocytes in miR‐126 knock‐down (KD) mice and wild‐type (WT) mice (8–10 weeks old, n = 6) respectively. Next, cells were cultured in the presence of anti‐CD3 (20 μg/ml)/anti‐CD28 (4 ng/ml) antibody plus interleukin (IL)‐2 (10 ng/ml) for 72 h. Then, the relative expression of miR‐126 was detected by real‐time polymerase chain reaction (RT–PCR) assay (a). The expression levels of CD69, CD62L and CD44 on CD4 + T cells were analysed by fluorescence activated cell sorting (FACS) (b) and calculated (c). Proliferation of CD4 + T cells from miR‐126KD mice and WT mice were assessed by Ki‐67 staining (d) and calculated (e). (f) The apoptosis of CD4 + T cells was also analysed by FACS and calculated (g). One representative example of three independent experiments is shown. * P

    Journal: Clinical and Experimental Immunology

    Article Title: MicroRNA‐126 deficiency enhanced the activation and function of CD4+T cells by elevating IRS‐1 pathway

    doi: 10.1111/cei.13067

    Figure Lengend Snippet: MicroRNA‐126 (Mir‐126) deficiency enhanced the activation and proliferation of CD4 + T cells. CD4 + CD62L + T cells purified by magnetic‐activated cell sorting (MACS) from splenocytes in miR‐126 knock‐down (KD) mice and wild‐type (WT) mice (8–10 weeks old, n = 6) respectively. Next, cells were cultured in the presence of anti‐CD3 (20 μg/ml)/anti‐CD28 (4 ng/ml) antibody plus interleukin (IL)‐2 (10 ng/ml) for 72 h. Then, the relative expression of miR‐126 was detected by real‐time polymerase chain reaction (RT–PCR) assay (a). The expression levels of CD69, CD62L and CD44 on CD4 + T cells were analysed by fluorescence activated cell sorting (FACS) (b) and calculated (c). Proliferation of CD4 + T cells from miR‐126KD mice and WT mice were assessed by Ki‐67 staining (d) and calculated (e). (f) The apoptosis of CD4 + T cells was also analysed by FACS and calculated (g). One representative example of three independent experiments is shown. * P

    Article Snippet: Coating with 20 μg/ml anti‐CD3e (eBioscience, San Diego, CA, USA; 16‐0031‐86) for 4°C overnight in plates and cells were co‐stimulated with 4 ng/ml anti‐CD28 (eBioscience; 16‐0281‐85) antibody and 10 ng/ml plus interleukin (IL)‐2 (ProSpec, cyt‐370‐b).

    Techniques: Activation Assay, Purification, FACS, Magnetic Cell Separation, Mouse Assay, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Fluorescence, Staining

    The effect of microRNA‐126 (Mir‐126) deficiency on the transduction of related signalling pathway. CD4 + CD62L + T cells purified by magnetic‐activated cell sorting (MACS) from miR‐126 knock‐down (KD) mice and wild‐type (WT) mice (8–10 weeks old, n = 6), respectively, Then, cells were cultured in the presence of anti‐CD3 (20 μg/ml)/anti‐CD28 (4 ng/ml) antibody plus interleukin (IL)‐2 (10 ng/ml) for 48 h. (a) The expression levels of phospho‐extracellular regulated kinase (p‐ERK), phospho‐protein kinase B (p‐AKT) and phospho‐nuclear factor kinase kappa B (p‐NF‐κB) were determined by fluorescence activated cell sorting (FACS) and calculated (b). (c) Schematic representation of the underlying mechanism of miR‐126 deficiency on the activation and function of CD4 + ]

    Journal: Clinical and Experimental Immunology

    Article Title: MicroRNA‐126 deficiency enhanced the activation and function of CD4+T cells by elevating IRS‐1 pathway

    doi: 10.1111/cei.13067

    Figure Lengend Snippet: The effect of microRNA‐126 (Mir‐126) deficiency on the transduction of related signalling pathway. CD4 + CD62L + T cells purified by magnetic‐activated cell sorting (MACS) from miR‐126 knock‐down (KD) mice and wild‐type (WT) mice (8–10 weeks old, n = 6), respectively, Then, cells were cultured in the presence of anti‐CD3 (20 μg/ml)/anti‐CD28 (4 ng/ml) antibody plus interleukin (IL)‐2 (10 ng/ml) for 48 h. (a) The expression levels of phospho‐extracellular regulated kinase (p‐ERK), phospho‐protein kinase B (p‐AKT) and phospho‐nuclear factor kinase kappa B (p‐NF‐κB) were determined by fluorescence activated cell sorting (FACS) and calculated (b). (c) Schematic representation of the underlying mechanism of miR‐126 deficiency on the activation and function of CD4 + ]

    Article Snippet: Coating with 20 μg/ml anti‐CD3e (eBioscience, San Diego, CA, USA; 16‐0031‐86) for 4°C overnight in plates and cells were co‐stimulated with 4 ng/ml anti‐CD28 (eBioscience; 16‐0281‐85) antibody and 10 ng/ml plus interleukin (IL)‐2 (ProSpec, cyt‐370‐b).

    Techniques: Transduction, Purification, FACS, Magnetic Cell Separation, Mouse Assay, Cell Culture, Expressing, Fluorescence, Activation Assay

    The expression of insulin receptor substrate 1 (IRS‐1) in CD4 + T cells. CD4 + CD62L + T cells were purified by magnetic‐activated cell sorting (MACS) from microRNA‐126 knock‐down (Mir‐126KD) mice and wild‐type (WT) mice (8–10 weeks old, n = 6), respectively. Then, cells were cultured in the presence of anti‐CD3 (20 μg/ml)/anti‐CD28 (4 ng/ml) antibody plus interleukin (IL)‐2 (10 ng/ml) for 48 h. The relative expression of indicated genes, including phosphomannomutase 1 (PMM1), target of Myb1 membrane trafficking protein (Tom1), phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit delta (PIK3CD), regulator of G protein signalling 3 (RGS3), tuberous sclerosis 1 (TSC1), insulin receptor substrate 1 (IRS‐1), ADAM metallopeptidase domain 9 (ADAM9), EGF‐like domain multiple 7 (EGFL7), vascular cell adhesion molecule 1 (VCAM1), PHD finger protein 7 (Phf7), solute carrier family 39 member 6 (SLC39a6), Huntingtin interacting protein 1 (HIP1) and poly(ADP‐ribose) polymerase family member 16 (PARP16P), were analysed by real‐time polymerase chain reaction (RT–PCR) assay and calculated (a). (b) Putative miR‐126‐binding sites in the 3' untranslated region (UTR) of murine IRS‐1. (c) CD4 + CD62L + T cells were purified by MACS from FVB/N6 miR‐126KD mice and WT mice, respectively. Then, cells were cultured in the presence of anti‐CD3 (20 μg/ml)/anti‐CD28 (4 ng/ml) antibody plus interleukin (IL)‐2 (10 ng/ml) for 48 h. The expression of IRS‐1 protein was determined by Western blot and calculated. CD4 + CD62L + T cells were purified by MACS from miR‐126KD mice, Then, cells were transfected with p‐IRS‐1 RNAi (5 ng) and cultured in the presence of anti‐CD3 (20 μg/ml)/anti‐CD28 (4 ng/ml) antibody plus IL‐2 (10 ng/ml) for 48 h. (d) The expression of IRS‐1 was analysed by real‐time polymerase chain reaction (RT–PCR). (e) Call counting kit‐8 assay. The expression levels of CD69 and CD44 were analysed by fluorescence activated cell sorting (FACS) and the percentage was calculated (f–i). (j) The expression levels of interferon (IFN)‐γ and IL‐4 were analysed by FACS and the percentage was calculated (k). One representative example of three independent experiments is shown. * P

    Journal: Clinical and Experimental Immunology

    Article Title: MicroRNA‐126 deficiency enhanced the activation and function of CD4+T cells by elevating IRS‐1 pathway

    doi: 10.1111/cei.13067

    Figure Lengend Snippet: The expression of insulin receptor substrate 1 (IRS‐1) in CD4 + T cells. CD4 + CD62L + T cells were purified by magnetic‐activated cell sorting (MACS) from microRNA‐126 knock‐down (Mir‐126KD) mice and wild‐type (WT) mice (8–10 weeks old, n = 6), respectively. Then, cells were cultured in the presence of anti‐CD3 (20 μg/ml)/anti‐CD28 (4 ng/ml) antibody plus interleukin (IL)‐2 (10 ng/ml) for 48 h. The relative expression of indicated genes, including phosphomannomutase 1 (PMM1), target of Myb1 membrane trafficking protein (Tom1), phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit delta (PIK3CD), regulator of G protein signalling 3 (RGS3), tuberous sclerosis 1 (TSC1), insulin receptor substrate 1 (IRS‐1), ADAM metallopeptidase domain 9 (ADAM9), EGF‐like domain multiple 7 (EGFL7), vascular cell adhesion molecule 1 (VCAM1), PHD finger protein 7 (Phf7), solute carrier family 39 member 6 (SLC39a6), Huntingtin interacting protein 1 (HIP1) and poly(ADP‐ribose) polymerase family member 16 (PARP16P), were analysed by real‐time polymerase chain reaction (RT–PCR) assay and calculated (a). (b) Putative miR‐126‐binding sites in the 3' untranslated region (UTR) of murine IRS‐1. (c) CD4 + CD62L + T cells were purified by MACS from FVB/N6 miR‐126KD mice and WT mice, respectively. Then, cells were cultured in the presence of anti‐CD3 (20 μg/ml)/anti‐CD28 (4 ng/ml) antibody plus interleukin (IL)‐2 (10 ng/ml) for 48 h. The expression of IRS‐1 protein was determined by Western blot and calculated. CD4 + CD62L + T cells were purified by MACS from miR‐126KD mice, Then, cells were transfected with p‐IRS‐1 RNAi (5 ng) and cultured in the presence of anti‐CD3 (20 μg/ml)/anti‐CD28 (4 ng/ml) antibody plus IL‐2 (10 ng/ml) for 48 h. (d) The expression of IRS‐1 was analysed by real‐time polymerase chain reaction (RT–PCR). (e) Call counting kit‐8 assay. The expression levels of CD69 and CD44 were analysed by fluorescence activated cell sorting (FACS) and the percentage was calculated (f–i). (j) The expression levels of interferon (IFN)‐γ and IL‐4 were analysed by FACS and the percentage was calculated (k). One representative example of three independent experiments is shown. * P

    Article Snippet: Coating with 20 μg/ml anti‐CD3e (eBioscience, San Diego, CA, USA; 16‐0031‐86) for 4°C overnight in plates and cells were co‐stimulated with 4 ng/ml anti‐CD28 (eBioscience; 16‐0281‐85) antibody and 10 ng/ml plus interleukin (IL)‐2 (ProSpec, cyt‐370‐b).

    Techniques: Expressing, Purification, FACS, Magnetic Cell Separation, Mouse Assay, Cell Culture, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Western Blot, Transfection, Fluorescence

    Analyzing the role of Treg cells in tolerant B6 mice. (A) Analysis of IFN-γ-secreting CD8+ T cells in tolerant mice using an ELISpot assay. CD8+ T cells stimulated with PMA (phorbol myristate acetate) (50 ng/ml) and ionomycin (1 μg/ml) were used as a positive control. The absolute number of IFN-γ-secreting CD8+ T cells was counted using an ELISpot Reader. (B) Schematic illustration of the experimental setup. Diphtheria toxin was given on days 28, 29, 31, and 32 posttransplantation at 1.5 µg per dosage. (C) Percentage of Treg cells in peripheral blood before and after diphtheria toxin treatment. (D) BGL was measured with a OneTouch Ultra device from day 0. The blood was obtained from snipped tail. (E) Immunohistochemical stain of liver tissues of hyperglycemic B6 mice. Section slides were triple-stained with anti-CD3 (brown), anti-insulin (red), and anti-FoxP3 (blue). Original magnification 100 μm. (F) The ratio of FoxP3+ Treg cells to CD3+ T cells near the graft sites was analyzed using Cell Counter Image J software. The ratio was obtained from three different areas, and each group expressed as mean ± SD (Fig. S3). (G) Schematic illustration of mixed lymphocyte reaction. Stimulator and responder cells were prepared as described in Materials and Methods. (H) Schematic illustration of cell coculture. In a coculture of CFSE-labeled naïve effector T cells with irradiated BALB/c or C3H splenocytes, Treg cells isolated from tolerant and naïve mice were added at a ratio of 2:1, 8:1, and 32:1, respectively. Cells were incubated in a 96-well round-bottom plate for 5 d. (I, J). The suppressive ability of Treg cells against the proliferation of naïve effector T cells in the coculture with irradiated BALB/c splenocytes was evaluated through FACS analysis. (K, L) The suppressive ability of Treg cells against the proliferation of naïve effector T cells in the coculture with irradiated C3H splenocytes was evaluated through FACS analysis. Naïve effector T cells stimulated with anti-CD3 and CD28 Abs were used as a positive control. The proliferation of each group was expressed as mean ± SD ( n = 3). Statistical significance was determined by paired Student’s t test. Asterisk (*) indicates statistical significance ( P

    Journal: Cell Transplantation

    Article Title: Donor-Specific Regulatory T Cell-Mediated Immune Tolerance in an Intrahepatic Murine Allogeneic Islet Transplantation Model with Short-Term Anti-CD154 mAb Single Treatment

    doi: 10.1177/0963689720913876

    Figure Lengend Snippet: Analyzing the role of Treg cells in tolerant B6 mice. (A) Analysis of IFN-γ-secreting CD8+ T cells in tolerant mice using an ELISpot assay. CD8+ T cells stimulated with PMA (phorbol myristate acetate) (50 ng/ml) and ionomycin (1 μg/ml) were used as a positive control. The absolute number of IFN-γ-secreting CD8+ T cells was counted using an ELISpot Reader. (B) Schematic illustration of the experimental setup. Diphtheria toxin was given on days 28, 29, 31, and 32 posttransplantation at 1.5 µg per dosage. (C) Percentage of Treg cells in peripheral blood before and after diphtheria toxin treatment. (D) BGL was measured with a OneTouch Ultra device from day 0. The blood was obtained from snipped tail. (E) Immunohistochemical stain of liver tissues of hyperglycemic B6 mice. Section slides were triple-stained with anti-CD3 (brown), anti-insulin (red), and anti-FoxP3 (blue). Original magnification 100 μm. (F) The ratio of FoxP3+ Treg cells to CD3+ T cells near the graft sites was analyzed using Cell Counter Image J software. The ratio was obtained from three different areas, and each group expressed as mean ± SD (Fig. S3). (G) Schematic illustration of mixed lymphocyte reaction. Stimulator and responder cells were prepared as described in Materials and Methods. (H) Schematic illustration of cell coculture. In a coculture of CFSE-labeled naïve effector T cells with irradiated BALB/c or C3H splenocytes, Treg cells isolated from tolerant and naïve mice were added at a ratio of 2:1, 8:1, and 32:1, respectively. Cells were incubated in a 96-well round-bottom plate for 5 d. (I, J). The suppressive ability of Treg cells against the proliferation of naïve effector T cells in the coculture with irradiated BALB/c splenocytes was evaluated through FACS analysis. (K, L) The suppressive ability of Treg cells against the proliferation of naïve effector T cells in the coculture with irradiated C3H splenocytes was evaluated through FACS analysis. Naïve effector T cells stimulated with anti-CD3 and CD28 Abs were used as a positive control. The proliferation of each group was expressed as mean ± SD ( n = 3). Statistical significance was determined by paired Student’s t test. Asterisk (*) indicates statistical significance ( P

    Article Snippet: GFP− Teff cells stimulated with anti-CD3 (eBioscience) and CD28 Abs (eBioscience) were used as a positive control.

    Techniques: Mouse Assay, Enzyme-linked Immunospot, Positive Control, Immunohistochemistry, Staining, Software, Labeling, Irradiation, Isolation, Incubation, FACS

    RFX1 inhibits IL-17A expression in CD4 + T cells. a CD4 + T cells from healthy controls were transfected with negative control siRNA (NC), RFX1-specific siRNA#1 or siRNA#2, along with anti-CD3 and -CD28 antibody stimulation. The RFX1 protein level was downregulated significantly in CD4 + T cells transfected with siRNA#1 or siRNA#2 compared with NC. Representative results of western blot analysis are shown in the left panel, and the quantification of three independent experiments is shown in the right panel. b mRNA expression levels of IL-17A in cells from three groups were quantified by RT-qPCR. c IL-17 protein levels in the supernatants of culture media of three groups were measured by ELISA. d CD4 + T cells from SLE patients were transfected with RFX1 expression plasmid or empty control plasmid. Representative results of western blot analysis are shown in the left panel and the quantification of three independent experiments is shown in the right panel. e mRNA expression levels of IL-17A in cells of two groups were quantified by RT-qPCR. f IL-17A protein levels in supernatants of culture media of two groups were measured by ELISA. Data are representative of three independent experiments (mean ± s.d.; n = 3). * P

    Journal: Nature Communications

    Article Title: IL-6/STAT3 pathway induced deficiency of RFX1 contributes to Th17-dependent autoimmune diseases via epigenetic regulation

    doi: 10.1038/s41467-018-02890-0

    Figure Lengend Snippet: RFX1 inhibits IL-17A expression in CD4 + T cells. a CD4 + T cells from healthy controls were transfected with negative control siRNA (NC), RFX1-specific siRNA#1 or siRNA#2, along with anti-CD3 and -CD28 antibody stimulation. The RFX1 protein level was downregulated significantly in CD4 + T cells transfected with siRNA#1 or siRNA#2 compared with NC. Representative results of western blot analysis are shown in the left panel, and the quantification of three independent experiments is shown in the right panel. b mRNA expression levels of IL-17A in cells from three groups were quantified by RT-qPCR. c IL-17 protein levels in the supernatants of culture media of three groups were measured by ELISA. d CD4 + T cells from SLE patients were transfected with RFX1 expression plasmid or empty control plasmid. Representative results of western blot analysis are shown in the left panel and the quantification of three independent experiments is shown in the right panel. e mRNA expression levels of IL-17A in cells of two groups were quantified by RT-qPCR. f IL-17A protein levels in supernatants of culture media of two groups were measured by ELISA. Data are representative of three independent experiments (mean ± s.d.; n = 3). * P

    Article Snippet: Purified naive CD4+ T cells were stimulated with plate-bound anti-CD3 (5 μg ml−1 , Calbiochem, clone: UCHT1) and anti-CD28 (2 μg ml−1 , Calbiochem, clone: ANC28.1/5D10) for 3 or 5 days under Th17-polarizing conditions: TGF-β (5 ng ml−1 , PeproTech), IL-6 (10 ng ml−1 , PeproTech), IL-1β (10 ng ml−1 , PeproTech), IL-23 (20 ng ml−1 , PeproTech), anti-IFN-γ (10 μg ml−1 , eBioscience, clone: MD-1), and anti-IL-4 (10 μg ml−1 , eBioscience, clone: MP4-25D2) for Th17.

    Techniques: Expressing, Transfection, Negative Control, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

    Western blot analysis of the levels of (active) tyrosine-phosphorylated (Y536) SHP-1. (A) Purified T cells of young and elderly donors were left non-stimulated (NS) or exposed to a mixture of anti-CD3 and anti-CD28 (5 μg/ml each) mAbs for 30 s or 5 min, as indicated. Cell lysates were sized by SDS-PAGE under reducing conditions, electrotransfered to nitrocellulose membranes and proteins revealed using an anti-Y536 SHP-1 mAb and the chemiluminescence technique. β-Actin was used as control of gel loading. The protein transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of protein had been loaded in each lane. (B) Semi-quantitative densitometric analysis of protein bands of young (filled columns) and elderly (empty columns) donors reported in arbitrary units. Data are represented as the mean ± SD and are representative of one of 20 independent experiments. (C) Flow cytometry measurements of pSHP-1-Y536 MFI in T cells of young and elderly subjects at non-stimulated (NS) and stimulated for 30 seconds (S) states as decribed in the Materials and Methods section. The non-stimulated is normalized as 100% and significant difference is shown by **, p

    Journal: Cell Communication and Signaling : CCS

    Article Title: Downregulation of inhibitory SRC Homology 2 Domain-containing Phosphatase-1 (SHP-1) leads to recovery of T cell responses in elderly

    doi: 10.1186/1478-811X-12-2

    Figure Lengend Snippet: Western blot analysis of the levels of (active) tyrosine-phosphorylated (Y536) SHP-1. (A) Purified T cells of young and elderly donors were left non-stimulated (NS) or exposed to a mixture of anti-CD3 and anti-CD28 (5 μg/ml each) mAbs for 30 s or 5 min, as indicated. Cell lysates were sized by SDS-PAGE under reducing conditions, electrotransfered to nitrocellulose membranes and proteins revealed using an anti-Y536 SHP-1 mAb and the chemiluminescence technique. β-Actin was used as control of gel loading. The protein transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of protein had been loaded in each lane. (B) Semi-quantitative densitometric analysis of protein bands of young (filled columns) and elderly (empty columns) donors reported in arbitrary units. Data are represented as the mean ± SD and are representative of one of 20 independent experiments. (C) Flow cytometry measurements of pSHP-1-Y536 MFI in T cells of young and elderly subjects at non-stimulated (NS) and stimulated for 30 seconds (S) states as decribed in the Materials and Methods section. The non-stimulated is normalized as 100% and significant difference is shown by **, p

    Article Snippet: Lymphocyte proliferation PBMCs (2 × 105 cells/well) were exposed to anti-CD3 (5 μg/ml) and/or anti-CD28 (5 μg/ml) for 72 h in 96 well flat-bottomed microcultures (Microtest, Becton Dickinson) in a final volume of 200 μl of RPMI 1640 medium containing 10% foetal bovine serum (FBS), streptomycin (100 μg/ml) and penicillin G (100 U/ml) at 37°C in an atmosphere of 95% air, 5% CO2 and 90% relative humidity.

    Techniques: Western Blot, Purification, SDS Page, Staining, Flow Cytometry, Cytometry

    Western blot analysis of the levels of Y505- and Y394-phosphorylated forms of Lck in untreated and PTP-1-treated T cells. (A) T cells were left non-stimulated (NS) or exposed to a mixture of anti-CD3 and anti-CD28 (5 μg/ml each) mAbs for the times indicated, in the absence of the SHP-1 inhibitor (I) PTP-1 or its presence (50 ng/ml). Cell lysates were sized by SDS-PAGE under reducing conditions, electrotransfered to nitrocellulose membranes and proteins revealed using relevant mAbs and the chemiluminescence technique. β-Actin was used as control of gel loading. The protein transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of protein had been loaded in each lane. (B) Densitometric semi-quantification of the data, shown in arbitrary units in the case of young and elderly donors. For sake of clarity, significant statistical differences are indicated with filled circles (●, pLck-Y505) or filled squares (■, pLck-Y394) with respect to resting cells in the case of young donors. In the case of elderly donors, significant statistical differences are indicated with filled diamonds (♦, pLck-Y505) or ddags (‡, pLck-Y394) with respect to resting cells. (C and D) Densitometric ratio with respect to resting cells were determined for the various times and conditions of experiments for young (C) and elderly (D) . Data are representative of one of 15 independent experiments.

    Journal: Cell Communication and Signaling : CCS

    Article Title: Downregulation of inhibitory SRC Homology 2 Domain-containing Phosphatase-1 (SHP-1) leads to recovery of T cell responses in elderly

    doi: 10.1186/1478-811X-12-2

    Figure Lengend Snippet: Western blot analysis of the levels of Y505- and Y394-phosphorylated forms of Lck in untreated and PTP-1-treated T cells. (A) T cells were left non-stimulated (NS) or exposed to a mixture of anti-CD3 and anti-CD28 (5 μg/ml each) mAbs for the times indicated, in the absence of the SHP-1 inhibitor (I) PTP-1 or its presence (50 ng/ml). Cell lysates were sized by SDS-PAGE under reducing conditions, electrotransfered to nitrocellulose membranes and proteins revealed using relevant mAbs and the chemiluminescence technique. β-Actin was used as control of gel loading. The protein transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of protein had been loaded in each lane. (B) Densitometric semi-quantification of the data, shown in arbitrary units in the case of young and elderly donors. For sake of clarity, significant statistical differences are indicated with filled circles (●, pLck-Y505) or filled squares (■, pLck-Y394) with respect to resting cells in the case of young donors. In the case of elderly donors, significant statistical differences are indicated with filled diamonds (♦, pLck-Y505) or ddags (‡, pLck-Y394) with respect to resting cells. (C and D) Densitometric ratio with respect to resting cells were determined for the various times and conditions of experiments for young (C) and elderly (D) . Data are representative of one of 15 independent experiments.

    Article Snippet: Lymphocyte proliferation PBMCs (2 × 105 cells/well) were exposed to anti-CD3 (5 μg/ml) and/or anti-CD28 (5 μg/ml) for 72 h in 96 well flat-bottomed microcultures (Microtest, Becton Dickinson) in a final volume of 200 μl of RPMI 1640 medium containing 10% foetal bovine serum (FBS), streptomycin (100 μg/ml) and penicillin G (100 U/ml) at 37°C in an atmosphere of 95% air, 5% CO2 and 90% relative humidity.

    Techniques: Western Blot, SDS Page, Staining

    Proliferation and interleukin-2 production in PBMCs exposed to the SHP-1 inhibitor PTP-1. (A) PBMCs were isolated from young (filled columns) and elderly (empty columns) donors, left untreated or exposed to the SHP-1 inhibitor PTP-1 (50 ng/ml) for the indicated times. The cells were non-stimulated (NS) or cultured in the presence of a mixture of anti-CD3 and anti-CD28 (5 μg/ml each) mAbs, or PHA (5 μg/ml) for 72 h. Cell proliferation was assayed by incorporation of [ 3 H]thymidine. Data are represented as the mean ± SD. Letter (a) corresponds to statistical significance (one way ANOVA) for p

    Journal: Cell Communication and Signaling : CCS

    Article Title: Downregulation of inhibitory SRC Homology 2 Domain-containing Phosphatase-1 (SHP-1) leads to recovery of T cell responses in elderly

    doi: 10.1186/1478-811X-12-2

    Figure Lengend Snippet: Proliferation and interleukin-2 production in PBMCs exposed to the SHP-1 inhibitor PTP-1. (A) PBMCs were isolated from young (filled columns) and elderly (empty columns) donors, left untreated or exposed to the SHP-1 inhibitor PTP-1 (50 ng/ml) for the indicated times. The cells were non-stimulated (NS) or cultured in the presence of a mixture of anti-CD3 and anti-CD28 (5 μg/ml each) mAbs, or PHA (5 μg/ml) for 72 h. Cell proliferation was assayed by incorporation of [ 3 H]thymidine. Data are represented as the mean ± SD. Letter (a) corresponds to statistical significance (one way ANOVA) for p

    Article Snippet: Lymphocyte proliferation PBMCs (2 × 105 cells/well) were exposed to anti-CD3 (5 μg/ml) and/or anti-CD28 (5 μg/ml) for 72 h in 96 well flat-bottomed microcultures (Microtest, Becton Dickinson) in a final volume of 200 μl of RPMI 1640 medium containing 10% foetal bovine serum (FBS), streptomycin (100 μg/ml) and penicillin G (100 U/ml) at 37°C in an atmosphere of 95% air, 5% CO2 and 90% relative humidity.

    Techniques: Isolation, Cell Culture

    Minimal model summarizing the roles of SHP-1 and PAG in negative regulation of T cell response in aging. A) The model proposes that in T cells o f young individuals, ligation of the TCR and CD28 leads to activation (TCR/CD28 * ) and concomitant up-regulation of Lck activity resulting from dephosphorylation of Y505, subsequent release of self-inhibition and activation following autophosphorylation at position Y394. Activation of pLck leads to phosphorylation of ITAM motifs of CD3 and the ζ homodimer, triggering the early events of signal transduction. Data reported here showed that the activity of pSHP-1 in T cells of young subjects was transiently low following T cell activation, whereas pPAG transiently migrated out of lipid rafts. These combined events would favor maintaining upregulation of Lck activity which would fulfill the requirements of Signal 1 of T cell activation. Initiation of the Signal 2 leads to full T cell response. B) A similar series of events occurs in T cells of elderly individuals. However, data reported here showed that the activity of pSHP-1 was sustained in these cells and that pPAG was retained in lipid rafts thus contributing to sustained elevated Csk activity. These combined events would lead to lowered activity of pLck, a decrease of the strength of Signal 1 and, an overall decrease of T cell activation. These conditions lead to a diminished T cell response that contributes to immunosenescence.

    Journal: Cell Communication and Signaling : CCS

    Article Title: Downregulation of inhibitory SRC Homology 2 Domain-containing Phosphatase-1 (SHP-1) leads to recovery of T cell responses in elderly

    doi: 10.1186/1478-811X-12-2

    Figure Lengend Snippet: Minimal model summarizing the roles of SHP-1 and PAG in negative regulation of T cell response in aging. A) The model proposes that in T cells o f young individuals, ligation of the TCR and CD28 leads to activation (TCR/CD28 * ) and concomitant up-regulation of Lck activity resulting from dephosphorylation of Y505, subsequent release of self-inhibition and activation following autophosphorylation at position Y394. Activation of pLck leads to phosphorylation of ITAM motifs of CD3 and the ζ homodimer, triggering the early events of signal transduction. Data reported here showed that the activity of pSHP-1 in T cells of young subjects was transiently low following T cell activation, whereas pPAG transiently migrated out of lipid rafts. These combined events would favor maintaining upregulation of Lck activity which would fulfill the requirements of Signal 1 of T cell activation. Initiation of the Signal 2 leads to full T cell response. B) A similar series of events occurs in T cells of elderly individuals. However, data reported here showed that the activity of pSHP-1 was sustained in these cells and that pPAG was retained in lipid rafts thus contributing to sustained elevated Csk activity. These combined events would lead to lowered activity of pLck, a decrease of the strength of Signal 1 and, an overall decrease of T cell activation. These conditions lead to a diminished T cell response that contributes to immunosenescence.

    Article Snippet: Lymphocyte proliferation PBMCs (2 × 105 cells/well) were exposed to anti-CD3 (5 μg/ml) and/or anti-CD28 (5 μg/ml) for 72 h in 96 well flat-bottomed microcultures (Microtest, Becton Dickinson) in a final volume of 200 μl of RPMI 1640 medium containing 10% foetal bovine serum (FBS), streptomycin (100 μg/ml) and penicillin G (100 U/ml) at 37°C in an atmosphere of 95% air, 5% CO2 and 90% relative humidity.

    Techniques: Ligation, Activation Assay, Activity Assay, De-Phosphorylation Assay, Inhibition, Transduction

    Western blot analysis of the levels of tyrosine-phosphorylated PAG. (A) Purified T cells from young and elderly donors were left non-stimulated (NS) or exposed to a mixture of anti-CD3 and anti-CD28 (5 μg/ml each) mAbs for 30 s or 5 min, as indicated. Cell lysates were sized by SDS-PAGE under reducing conditions, electrotransfered to nitrocellulose membranes and proteins revealed using appropriate mAbs and the chemiluminescence technique. β-Actin was used as control of gel loading. The protein transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of protein had been loaded in each lane. (B) Protein bands were analyzed by semi-quantitative densitometry and are reported in arbitrary units. Results of T cells of young (filled columns) and elderly (empty columns) donors are shown. Data are represented as the mean ± SD. Asterisks correspond to statistical significance (Student’s t -test) for p

    Journal: Cell Communication and Signaling : CCS

    Article Title: Downregulation of inhibitory SRC Homology 2 Domain-containing Phosphatase-1 (SHP-1) leads to recovery of T cell responses in elderly

    doi: 10.1186/1478-811X-12-2

    Figure Lengend Snippet: Western blot analysis of the levels of tyrosine-phosphorylated PAG. (A) Purified T cells from young and elderly donors were left non-stimulated (NS) or exposed to a mixture of anti-CD3 and anti-CD28 (5 μg/ml each) mAbs for 30 s or 5 min, as indicated. Cell lysates were sized by SDS-PAGE under reducing conditions, electrotransfered to nitrocellulose membranes and proteins revealed using appropriate mAbs and the chemiluminescence technique. β-Actin was used as control of gel loading. The protein transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of protein had been loaded in each lane. (B) Protein bands were analyzed by semi-quantitative densitometry and are reported in arbitrary units. Results of T cells of young (filled columns) and elderly (empty columns) donors are shown. Data are represented as the mean ± SD. Asterisks correspond to statistical significance (Student’s t -test) for p

    Article Snippet: Lymphocyte proliferation PBMCs (2 × 105 cells/well) were exposed to anti-CD3 (5 μg/ml) and/or anti-CD28 (5 μg/ml) for 72 h in 96 well flat-bottomed microcultures (Microtest, Becton Dickinson) in a final volume of 200 μl of RPMI 1640 medium containing 10% foetal bovine serum (FBS), streptomycin (100 μg/ml) and penicillin G (100 U/ml) at 37°C in an atmosphere of 95% air, 5% CO2 and 90% relative humidity.

    Techniques: Western Blot, Purification, SDS Page, Staining

    Western blot and confocal analysis of Csk and tyrosine-phosphorylated PAG in plasma membrane lipid rafts (LR). (A) Purified T cells were left non-stimulated (NS) or exposed to a mixture of anti-CD3 and anti-CD28 (5 μg/ml each) mAbs for various periods of time, as indicated. Cell lysates were separated on sucrose density gradients and fractions corresponding to lipid rafts (LR) and non-lipid rafts (NLR) were isolated, sized by SDS-PAGE under reducing conditions, electrotransfered to nitrocellulose membranes and proteins revealed using appropriate mAbs and the chemiluminescence technique. β-Actin was used as control of gel loading. The protein transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of protein had been loaded in each lane. (B) Protein bands were analyzed by semi-quantitative densitometry and are reported in arbitrary units. Results of T cells of young (filled columns) and elderly (empty columns) donors are shown. Data are represented as the mean ± SD. Asterisks indicate statistical significance (Student’s t -test) for p

    Journal: Cell Communication and Signaling : CCS

    Article Title: Downregulation of inhibitory SRC Homology 2 Domain-containing Phosphatase-1 (SHP-1) leads to recovery of T cell responses in elderly

    doi: 10.1186/1478-811X-12-2

    Figure Lengend Snippet: Western blot and confocal analysis of Csk and tyrosine-phosphorylated PAG in plasma membrane lipid rafts (LR). (A) Purified T cells were left non-stimulated (NS) or exposed to a mixture of anti-CD3 and anti-CD28 (5 μg/ml each) mAbs for various periods of time, as indicated. Cell lysates were separated on sucrose density gradients and fractions corresponding to lipid rafts (LR) and non-lipid rafts (NLR) were isolated, sized by SDS-PAGE under reducing conditions, electrotransfered to nitrocellulose membranes and proteins revealed using appropriate mAbs and the chemiluminescence technique. β-Actin was used as control of gel loading. The protein transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of protein had been loaded in each lane. (B) Protein bands were analyzed by semi-quantitative densitometry and are reported in arbitrary units. Results of T cells of young (filled columns) and elderly (empty columns) donors are shown. Data are represented as the mean ± SD. Asterisks indicate statistical significance (Student’s t -test) for p

    Article Snippet: Lymphocyte proliferation PBMCs (2 × 105 cells/well) were exposed to anti-CD3 (5 μg/ml) and/or anti-CD28 (5 μg/ml) for 72 h in 96 well flat-bottomed microcultures (Microtest, Becton Dickinson) in a final volume of 200 μl of RPMI 1640 medium containing 10% foetal bovine serum (FBS), streptomycin (100 μg/ml) and penicillin G (100 U/ml) at 37°C in an atmosphere of 95% air, 5% CO2 and 90% relative humidity.

    Techniques: Western Blot, Purification, Isolation, SDS Page, Staining

    Western blot analysis and measurement of CD45 and CD45RA and CD45RO activities. (A) Purified T cells from young and elderly donors were left non-stimulated (NS) or exposed to a mixture of anti-CD3 and anti-CD28 (5 μg/ml each) mAbs for various periods of time, as indicated. Cell lysates were sized by SDS-PAGE under reducing conditions, electrotransfered to nitrocellulose membranes and proteins revealed using an anti-CD45 mAb and the chemiluminescence technique. β-Actin was used as control of gel loading. The protein transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of protein had been loaded in each lane. (B) Results of T cells of young (empty columns) and elderly (filled columns) donors are shown. Protein bands were analyzed by semi-quantitative densitometry and are reported in arbitrary units. Left panel shows the CD45/β-actin expression (**, p

    Journal: Cell Communication and Signaling : CCS

    Article Title: Downregulation of inhibitory SRC Homology 2 Domain-containing Phosphatase-1 (SHP-1) leads to recovery of T cell responses in elderly

    doi: 10.1186/1478-811X-12-2

    Figure Lengend Snippet: Western blot analysis and measurement of CD45 and CD45RA and CD45RO activities. (A) Purified T cells from young and elderly donors were left non-stimulated (NS) or exposed to a mixture of anti-CD3 and anti-CD28 (5 μg/ml each) mAbs for various periods of time, as indicated. Cell lysates were sized by SDS-PAGE under reducing conditions, electrotransfered to nitrocellulose membranes and proteins revealed using an anti-CD45 mAb and the chemiluminescence technique. β-Actin was used as control of gel loading. The protein transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of protein had been loaded in each lane. (B) Results of T cells of young (empty columns) and elderly (filled columns) donors are shown. Protein bands were analyzed by semi-quantitative densitometry and are reported in arbitrary units. Left panel shows the CD45/β-actin expression (**, p

    Article Snippet: Lymphocyte proliferation PBMCs (2 × 105 cells/well) were exposed to anti-CD3 (5 μg/ml) and/or anti-CD28 (5 μg/ml) for 72 h in 96 well flat-bottomed microcultures (Microtest, Becton Dickinson) in a final volume of 200 μl of RPMI 1640 medium containing 10% foetal bovine serum (FBS), streptomycin (100 μg/ml) and penicillin G (100 U/ml) at 37°C in an atmosphere of 95% air, 5% CO2 and 90% relative humidity.

    Techniques: Western Blot, Purification, SDS Page, Staining, Expressing

    Confocal analysis of the distribution of SHP-1 in plasma membrane lipid rafts. (A) Analysis of the distribution in lipid rafts of cholera toxin B subunit (CTx) and SHP-1 of non-stimulated (NS) and activated (anti-TCR/anti-CD28 mAbs, 5 μg/ml each) T cells of young and elderly donors for various periods of time. An illustrative example of individual, merged images and masking is shown. (B) Colocalization data showing results of T cells of young (filled columns) and elderly (empty columns) donors. Data are represented as the mean ± SD of pixel intensities determined by masking. The asterisks indicate significance (Student’s t -test) for p

    Journal: Cell Communication and Signaling : CCS

    Article Title: Downregulation of inhibitory SRC Homology 2 Domain-containing Phosphatase-1 (SHP-1) leads to recovery of T cell responses in elderly

    doi: 10.1186/1478-811X-12-2

    Figure Lengend Snippet: Confocal analysis of the distribution of SHP-1 in plasma membrane lipid rafts. (A) Analysis of the distribution in lipid rafts of cholera toxin B subunit (CTx) and SHP-1 of non-stimulated (NS) and activated (anti-TCR/anti-CD28 mAbs, 5 μg/ml each) T cells of young and elderly donors for various periods of time. An illustrative example of individual, merged images and masking is shown. (B) Colocalization data showing results of T cells of young (filled columns) and elderly (empty columns) donors. Data are represented as the mean ± SD of pixel intensities determined by masking. The asterisks indicate significance (Student’s t -test) for p

    Article Snippet: Lymphocyte proliferation PBMCs (2 × 105 cells/well) were exposed to anti-CD3 (5 μg/ml) and/or anti-CD28 (5 μg/ml) for 72 h in 96 well flat-bottomed microcultures (Microtest, Becton Dickinson) in a final volume of 200 μl of RPMI 1640 medium containing 10% foetal bovine serum (FBS), streptomycin (100 μg/ml) and penicillin G (100 U/ml) at 37°C in an atmosphere of 95% air, 5% CO2 and 90% relative humidity.

    Techniques:

    Western blot analysis of the levels of tyrosine-phosphorylated (Y505) Lck. (A) Purified T cells from young and elderly donors were left non-stimulated (NS) or exposed to a mixture of anti-CD3 (5 μg/ml) and anti-CD28 (5 μg/ml) mAbs for 30 s or 5 min, as indicated. Cell lysates were sized by SDS-PAGE under reducing conditions, electrotransfered to nitrocellulose membranes and proteins revealed using appropriate mAbs and the chemiluminescence technique. β-Actin was used as control of gel loading. The protein transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of protein had been loaded in each lane. (B) Time-related Y505-phosphorylated Lck analyzed by semi-quantitative densitometry and reported in arbitrary units in the case of stimulated (anti-CD3/anti-CD28, 5 μg/ml each) T cells of young (filled columns) and elderly (empty columns). Data are represented as the mean ± SD. Asterisks correspond to statistical significance (Student’s t -test) for p

    Journal: Cell Communication and Signaling : CCS

    Article Title: Downregulation of inhibitory SRC Homology 2 Domain-containing Phosphatase-1 (SHP-1) leads to recovery of T cell responses in elderly

    doi: 10.1186/1478-811X-12-2

    Figure Lengend Snippet: Western blot analysis of the levels of tyrosine-phosphorylated (Y505) Lck. (A) Purified T cells from young and elderly donors were left non-stimulated (NS) or exposed to a mixture of anti-CD3 (5 μg/ml) and anti-CD28 (5 μg/ml) mAbs for 30 s or 5 min, as indicated. Cell lysates were sized by SDS-PAGE under reducing conditions, electrotransfered to nitrocellulose membranes and proteins revealed using appropriate mAbs and the chemiluminescence technique. β-Actin was used as control of gel loading. The protein transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of protein had been loaded in each lane. (B) Time-related Y505-phosphorylated Lck analyzed by semi-quantitative densitometry and reported in arbitrary units in the case of stimulated (anti-CD3/anti-CD28, 5 μg/ml each) T cells of young (filled columns) and elderly (empty columns). Data are represented as the mean ± SD. Asterisks correspond to statistical significance (Student’s t -test) for p

    Article Snippet: Lymphocyte proliferation PBMCs (2 × 105 cells/well) were exposed to anti-CD3 (5 μg/ml) and/or anti-CD28 (5 μg/ml) for 72 h in 96 well flat-bottomed microcultures (Microtest, Becton Dickinson) in a final volume of 200 μl of RPMI 1640 medium containing 10% foetal bovine serum (FBS), streptomycin (100 μg/ml) and penicillin G (100 U/ml) at 37°C in an atmosphere of 95% air, 5% CO2 and 90% relative humidity.

    Techniques: Western Blot, Purification, SDS Page, Staining

    Effects of anti-cytokine antibodies on the frequency of GM-CSF-expressing PBMC subsets: Frozen/thawed cultured PBMC from healthy controls and MS patients were cultured either unstimulated (US) or stimulated (Stim) with anti-CD3/anti-CD28 for 5 days. Stimulated cells were left with or without adding one of the antibodies to block the following cytokine (a-cytokine: cytokine antibody): IL-2, IL-12, IL-2 + IL-12 (IL-2/IL-12), and IL-1β. In addition, stimulated cells were cultured with an antibody isotype control (ic) as the controls. The left panel represents healthy control results, and the right panel represents MS patient results. ( A , B ) Th-GM percentage; ( C , D ) percentage of cells expressing GM-CSF in Tc cells; ( E , F ) percentage of cells expressing GM-CSF in NK cells; ( G , H ) percentage of cells expressing GM-CSF in B cells. * p

    Journal: Biomedicines

    Article Title: Increased IL-2 and Reduced TGF-β Upon T-Cell Stimulation are Associated with GM-CSF Upregulation in Multiple Immune Cell Types in Multiple Sclerosis

    doi: 10.3390/biomedicines8070226

    Figure Lengend Snippet: Effects of anti-cytokine antibodies on the frequency of GM-CSF-expressing PBMC subsets: Frozen/thawed cultured PBMC from healthy controls and MS patients were cultured either unstimulated (US) or stimulated (Stim) with anti-CD3/anti-CD28 for 5 days. Stimulated cells were left with or without adding one of the antibodies to block the following cytokine (a-cytokine: cytokine antibody): IL-2, IL-12, IL-2 + IL-12 (IL-2/IL-12), and IL-1β. In addition, stimulated cells were cultured with an antibody isotype control (ic) as the controls. The left panel represents healthy control results, and the right panel represents MS patient results. ( A , B ) Th-GM percentage; ( C , D ) percentage of cells expressing GM-CSF in Tc cells; ( E , F ) percentage of cells expressing GM-CSF in NK cells; ( G , H ) percentage of cells expressing GM-CSF in B cells. * p

    Article Snippet: Cells were either left unstimulated or stimulated with soluble anti-CD3 and anti-CD28 antibodies (1 μg/mL each; BD Biosciences, Franklin Lakes, NJ, USA) and incubated for 5 days in a 37 °C incubator with humidified atmosphere and 5% CO2.

    Techniques: Expressing, Cell Culture, Blocking Assay

    GM-CSF expression in natural killer (NK) cells: Fresh isolated PBMC were stimulated with anti-CD3/anti-CD28 for 5 days and restimulated with PMA/I in the presence of brefeldin A during the last 5 h. The left panel shows a representative flow cytometry analysis in which cells were gated for live NK cells (full details on the gating strategy can be found in Figure S2 ). Then, the percentage of GM-CSF-producing cells was counted, and the collective results in healthy controls (H) and MS patients are shown (right panel). US: unstimulated cells; Stim: stimulated cells. Horizontal lines are medians. *** p

    Journal: Biomedicines

    Article Title: Increased IL-2 and Reduced TGF-β Upon T-Cell Stimulation are Associated with GM-CSF Upregulation in Multiple Immune Cell Types in Multiple Sclerosis

    doi: 10.3390/biomedicines8070226

    Figure Lengend Snippet: GM-CSF expression in natural killer (NK) cells: Fresh isolated PBMC were stimulated with anti-CD3/anti-CD28 for 5 days and restimulated with PMA/I in the presence of brefeldin A during the last 5 h. The left panel shows a representative flow cytometry analysis in which cells were gated for live NK cells (full details on the gating strategy can be found in Figure S2 ). Then, the percentage of GM-CSF-producing cells was counted, and the collective results in healthy controls (H) and MS patients are shown (right panel). US: unstimulated cells; Stim: stimulated cells. Horizontal lines are medians. *** p

    Article Snippet: Cells were either left unstimulated or stimulated with soluble anti-CD3 and anti-CD28 antibodies (1 μg/mL each; BD Biosciences, Franklin Lakes, NJ, USA) and incubated for 5 days in a 37 °C incubator with humidified atmosphere and 5% CO2.

    Techniques: Expressing, Isolation, Flow Cytometry

    Frequency of cytokine-producing CD4 + T cells differentiated from naïve CD4 T cells in MS patient samples. Representative plots are shown in this figure. Freshly isolated (≥90% pure) naïve CD4 T cells (CD4 + CD45RA + ) from healthy controls (H) and MS patients (MS) were left either unstimulated (US) or stimulated (Stim) with anti-CD3/anti-CD28 alone or with IL-2 and/or IL-7 for 7 days. ( A ) Identification of GM-CSF-expressing naïve CD4 cells (labelled as population X). ( B ) Using gating on population X, GM-CSF-only expressing cells were identified (labelled as population Y). The frequency of Th-GM cells among CD4 T cells was calculated according to the equation Th-GM (%) = X × Y × 100% (where X and Y are expressed as decimal values). ( C ) cumulative data from healthy controls and MS patients showing the frequency of Th-GM cells among CD4 T cells. ( D , E ) cumulative data from healthy controls and MS patients showing the frequency of IFN-γ − and IL-17-producing CD4 T cells, respectively. Bars are medians. * p

    Journal: Biomedicines

    Article Title: Increased IL-2 and Reduced TGF-β Upon T-Cell Stimulation are Associated with GM-CSF Upregulation in Multiple Immune Cell Types in Multiple Sclerosis

    doi: 10.3390/biomedicines8070226

    Figure Lengend Snippet: Frequency of cytokine-producing CD4 + T cells differentiated from naïve CD4 T cells in MS patient samples. Representative plots are shown in this figure. Freshly isolated (≥90% pure) naïve CD4 T cells (CD4 + CD45RA + ) from healthy controls (H) and MS patients (MS) were left either unstimulated (US) or stimulated (Stim) with anti-CD3/anti-CD28 alone or with IL-2 and/or IL-7 for 7 days. ( A ) Identification of GM-CSF-expressing naïve CD4 cells (labelled as population X). ( B ) Using gating on population X, GM-CSF-only expressing cells were identified (labelled as population Y). The frequency of Th-GM cells among CD4 T cells was calculated according to the equation Th-GM (%) = X × Y × 100% (where X and Y are expressed as decimal values). ( C ) cumulative data from healthy controls and MS patients showing the frequency of Th-GM cells among CD4 T cells. ( D , E ) cumulative data from healthy controls and MS patients showing the frequency of IFN-γ − and IL-17-producing CD4 T cells, respectively. Bars are medians. * p

    Article Snippet: Cells were either left unstimulated or stimulated with soluble anti-CD3 and anti-CD28 antibodies (1 μg/mL each; BD Biosciences, Franklin Lakes, NJ, USA) and incubated for 5 days in a 37 °C incubator with humidified atmosphere and 5% CO2.

    Techniques: Isolation, Expressing

    Cytokine profile in PBMC culture supernatants: Fresh isolated PBMC were cultured following anti-CD3/anti-CD28 protocol for 5 days. Culture supernatants were taken on day 5 from healthy controls ( n = 10) and MS patients ( n = 14), and profiles of cytokines both in unstimulated and stimulated conditions were determined as explained in the Subjects and Methods section. H-US and MS-US: unstimulated samples from healthy controls (H) and MS patients, respectively. H-Stim and MS-Stim: anti-CD3/anti-CD28-stimulated samples from healthy controls and MS patients, respectively. * p

    Journal: Biomedicines

    Article Title: Increased IL-2 and Reduced TGF-β Upon T-Cell Stimulation are Associated with GM-CSF Upregulation in Multiple Immune Cell Types in Multiple Sclerosis

    doi: 10.3390/biomedicines8070226

    Figure Lengend Snippet: Cytokine profile in PBMC culture supernatants: Fresh isolated PBMC were cultured following anti-CD3/anti-CD28 protocol for 5 days. Culture supernatants were taken on day 5 from healthy controls ( n = 10) and MS patients ( n = 14), and profiles of cytokines both in unstimulated and stimulated conditions were determined as explained in the Subjects and Methods section. H-US and MS-US: unstimulated samples from healthy controls (H) and MS patients, respectively. H-Stim and MS-Stim: anti-CD3/anti-CD28-stimulated samples from healthy controls and MS patients, respectively. * p

    Article Snippet: Cells were either left unstimulated or stimulated with soluble anti-CD3 and anti-CD28 antibodies (1 μg/mL each; BD Biosciences, Franklin Lakes, NJ, USA) and incubated for 5 days in a 37 °C incubator with humidified atmosphere and 5% CO2.

    Techniques: Isolation, Cell Culture

    Granulocyte macrophage colony stimulating factor (GM-CSF) expression in Th1, Th17, Th, and Th-GM cells: Frozen isolated peripheral blood mononuclear cells (PBMC) were thawed and stimulated with anti-CD3/anti-CD28 for 5 days and restimulated with phorbol myristate acetate (PMA)/ionomycin (I) in the presence of brefeldin A for the last 5 h. ( A ) Live CD4 + cells were gated for Th1 (middle left panel) and Th17 expression (middle right panel). Then, the corresponding percentage of GM-CSF-producing cells were evaluated (bottom panels). ( B ) Global analysis of Th1 GM-CSF-producing cells, both in unstimulated and stimulated healthy control and multiple sclerosis (MS) samples: The paired unstimuated/stimulated (US/S)data for each sample are shown in Figure S1 . ( C ) Global analysis of Th17 GM-CSF-producing cells, both in unstimulated and stimulated HC and MS samples. ( D ) Live CD4 + (Th) were gated for GM-CSF expression (top panel, population X) and analysed both in unstimulated and stimulated HC and MS samples (lower panel). ( E ) Population X gated cells were used to identify double negatives for IL17 and IFN-γ (population Y). ( F ) Global analysis of non-Th1, non-Th17 Th cells expressing GM-CSF (Th-GM), both in unstimulated and stimulated HC and MS samples, was done using the equation Th-GM (%) = X × Y × 100% (where X and Y are expressed as decimal values). H-US: unstimulated cells from healthy controls; H-Stim: stimulated cells from healthy controls; MS-US: unstimulated cells from MS patients; MS-Stim: stimulated cells from MS patients; and SSC: side scattered. Horizontal lines are medians. * p

    Journal: Biomedicines

    Article Title: Increased IL-2 and Reduced TGF-β Upon T-Cell Stimulation are Associated with GM-CSF Upregulation in Multiple Immune Cell Types in Multiple Sclerosis

    doi: 10.3390/biomedicines8070226

    Figure Lengend Snippet: Granulocyte macrophage colony stimulating factor (GM-CSF) expression in Th1, Th17, Th, and Th-GM cells: Frozen isolated peripheral blood mononuclear cells (PBMC) were thawed and stimulated with anti-CD3/anti-CD28 for 5 days and restimulated with phorbol myristate acetate (PMA)/ionomycin (I) in the presence of brefeldin A for the last 5 h. ( A ) Live CD4 + cells were gated for Th1 (middle left panel) and Th17 expression (middle right panel). Then, the corresponding percentage of GM-CSF-producing cells were evaluated (bottom panels). ( B ) Global analysis of Th1 GM-CSF-producing cells, both in unstimulated and stimulated healthy control and multiple sclerosis (MS) samples: The paired unstimuated/stimulated (US/S)data for each sample are shown in Figure S1 . ( C ) Global analysis of Th17 GM-CSF-producing cells, both in unstimulated and stimulated HC and MS samples. ( D ) Live CD4 + (Th) were gated for GM-CSF expression (top panel, population X) and analysed both in unstimulated and stimulated HC and MS samples (lower panel). ( E ) Population X gated cells were used to identify double negatives for IL17 and IFN-γ (population Y). ( F ) Global analysis of non-Th1, non-Th17 Th cells expressing GM-CSF (Th-GM), both in unstimulated and stimulated HC and MS samples, was done using the equation Th-GM (%) = X × Y × 100% (where X and Y are expressed as decimal values). H-US: unstimulated cells from healthy controls; H-Stim: stimulated cells from healthy controls; MS-US: unstimulated cells from MS patients; MS-Stim: stimulated cells from MS patients; and SSC: side scattered. Horizontal lines are medians. * p

    Article Snippet: Cells were either left unstimulated or stimulated with soluble anti-CD3 and anti-CD28 antibodies (1 μg/mL each; BD Biosciences, Franklin Lakes, NJ, USA) and incubated for 5 days in a 37 °C incubator with humidified atmosphere and 5% CO2.

    Techniques: Expressing, Isolation

    GM-CSF expression in cytotoxic T (Tc) cells: Fresh isolated PBMC were stimulated with anti-CD3/anti-CD28 for 5 days and restimulated with PMA/I in the presence of brefeldin A during the last 5 h. The left panel shows a representative flow cytometry plot analysis in which cells were gated for live Tc cells (full details on the gating strategy can be found in Figure S2 ). Then, the percentage of GM-CSF-producing cells was counted, and the collective results in healthy controls (H) and MS patients are shown (right panel). US: unstimulated cells; Stim: stimulated. Horizontal lines are medians. *** p

    Journal: Biomedicines

    Article Title: Increased IL-2 and Reduced TGF-β Upon T-Cell Stimulation are Associated with GM-CSF Upregulation in Multiple Immune Cell Types in Multiple Sclerosis

    doi: 10.3390/biomedicines8070226

    Figure Lengend Snippet: GM-CSF expression in cytotoxic T (Tc) cells: Fresh isolated PBMC were stimulated with anti-CD3/anti-CD28 for 5 days and restimulated with PMA/I in the presence of brefeldin A during the last 5 h. The left panel shows a representative flow cytometry plot analysis in which cells were gated for live Tc cells (full details on the gating strategy can be found in Figure S2 ). Then, the percentage of GM-CSF-producing cells was counted, and the collective results in healthy controls (H) and MS patients are shown (right panel). US: unstimulated cells; Stim: stimulated. Horizontal lines are medians. *** p

    Article Snippet: Cells were either left unstimulated or stimulated with soluble anti-CD3 and anti-CD28 antibodies (1 μg/mL each; BD Biosciences, Franklin Lakes, NJ, USA) and incubated for 5 days in a 37 °C incubator with humidified atmosphere and 5% CO2.

    Techniques: Expressing, Isolation, Flow Cytometry

    GM-CSF expression in B cells: Frozen isolated PBMC were thawed and stimulated with anti-CD3/anti-CD28 for 5 days and restimulated with PMA/I in the presence of brefeldin A during the last 5 h. The left panel shows a representative flow cytometry analysis in which cells were gated for live B cells (full details on gating in Figure S2 ). Then, the percentage of GM-CSF-producing cells was counted, and the collective results in healthy controls (H) and MS patients are shown (right panel). US: unstimulated cells; Stim: stimulated cells. Horizontal lines are medians. * p

    Journal: Biomedicines

    Article Title: Increased IL-2 and Reduced TGF-β Upon T-Cell Stimulation are Associated with GM-CSF Upregulation in Multiple Immune Cell Types in Multiple Sclerosis

    doi: 10.3390/biomedicines8070226

    Figure Lengend Snippet: GM-CSF expression in B cells: Frozen isolated PBMC were thawed and stimulated with anti-CD3/anti-CD28 for 5 days and restimulated with PMA/I in the presence of brefeldin A during the last 5 h. The left panel shows a representative flow cytometry analysis in which cells were gated for live B cells (full details on gating in Figure S2 ). Then, the percentage of GM-CSF-producing cells was counted, and the collective results in healthy controls (H) and MS patients are shown (right panel). US: unstimulated cells; Stim: stimulated cells. Horizontal lines are medians. * p

    Article Snippet: Cells were either left unstimulated or stimulated with soluble anti-CD3 and anti-CD28 antibodies (1 μg/mL each; BD Biosciences, Franklin Lakes, NJ, USA) and incubated for 5 days in a 37 °C incubator with humidified atmosphere and 5% CO2.

    Techniques: Expressing, Isolation, Flow Cytometry

    SIRT5 deficiency does not affect proliferation of and IL-2 production by splenocytes. SIRT5 +/+ and SIRT5 −/− splenocytes were incubated for 48 h with LPS (5 μg/ml), CpG (2 μg/ml), Pam 3 CSK 4 (5 μg/ml), TSST-1 (2 μg/ml), anti-CD3/CD28 antibodies (1μg/ml) and PMA + ionomycin (PMA/iono, 10 ng/ml/100 ng/ml). (A) Proliferation was measured by 3 H-thymidine incorporation. (B) IL-2 concentrations in cell culture supernatants were quantified by ELISA. Data are means ± SD of one experiment performed with three mice and are representative of two experiments. P > 0.05 for all conditions.

    Journal: Frontiers in Immunology

    Article Title: Sirtuin 5 Deficiency Does Not Compromise Innate Immune Responses to Bacterial Infections

    doi: 10.3389/fimmu.2018.02675

    Figure Lengend Snippet: SIRT5 deficiency does not affect proliferation of and IL-2 production by splenocytes. SIRT5 +/+ and SIRT5 −/− splenocytes were incubated for 48 h with LPS (5 μg/ml), CpG (2 μg/ml), Pam 3 CSK 4 (5 μg/ml), TSST-1 (2 μg/ml), anti-CD3/CD28 antibodies (1μg/ml) and PMA + ionomycin (PMA/iono, 10 ng/ml/100 ng/ml). (A) Proliferation was measured by 3 H-thymidine incorporation. (B) IL-2 concentrations in cell culture supernatants were quantified by ELISA. Data are means ± SD of one experiment performed with three mice and are representative of two experiments. P > 0.05 for all conditions.

    Article Snippet: Stimuli were Salmonella minnesota ultra pure LPS (InvivoGen, San Diego, CA), Pam3 CSK4 (EMC microcollections, Tübingen, Germany), CpG ODN 1826 (CpG, InvivoGen), toxic shock syndrome toxin-1 (TSST-1, Toxin Technology, Sarasota, FL), concanavalin A (Sigma-Aldrich, St. Louis, MI), anti-CD3ε, and anti-CD28 antibodies (clones 145-2C11 and 37.51, eBioscience, San Diego, CA) and phorbol-12-myristate-13-acetate (PMA) plus ionomycin (Sigma-Aldrich) or bacteria.

    Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Mouse Assay

    Microarray analysis of CD4+ cells treated with CP655 (7‐diethylamino‐ N ‐((5‐hydroxy‐6‐methyl‐4‐oxo‐1,4‐dihydropyridin‐3‐yl)methyl)‐ N ‐methyl‐2‐oxo‐chromen‐3‐carboxamide) vs CP655OMe. Cells from five individual donors were either left unstimulated or stimulated with anti‐CD3/CD28 beads and left untreated or treated with either CP655 or CP655OMe for 18 hours. Extracted messenger RNA was used for microarray hybridization and analysis. A, Proliferation of CD4+ T‐cell samples used for microarray. CD4+ T cells were isolated from peripheral blood mononuclear cells of healthy donors. Cells were stimulated with either anti‐CD3/CD28 beads (1:20) and left untreated or treated with either CP655 (5 µM) or the control CP655OMe (5 µM) for 18 hours. Proliferation was measured by 3 H‐thymidine incorporation. * P

    Journal: Immunity, Inflammation and Disease

    Article Title: HPO iron chelator, CP655, causes the G1/S phase cell cycle block via p21 upregulation. HPO iron chelator, CP655, causes the G1/S phase cell cycle block via p21 upregulation

    doi: 10.1002/iid3.342

    Figure Lengend Snippet: Microarray analysis of CD4+ cells treated with CP655 (7‐diethylamino‐ N ‐((5‐hydroxy‐6‐methyl‐4‐oxo‐1,4‐dihydropyridin‐3‐yl)methyl)‐ N ‐methyl‐2‐oxo‐chromen‐3‐carboxamide) vs CP655OMe. Cells from five individual donors were either left unstimulated or stimulated with anti‐CD3/CD28 beads and left untreated or treated with either CP655 or CP655OMe for 18 hours. Extracted messenger RNA was used for microarray hybridization and analysis. A, Proliferation of CD4+ T‐cell samples used for microarray. CD4+ T cells were isolated from peripheral blood mononuclear cells of healthy donors. Cells were stimulated with either anti‐CD3/CD28 beads (1:20) and left untreated or treated with either CP655 (5 µM) or the control CP655OMe (5 µM) for 18 hours. Proliferation was measured by 3 H‐thymidine incorporation. * P

    Article Snippet: Cells were stimulated with anti‐CD3/CD28 beads (Invitrogen, UK) in a ratio of 1:20 (bead:CD4+ T cells) and treated with or without iron chelator CP655 (5 µM) or control compound CP655OMe (5 µM) for various time points ranging from 18 hours to 6 days posttreatment.

    Techniques: Microarray, Hybridization, Isolation

    Cell cycle arrest of CD4+ T cells following CP655 (7‐diethylamino‐ N ‐((5‐hydroxy‐6‐methyl‐4‐oxo‐1,4‐dihydropyridin‐3‐yl)methyl)‐ N ‐methyl‐2‐oxo‐chromen‐3‐carboxamide) treatment. CD4+ T cells were isolated from fresh peripheral blood mononuclear cells of healthy donors. Cells were either left unstimulated or stimulated with 1:5 bead:cells ratio of anti‐CD3/CD28 beads in the presence or absence of either CP655 (5 µM) or CP655OMe (5 µM) for 48 hours. Cells were lysed with cold 100% ethanol and stained with propidium iodide and ribonuclease A, before analysis by flow cytometry. A, Representative Fluorescence‐activated cell sorting results from one experiment. B, Cumulative data showing cell cycle arrest of CD4+ T cells from n = 4 donor. Results shown as mean ± standard error of the means. * P

    Journal: Immunity, Inflammation and Disease

    Article Title: HPO iron chelator, CP655, causes the G1/S phase cell cycle block via p21 upregulation. HPO iron chelator, CP655, causes the G1/S phase cell cycle block via p21 upregulation

    doi: 10.1002/iid3.342

    Figure Lengend Snippet: Cell cycle arrest of CD4+ T cells following CP655 (7‐diethylamino‐ N ‐((5‐hydroxy‐6‐methyl‐4‐oxo‐1,4‐dihydropyridin‐3‐yl)methyl)‐ N ‐methyl‐2‐oxo‐chromen‐3‐carboxamide) treatment. CD4+ T cells were isolated from fresh peripheral blood mononuclear cells of healthy donors. Cells were either left unstimulated or stimulated with 1:5 bead:cells ratio of anti‐CD3/CD28 beads in the presence or absence of either CP655 (5 µM) or CP655OMe (5 µM) for 48 hours. Cells were lysed with cold 100% ethanol and stained with propidium iodide and ribonuclease A, before analysis by flow cytometry. A, Representative Fluorescence‐activated cell sorting results from one experiment. B, Cumulative data showing cell cycle arrest of CD4+ T cells from n = 4 donor. Results shown as mean ± standard error of the means. * P

    Article Snippet: Cells were stimulated with anti‐CD3/CD28 beads (Invitrogen, UK) in a ratio of 1:20 (bead:CD4+ T cells) and treated with or without iron chelator CP655 (5 µM) or control compound CP655OMe (5 µM) for various time points ranging from 18 hours to 6 days posttreatment.

    Techniques: Isolation, Staining, Flow Cytometry, Fluorescence, FACS

    Effect of CP655 (7‐diethylamino‐ N ‐((5‐hydroxy‐6‐methyl‐4‐oxo‐1,4‐dihydropyridin‐3‐yl)methyl)‐ N ‐methyl‐2‐oxo‐chromen‐3‐carboxamide) treatment on p21 protein expression in CD4+ T cells. CD4+ T cells were isolated from peripheral blood mononuclear cells of healthy donors and either left unstimulated (USUT) or stimulated with anti‐CD3/CD28 beads (1:20) in the presence or absence of either CP655 (5 µM) or CP655OMe (5 µM). After 4 hours of culture, p21 expression was analyzed by Western blot analysis. A, Results from one representative experiment. B, Results represented as mean ± standard error of the mean from n = 4 individual donors

    Journal: Immunity, Inflammation and Disease

    Article Title: HPO iron chelator, CP655, causes the G1/S phase cell cycle block via p21 upregulation. HPO iron chelator, CP655, causes the G1/S phase cell cycle block via p21 upregulation

    doi: 10.1002/iid3.342

    Figure Lengend Snippet: Effect of CP655 (7‐diethylamino‐ N ‐((5‐hydroxy‐6‐methyl‐4‐oxo‐1,4‐dihydropyridin‐3‐yl)methyl)‐ N ‐methyl‐2‐oxo‐chromen‐3‐carboxamide) treatment on p21 protein expression in CD4+ T cells. CD4+ T cells were isolated from peripheral blood mononuclear cells of healthy donors and either left unstimulated (USUT) or stimulated with anti‐CD3/CD28 beads (1:20) in the presence or absence of either CP655 (5 µM) or CP655OMe (5 µM). After 4 hours of culture, p21 expression was analyzed by Western blot analysis. A, Results from one representative experiment. B, Results represented as mean ± standard error of the mean from n = 4 individual donors

    Article Snippet: Cells were stimulated with anti‐CD3/CD28 beads (Invitrogen, UK) in a ratio of 1:20 (bead:CD4+ T cells) and treated with or without iron chelator CP655 (5 µM) or control compound CP655OMe (5 µM) for various time points ranging from 18 hours to 6 days posttreatment.

    Techniques: Expressing, Isolation, Western Blot

    Compare platelet-derived mitochondria with other cell-derived mitochondria. ( A , B ) Compare the SDF-1 expression among platelet-derived mitochondria, PBMC-derived mitochondria and human peripheral blood insulin-producing cells (PB-IPC)-derived mitochondria. The mitochondria were isolated from peripheral blood (PB)-platelets, PBMC and PB-IPC, respectively, using the Mitochondria Isolation kit (Thermo scientific) according to the manufacturer’s recommended protocol. Culture of PB-IPC from adult peripheral blood was performed as previously described [ 21 , 22 ]. ( A ) Flow cytometry show the expression of SDF-1 on the platelet-derived mitochondria and PBMC-derived mitochondria and PB-IPC-derived mitochondria. Isotype-matched IgGs served as controls. Data were representative from three experiments. ( B ) Compare the level of SDF-1 expression between platelet-derived mitochondria (Plt-Mito) and PBMC-derived mitochondria (PBMC-Mito) with no marked difference, but much lower on PB-IPC-Mito. Data represent mean ± SD, n = 3. ( C – E ) Effects of PBMC-derived mitochondria on the differentiation of memory CD4 + T cells. The purified CD4 + T cells (1 × 10 5 cells/well) from PBMC of healthy donors ( n = 3) were activated with T-cell activator anti-CD3/CD28 Dynabeads in the presence or absence of 100 µg/mL PBMC-derived mitochondria ( n = 3). Untreated CD4 + T cells served as negative control. ( C ) Upregulation of the percentage of naïve CD4 + T cells by PBMC-derived mitochondria. ( D ) Increase in the percentage of CD4 + T CM cells by PBMC-derived mitochondria. ( E ) Decrease in the percentage of CD4 + T EM cells by PBMC-derived mitochondria. Data represent mean ± SD. n = 3.

    Journal: International Journal of Molecular Sciences

    Article Title: Immune Modulation of Platelet-Derived Mitochondria on Memory CD4+ T Cells in Humans

    doi: 10.3390/ijms21176295

    Figure Lengend Snippet: Compare platelet-derived mitochondria with other cell-derived mitochondria. ( A , B ) Compare the SDF-1 expression among platelet-derived mitochondria, PBMC-derived mitochondria and human peripheral blood insulin-producing cells (PB-IPC)-derived mitochondria. The mitochondria were isolated from peripheral blood (PB)-platelets, PBMC and PB-IPC, respectively, using the Mitochondria Isolation kit (Thermo scientific) according to the manufacturer’s recommended protocol. Culture of PB-IPC from adult peripheral blood was performed as previously described [ 21 , 22 ]. ( A ) Flow cytometry show the expression of SDF-1 on the platelet-derived mitochondria and PBMC-derived mitochondria and PB-IPC-derived mitochondria. Isotype-matched IgGs served as controls. Data were representative from three experiments. ( B ) Compare the level of SDF-1 expression between platelet-derived mitochondria (Plt-Mito) and PBMC-derived mitochondria (PBMC-Mito) with no marked difference, but much lower on PB-IPC-Mito. Data represent mean ± SD, n = 3. ( C – E ) Effects of PBMC-derived mitochondria on the differentiation of memory CD4 + T cells. The purified CD4 + T cells (1 × 10 5 cells/well) from PBMC of healthy donors ( n = 3) were activated with T-cell activator anti-CD3/CD28 Dynabeads in the presence or absence of 100 µg/mL PBMC-derived mitochondria ( n = 3). Untreated CD4 + T cells served as negative control. ( C ) Upregulation of the percentage of naïve CD4 + T cells by PBMC-derived mitochondria. ( D ) Increase in the percentage of CD4 + T CM cells by PBMC-derived mitochondria. ( E ) Decrease in the percentage of CD4 + T EM cells by PBMC-derived mitochondria. Data represent mean ± SD. n = 3.

    Article Snippet: Evaluation of Cytokine Levels in Serum To detect the cytokine production by CD4+ T cells, 1 × 105 CD4+ T cells were stimulated with anti-CD3/anti-CD28 beads (Thermo Fisher Scientific, Waltham, MA, USA) in the presence or absence of mitochondria at 100 μg/mL in a 96-well plate with a total of 200 μL X-VIVO 15 serum-free culture medium (Lonza, Walkersville, MD, USA) per well.

    Techniques: Derivative Assay, Expressing, Isolation, Flow Cytometry, Purification, Negative Control

    Blocking effects of SDF-1 Ab on the immune modulation of platelet-derived mitochondria. The purified CD4 + T cells (1 × 10 5 cells/well) from PBMC of healthy donors ( n = 3) were activated with T-cell activator CD3/CD28 Dynabeads in the presence or absence of 100 μg/mL platelet-derived mitochondria or 20 μg/mL SDF-1 Ab. Untreated CD4 + T cells served as negative control. Cells were collected for flow cytometry analysis after the treatment at 37 °C and 5% CO 2 for 48 h. ( A ) The percentage of CD45RO − CCR7 + Naïve T cells were down-regulated in the presence of SDF-1 Ab. ( B ) The percentage of CD45RO + CCR7 + T CM cells were decreased after blocking with SDF-1 Ab. ( C ) The percentage of CD45RO + CCR7 − T EM cells were up-regulated in the presence of SDF-1 Ab. Data represent mean ± SD. n = 3.

    Journal: International Journal of Molecular Sciences

    Article Title: Immune Modulation of Platelet-Derived Mitochondria on Memory CD4+ T Cells in Humans

    doi: 10.3390/ijms21176295

    Figure Lengend Snippet: Blocking effects of SDF-1 Ab on the immune modulation of platelet-derived mitochondria. The purified CD4 + T cells (1 × 10 5 cells/well) from PBMC of healthy donors ( n = 3) were activated with T-cell activator CD3/CD28 Dynabeads in the presence or absence of 100 μg/mL platelet-derived mitochondria or 20 μg/mL SDF-1 Ab. Untreated CD4 + T cells served as negative control. Cells were collected for flow cytometry analysis after the treatment at 37 °C and 5% CO 2 for 48 h. ( A ) The percentage of CD45RO − CCR7 + Naïve T cells were down-regulated in the presence of SDF-1 Ab. ( B ) The percentage of CD45RO + CCR7 + T CM cells were decreased after blocking with SDF-1 Ab. ( C ) The percentage of CD45RO + CCR7 − T EM cells were up-regulated in the presence of SDF-1 Ab. Data represent mean ± SD. n = 3.

    Article Snippet: Evaluation of Cytokine Levels in Serum To detect the cytokine production by CD4+ T cells, 1 × 105 CD4+ T cells were stimulated with anti-CD3/anti-CD28 beads (Thermo Fisher Scientific, Waltham, MA, USA) in the presence or absence of mitochondria at 100 μg/mL in a 96-well plate with a total of 200 μL X-VIVO 15 serum-free culture medium (Lonza, Walkersville, MD, USA) per well.

    Techniques: Blocking Assay, Derivative Assay, Purification, Negative Control, Flow Cytometry

    Effects of platelet-derived mitochondria on the differentiation of memory CD4 + T cells. The purified CD4 + T cells (1 × 10 5 cells/well) from PBMC of healthy donors ( n = 3) were activated with T-cell activator CD3/CD28 Dynabeads in the presence or absence of 100 μg/mL platelet-derived mitochondria ( n = 3). Untreated CD4 + T cells served as negative control. ( A ) Flow cytometry analysis with human memory CD4 T cell-associated markers CD45RO and CCR7, with three subsets including CD45RO − CCR7 + Naïve T cells, CD45RO + CCR7 + T CM cells, and CD45RO + CCR7 − T EM cells. Dot plots of flow cytometry were representative of three experiments with similar results. ( B ) Upregulation of the percentage of naïve CD4 + T cells by platelet-derived mitochondria. ( C ) Increase in the percentage of CD4 + T CM cells by platelet-derived mitochondria. ( D ) Decrease in the percentage of CD4 + T EM cells by platelet-derived mitochondria. Data represent mean ± SD. n = 3.

    Journal: International Journal of Molecular Sciences

    Article Title: Immune Modulation of Platelet-Derived Mitochondria on Memory CD4+ T Cells in Humans

    doi: 10.3390/ijms21176295

    Figure Lengend Snippet: Effects of platelet-derived mitochondria on the differentiation of memory CD4 + T cells. The purified CD4 + T cells (1 × 10 5 cells/well) from PBMC of healthy donors ( n = 3) were activated with T-cell activator CD3/CD28 Dynabeads in the presence or absence of 100 μg/mL platelet-derived mitochondria ( n = 3). Untreated CD4 + T cells served as negative control. ( A ) Flow cytometry analysis with human memory CD4 T cell-associated markers CD45RO and CCR7, with three subsets including CD45RO − CCR7 + Naïve T cells, CD45RO + CCR7 + T CM cells, and CD45RO + CCR7 − T EM cells. Dot plots of flow cytometry were representative of three experiments with similar results. ( B ) Upregulation of the percentage of naïve CD4 + T cells by platelet-derived mitochondria. ( C ) Increase in the percentage of CD4 + T CM cells by platelet-derived mitochondria. ( D ) Decrease in the percentage of CD4 + T EM cells by platelet-derived mitochondria. Data represent mean ± SD. n = 3.

    Article Snippet: Evaluation of Cytokine Levels in Serum To detect the cytokine production by CD4+ T cells, 1 × 105 CD4+ T cells were stimulated with anti-CD3/anti-CD28 beads (Thermo Fisher Scientific, Waltham, MA, USA) in the presence or absence of mitochondria at 100 μg/mL in a 96-well plate with a total of 200 μL X-VIVO 15 serum-free culture medium (Lonza, Walkersville, MD, USA) per well.

    Techniques: Derivative Assay, Purification, Negative Control, Flow Cytometry

    Modulation of cytokine productions in CD4 + T cells by platelet-derived mitochondria. The purified CD4 + T cells (1 × 10 5 cells/well) from PBMC of healthy donors ( n = 3) were activated with T-cell activator anti-CD3/CD28 Dynabeads in the presence or absence of 100 μg/mL platelet-derived mitochondria ( n = 3). Untreated CD4 + T cells served as negative control. After the treatment for 2 days, supernatants were collected to measure the levels of cytokines (IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17F, IL-21, IL-22, IFN-γ, and TNF-α) by using a LEGENDplex™ Human Th Panel (Biolegend) with Gallios Flow Cytometer. ( A ) The level of IL-6 was markedly decreased after the mitochondrial treatment. ( B ) The level of IL-5 was markedly decreased after the mitochondrial treatment. ( C ) The level of IL-21 was significantly decreased after the mitochondrial treatment. ( D ) The level of IL-4 was significantly increased after the mitochondrial treatment. ( E – L ) There were no marked differences in the levels of IL-12 ( E ), IL-9 ( F ), IL-10 ( G ), IL-13 ( H ), IL-17F ( I ), IL-22 ( J ), IFN-γ ( K ), and TNF-α ( L ) after the treatment with platelet-derived mitochondria. Data were given as mean ± SD ( n = 3).

    Journal: International Journal of Molecular Sciences

    Article Title: Immune Modulation of Platelet-Derived Mitochondria on Memory CD4+ T Cells in Humans

    doi: 10.3390/ijms21176295

    Figure Lengend Snippet: Modulation of cytokine productions in CD4 + T cells by platelet-derived mitochondria. The purified CD4 + T cells (1 × 10 5 cells/well) from PBMC of healthy donors ( n = 3) were activated with T-cell activator anti-CD3/CD28 Dynabeads in the presence or absence of 100 μg/mL platelet-derived mitochondria ( n = 3). Untreated CD4 + T cells served as negative control. After the treatment for 2 days, supernatants were collected to measure the levels of cytokines (IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17F, IL-21, IL-22, IFN-γ, and TNF-α) by using a LEGENDplex™ Human Th Panel (Biolegend) with Gallios Flow Cytometer. ( A ) The level of IL-6 was markedly decreased after the mitochondrial treatment. ( B ) The level of IL-5 was markedly decreased after the mitochondrial treatment. ( C ) The level of IL-21 was significantly decreased after the mitochondrial treatment. ( D ) The level of IL-4 was significantly increased after the mitochondrial treatment. ( E – L ) There were no marked differences in the levels of IL-12 ( E ), IL-9 ( F ), IL-10 ( G ), IL-13 ( H ), IL-17F ( I ), IL-22 ( J ), IFN-γ ( K ), and TNF-α ( L ) after the treatment with platelet-derived mitochondria. Data were given as mean ± SD ( n = 3).

    Article Snippet: Evaluation of Cytokine Levels in Serum To detect the cytokine production by CD4+ T cells, 1 × 105 CD4+ T cells were stimulated with anti-CD3/anti-CD28 beads (Thermo Fisher Scientific, Waltham, MA, USA) in the presence or absence of mitochondria at 100 μg/mL in a 96-well plate with a total of 200 μL X-VIVO 15 serum-free culture medium (Lonza, Walkersville, MD, USA) per well.

    Techniques: Derivative Assay, Purification, Negative Control, Flow Cytometry

    Suppression of PBMC proliferation by platelet-derived mitochondria ( A ) Apoptotic effects of PBMC after the treatment with different dosages of platelet-derived mitochondria. ( B ) Suppression of PBMC proliferation by platelet-derived mitochondria. The carboxyfluorescein succinimidyl ester (CFSE)-labeled PBMC were stimulated to proliferate with T-cell activator anti-CD3/CD28 Dynabeads in the presence of different dosages of platelet-derived mitochondria. Untreated PBMC served as negative control. Histograms of flow cytometry were representative of three experiments with similar results. ( C ) Gating strategy for flow cytometry analysis with the lineage-specific surface markers for different cell populations in PBMC ( n = 3), including CD3/CD4/CD8 for subsets of T cells, CD19 for B cells, CD14 for monocytes, CD11c for dendritic cells (DCs), and CD56 for NK cells. ( D ) Flow cytometry revealed the distributions of MitoTracker Deep Red-labeled mitochondria ( n = 3) among different cell populations. ( E ) Different types of immune cells displayed different levels of median fluorescence intensity. The data were given as mean ± SD of three PBMC ( n = 3) treated with two preparations of platelet-derived mitochondria ( n = 2).

    Journal: International Journal of Molecular Sciences

    Article Title: Immune Modulation of Platelet-Derived Mitochondria on Memory CD4+ T Cells in Humans

    doi: 10.3390/ijms21176295

    Figure Lengend Snippet: Suppression of PBMC proliferation by platelet-derived mitochondria ( A ) Apoptotic effects of PBMC after the treatment with different dosages of platelet-derived mitochondria. ( B ) Suppression of PBMC proliferation by platelet-derived mitochondria. The carboxyfluorescein succinimidyl ester (CFSE)-labeled PBMC were stimulated to proliferate with T-cell activator anti-CD3/CD28 Dynabeads in the presence of different dosages of platelet-derived mitochondria. Untreated PBMC served as negative control. Histograms of flow cytometry were representative of three experiments with similar results. ( C ) Gating strategy for flow cytometry analysis with the lineage-specific surface markers for different cell populations in PBMC ( n = 3), including CD3/CD4/CD8 for subsets of T cells, CD19 for B cells, CD14 for monocytes, CD11c for dendritic cells (DCs), and CD56 for NK cells. ( D ) Flow cytometry revealed the distributions of MitoTracker Deep Red-labeled mitochondria ( n = 3) among different cell populations. ( E ) Different types of immune cells displayed different levels of median fluorescence intensity. The data were given as mean ± SD of three PBMC ( n = 3) treated with two preparations of platelet-derived mitochondria ( n = 2).

    Article Snippet: Evaluation of Cytokine Levels in Serum To detect the cytokine production by CD4+ T cells, 1 × 105 CD4+ T cells were stimulated with anti-CD3/anti-CD28 beads (Thermo Fisher Scientific, Waltham, MA, USA) in the presence or absence of mitochondria at 100 μg/mL in a 96-well plate with a total of 200 μL X-VIVO 15 serum-free culture medium (Lonza, Walkersville, MD, USA) per well.

    Techniques: Derivative Assay, Labeling, Negative Control, Flow Cytometry, Fluorescence

    RNA-seq for gene expression profiling of CD4 + T cells after the treatment with platelet-derived mitochondria. ( A ) The heatmap showed differentially expressed genes shown by the RNA-seq in three anti-CD3/CD28 bead-activated CD4 + T cells in the absence (control, left three columns) and presence of platelet-derived mitochondria (mitoTreat, right three columns). ( B ) The heatmap revealed 25 up-regulated genes in CD4 + T cells post the treatment with platelet-derived mitochondria. ( C ) Fifty-three down-regulated genes in CD4 + T cells post the treatment with platelet-derived mitochondria. ( D , E ) The purified CD4 + T cells (1 × 10 6 cells/mL in 6-well tissue culture plates) from PBMC of healthy donors ( n = 3) were activated with T-cell activator anti-CD3/CD28 Dynabeads in the presence or absence of 100 μg/mL platelet-derived mitochondria. Untreated CD4 + T cells served as negative control. Cells were collected for testing after the treatment at 37 °C and 5% CO 2 for 48 h. ( D ) Down-regulation of Carboxypeptidase M (CPM) mRNA level, as indicated by real time PCR. ( E ) Flow cytometry demonstrated the decrease in the percentage of CPM-positive cells. Data represent mean ± SD. n = 3.

    Journal: International Journal of Molecular Sciences

    Article Title: Immune Modulation of Platelet-Derived Mitochondria on Memory CD4+ T Cells in Humans

    doi: 10.3390/ijms21176295

    Figure Lengend Snippet: RNA-seq for gene expression profiling of CD4 + T cells after the treatment with platelet-derived mitochondria. ( A ) The heatmap showed differentially expressed genes shown by the RNA-seq in three anti-CD3/CD28 bead-activated CD4 + T cells in the absence (control, left three columns) and presence of platelet-derived mitochondria (mitoTreat, right three columns). ( B ) The heatmap revealed 25 up-regulated genes in CD4 + T cells post the treatment with platelet-derived mitochondria. ( C ) Fifty-three down-regulated genes in CD4 + T cells post the treatment with platelet-derived mitochondria. ( D , E ) The purified CD4 + T cells (1 × 10 6 cells/mL in 6-well tissue culture plates) from PBMC of healthy donors ( n = 3) were activated with T-cell activator anti-CD3/CD28 Dynabeads in the presence or absence of 100 μg/mL platelet-derived mitochondria. Untreated CD4 + T cells served as negative control. Cells were collected for testing after the treatment at 37 °C and 5% CO 2 for 48 h. ( D ) Down-regulation of Carboxypeptidase M (CPM) mRNA level, as indicated by real time PCR. ( E ) Flow cytometry demonstrated the decrease in the percentage of CPM-positive cells. Data represent mean ± SD. n = 3.

    Article Snippet: Evaluation of Cytokine Levels in Serum To detect the cytokine production by CD4+ T cells, 1 × 105 CD4+ T cells were stimulated with anti-CD3/anti-CD28 beads (Thermo Fisher Scientific, Waltham, MA, USA) in the presence or absence of mitochondria at 100 μg/mL in a 96-well plate with a total of 200 μL X-VIVO 15 serum-free culture medium (Lonza, Walkersville, MD, USA) per well.

    Techniques: RNA Sequencing Assay, Expressing, Derivative Assay, Purification, Negative Control, Real-time Polymerase Chain Reaction, Flow Cytometry

    ILDR2 inhibits activation of human and murine T cells. ( A ) Membrane-expressed ILDR2 inhibits human T cell activation. Stimulator cells (a murine thymoma cell line engineered to express membrane-bound anti-human CD3 single-chain Ab fragments) were transfected with expression constructs encoding the long or the short splice variant of human ILDR2. Cells transfected with the empty vector served as negative control. Proliferation of human CD3 + T cells was evaluated upon coculture with stimulator cells expressing the ILDR2 short and long variants, as well as ICOSL or PD-L1, as reference. Shown is a representative experiment of four experiments carried out with a total of three donors. Data are mean ± SEM of triplicate samples. ( B ) Soluble ILDR2-hFc inhibits activation of human T cells. Naive CD4 + T cells obtained from human PBMCs were activated by soluble anti-CD3 and anti-CD28 in the presence of irradiated autologous PBMCs. ILDR2-hFc or isotype control (human IgG1; Synagis) was added at the specified final concentrations. Cell proliferation was evaluated by [ 3 H]thymidine incorporation at 72 h. Shown are representative results obtained with three human donors. ( C . IL-2 secretion was measured by ELISA after 24 h. PD-L1–Fc (recombinant human B7-H1/Fc) was used as positive control. Shown are representative results for one of three experiments. ( D ) Bead-immobilized ILDR2-mFc reduces polyclonal murine T cell proliferation. Naive CD4 + . The beads were also coated with the indicated concentrations of ILDR2-mFc or isotype control (mIgG2a). Cells were activated with the coated beads at a 1:1 ratio, and cell proliferation was evaluated by [ 3 H]thymidine incorporation after 72 h. ( E ) Soluble ILDR2-mFc reduces Ag-specific murine T cell activation. Naive CD4 + T cells from DO11.1 mice were activated by 20 μg/ml OVA 323–339 peptide and coincubated with irradiated APCs at a 1:1 ratio in the presence of different concentrations of ILDR2-mFc or isotype control (mIgG2a). Proliferation was evaluated by [ 3 H] thymidine incorporation after 72 h. ** p

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

    Article Title: ILDR2 Is a Novel B7-like Protein That Negatively Regulates T Cell Responses

    doi: 10.4049/jimmunol.1700325

    Figure Lengend Snippet: ILDR2 inhibits activation of human and murine T cells. ( A ) Membrane-expressed ILDR2 inhibits human T cell activation. Stimulator cells (a murine thymoma cell line engineered to express membrane-bound anti-human CD3 single-chain Ab fragments) were transfected with expression constructs encoding the long or the short splice variant of human ILDR2. Cells transfected with the empty vector served as negative control. Proliferation of human CD3 + T cells was evaluated upon coculture with stimulator cells expressing the ILDR2 short and long variants, as well as ICOSL or PD-L1, as reference. Shown is a representative experiment of four experiments carried out with a total of three donors. Data are mean ± SEM of triplicate samples. ( B ) Soluble ILDR2-hFc inhibits activation of human T cells. Naive CD4 + T cells obtained from human PBMCs were activated by soluble anti-CD3 and anti-CD28 in the presence of irradiated autologous PBMCs. ILDR2-hFc or isotype control (human IgG1; Synagis) was added at the specified final concentrations. Cell proliferation was evaluated by [ 3 H]thymidine incorporation at 72 h. Shown are representative results obtained with three human donors. ( C . IL-2 secretion was measured by ELISA after 24 h. PD-L1–Fc (recombinant human B7-H1/Fc) was used as positive control. Shown are representative results for one of three experiments. ( D ) Bead-immobilized ILDR2-mFc reduces polyclonal murine T cell proliferation. Naive CD4 + . The beads were also coated with the indicated concentrations of ILDR2-mFc or isotype control (mIgG2a). Cells were activated with the coated beads at a 1:1 ratio, and cell proliferation was evaluated by [ 3 H]thymidine incorporation after 72 h. ( E ) Soluble ILDR2-mFc reduces Ag-specific murine T cell activation. Naive CD4 + T cells from DO11.1 mice were activated by 20 μg/ml OVA 323–339 peptide and coincubated with irradiated APCs at a 1:1 ratio in the presence of different concentrations of ILDR2-mFc or isotype control (mIgG2a). Proliferation was evaluated by [ 3 H] thymidine incorporation after 72 h. ** p

    Article Snippet: Following isolation, cells were left untreated (resting) or were activated using polyclonal activation of T cells, with plate-bound anti-CD3 Ab (clone-145-2C11; BD Pharmingen, coated on 96-well plates, at 2 μg/ml, for 4 h at 37°C) and 2 μg/ml soluble anti-CD28 Ab (clone 37.51; eBioscience).

    Techniques: Activation Assay, Transfection, Expressing, Construct, Variant Assay, Plasmid Preparation, Negative Control, Irradiation, Enzyme-linked Immunosorbent Assay, Recombinant, Positive Control, Mouse Assay

    Different TNFRSF agonists induced a similar transcriptomic signature. RNA sequencing was performed on Tregs stimulated with anti‐CD3/CD28 mAbs and agonists of TNFRSF for 18 h. Biological triplicates of one experiment is shown. (A) Principal component analysis. (B) FC/FC plots (expressed in log2) of differentially expressed genes (DEG) compared to controls (FDR

    Journal: European Journal of Immunology

    Article Title: Tumor necrosis factor receptor family costimulation increases regulatory T‐cell activation and function via NF‐κB

    doi: 10.1002/eji.201948393

    Figure Lengend Snippet: Different TNFRSF agonists induced a similar transcriptomic signature. RNA sequencing was performed on Tregs stimulated with anti‐CD3/CD28 mAbs and agonists of TNFRSF for 18 h. Biological triplicates of one experiment is shown. (A) Principal component analysis. (B) FC/FC plots (expressed in log2) of differentially expressed genes (DEG) compared to controls (FDR

    Article Snippet: Tregs were stimulated either with coated anti‐CD3 (coating at 1 or 2 μg/mL, 2C11; BioXcell) and anti‐CD28 (coating at 2 μg/mL) mAbs in 96‐well flat plates (15 x 104 cells/well) or with irradiated splenocytes from Cd3−/− mice (7.5 x 104 cells/well) used as APCs and soluble anti‐CD3 (0.05 μg/mL) mAb in 96‐well round plate (2.5 x 104 cells/well).

    Techniques: RNA Sequencing Assay

    Costimulation with agonists of TNFR2, 4‐1BB, GITR, and DR3 increased Treg proliferation and survival in vitro. (A) Expression of TNFR2, 4‐1BB, GITR, and OX40 was assessed by flow cytometry on freshly purified Tregs (d0) and after 24, 48, and 72 h of culture with agonists and beads coated with anti‐CD3/CD28 mAbs. Representative experiment of three independent experiments with biological duplicates. (B–D) Purified Tregs were stimulated by APCs, anti‐CD3 mAb, and agonists of TNFRSF receptors for 3 days to quantify proliferation and survival. (B) Representative cell proliferation profile of at least nine independent experiments with biological duplicates. Increased proliferation (C) and fold change (FC) of living cells (D) relative to the control culture (without agonist, dotted lines), with each symbol representing the mean of biological duplicates of independent experiments (at least nine). For each agonist, the horizontal bar represents the mean. In (A) and (B), around 20 000 cells were analyzed per gated Tregs.

    Journal: European Journal of Immunology

    Article Title: Tumor necrosis factor receptor family costimulation increases regulatory T‐cell activation and function via NF‐κB

    doi: 10.1002/eji.201948393

    Figure Lengend Snippet: Costimulation with agonists of TNFR2, 4‐1BB, GITR, and DR3 increased Treg proliferation and survival in vitro. (A) Expression of TNFR2, 4‐1BB, GITR, and OX40 was assessed by flow cytometry on freshly purified Tregs (d0) and after 24, 48, and 72 h of culture with agonists and beads coated with anti‐CD3/CD28 mAbs. Representative experiment of three independent experiments with biological duplicates. (B–D) Purified Tregs were stimulated by APCs, anti‐CD3 mAb, and agonists of TNFRSF receptors for 3 days to quantify proliferation and survival. (B) Representative cell proliferation profile of at least nine independent experiments with biological duplicates. Increased proliferation (C) and fold change (FC) of living cells (D) relative to the control culture (without agonist, dotted lines), with each symbol representing the mean of biological duplicates of independent experiments (at least nine). For each agonist, the horizontal bar represents the mean. In (A) and (B), around 20 000 cells were analyzed per gated Tregs.

    Article Snippet: Tregs were stimulated either with coated anti‐CD3 (coating at 1 or 2 μg/mL, 2C11; BioXcell) and anti‐CD28 (coating at 2 μg/mL) mAbs in 96‐well flat plates (15 x 104 cells/well) or with irradiated splenocytes from Cd3−/− mice (7.5 x 104 cells/well) used as APCs and soluble anti‐CD3 (0.05 μg/mL) mAb in 96‐well round plate (2.5 x 104 cells/well).

    Techniques: In Vitro, Expressing, Flow Cytometry, Purification

    TNFRSF receptor costimulation activated the canonical NF‐κB pathway in Tregs. (A, B) Tregs, pre‐activated with anti‐CD3/CD28 mAbs for 24 h, were restimulated with TNFRSF agonists alone. The EMSA (A) was performed at 0, 0.5, 1, and 4 h of restimulation. The supershift (B) was performed at 0.5 h of restimulation to investigate the subset composition of the NF‐κB containing complex. Arrowheads indicate the position of the NF‐κB containing complex and arrows indicate the positions of the supershifting complexes bound to mAb specific to RelA and c‐Rel. A representative of three experiments is shown. Proliferation (C, D) and survival (E) of Rela KO and control Tregs, stimulated and analyzed as in Fig. 1 . One representative proliferation profile (C) and increased proliferation and FC living cell numbers relative to the control culture (D, E) from the pool of two independent experiments with two mice per experiment. Each circle represents a mouse (two‐way ANOVA, * p

    Journal: European Journal of Immunology

    Article Title: Tumor necrosis factor receptor family costimulation increases regulatory T‐cell activation and function via NF‐κB

    doi: 10.1002/eji.201948393

    Figure Lengend Snippet: TNFRSF receptor costimulation activated the canonical NF‐κB pathway in Tregs. (A, B) Tregs, pre‐activated with anti‐CD3/CD28 mAbs for 24 h, were restimulated with TNFRSF agonists alone. The EMSA (A) was performed at 0, 0.5, 1, and 4 h of restimulation. The supershift (B) was performed at 0.5 h of restimulation to investigate the subset composition of the NF‐κB containing complex. Arrowheads indicate the position of the NF‐κB containing complex and arrows indicate the positions of the supershifting complexes bound to mAb specific to RelA and c‐Rel. A representative of three experiments is shown. Proliferation (C, D) and survival (E) of Rela KO and control Tregs, stimulated and analyzed as in Fig. 1 . One representative proliferation profile (C) and increased proliferation and FC living cell numbers relative to the control culture (D, E) from the pool of two independent experiments with two mice per experiment. Each circle represents a mouse (two‐way ANOVA, * p

    Article Snippet: Tregs were stimulated either with coated anti‐CD3 (coating at 1 or 2 μg/mL, 2C11; BioXcell) and anti‐CD28 (coating at 2 μg/mL) mAbs in 96‐well flat plates (15 x 104 cells/well) or with irradiated splenocytes from Cd3−/− mice (7.5 x 104 cells/well) used as APCs and soluble anti‐CD3 (0.05 μg/mL) mAb in 96‐well round plate (2.5 x 104 cells/well).

    Techniques: Mouse Assay

    TNFRSF costimulation increased Treg in vivo expansion and modified Treg tissue recirculation. Tregs, preactivated for 3 days with anti‐CD3/CD28 mAbs alone (control Tregs) or combined with TNFRSF costimulation (co‐stimulated Tregs), were cotransferred in equal numbers to assess their in vivo homeostasis 7 days later. (A) Experimental design (upper panel) and representative plot showing cotransferred Tregs identified with congenic markers (lower panel). (B) Proportions of coinjected Tregs in spleen, peripheral lymph nodes (pLN), liver, and lung. Each dot is a mouse and lines connect cells from the same mouse. Unpaired Mann–Whitney test was used. (C, D) Ki67, CD62L, CXCR3, and CCR6 expression among injected cells. Upper panels show representative histograms for control and TNFR2 costimulated Tregs in the spleen. Lower panels show the mean (±SD) of FC MFI expression of costimulated compared to control Tregs in spleen, pLN, and mesenteric (MLN) LN. Data were obtained from two independent experiments with six mice per group in total. From 100 to 500 cells were analyzed per gated Tregs.

    Journal: European Journal of Immunology

    Article Title: Tumor necrosis factor receptor family costimulation increases regulatory T‐cell activation and function via NF‐κB

    doi: 10.1002/eji.201948393

    Figure Lengend Snippet: TNFRSF costimulation increased Treg in vivo expansion and modified Treg tissue recirculation. Tregs, preactivated for 3 days with anti‐CD3/CD28 mAbs alone (control Tregs) or combined with TNFRSF costimulation (co‐stimulated Tregs), were cotransferred in equal numbers to assess their in vivo homeostasis 7 days later. (A) Experimental design (upper panel) and representative plot showing cotransferred Tregs identified with congenic markers (lower panel). (B) Proportions of coinjected Tregs in spleen, peripheral lymph nodes (pLN), liver, and lung. Each dot is a mouse and lines connect cells from the same mouse. Unpaired Mann–Whitney test was used. (C, D) Ki67, CD62L, CXCR3, and CCR6 expression among injected cells. Upper panels show representative histograms for control and TNFR2 costimulated Tregs in the spleen. Lower panels show the mean (±SD) of FC MFI expression of costimulated compared to control Tregs in spleen, pLN, and mesenteric (MLN) LN. Data were obtained from two independent experiments with six mice per group in total. From 100 to 500 cells were analyzed per gated Tregs.

    Article Snippet: Tregs were stimulated either with coated anti‐CD3 (coating at 1 or 2 μg/mL, 2C11; BioXcell) and anti‐CD28 (coating at 2 μg/mL) mAbs in 96‐well flat plates (15 x 104 cells/well) or with irradiated splenocytes from Cd3−/− mice (7.5 x 104 cells/well) used as APCs and soluble anti‐CD3 (0.05 μg/mL) mAb in 96‐well round plate (2.5 x 104 cells/well).

    Techniques: In Vivo, Modification, MANN-WHITNEY, Expressing, Injection, Mouse Assay

    TNFRSF agonists favored polarization toward type 2 and type 17 Tregs. (A) Cytokine production (measured in the supernatant) by Tregs activated by anti‐CD3/CD28 mAb and TNFRSF agonists for 3 days. Each symbol represents mean values of biological triplicates from an independent experiment. (B) DEG (FDR

    Journal: European Journal of Immunology

    Article Title: Tumor necrosis factor receptor family costimulation increases regulatory T‐cell activation and function via NF‐κB

    doi: 10.1002/eji.201948393

    Figure Lengend Snippet: TNFRSF agonists favored polarization toward type 2 and type 17 Tregs. (A) Cytokine production (measured in the supernatant) by Tregs activated by anti‐CD3/CD28 mAb and TNFRSF agonists for 3 days. Each symbol represents mean values of biological triplicates from an independent experiment. (B) DEG (FDR

    Article Snippet: Tregs were stimulated either with coated anti‐CD3 (coating at 1 or 2 μg/mL, 2C11; BioXcell) and anti‐CD28 (coating at 2 μg/mL) mAbs in 96‐well flat plates (15 x 104 cells/well) or with irradiated splenocytes from Cd3−/− mice (7.5 x 104 cells/well) used as APCs and soluble anti‐CD3 (0.05 μg/mL) mAb in 96‐well round plate (2.5 x 104 cells/well).

    Techniques:

    Costimulation of Tregs by TNFRSF receptors modified their expression of suppressive molecules and increased their capacity to suppress colitis. (A) Tregs, preactivated with anti‐CD3/CD28 mAb and TNFRSF agonists, were tested for their in vitro suppressive function. Mean (±SD) values from one representative of three independent experiments is shown. (B) DEG (FDR

    Journal: European Journal of Immunology

    Article Title: Tumor necrosis factor receptor family costimulation increases regulatory T‐cell activation and function via NF‐κB

    doi: 10.1002/eji.201948393

    Figure Lengend Snippet: Costimulation of Tregs by TNFRSF receptors modified their expression of suppressive molecules and increased their capacity to suppress colitis. (A) Tregs, preactivated with anti‐CD3/CD28 mAb and TNFRSF agonists, were tested for their in vitro suppressive function. Mean (±SD) values from one representative of three independent experiments is shown. (B) DEG (FDR

    Article Snippet: Tregs were stimulated either with coated anti‐CD3 (coating at 1 or 2 μg/mL, 2C11; BioXcell) and anti‐CD28 (coating at 2 μg/mL) mAbs in 96‐well flat plates (15 x 104 cells/well) or with irradiated splenocytes from Cd3−/− mice (7.5 x 104 cells/well) used as APCs and soluble anti‐CD3 (0.05 μg/mL) mAb in 96‐well round plate (2.5 x 104 cells/well).

    Techniques: Modification, Expressing, In Vitro

    Construction of CAR-T cells secreting α-PD-1 scFv. (A) The surface expressions of CAR were determined by protein-L staining using FACS test. (B,C) The secreted α-PD-1 scFv with His tag by CAR-T cells cold bind with CAR-T cells. CAR-T cells were activated by anti-CD3/CD28 beads for 24 h. Then the supernatants were subjected to western blot assay and scFv secretion from CAR-meso-α-PD-1 cells were confirmed (B) . CAR-meso and CAR-meso-α-PD-1 cells were further stained with AF488-labeled antibody against His tag, and checked on image flow cytometer (C) . CAR-T cells derived from 5 different donors were tested and the representative results were shown.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Augmenting the Effectiveness of CAR-T Cells by Enhanced Self-Delivery of PD-1-Neutralizing scFv

    doi: 10.3389/fcell.2020.00803

    Figure Lengend Snippet: Construction of CAR-T cells secreting α-PD-1 scFv. (A) The surface expressions of CAR were determined by protein-L staining using FACS test. (B,C) The secreted α-PD-1 scFv with His tag by CAR-T cells cold bind with CAR-T cells. CAR-T cells were activated by anti-CD3/CD28 beads for 24 h. Then the supernatants were subjected to western blot assay and scFv secretion from CAR-meso-α-PD-1 cells were confirmed (B) . CAR-meso and CAR-meso-α-PD-1 cells were further stained with AF488-labeled antibody against His tag, and checked on image flow cytometer (C) . CAR-T cells derived from 5 different donors were tested and the representative results were shown.

    Article Snippet: CD3+ T cells were magnetically purified from the peripheral blood mononuclear cells (PBMCs) of health donors and then activated with anti-CD3/CD28 Dynabeads (Thermo Fisher, Cat. No. 111.32D) for 48 h. Spinfection was used to enhance the transduction efficacies of T cells by lentivirus.

    Techniques: Staining, FACS, Western Blot, Labeling, Flow Cytometry, Derivative Assay

    Effects of secreted α-PD-1 scFv on T cell activation. CD3+ T cells from healthy donors were stained with CFSE and then activated by anti-CD3/CD28 beads for 2 days. (A) PD-1 expressions were determined in resting and activated T cells by FACS assay. (B) 293T cells transfected with mock or α-PD-1 scFv-expressing vectors were plated at the bottom. 48 h later, activated T cells were added into the upper transwell chambers with fixed PD-L1-overexpressing A549 cells at the ratio of 1:1 for 24 h. (C) Then the dilutions of CFSE in CD3+ T cells were determined using FACS assay. (D–F) The expressions of Ki67 (D) , CD107a (E) and IFN-γ (F) were further detected in CD3+ T cells after incubation. T cells from 5 donors were tested and the representative results were depicted. * indicates P

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Augmenting the Effectiveness of CAR-T Cells by Enhanced Self-Delivery of PD-1-Neutralizing scFv

    doi: 10.3389/fcell.2020.00803

    Figure Lengend Snippet: Effects of secreted α-PD-1 scFv on T cell activation. CD3+ T cells from healthy donors were stained with CFSE and then activated by anti-CD3/CD28 beads for 2 days. (A) PD-1 expressions were determined in resting and activated T cells by FACS assay. (B) 293T cells transfected with mock or α-PD-1 scFv-expressing vectors were plated at the bottom. 48 h later, activated T cells were added into the upper transwell chambers with fixed PD-L1-overexpressing A549 cells at the ratio of 1:1 for 24 h. (C) Then the dilutions of CFSE in CD3+ T cells were determined using FACS assay. (D–F) The expressions of Ki67 (D) , CD107a (E) and IFN-γ (F) were further detected in CD3+ T cells after incubation. T cells from 5 donors were tested and the representative results were depicted. * indicates P

    Article Snippet: CD3+ T cells were magnetically purified from the peripheral blood mononuclear cells (PBMCs) of health donors and then activated with anti-CD3/CD28 Dynabeads (Thermo Fisher, Cat. No. 111.32D) for 48 h. Spinfection was used to enhance the transduction efficacies of T cells by lentivirus.

    Techniques: Activation Assay, Staining, FACS, Transfection, Expressing, Incubation

    Blockade of PD-1 suppression by self-delivered α-PD-1 scFv. (A,B) Activated CAR-T cells were stained with anti-PD-1 antibody and checked on image flow cytometer (A) and the expressing levels of PD-1 were determined (B) . (C–E) To check the effects of scFv on the cytotoxic activities, CAR-T cells were directly incubated with H322 cells without pre-activation by anti-CD3/CD28 Dynabeads. CAR-T cells were co-cultured with luciferase-expressing H322 cells at indicated E:T ratios for 18 h. Then the relative viabilities of tumor cells were determined and compared between treatments with CAR-meso or CAR-meso-α-PD-1 cells (C) . Additionally, CAR-T cells were co-incubated with H322 cells at the ratio of 1:1 for 24 h. Then the supernatants were checked for the secretions of IFN-γ (D) and IL2 (E) . The cells were collected and Ki67 expressions were determined in CD3+ T cells using FACS test (F) . CAR-T cells derived from 5 different donors were tested and the representative data were demonstrated. * indicates P

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Augmenting the Effectiveness of CAR-T Cells by Enhanced Self-Delivery of PD-1-Neutralizing scFv

    doi: 10.3389/fcell.2020.00803

    Figure Lengend Snippet: Blockade of PD-1 suppression by self-delivered α-PD-1 scFv. (A,B) Activated CAR-T cells were stained with anti-PD-1 antibody and checked on image flow cytometer (A) and the expressing levels of PD-1 were determined (B) . (C–E) To check the effects of scFv on the cytotoxic activities, CAR-T cells were directly incubated with H322 cells without pre-activation by anti-CD3/CD28 Dynabeads. CAR-T cells were co-cultured with luciferase-expressing H322 cells at indicated E:T ratios for 18 h. Then the relative viabilities of tumor cells were determined and compared between treatments with CAR-meso or CAR-meso-α-PD-1 cells (C) . Additionally, CAR-T cells were co-incubated with H322 cells at the ratio of 1:1 for 24 h. Then the supernatants were checked for the secretions of IFN-γ (D) and IL2 (E) . The cells were collected and Ki67 expressions were determined in CD3+ T cells using FACS test (F) . CAR-T cells derived from 5 different donors were tested and the representative data were demonstrated. * indicates P

    Article Snippet: CD3+ T cells were magnetically purified from the peripheral blood mononuclear cells (PBMCs) of health donors and then activated with anti-CD3/CD28 Dynabeads (Thermo Fisher, Cat. No. 111.32D) for 48 h. Spinfection was used to enhance the transduction efficacies of T cells by lentivirus.

    Techniques: Staining, Flow Cytometry, Expressing, Incubation, Activation Assay, Cell Culture, Luciferase, FACS, Derivative Assay

    Analysis of STAT5 binding to the Il4ra and Il4 loci. (a-f) ChIP-Seq analysis was performed to analyze STAT5 binding at the Il4ra ( a-c) and Il4-Il13-Il5 (d-f) loci in CD4 + T cells cultured under T H 2 conditions (anti-CD3 + anti-CD28 + 10 ng/ml IL-4 + 10 ug/ml anti—IFN-γ) for the indicated amounts of time. Cells subjected to two rounds of T H 2 differentiation refers to cells cultured under T H 2 conditions for 3 days, expanded with IL-4 and anti—IFN-γ for 2 days, washed, re-cultured under T H 2 conditions for another 3 days and then analyzed without further cytokine stimulation. These cells were not exposed to exogenous IL-2. Unique sequence reads were first adjusted to center them on the corresponding chromatin fragments. The adjusted reads were then summed in 400 bp windows and displayed as custom tracks on the UCSC genome browser. ChIP was performed with IgG as a control for STAT5A- and STAT5B-specific antibodies. Schematics of the Il4ra ( a-c ) and Il4-Il13-Il5 ( d-f ) loci with standard conservation tracks from the UCSC genome browser indicating the areas of highest conservation among 17 vertebrate species are shown in blue at the bottom of each panel. The experiment was preformed three independent times, with similar results. ( g ) T H 2 cells polarized for 2 rounds were incubated in the presence of 10 μg/ml each of anti-IL-2 (S4B6), anti-IL-2Rα (PC61) and anti-IL-2Rβ, all from BD Bioscience, for an extra 18 h and ChIP was preformed to assess STAT5B binding to indicated gene regions. (h) IL-2-induced binding of STAT5A and STAT5B to indicated gene regions, as measured by ChIP. This is representative of two similar experiments. See Supplementary Table 10 for sequences or primers used in ChIP experiments.

    Journal: Nature immunology

    Article Title: Priming for T helper type 2 differentiation by interleukin 2-mediated induction of IL-4 receptor ? chain expression

    doi: 10.1038/ni.1656

    Figure Lengend Snippet: Analysis of STAT5 binding to the Il4ra and Il4 loci. (a-f) ChIP-Seq analysis was performed to analyze STAT5 binding at the Il4ra ( a-c) and Il4-Il13-Il5 (d-f) loci in CD4 + T cells cultured under T H 2 conditions (anti-CD3 + anti-CD28 + 10 ng/ml IL-4 + 10 ug/ml anti—IFN-γ) for the indicated amounts of time. Cells subjected to two rounds of T H 2 differentiation refers to cells cultured under T H 2 conditions for 3 days, expanded with IL-4 and anti—IFN-γ for 2 days, washed, re-cultured under T H 2 conditions for another 3 days and then analyzed without further cytokine stimulation. These cells were not exposed to exogenous IL-2. Unique sequence reads were first adjusted to center them on the corresponding chromatin fragments. The adjusted reads were then summed in 400 bp windows and displayed as custom tracks on the UCSC genome browser. ChIP was performed with IgG as a control for STAT5A- and STAT5B-specific antibodies. Schematics of the Il4ra ( a-c ) and Il4-Il13-Il5 ( d-f ) loci with standard conservation tracks from the UCSC genome browser indicating the areas of highest conservation among 17 vertebrate species are shown in blue at the bottom of each panel. The experiment was preformed three independent times, with similar results. ( g ) T H 2 cells polarized for 2 rounds were incubated in the presence of 10 μg/ml each of anti-IL-2 (S4B6), anti-IL-2Rα (PC61) and anti-IL-2Rβ, all from BD Bioscience, for an extra 18 h and ChIP was preformed to assess STAT5B binding to indicated gene regions. (h) IL-2-induced binding of STAT5A and STAT5B to indicated gene regions, as measured by ChIP. This is representative of two similar experiments. See Supplementary Table 10 for sequences or primers used in ChIP experiments.

    Article Snippet: Animal protocols were approved by the NHLBI Animal Care and Use Committee and followed the NIH Guidelines “Using Animals in Intramural Research.” Splenic total T cells or CD4+ subpopulations were purified from 5-12 week old mice by negative or positive selection using magnetic beads (Miltenyi), cultured in RPMI 1640 medium containing 10 mM Hepes (pH 7.0), 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics (complete medium), and activated for 1.5 h at 37°C in dishes pre-coated with anti-CD3 (2 μg/ml in PBS) in complete medium containing 1 μg/ml anti-CD28 (PharMingen).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Sequencing, Incubation

    Extent of IL-4Rα expression influences T H 2 cell differentiation. CD4 + T cells from Il4ra +/+ , Il4ra +/- , and Il4ra -/- Balb/c mice were activated with 2 μg/ml anti-CD3 and 1 μg/ml anti-CD28 for 92 h. ( a,b ) IL-4Rα surface expression was analyzed by flow cytometry ( a ) or cells were cultured under T H 2 conditions for 92 h and intracellular IL-4 and IFN-g were measured ( b ). In ( a ), the numbers indicate MFI and in ( b ) they indicate the percent of cells producing IL-4. The experiment was performed three times with 2 to 4 mice per group in each experiment.

    Journal: Nature immunology

    Article Title: Priming for T helper type 2 differentiation by interleukin 2-mediated induction of IL-4 receptor ? chain expression

    doi: 10.1038/ni.1656

    Figure Lengend Snippet: Extent of IL-4Rα expression influences T H 2 cell differentiation. CD4 + T cells from Il4ra +/+ , Il4ra +/- , and Il4ra -/- Balb/c mice were activated with 2 μg/ml anti-CD3 and 1 μg/ml anti-CD28 for 92 h. ( a,b ) IL-4Rα surface expression was analyzed by flow cytometry ( a ) or cells were cultured under T H 2 conditions for 92 h and intracellular IL-4 and IFN-g were measured ( b ). In ( a ), the numbers indicate MFI and in ( b ) they indicate the percent of cells producing IL-4. The experiment was performed three times with 2 to 4 mice per group in each experiment.

    Article Snippet: Animal protocols were approved by the NHLBI Animal Care and Use Committee and followed the NIH Guidelines “Using Animals in Intramural Research.” Splenic total T cells or CD4+ subpopulations were purified from 5-12 week old mice by negative or positive selection using magnetic beads (Miltenyi), cultured in RPMI 1640 medium containing 10 mM Hepes (pH 7.0), 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics (complete medium), and activated for 1.5 h at 37°C in dishes pre-coated with anti-CD3 (2 μg/ml in PBS) in complete medium containing 1 μg/ml anti-CD28 (PharMingen).

    Techniques: Expressing, Cell Differentiation, Mouse Assay, Flow Cytometry, Cytometry, Cell Culture

    Other STAT5-activatingn cytokines can increase IL-4Rα expression. Freshly isolated T cells (top) and T cells pre-activated with anti-CD3 and anti-CD28 (bottom) were treated with the indicated cytokines and stained for IL-4Rα surface expression (thin line, no cytokine; thick line, cytokine). The experiment shown is representative of four independent experiments.

    Journal: Nature immunology

    Article Title: Priming for T helper type 2 differentiation by interleukin 2-mediated induction of IL-4 receptor ? chain expression

    doi: 10.1038/ni.1656

    Figure Lengend Snippet: Other STAT5-activatingn cytokines can increase IL-4Rα expression. Freshly isolated T cells (top) and T cells pre-activated with anti-CD3 and anti-CD28 (bottom) were treated with the indicated cytokines and stained for IL-4Rα surface expression (thin line, no cytokine; thick line, cytokine). The experiment shown is representative of four independent experiments.

    Article Snippet: Animal protocols were approved by the NHLBI Animal Care and Use Committee and followed the NIH Guidelines “Using Animals in Intramural Research.” Splenic total T cells or CD4+ subpopulations were purified from 5-12 week old mice by negative or positive selection using magnetic beads (Miltenyi), cultured in RPMI 1640 medium containing 10 mM Hepes (pH 7.0), 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics (complete medium), and activated for 1.5 h at 37°C in dishes pre-coated with anti-CD3 (2 μg/ml in PBS) in complete medium containing 1 μg/ml anti-CD28 (PharMingen).

    Techniques: Expressing, Isolation, Staining

    IL-2-induced IL-4Rα expression is independent of IL-4. ( a,b ) Splenic CD4 + T cells from Il4 -/- mice were pre-activated with anti-CD3 and anti-CD28 for 48 h, washed, and then 0 or 100 U/ml of IL-2 was added. IL-4Rα expression was measured by flow cytometry ( a ) or immunoblotting ( b ). Shown are results representative of five (a) or three (b) independent experiments.

    Journal: Nature immunology

    Article Title: Priming for T helper type 2 differentiation by interleukin 2-mediated induction of IL-4 receptor ? chain expression

    doi: 10.1038/ni.1656

    Figure Lengend Snippet: IL-2-induced IL-4Rα expression is independent of IL-4. ( a,b ) Splenic CD4 + T cells from Il4 -/- mice were pre-activated with anti-CD3 and anti-CD28 for 48 h, washed, and then 0 or 100 U/ml of IL-2 was added. IL-4Rα expression was measured by flow cytometry ( a ) or immunoblotting ( b ). Shown are results representative of five (a) or three (b) independent experiments.

    Article Snippet: Animal protocols were approved by the NHLBI Animal Care and Use Committee and followed the NIH Guidelines “Using Animals in Intramural Research.” Splenic total T cells or CD4+ subpopulations were purified from 5-12 week old mice by negative or positive selection using magnetic beads (Miltenyi), cultured in RPMI 1640 medium containing 10 mM Hepes (pH 7.0), 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics (complete medium), and activated for 1.5 h at 37°C in dishes pre-coated with anti-CD3 (2 μg/ml in PBS) in complete medium containing 1 μg/ml anti-CD28 (PharMingen).

    Techniques: Expressing, Mouse Assay, Flow Cytometry, Cytometry

    STAT5-dependent regulation of IL-4Rα expression. ( a ) IL-4Rα expression on splenic CD4 + T cells from wild-type and Stat5b transgenic mice 40 , as evaluated by flow cytometry. The experiment shown is representative of two independent experiments with 2 to 3 mice in each group. ( b ) Expression of indicated transcripts, after Cre recombinase-mediated deletion of the LoxP-flanked Stat5a / Stat5b locus 29 in splenic T cells that then were cultured with IL-2. See Supplementary Table 1 , online for the entire list of genes. Three independent experiments were performed. ( c ) Schematic of five TTCN 3 GAA potential GAS motifs in the mouse Il4ra gene 5′ regulatory region and first intron. GAS1 is approximately 3.5 kb (mouse) or 1.5 kb (human) 5′ of the Il4ra transcription initiation site (TIS), whereas GAS2, GAS3, GAS4, and GAS5 are in the first intron. ( d ) Indicated PCR-generated constructs (left; luc , luciferase) were transfected into YT cells not treated or treated with 100 U/ml of IL-2 and cell lysates were analyzed for luciferase activity (right). Three independent experiments were performed. ( e ) EMSA 41 using an Il4ra probe spanning GAS3 and nuclear extracts 41 from human peripheral T cells. Cells were untreated or treated with IL-2 or IL-6. For supershifting assays, each antiserum was pre-incubated with nuclear extracts before adding labeled probe. In lane 6, a probe mutated at GAS3 was the control. The experiment shown is representative of three independent experiments. ( f ) ChIP assays 41 of STAT5A and STAT5B binding using CD4 + splenic T cells from Balb/c mice pre-activated with anti-CD3 and anti-CD28 for 3 days, rested overnight, not treated or treated with 100 U/ml IL-2 for 4-5 h at 37 °C, followed by cross-linking with formaldehyde. Nuclear lysates were immunoprecipitated at 4°C overnight with anti-STAT5A, anti-STAT5B (R D Systems) or an isotype control antibody to allow normalization of the fold induction by IL-2. After deproteination and cross-link reversal, selected DNA sequences were assessed by real-time PCR. Primers spanning the Socs3 STAT binding site were used as a positive control and Gapdh as a negative control. See Supplementary Table 10 for sequences or primers used in ChIP experiments. The experiment shown is representative of three independent experiments.

    Journal: Nature immunology

    Article Title: Priming for T helper type 2 differentiation by interleukin 2-mediated induction of IL-4 receptor ? chain expression

    doi: 10.1038/ni.1656

    Figure Lengend Snippet: STAT5-dependent regulation of IL-4Rα expression. ( a ) IL-4Rα expression on splenic CD4 + T cells from wild-type and Stat5b transgenic mice 40 , as evaluated by flow cytometry. The experiment shown is representative of two independent experiments with 2 to 3 mice in each group. ( b ) Expression of indicated transcripts, after Cre recombinase-mediated deletion of the LoxP-flanked Stat5a / Stat5b locus 29 in splenic T cells that then were cultured with IL-2. See Supplementary Table 1 , online for the entire list of genes. Three independent experiments were performed. ( c ) Schematic of five TTCN 3 GAA potential GAS motifs in the mouse Il4ra gene 5′ regulatory region and first intron. GAS1 is approximately 3.5 kb (mouse) or 1.5 kb (human) 5′ of the Il4ra transcription initiation site (TIS), whereas GAS2, GAS3, GAS4, and GAS5 are in the first intron. ( d ) Indicated PCR-generated constructs (left; luc , luciferase) were transfected into YT cells not treated or treated with 100 U/ml of IL-2 and cell lysates were analyzed for luciferase activity (right). Three independent experiments were performed. ( e ) EMSA 41 using an Il4ra probe spanning GAS3 and nuclear extracts 41 from human peripheral T cells. Cells were untreated or treated with IL-2 or IL-6. For supershifting assays, each antiserum was pre-incubated with nuclear extracts before adding labeled probe. In lane 6, a probe mutated at GAS3 was the control. The experiment shown is representative of three independent experiments. ( f ) ChIP assays 41 of STAT5A and STAT5B binding using CD4 + splenic T cells from Balb/c mice pre-activated with anti-CD3 and anti-CD28 for 3 days, rested overnight, not treated or treated with 100 U/ml IL-2 for 4-5 h at 37 °C, followed by cross-linking with formaldehyde. Nuclear lysates were immunoprecipitated at 4°C overnight with anti-STAT5A, anti-STAT5B (R D Systems) or an isotype control antibody to allow normalization of the fold induction by IL-2. After deproteination and cross-link reversal, selected DNA sequences were assessed by real-time PCR. Primers spanning the Socs3 STAT binding site were used as a positive control and Gapdh as a negative control. See Supplementary Table 10 for sequences or primers used in ChIP experiments. The experiment shown is representative of three independent experiments.

    Article Snippet: Animal protocols were approved by the NHLBI Animal Care and Use Committee and followed the NIH Guidelines “Using Animals in Intramural Research.” Splenic total T cells or CD4+ subpopulations were purified from 5-12 week old mice by negative or positive selection using magnetic beads (Miltenyi), cultured in RPMI 1640 medium containing 10 mM Hepes (pH 7.0), 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics (complete medium), and activated for 1.5 h at 37°C in dishes pre-coated with anti-CD3 (2 μg/ml in PBS) in complete medium containing 1 μg/ml anti-CD28 (PharMingen).

    Techniques: Expressing, Transgenic Assay, Mouse Assay, Flow Cytometry, Cytometry, Cell Culture, Polymerase Chain Reaction, Generated, Construct, Luciferase, Transfection, Activity Assay, Incubation, Labeling, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Negative Control

    IL-2 is important for TCR-induced IL-4Rα expression. ( a ) Basal IL-4Rα expression in splenic CD4 + T cells freshly isolated from Il4 +/+ and Il4 -/- mice (top) and after activation with anti-CD3 and anti-CD28 for approximately 20 h (bottom). ( b ) As in ( a ) except Il2 -/- instead of Il4 -/- mice were used and cells were stimulated with anti-CD3 and anti-CD28 alone or with 100 U/ml of IL-2. Experiments were repeated three times with six mice each, with similar results in each case. ( c ) Left, time course of IL-2 protein production by splenic CD4 + T cells from Balb/c mice that were treated with anti-CD3 and anti-CD28. IL-2 was measured by double antibody ELISA. Right, purified splenic CD4 + T cells from C57BL/6 mice were not treated or treated with anti-CD3 and anti-CD28 for 4 h, after which RNA was extracted, and Il4ra and Il2ra mRNA expression was measured by real-time PCR. The experiment shown is representative of three independent experiments.

    Journal: Nature immunology

    Article Title: Priming for T helper type 2 differentiation by interleukin 2-mediated induction of IL-4 receptor ? chain expression

    doi: 10.1038/ni.1656

    Figure Lengend Snippet: IL-2 is important for TCR-induced IL-4Rα expression. ( a ) Basal IL-4Rα expression in splenic CD4 + T cells freshly isolated from Il4 +/+ and Il4 -/- mice (top) and after activation with anti-CD3 and anti-CD28 for approximately 20 h (bottom). ( b ) As in ( a ) except Il2 -/- instead of Il4 -/- mice were used and cells were stimulated with anti-CD3 and anti-CD28 alone or with 100 U/ml of IL-2. Experiments were repeated three times with six mice each, with similar results in each case. ( c ) Left, time course of IL-2 protein production by splenic CD4 + T cells from Balb/c mice that were treated with anti-CD3 and anti-CD28. IL-2 was measured by double antibody ELISA. Right, purified splenic CD4 + T cells from C57BL/6 mice were not treated or treated with anti-CD3 and anti-CD28 for 4 h, after which RNA was extracted, and Il4ra and Il2ra mRNA expression was measured by real-time PCR. The experiment shown is representative of three independent experiments.

    Article Snippet: Animal protocols were approved by the NHLBI Animal Care and Use Committee and followed the NIH Guidelines “Using Animals in Intramural Research.” Splenic total T cells or CD4+ subpopulations were purified from 5-12 week old mice by negative or positive selection using magnetic beads (Miltenyi), cultured in RPMI 1640 medium containing 10 mM Hepes (pH 7.0), 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics (complete medium), and activated for 1.5 h at 37°C in dishes pre-coated with anti-CD3 (2 μg/ml in PBS) in complete medium containing 1 μg/ml anti-CD28 (PharMingen).

    Techniques: Expressing, Isolation, Mouse Assay, Activation Assay, Enzyme-linked Immunosorbent Assay, Purification, Real-time Polymerase Chain Reaction

    IL-2 potently induces IL-4Rα expression. ( a—c ) Mouse splenic T lymphocytes were pre-activated by anti-CD3 and anti-CD28 for 48 h, rested overnight, and then 0, 10, or 100 U/ml of IL-2 was added for 4 h. ( a ) Real time PCR was used to measure expression of the indicated mRNA transcripts. ( b,c ) Flow cytometry ( b ) and immunoblotting ( c ) were used to measure cell surface ( b ) and total ( c ) IL-4Rα expression. ( d ) Increased IL4R mRNA expression in human peripheral blood T cells pre-a ctivated with anti-CD3 and anti-CD28 and then stimulated with IL-2 or IL-4 for 4 h. ( e ) Increased IL-4Rα protein expression in human T cells pre-activated with anti-CD3 and anti-CD28 and then treated with IL-2 for 16 h. ( f ) Purified splenic CD4 + T cells were pre-activated with anti-CD3 and anti-CD28 for 72 h, washed and incubated without or with 10 U/ml IL-2 for 16 h, then washed twice with PBS, rested 18 h, and cultured without or with 10 ng/ml IL-4 for 4 h. Gfi1 mRNA was measured by RT-PCR. For each panel, 3-5 independent experiments were performed.

    Journal: Nature immunology

    Article Title: Priming for T helper type 2 differentiation by interleukin 2-mediated induction of IL-4 receptor ? chain expression

    doi: 10.1038/ni.1656

    Figure Lengend Snippet: IL-2 potently induces IL-4Rα expression. ( a—c ) Mouse splenic T lymphocytes were pre-activated by anti-CD3 and anti-CD28 for 48 h, rested overnight, and then 0, 10, or 100 U/ml of IL-2 was added for 4 h. ( a ) Real time PCR was used to measure expression of the indicated mRNA transcripts. ( b,c ) Flow cytometry ( b ) and immunoblotting ( c ) were used to measure cell surface ( b ) and total ( c ) IL-4Rα expression. ( d ) Increased IL4R mRNA expression in human peripheral blood T cells pre-a ctivated with anti-CD3 and anti-CD28 and then stimulated with IL-2 or IL-4 for 4 h. ( e ) Increased IL-4Rα protein expression in human T cells pre-activated with anti-CD3 and anti-CD28 and then treated with IL-2 for 16 h. ( f ) Purified splenic CD4 + T cells were pre-activated with anti-CD3 and anti-CD28 for 72 h, washed and incubated without or with 10 U/ml IL-2 for 16 h, then washed twice with PBS, rested 18 h, and cultured without or with 10 ng/ml IL-4 for 4 h. Gfi1 mRNA was measured by RT-PCR. For each panel, 3-5 independent experiments were performed.

    Article Snippet: Animal protocols were approved by the NHLBI Animal Care and Use Committee and followed the NIH Guidelines “Using Animals in Intramural Research.” Splenic total T cells or CD4+ subpopulations were purified from 5-12 week old mice by negative or positive selection using magnetic beads (Miltenyi), cultured in RPMI 1640 medium containing 10 mM Hepes (pH 7.0), 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics (complete medium), and activated for 1.5 h at 37°C in dishes pre-coated with anti-CD3 (2 μg/ml in PBS) in complete medium containing 1 μg/ml anti-CD28 (PharMingen).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Purification, Incubation, Cell Culture, Reverse Transcription Polymerase Chain Reaction

    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: Ten million T cells were then incubated with 5 μg of anti-CD3 antibody (145-2C11; Pharmingen) with or without costimulation with 10 μg of anti-CD28 antibody (37.51; Pharmingen) on ice for 30 min, washed, and then incubated with 20 μg of goat anti-hamster immunoglobulin (Fisher) for 5 min at 37°C and lysed for the RalGDS assay, as described below.

    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: Ten million T cells were then incubated with 5 μg of anti-CD3 antibody (145-2C11; Pharmingen) with or without costimulation with 10 μg of anti-CD28 antibody (37.51; Pharmingen) on ice for 30 min, washed, and then incubated with 20 μg of goat anti-hamster immunoglobulin (Fisher) for 5 min at 37°C and lysed for the RalGDS assay, as described below.

    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: Ten million T cells were then incubated with 5 μg of anti-CD3 antibody (145-2C11; Pharmingen) with or without costimulation with 10 μg of anti-CD28 antibody (37.51; Pharmingen) on ice for 30 min, washed, and then incubated with 20 μg of goat anti-hamster immunoglobulin (Fisher) for 5 min at 37°C and lysed for the RalGDS assay, as described below.

    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: Ten million T cells were then incubated with 5 μg of anti-CD3 antibody (145-2C11; Pharmingen) with or without costimulation with 10 μg of anti-CD28 antibody (37.51; Pharmingen) on ice for 30 min, washed, and then incubated with 20 μg of goat anti-hamster immunoglobulin (Fisher) for 5 min at 37°C and lysed for the RalGDS assay, as described below.

    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: Ten million T cells were then incubated with 5 μg of anti-CD3 antibody (145-2C11; Pharmingen) with or without costimulation with 10 μg of anti-CD28 antibody (37.51; Pharmingen) on ice for 30 min, washed, and then incubated with 20 μg of goat anti-hamster immunoglobulin (Fisher) for 5 min at 37°C and lysed for the RalGDS assay, as described below.

    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: Ten million T cells were then incubated with 5 μg of anti-CD3 antibody (145-2C11; Pharmingen) with or without costimulation with 10 μg of anti-CD28 antibody (37.51; Pharmingen) on ice for 30 min, washed, and then incubated with 20 μg of goat anti-hamster immunoglobulin (Fisher) for 5 min at 37°C and lysed for the RalGDS assay, as described below.

    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: Ten million T cells were then incubated with 5 μg of anti-CD3 antibody (145-2C11; Pharmingen) with or without costimulation with 10 μg of anti-CD28 antibody (37.51; Pharmingen) on ice for 30 min, washed, and then incubated with 20 μg of goat anti-hamster immunoglobulin (Fisher) for 5 min at 37°C and lysed for the RalGDS assay, as described below.

    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: Ten million T cells were then incubated with 5 μg of anti-CD3 antibody (145-2C11; Pharmingen) with or without costimulation with 10 μg of anti-CD28 antibody (37.51; Pharmingen) on ice for 30 min, washed, and then incubated with 20 μg of goat anti-hamster immunoglobulin (Fisher) for 5 min at 37°C and lysed for the RalGDS assay, as described below.

    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: Ten million T cells were then incubated with 5 μg of anti-CD3 antibody (145-2C11; Pharmingen) with or without costimulation with 10 μg of anti-CD28 antibody (37.51; Pharmingen) on ice for 30 min, washed, and then incubated with 20 μg of goat anti-hamster immunoglobulin (Fisher) for 5 min at 37°C and lysed for the RalGDS assay, as described below.

    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: Ten million T cells were then incubated with 5 μg of anti-CD3 antibody (145-2C11; Pharmingen) with or without costimulation with 10 μg of anti-CD28 antibody (37.51; Pharmingen) on ice for 30 min, washed, and then incubated with 20 μg of goat anti-hamster immunoglobulin (Fisher) for 5 min at 37°C and lysed for the RalGDS assay, as described below.

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

    The Effect of Protocol A and Protocol B on the Expansion and Function of Treg Lines (A) Tregs were expanded by stimulation with anti-CD3/CD28 beads and expanded for 36 days with IL-2 in the presence (rapa) or absence (untreated) of rapamycin. There was no difference in the fold expansion of Tregs between the different isolation strategies (protocol A and protocol B) or culture conditions. (B) The suppressive function of freshly isolated Tregs and expanded Treg lines. Suppressive ability of Tregs was measured as a decrease of proliferation of effector T cells in the presence of different concentrations of Tregs (Treg:Teff 1:1 and 1:10). Data represent the percentage Treg suppression at a Treg:Teff ratio of 1:1. Data represent mean ± SD of 3 independent experiments. Statistical analysis was performed using 1-way ANOVA and revealed no significant difference.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: A Rapamycin-Based GMP-Compatible Process for the Isolation and Expansion of Regulatory T Cells for Clinical Trials

    doi: 10.1016/j.omtm.2018.01.006

    Figure Lengend Snippet: The Effect of Protocol A and Protocol B on the Expansion and Function of Treg Lines (A) Tregs were expanded by stimulation with anti-CD3/CD28 beads and expanded for 36 days with IL-2 in the presence (rapa) or absence (untreated) of rapamycin. There was no difference in the fold expansion of Tregs between the different isolation strategies (protocol A and protocol B) or culture conditions. (B) The suppressive function of freshly isolated Tregs and expanded Treg lines. Suppressive ability of Tregs was measured as a decrease of proliferation of effector T cells in the presence of different concentrations of Tregs (Treg:Teff 1:1 and 1:10). Data represent the percentage Treg suppression at a Treg:Teff ratio of 1:1. Data represent mean ± SD of 3 independent experiments. Statistical analysis was performed using 1-way ANOVA and revealed no significant difference.

    Article Snippet: 1 × 105 /well of responder T cells were co-cultured with Tregs at different ratios (Treg:Teff 1:1, 1:5, and 1:10) in X-vivo 15 medium supplemented with 5% HS and activated by anti-CD3/CD28-coated beads (Invitrogen, Paisley, UK) in U-bottom 96-well plates.

    Techniques: Isolation

    Purity, Expansion, and Phenotypic Characterization of Tregs on Isolation and Expansion Tregs isolated in accordance with protocol B, stimulated with anti-CD3/CD28 beads, and expanded for 36 days with IL-2 in the presence (rapa) or absence (untreated) of rapamycin. (A) CD4 + CD25 + FOXP3 + expression of freshly isolated cells and expanded Treg lines. (B) Treg fold expansion by day 36 of culture. Rapa-treated and untreated cultures exhibited similar expansion profiles. (C) Total Treg numbers post expansion. (D) Expression of regulatory markers and homing receptor expression on CD4 + CD25 + FOXP3 + cells throughout culture. S- stimulation: S1, day 0; S2, day 12; S3, day 24; and final harvest, day 36. Data represent the mean ± SD of 25 independent experiments. Statistical analysis was performed using 1-way ANOVA, and where there was a significant difference, this is indicated. *p

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: A Rapamycin-Based GMP-Compatible Process for the Isolation and Expansion of Regulatory T Cells for Clinical Trials

    doi: 10.1016/j.omtm.2018.01.006

    Figure Lengend Snippet: Purity, Expansion, and Phenotypic Characterization of Tregs on Isolation and Expansion Tregs isolated in accordance with protocol B, stimulated with anti-CD3/CD28 beads, and expanded for 36 days with IL-2 in the presence (rapa) or absence (untreated) of rapamycin. (A) CD4 + CD25 + FOXP3 + expression of freshly isolated cells and expanded Treg lines. (B) Treg fold expansion by day 36 of culture. Rapa-treated and untreated cultures exhibited similar expansion profiles. (C) Total Treg numbers post expansion. (D) Expression of regulatory markers and homing receptor expression on CD4 + CD25 + FOXP3 + cells throughout culture. S- stimulation: S1, day 0; S2, day 12; S3, day 24; and final harvest, day 36. Data represent the mean ± SD of 25 independent experiments. Statistical analysis was performed using 1-way ANOVA, and where there was a significant difference, this is indicated. *p

    Article Snippet: 1 × 105 /well of responder T cells were co-cultured with Tregs at different ratios (Treg:Teff 1:1, 1:5, and 1:10) in X-vivo 15 medium supplemented with 5% HS and activated by anti-CD3/CD28-coated beads (Invitrogen, Paisley, UK) in U-bottom 96-well plates.

    Techniques: Isolation, Expressing

    Schematic Representation of the GMP-Compliant Protocol for the Isolation and Expansion of Tregs for Clinical Application Blood is volume reduced using the Sepax 2 device (Biosafe) prior to Treg isolation. CD4 + CD25 + T cells are isolated using a combination of CD8 + depletion (CD8 reagent, Miltenyi Biotec) and enrichment step for CD25 + cells (CD25 reagent, Miltenyi Biotec) using the automated CliniMACS Plus System (Miltenyi Biotec). All processing steps are performed in closed systems. Tregs are stimulated with anti-CD3/CD28 beads in a 4:1 ratio (ExpAct Treg kit, Miltenyi Biotec) and cultured in TexMACS GMP media (Miltenyi Biotec) supplemented with 5% human serum (Lonza/Seralab). Rapamycin (Pfizer) (100 nM) is added at the beginning of the culture, whereas IL-2 (Novartis) (500 IU/mL) is added after 4 days. Both rapamycin and IL-2 are replenished every 2 to 3 days, and cells are rested for 4 days prior to restimulation. Cells are restimulated every 12 days by adding new activation beads, rapamycin, and IL-2. Phenotypic and functional characterization of the Tregs is carried out following final harvest at day 36 to ensure all final products meet the specified release criteria prior to cryopreservation and subsequent clinical application.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: A Rapamycin-Based GMP-Compatible Process for the Isolation and Expansion of Regulatory T Cells for Clinical Trials

    doi: 10.1016/j.omtm.2018.01.006

    Figure Lengend Snippet: Schematic Representation of the GMP-Compliant Protocol for the Isolation and Expansion of Tregs for Clinical Application Blood is volume reduced using the Sepax 2 device (Biosafe) prior to Treg isolation. CD4 + CD25 + T cells are isolated using a combination of CD8 + depletion (CD8 reagent, Miltenyi Biotec) and enrichment step for CD25 + cells (CD25 reagent, Miltenyi Biotec) using the automated CliniMACS Plus System (Miltenyi Biotec). All processing steps are performed in closed systems. Tregs are stimulated with anti-CD3/CD28 beads in a 4:1 ratio (ExpAct Treg kit, Miltenyi Biotec) and cultured in TexMACS GMP media (Miltenyi Biotec) supplemented with 5% human serum (Lonza/Seralab). Rapamycin (Pfizer) (100 nM) is added at the beginning of the culture, whereas IL-2 (Novartis) (500 IU/mL) is added after 4 days. Both rapamycin and IL-2 are replenished every 2 to 3 days, and cells are rested for 4 days prior to restimulation. Cells are restimulated every 12 days by adding new activation beads, rapamycin, and IL-2. Phenotypic and functional characterization of the Tregs is carried out following final harvest at day 36 to ensure all final products meet the specified release criteria prior to cryopreservation and subsequent clinical application.

    Article Snippet: 1 × 105 /well of responder T cells were co-cultured with Tregs at different ratios (Treg:Teff 1:1, 1:5, and 1:10) in X-vivo 15 medium supplemented with 5% HS and activated by anti-CD3/CD28-coated beads (Invitrogen, Paisley, UK) in U-bottom 96-well plates.

    Techniques: Isolation, Cell Culture, Activation Assay, Functional Assay

    The frequencies and function of ICOS + Tregs in patients with AML. (A,B) Representative plots (left panel) and statistical data (right panel) showed that the frequencies of CD4 + CD25 + FoxP3 + cells and CD4 + FoxP3 + ICOS + cells in 11 healthy donors and 121 patients with AML. Unpaired t -test was used to determine the difference. (C) The CD4 + CD25 high ICOS + cells and CD4 + CD25 high ICOS − cells were sorted from bone marrow mononuclear cells of patients with AML using flow cytometry, and then incubated for 5 days with PBMCs treated with 20 μg/ml mitomycin and CFSE-labeled CD4 + CD25 − T cells, with stimulation with plate-coated anti-CD3 (1 μg/ml) and soluble anti-CD28 (3 μg/ml) and IL-2 (20 ng/ml). The cell division was measured by levels of CFSE dilution by flow cytometry. Histograms were representatives of four independent experiments and ANOVA was used to determine the differences.

    Journal: Frontiers in Immunology

    Article Title: Acute Myeloid Leukemia Cells Express ICOS Ligand to Promote the Expansion of Regulatory T Cells

    doi: 10.3389/fimmu.2018.02227

    Figure Lengend Snippet: The frequencies and function of ICOS + Tregs in patients with AML. (A,B) Representative plots (left panel) and statistical data (right panel) showed that the frequencies of CD4 + CD25 + FoxP3 + cells and CD4 + FoxP3 + ICOS + cells in 11 healthy donors and 121 patients with AML. Unpaired t -test was used to determine the difference. (C) The CD4 + CD25 high ICOS + cells and CD4 + CD25 high ICOS − cells were sorted from bone marrow mononuclear cells of patients with AML using flow cytometry, and then incubated for 5 days with PBMCs treated with 20 μg/ml mitomycin and CFSE-labeled CD4 + CD25 − T cells, with stimulation with plate-coated anti-CD3 (1 μg/ml) and soluble anti-CD28 (3 μg/ml) and IL-2 (20 ng/ml). The cell division was measured by levels of CFSE dilution by flow cytometry. Histograms were representatives of four independent experiments and ANOVA was used to determine the differences.

    Article Snippet: Purified CD4+ T cells, isolated from PBMNCs of healthy donors using MACS CD4+ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany), were seeded at 5 × 104 cells/well on 96-well plates coated with 1 μg/ml anti-CD3 monoclonal antibody (eBiosciences, San Diego, CA, USA) and stimulated with 3 μg/ml anti-CD28 monoclonal antibody (eBiosciences) and 20 ng/ml IL-2 for 5 days.

    Techniques: Flow Cytometry, Cytometry, Incubation, Labeling

    CAR-CD28 heterodimers are B7-unresponsive but reduce CD28 expression. ( A ) Representative example showing CD71 upregulation in CAR T cells containing an IgG 4 -HD/CD28-TMD co-cultured for 48h with irradiated (4000Rad) CD19-wild type or deficient Raji cells with or without CTLA-4 Ig. ( B ) CD25 + CD71 + T cells were analyzed in low, intermediate (int) or high mCherry-expressing CAR-T cells using the gating strategy described in Supplementary Figure 5. Data were pooled from 4 independent experiments using T cells from 4-5 unrelated donors. ( C ) Editing strategy and homology-directed repair-mediated integration into the TRAC locus of various CD19 CARs using an AAV-6 transduction protocol. ( D ) Myc and CD28 expression in a representative example analyzed 6 days after editing and beads removal. ( E ) CD28 MFI ratio was calculated by dividing CD28 MFI of Myc + cells by Myc - cells in the same culture. Pooled data from 3-4 independent experiments across 5 unrelated donors are shown. Each dot represents one independent editing condition. Two-way ANOVA was used for statistical analysis. *p

    Journal: bioRxiv

    Article Title: The CD28-transmembrane domain mediates chimeric antigen receptor heterodimerization with CD28

    doi: 10.1101/2020.09.18.296913

    Figure Lengend Snippet: CAR-CD28 heterodimers are B7-unresponsive but reduce CD28 expression. ( A ) Representative example showing CD71 upregulation in CAR T cells containing an IgG 4 -HD/CD28-TMD co-cultured for 48h with irradiated (4000Rad) CD19-wild type or deficient Raji cells with or without CTLA-4 Ig. ( B ) CD25 + CD71 + T cells were analyzed in low, intermediate (int) or high mCherry-expressing CAR-T cells using the gating strategy described in Supplementary Figure 5. Data were pooled from 4 independent experiments using T cells from 4-5 unrelated donors. ( C ) Editing strategy and homology-directed repair-mediated integration into the TRAC locus of various CD19 CARs using an AAV-6 transduction protocol. ( D ) Myc and CD28 expression in a representative example analyzed 6 days after editing and beads removal. ( E ) CD28 MFI ratio was calculated by dividing CD28 MFI of Myc + cells by Myc - cells in the same culture. Pooled data from 3-4 independent experiments across 5 unrelated donors are shown. Each dot represents one independent editing condition. Two-way ANOVA was used for statistical analysis. *p

    Article Snippet: For assessing early T cell activation, purified CAR T cells were stimulated with soluble anti-CD28 (clone CD28.2, 1 µg/mL, BD Pharmigen), parental CD19+ Raji cells, or CD19deficient Raji cells for 2 days.

    Techniques: Expressing, Cell Culture, Irradiation, Transduction

    The dimerization of the CD28-TMD depends on a core of four amino acids ( A ) Diagram representing the amino acid sequence of the wild type and four mutants of the CD28-TMD. A representative example of MYC and mCherry expression for each mutant is shown. ( B ) A representative example of CFSE dilution of a mixed population of CD3 +/- CAR +/- T cells re-stimulated with anti-CD3/CD28 beads. ( C ) Normalized CFSE MFI ratio for CD3-CAR low/int/high was calculated by dividing the MFI of each of these population with the MFI of CD3 - mCherry - cells within the same culture. A summary of results using T cells from 4 unrelated donors in 2 independent experiments is shown. ( D ) Proliferation of purified CD3 - CAR + T cells in response to plate-bound anti-CD28 stimulation. Results represent the mean of 3 independent experiments. ( E ) CD28 or the Myc-tag of CD3 - CAR + T cells were immunoprecipitated. Western blot analysis of the input (5% of the whole cell lysate) as well as of the precipitated was performed using anti-CD28 (clone D2Z4E) and anti-Myc (clone 9B11). Results are representative of 2 independent experiments for each condition. Two-way ANOVA were used for statistical analysis. * p

    Journal: bioRxiv

    Article Title: The CD28-transmembrane domain mediates chimeric antigen receptor heterodimerization with CD28

    doi: 10.1101/2020.09.18.296913

    Figure Lengend Snippet: The dimerization of the CD28-TMD depends on a core of four amino acids ( A ) Diagram representing the amino acid sequence of the wild type and four mutants of the CD28-TMD. A representative example of MYC and mCherry expression for each mutant is shown. ( B ) A representative example of CFSE dilution of a mixed population of CD3 +/- CAR +/- T cells re-stimulated with anti-CD3/CD28 beads. ( C ) Normalized CFSE MFI ratio for CD3-CAR low/int/high was calculated by dividing the MFI of each of these population with the MFI of CD3 - mCherry - cells within the same culture. A summary of results using T cells from 4 unrelated donors in 2 independent experiments is shown. ( D ) Proliferation of purified CD3 - CAR + T cells in response to plate-bound anti-CD28 stimulation. Results represent the mean of 3 independent experiments. ( E ) CD28 or the Myc-tag of CD3 - CAR + T cells were immunoprecipitated. Western blot analysis of the input (5% of the whole cell lysate) as well as of the precipitated was performed using anti-CD28 (clone D2Z4E) and anti-Myc (clone 9B11). Results are representative of 2 independent experiments for each condition. Two-way ANOVA were used for statistical analysis. * p

    Article Snippet: For assessing early T cell activation, purified CAR T cells were stimulated with soluble anti-CD28 (clone CD28.2, 1 µg/mL, BD Pharmigen), parental CD19+ Raji cells, or CD19deficient Raji cells for 2 days.

    Techniques: Sequencing, Expressing, Mutagenesis, Purification, Immunoprecipitation, Western Blot

    Anti-CD28 stimulation of CD19-CAR T cells is TMD dependent. (A) Designs of five chimeric antigen receptors (CAR) against CD19 bearing a 4-1BB co-stimulatory domain and differing by their hinge and transmembrane domain. ( B ) FACS sorted CD4 + CD127 + CD25 low T cells were electroporated with a CRISPR-Cas9 ribonucleoprotein complex (RNP) targeting the constant region of the TCR β chain gene ( TRBC ), followed by stimulation with anti-CD3/CD28 beads (1:1 ratio). ( C ) Representative results of flow cytometric analysis of CD3 expression over time of cells electroporated with or without RNP. Percentages of residual CD3 + population and fold-expansion after 9 days of culture of CD4 + T cells electroporated with or without RNPs targeting TRBC are shown. Results from 4 independent experiments. ( D ) A representative example of CFSE dilution of a mixed population of CD3 +/- CAR +/- T re-stimulated with anti-CD3/28 beads. ( E ) Normalized CFSE MFI ratio for CD3 - mCherry + , CD3 + mCherry - and CD3 + mCherry + cells was calculated by dividing CFSE MFI of these populations with the MFI of the CD3 - mCherry - cells in the same culture. Two-way ANOVA was used for statistical analysis (bold line set as reference). ( F ) Percentages of CD3 + and mCherry + cells before and 5 days after re-stimulation of edited T cells with anti-CD3/CD28 beads. Unpaired t-test was performed comparing CD8-TMD and CD28-TMD-containing CARs on D14. For E and F , results shown are a summary of 2 independent experiments using T cells from 5 unrelated donors for each construct. *** p

    Journal: bioRxiv

    Article Title: The CD28-transmembrane domain mediates chimeric antigen receptor heterodimerization with CD28

    doi: 10.1101/2020.09.18.296913

    Figure Lengend Snippet: Anti-CD28 stimulation of CD19-CAR T cells is TMD dependent. (A) Designs of five chimeric antigen receptors (CAR) against CD19 bearing a 4-1BB co-stimulatory domain and differing by their hinge and transmembrane domain. ( B ) FACS sorted CD4 + CD127 + CD25 low T cells were electroporated with a CRISPR-Cas9 ribonucleoprotein complex (RNP) targeting the constant region of the TCR β chain gene ( TRBC ), followed by stimulation with anti-CD3/CD28 beads (1:1 ratio). ( C ) Representative results of flow cytometric analysis of CD3 expression over time of cells electroporated with or without RNP. Percentages of residual CD3 + population and fold-expansion after 9 days of culture of CD4 + T cells electroporated with or without RNPs targeting TRBC are shown. Results from 4 independent experiments. ( D ) A representative example of CFSE dilution of a mixed population of CD3 +/- CAR +/- T re-stimulated with anti-CD3/28 beads. ( E ) Normalized CFSE MFI ratio for CD3 - mCherry + , CD3 + mCherry - and CD3 + mCherry + cells was calculated by dividing CFSE MFI of these populations with the MFI of the CD3 - mCherry - cells in the same culture. Two-way ANOVA was used for statistical analysis (bold line set as reference). ( F ) Percentages of CD3 + and mCherry + cells before and 5 days after re-stimulation of edited T cells with anti-CD3/CD28 beads. Unpaired t-test was performed comparing CD8-TMD and CD28-TMD-containing CARs on D14. For E and F , results shown are a summary of 2 independent experiments using T cells from 5 unrelated donors for each construct. *** p

    Article Snippet: For assessing early T cell activation, purified CAR T cells were stimulated with soluble anti-CD28 (clone CD28.2, 1 µg/mL, BD Pharmigen), parental CD19+ Raji cells, or CD19deficient Raji cells for 2 days.

    Techniques: FACS, CRISPR, Expressing, Construct

    CD28-TMD-containing CARs interact with CD28 (A) A mixture of CFSE-labeled CD4 + T cells with or without CD3, CD28, and CAR expression. CFSE MFI of 5 independent donors in 2 independent experiments is reported. One-way ANOVA was used for statistical analysis. ( B ) Proliferation of purified CD3 - CAR + CD4 + T cells in response to plate-bound or soluble anti-CD28 stimulation. Results are representative of 3 independent experiments. Two-way ANOVA was used for statistical analysis. (C) CD28 or the Myc-tag of CD3 - CAR + T cells were immunoprecipitated. Western blot analysis of the input (5% of the whole cell lysate) as well as of the precipitated was performed using anti-CD28 (clone D2Z4E) and anti-Myc (clone 9B11). Results are representative of 2-3 independent experiments for each condition. ** p

    Journal: bioRxiv

    Article Title: The CD28-transmembrane domain mediates chimeric antigen receptor heterodimerization with CD28

    doi: 10.1101/2020.09.18.296913

    Figure Lengend Snippet: CD28-TMD-containing CARs interact with CD28 (A) A mixture of CFSE-labeled CD4 + T cells with or without CD3, CD28, and CAR expression. CFSE MFI of 5 independent donors in 2 independent experiments is reported. One-way ANOVA was used for statistical analysis. ( B ) Proliferation of purified CD3 - CAR + CD4 + T cells in response to plate-bound or soluble anti-CD28 stimulation. Results are representative of 3 independent experiments. Two-way ANOVA was used for statistical analysis. (C) CD28 or the Myc-tag of CD3 - CAR + T cells were immunoprecipitated. Western blot analysis of the input (5% of the whole cell lysate) as well as of the precipitated was performed using anti-CD28 (clone D2Z4E) and anti-Myc (clone 9B11). Results are representative of 2-3 independent experiments for each condition. ** p

    Article Snippet: For assessing early T cell activation, purified CAR T cells were stimulated with soluble anti-CD28 (clone CD28.2, 1 µg/mL, BD Pharmigen), parental CD19+ Raji cells, or CD19deficient Raji cells for 2 days.

    Techniques: Labeling, Expressing, Purification, Immunoprecipitation, Western Blot

    The effect of compounds of regulatory and inflammatory protein and gene expression basal levels of FOXP3 were measured by flow cytometry in unstimulated perientheseal bone (PEB) and peripheral blood mononuclear cell (PBMC) and show a clear population of Tregs in blood but not the enthesis (A). CD4+ and CD8T cells were isolated from PEB or matched peripheral blood and stimulated with anti-CD3/CD28 for 48 hours with and without therapeutic agents indicated. qRT-PCR was used to determine the expression of FOXP3 and TGFβ1. (B–E) 2 -ΔΔCt was used to measure relative expression fold change. IL-17A and TNF protein secretion was measured by ELISA (F–G) (n=8). The effect of therapeutic agents on CD4+ and CD8+ viability following a 48-hour incubation (H), n=3. The difference in efficacy between phosphodiesterase type 4 inhibitor (PDE4i) and retinoic acid receptor-related orphan nuclear receptor gamma t inhibitor (RORγti) in attenuating cytokine secretion in enthesis (PEB) compared with blood in CD4+ populations and CD8+ populations (I). One-way t test, *p

    Journal: Annals of the Rheumatic Diseases

    Article Title: Normal human enthesis harbours conventional CD4+ and CD8+ T cells with regulatory features and inducible IL-17A and TNF expression

    doi: 10.1136/annrheumdis-2020-217309

    Figure Lengend Snippet: The effect of compounds of regulatory and inflammatory protein and gene expression basal levels of FOXP3 were measured by flow cytometry in unstimulated perientheseal bone (PEB) and peripheral blood mononuclear cell (PBMC) and show a clear population of Tregs in blood but not the enthesis (A). CD4+ and CD8T cells were isolated from PEB or matched peripheral blood and stimulated with anti-CD3/CD28 for 48 hours with and without therapeutic agents indicated. qRT-PCR was used to determine the expression of FOXP3 and TGFβ1. (B–E) 2 -ΔΔCt was used to measure relative expression fold change. IL-17A and TNF protein secretion was measured by ELISA (F–G) (n=8). The effect of therapeutic agents on CD4+ and CD8+ viability following a 48-hour incubation (H), n=3. The difference in efficacy between phosphodiesterase type 4 inhibitor (PDE4i) and retinoic acid receptor-related orphan nuclear receptor gamma t inhibitor (RORγti) in attenuating cytokine secretion in enthesis (PEB) compared with blood in CD4+ populations and CD8+ populations (I). One-way t test, *p

    Article Snippet: Cells were stimulated using anti-CD3/CD28 (GIBCO) for 48 hours.

    Techniques: Expressing, Flow Cytometry, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation

    Intracellular interleukin (IL)-17A and tumour necrosis factor (TNF) induction in entheseal CD4+ and CD8+ T cells. Following digestion of human entheseal samples (PEB), cells were stimulated with anti-CD3/CD28 in the presence of Golgi plug for 3 hours. CD4+ and CD8+ T cells were subsequently stained intracellularly for TNF and IL-17A. TNF or IL-17A was quantified (A) following assessment by flow cytometry (B). n=3. Paired t tests. *p

    Journal: Annals of the Rheumatic Diseases

    Article Title: Normal human enthesis harbours conventional CD4+ and CD8+ T cells with regulatory features and inducible IL-17A and TNF expression

    doi: 10.1136/annrheumdis-2020-217309

    Figure Lengend Snippet: Intracellular interleukin (IL)-17A and tumour necrosis factor (TNF) induction in entheseal CD4+ and CD8+ T cells. Following digestion of human entheseal samples (PEB), cells were stimulated with anti-CD3/CD28 in the presence of Golgi plug for 3 hours. CD4+ and CD8+ T cells were subsequently stained intracellularly for TNF and IL-17A. TNF or IL-17A was quantified (A) following assessment by flow cytometry (B). n=3. Paired t tests. *p

    Article Snippet: Cells were stimulated using anti-CD3/CD28 (GIBCO) for 48 hours.

    Techniques: Staining, Flow Cytometry

    Splenocyte population levels in LGI model. CD3/CD4-positive cells and subsets detected by flow cytometry ( A , B ) and cytokine production in splenocyte cultures stimulated with CD3 + /CD28 +  or PMA/IO ( C , D ). Control non-inflamed (vehicle-PBS), control inflamed (DNBS-PBS),  B. bifidum  CNCM I-4319 strain (DNBS-CNCM-I4319). *:  p

    Journal: Microorganisms

    Article Title: The Infant-Derived Bifidobacterium bifidum Strain CNCM I-4319 Strengthens Gut Functionality

    doi: 10.3390/microorganisms8091313

    Figure Lengend Snippet: Splenocyte population levels in LGI model. CD3/CD4-positive cells and subsets detected by flow cytometry ( A , B ) and cytokine production in splenocyte cultures stimulated with CD3 + /CD28 + or PMA/IO ( C , D ). Control non-inflamed (vehicle-PBS), control inflamed (DNBS-PBS), B. bifidum CNCM I-4319 strain (DNBS-CNCM-I4319). *: p

    Article Snippet: For stimulation experiments, 2 × 105 cells isolated from spleens or MLN were cultured for 48 h (37 °C, 10% CO2) in DMEM medium in P24 plates pre-coated with anti-CD3/CD28 antibodies (4 µg/mL each; eBioscience) or phorbol 12-myristate 13-acetate (PMA)/ionomycin (cell stimulation cocktail, 1×, ebioscience).

    Techniques: Flow Cytometry

    MLN population levels in LGI model. CD3/CD4-positive cells and subsets detected by flow cytometry ( A – C ) and cytokine production in MLN cultures stimulated with CD3 + /CD28 +  or PMA/IO ( D , E ). Control non-inflamed (vehicle-PBS), control inflamed (DNBS-PBS),  B. bifidum  CNCM I-4319 strain (DNBS-CNCM-I4319). *  p

    Journal: Microorganisms

    Article Title: The Infant-Derived Bifidobacterium bifidum Strain CNCM I-4319 Strengthens Gut Functionality

    doi: 10.3390/microorganisms8091313

    Figure Lengend Snippet: MLN population levels in LGI model. CD3/CD4-positive cells and subsets detected by flow cytometry ( A – C ) and cytokine production in MLN cultures stimulated with CD3 + /CD28 + or PMA/IO ( D , E ). Control non-inflamed (vehicle-PBS), control inflamed (DNBS-PBS), B. bifidum CNCM I-4319 strain (DNBS-CNCM-I4319). * p

    Article Snippet: For stimulation experiments, 2 × 105 cells isolated from spleens or MLN were cultured for 48 h (37 °C, 10% CO2) in DMEM medium in P24 plates pre-coated with anti-CD3/CD28 antibodies (4 µg/mL each; eBioscience) or phorbol 12-myristate 13-acetate (PMA)/ionomycin (cell stimulation cocktail, 1×, ebioscience).

    Techniques: Flow Cytometry

    Inhibition of lymphocyte proliferation with Imatinib . Imatinib inhibits proliferation of splenic lymphocytes from naïve BR.RIII- Eae27 and B10.RIII mice upon in vitro stimulation with ( a ) LPS (10 μg/ml), ( b ) anti-IgM (40 μg/ml), or ( c ) anti-CD3/anti-CD28 (1 μg/ml and 3 μg/ml, respectively). Proliferation was measured as 3 H-thymidine incorporation. Data represent mean CPM ± SEM

    Journal: Journal of Neuroimmune Pharmacology

    Article Title: A Role for the Non-Receptor Tyrosine Kinase Abl2/Arg in Experimental Neuroinflammation

    doi: 10.1007/s11481-018-9783-8

    Figure Lengend Snippet: Inhibition of lymphocyte proliferation with Imatinib . Imatinib inhibits proliferation of splenic lymphocytes from naïve BR.RIII- Eae27 and B10.RIII mice upon in vitro stimulation with ( a ) LPS (10 μg/ml), ( b ) anti-IgM (40 μg/ml), or ( c ) anti-CD3/anti-CD28 (1 μg/ml and 3 μg/ml, respectively). Proliferation was measured as 3 H-thymidine incorporation. Data represent mean CPM ± SEM

    Article Snippet: Single-cell suspensions (2 × 105 cells/well) were stimulated in vitro with purified anti-mouse CD3 (BD Biosciences, San Jose, CA) and anti-mouse CD28 (eBioscience, San Diego, CA); Concanavalin A (ConA) (Sigma-Aldrich, St. Louis, MO); lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, MO); or F(ab’)2 Fragment Goat Anti-Mouse IgM (Jackson Immuno Research, West Grove, PA) for 48 h. Cells were subsequently pulsed with 1 μCi 3H-thymidine (Perkin Elmer, Waltham, MA) per well for 16–18 h, and 3 H-thymidine incorporation monitored on a TopCount NXT (Perkin Elmer, Waltham, MA).

    Techniques: Inhibition, Mouse Assay, In Vitro

    Proliferation of spleen cells from mice immunized with MBP 89–101 upon activation in vitro . 3 H-thymidine incorporation in in vitro stimulated splenic lymphocytes from MBP 89–101 -immunized BR.RIIIS/J- Eae27 and B10.RIII mice. The graphs show T-cell proliferation in response to ( a ) anti-CD3/anti-CD28 (3 μg/ml) and ( b ) ConA, and B-cell proliferation in response to ( c ) LPS, and ( d ) anti-IgM stimulation. Data are reported as mean stimulation index ratio (SI) ± SEM. Results are based on data from six mice in each group and triplicate measurements for each point

    Journal: Journal of Neuroimmune Pharmacology

    Article Title: A Role for the Non-Receptor Tyrosine Kinase Abl2/Arg in Experimental Neuroinflammation

    doi: 10.1007/s11481-018-9783-8

    Figure Lengend Snippet: Proliferation of spleen cells from mice immunized with MBP 89–101 upon activation in vitro . 3 H-thymidine incorporation in in vitro stimulated splenic lymphocytes from MBP 89–101 -immunized BR.RIIIS/J- Eae27 and B10.RIII mice. The graphs show T-cell proliferation in response to ( a ) anti-CD3/anti-CD28 (3 μg/ml) and ( b ) ConA, and B-cell proliferation in response to ( c ) LPS, and ( d ) anti-IgM stimulation. Data are reported as mean stimulation index ratio (SI) ± SEM. Results are based on data from six mice in each group and triplicate measurements for each point

    Article Snippet: Single-cell suspensions (2 × 105 cells/well) were stimulated in vitro with purified anti-mouse CD3 (BD Biosciences, San Jose, CA) and anti-mouse CD28 (eBioscience, San Diego, CA); Concanavalin A (ConA) (Sigma-Aldrich, St. Louis, MO); lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, MO); or F(ab’)2 Fragment Goat Anti-Mouse IgM (Jackson Immuno Research, West Grove, PA) for 48 h. Cells were subsequently pulsed with 1 μCi 3H-thymidine (Perkin Elmer, Waltham, MA) per well for 16–18 h, and 3 H-thymidine incorporation monitored on a TopCount NXT (Perkin Elmer, Waltham, MA).

    Techniques: Mouse Assay, Activation Assay, In Vitro

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-020-18262-6

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

    Article Snippet: Cells were activated with 5 µg/ml anti-CD3 (Clone#HIT3a, Cat#300302, Biolegend) and 2 µg/ml anti-CD28 (Clone#CD28.2, Cat#302901, Biolegend) in the presence or absence of butyrate (0.5 mM).

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

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-020-18262-6

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

    Article Snippet: Cells were activated with 5 µg/ml anti-CD3 (Clone#HIT3a, Cat#300302, Biolegend) and 2 µg/ml anti-CD28 (Clone#CD28.2, Cat#302901, Biolegend) in the presence or absence of butyrate (0.5 mM).

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

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-020-18262-6

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

    Article Snippet: Cells were activated with 5 µg/ml anti-CD3 (Clone#HIT3a, Cat#300302, Biolegend) and 2 µg/ml anti-CD28 (Clone#CD28.2, Cat#302901, Biolegend) in the presence or absence of butyrate (0.5 mM).

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

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-020-18262-6

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

    Article Snippet: Cells were activated with 5 µg/ml anti-CD3 (Clone#HIT3a, Cat#300302, Biolegend) and 2 µg/ml anti-CD28 (Clone#CD28.2, Cat#302901, Biolegend) in the presence or absence of butyrate (0.5 mM).

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

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-020-18262-6

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

    Article Snippet: Cells were activated with 5 µg/ml anti-CD3 (Clone#HIT3a, Cat#300302, Biolegend) and 2 µg/ml anti-CD28 (Clone#CD28.2, Cat#302901, Biolegend) in the presence or absence of butyrate (0.5 mM).

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

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-020-18262-6

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

    Article Snippet: Cells were activated with 5 µg/ml anti-CD3 (Clone#HIT3a, Cat#300302, Biolegend) and 2 µg/ml anti-CD28 (Clone#CD28.2, Cat#302901, Biolegend) in the presence or absence of butyrate (0.5 mM).

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

    RORα and IL-1β mediate the enhanced pathogenic Th17 cell differentiation of RIP2 deficient T cells. Naïve CD4 + T cells were isolated from the spleens of WT and Rip2 −/− mice and differentiated in vitro with anti-CD3 and anti-CD28 Ab in the presence of pTh17 cell differentiation cytokines. (A-C) qPCR of Il17a (A), Rora (B) and Rorc (C) mRNA expression from pTh17 cells (n=5–6). (D) Flow cytometry analysis of IL-17A expression in in vitro derived pTh17 cells differentiated following transduction with control or Rip2 shRNA retrovirus (n=6). (E) qPCR of Rora mRNA expression in in vitro derived pTh17 cells differentiated following transduction with control or Rip2 shRNA retrovirus (n=5). (F-I) Percent of in vitro derived pTh17 cells expressing RORα (F) and IL-17A (G). Percent of in vitro derived pTh17 cells expressing IL-17A and RORα (H). MFI of RORα expression in IL-17A + pTh17 cells (I) (n=5). (J) RORα binding to CNS1 and CNS2 in the Il17a locus as measured by ChIP with anti-RORα antibody followed by qPCR (n=5). (K) IL-17A expression in in vitro derived WT and Rip2 −/− pTh17 cells differentiated in the presence of control siRNA or siRNA targeting Rora (n=5–6). (L) IL-17A expression in in vitro derived pTh17 cells differentiated in the presence of control siRNA or siRNA targeting Rora and/or Rip2 (n=7–9). (M) qPCR of Tbx21 , Ifng , Il23r , Il1r1 , Csf2 and Il10 expression in pTh17 cells (n=5). (N) qPCR or Il1r1 expression in Rip2 −/− pTh17 cells differentiated in the presence of control siRNA or siRNA targeting Rora (n=8). (O) IL-17A expression in in vitro derived Th17 cells differentiated in the presence of indicated cytokines (n=10–11). (P) qPCR of Rora expression in in vitro derived Th17 cells differentiated in the presence of indicated cytokines (n=11–12). Data are representative of two to three independent experiments (A-L, N-P) or one independent experiment (M). Statistical analyses: Student’s t -test (A-B, E-J, M, N-P), paired Student’s t -test (D-E) or one-way ANOVA followed by Tukey’s post-hoc test (K-L). * p

    Journal: Immunity

    Article Title: T cell intrinsic Receptor Interacting Protein 2 regulates pathogenic T helper-17 cell differentiation

    doi: 10.1016/j.immuni.2018.08.022

    Figure Lengend Snippet: RORα and IL-1β mediate the enhanced pathogenic Th17 cell differentiation of RIP2 deficient T cells. Naïve CD4 + T cells were isolated from the spleens of WT and Rip2 −/− mice and differentiated in vitro with anti-CD3 and anti-CD28 Ab in the presence of pTh17 cell differentiation cytokines. (A-C) qPCR of Il17a (A), Rora (B) and Rorc (C) mRNA expression from pTh17 cells (n=5–6). (D) Flow cytometry analysis of IL-17A expression in in vitro derived pTh17 cells differentiated following transduction with control or Rip2 shRNA retrovirus (n=6). (E) qPCR of Rora mRNA expression in in vitro derived pTh17 cells differentiated following transduction with control or Rip2 shRNA retrovirus (n=5). (F-I) Percent of in vitro derived pTh17 cells expressing RORα (F) and IL-17A (G). Percent of in vitro derived pTh17 cells expressing IL-17A and RORα (H). MFI of RORα expression in IL-17A + pTh17 cells (I) (n=5). (J) RORα binding to CNS1 and CNS2 in the Il17a locus as measured by ChIP with anti-RORα antibody followed by qPCR (n=5). (K) IL-17A expression in in vitro derived WT and Rip2 −/− pTh17 cells differentiated in the presence of control siRNA or siRNA targeting Rora (n=5–6). (L) IL-17A expression in in vitro derived pTh17 cells differentiated in the presence of control siRNA or siRNA targeting Rora and/or Rip2 (n=7–9). (M) qPCR of Tbx21 , Ifng , Il23r , Il1r1 , Csf2 and Il10 expression in pTh17 cells (n=5). (N) qPCR or Il1r1 expression in Rip2 −/− pTh17 cells differentiated in the presence of control siRNA or siRNA targeting Rora (n=8). (O) IL-17A expression in in vitro derived Th17 cells differentiated in the presence of indicated cytokines (n=10–11). (P) qPCR of Rora expression in in vitro derived Th17 cells differentiated in the presence of indicated cytokines (n=11–12). Data are representative of two to three independent experiments (A-L, N-P) or one independent experiment (M). Statistical analyses: Student’s t -test (A-B, E-J, M, N-P), paired Student’s t -test (D-E) or one-way ANOVA followed by Tukey’s post-hoc test (K-L). * p

    Article Snippet: Purified cells were cultured in RPMI media containing 10% FBS and activated with anti-CD3 and anti-CD28 coated beads (Invitrogen) at a bead: cell ratio of 1:4 in media containing human rIL-2.

    Techniques: Cell Differentiation, Isolation, Mouse Assay, In Vitro, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Derivative Assay, Transduction, shRNA, Binding Assay, Chromatin Immunoprecipitation

    RIP2 deficiency enhances IL-17A production in CD4 + T cells. WT and Rip2 −/− mice were intratracheally infected with C. pneumoniae (1×10 6 IFU). (A) Flow cytometry plots of IL-17A and IFN-γ producing lung CD4 + T cells harvested 21 days p.i.. (B) The number of lung IL-17A producing cells harvested 21 days p.i.. (C and D) IL-17A and IFN-γ production in culture supernatant of MLN harvested 21 days p.i. and stimulated ex vivo with anti-CD3 and anti-CD28 Ab, UVCP (MOI 5, or as indicated) or LPS (100 ng/ml). (E) IL-17A and IFN-γ production in culture supernatant of splenocytes harvested 5 days p.i. and co-cultured with UVCP (MOI 5) pre-loaded or LPS (100 ng/ml) pre-stimulated WT BMDC. (F) Bacterial load in lungs of C. pneumoniae infected Rag1 −/− mice adoptively transferred with WT or Rip2 −/− naïve CD4 + T cells. (G and H) IL-17A and IFN-γ production in culture supernatant of MLN, harvested 5 days p.i. from Rag1 −/− mice adoptively transferred with WT or Rip2 −/− naïve CD4 + T cells and stimulated ex vivo with anti-CD3 and anti-CD28 Ab or UVCP. Data are representative of three independent experiments (n=5–7 mice/group). Statistical analyses: Student’s t -test. * p

    Journal: Immunity

    Article Title: T cell intrinsic Receptor Interacting Protein 2 regulates pathogenic T helper-17 cell differentiation

    doi: 10.1016/j.immuni.2018.08.022

    Figure Lengend Snippet: RIP2 deficiency enhances IL-17A production in CD4 + T cells. WT and Rip2 −/− mice were intratracheally infected with C. pneumoniae (1×10 6 IFU). (A) Flow cytometry plots of IL-17A and IFN-γ producing lung CD4 + T cells harvested 21 days p.i.. (B) The number of lung IL-17A producing cells harvested 21 days p.i.. (C and D) IL-17A and IFN-γ production in culture supernatant of MLN harvested 21 days p.i. and stimulated ex vivo with anti-CD3 and anti-CD28 Ab, UVCP (MOI 5, or as indicated) or LPS (100 ng/ml). (E) IL-17A and IFN-γ production in culture supernatant of splenocytes harvested 5 days p.i. and co-cultured with UVCP (MOI 5) pre-loaded or LPS (100 ng/ml) pre-stimulated WT BMDC. (F) Bacterial load in lungs of C. pneumoniae infected Rag1 −/− mice adoptively transferred with WT or Rip2 −/− naïve CD4 + T cells. (G and H) IL-17A and IFN-γ production in culture supernatant of MLN, harvested 5 days p.i. from Rag1 −/− mice adoptively transferred with WT or Rip2 −/− naïve CD4 + T cells and stimulated ex vivo with anti-CD3 and anti-CD28 Ab or UVCP. Data are representative of three independent experiments (n=5–7 mice/group). Statistical analyses: Student’s t -test. * p

    Article Snippet: Purified cells were cultured in RPMI media containing 10% FBS and activated with anti-CD3 and anti-CD28 coated beads (Invitrogen) at a bead: cell ratio of 1:4 in media containing human rIL-2.

    Techniques: Mouse Assay, Infection, Flow Cytometry, Ex Vivo, Cell Culture

    RIP2 CARD domain suppresses pathogenic Th17 cell differentiation. (A) qPCR of Rip2 expression in splenic naïve CD4 + T cells differentiated in vitro with anti-CD3 and anti-CD28 Ab alone or in the presence of cTh17 cell or pTh17 cell differentiation cytokines. (B) qPCR of Rip2 expression in flow cytometry sorted IL-17A − IFN-γ − , IL-17A − IFN-γ + and IL-17A + IFN-γ − cells from IL-17A and IFN-γ reporter mice infected intratracheally with C. pneumoniae (1×10 6 IFU) for 21 days. (C) IL-17A expression in in vitro derived pTh17 cells treated with RIP2 kinase inhibitors GSK583 or OD36. (D) IL-17A and IFN-γ expression in in vitro derived pTh17 cells following transduction with control, RIP2, RIP2K47A or RIP2ΔCARD retrovirus. (E) Percent of IL-17A inhibition in in vitro derived pTh17 cells following transduction with control or RIP2 CARD domain (RCD) retrovirus. (F) Percent of IL-17A expression in in vitro derived pTh17 cells treated with indicated concentrations of cell penetrating peptides; dNP2-FLAG (control) or dNP2-FLAG-CARD (RCD). (G) qPCR of Rora expression in in vitro derived pTh17 cells treated with dNP2-FLAG (control) or dNP2-FLAG-CARD (RCD) peptides. Data are representative of two to three independent experiments (A-D, F-G) or one independent experiment (E) (n=4–5). Statistical analyses: one-way ANOVA followed by Tukey’s post-hoc test (A), Student’s t -test (C, F-G) or paired Student’s t -test (B and E). * p

    Journal: Immunity

    Article Title: T cell intrinsic Receptor Interacting Protein 2 regulates pathogenic T helper-17 cell differentiation

    doi: 10.1016/j.immuni.2018.08.022

    Figure Lengend Snippet: RIP2 CARD domain suppresses pathogenic Th17 cell differentiation. (A) qPCR of Rip2 expression in splenic naïve CD4 + T cells differentiated in vitro with anti-CD3 and anti-CD28 Ab alone or in the presence of cTh17 cell or pTh17 cell differentiation cytokines. (B) qPCR of Rip2 expression in flow cytometry sorted IL-17A − IFN-γ − , IL-17A − IFN-γ + and IL-17A + IFN-γ − cells from IL-17A and IFN-γ reporter mice infected intratracheally with C. pneumoniae (1×10 6 IFU) for 21 days. (C) IL-17A expression in in vitro derived pTh17 cells treated with RIP2 kinase inhibitors GSK583 or OD36. (D) IL-17A and IFN-γ expression in in vitro derived pTh17 cells following transduction with control, RIP2, RIP2K47A or RIP2ΔCARD retrovirus. (E) Percent of IL-17A inhibition in in vitro derived pTh17 cells following transduction with control or RIP2 CARD domain (RCD) retrovirus. (F) Percent of IL-17A expression in in vitro derived pTh17 cells treated with indicated concentrations of cell penetrating peptides; dNP2-FLAG (control) or dNP2-FLAG-CARD (RCD). (G) qPCR of Rora expression in in vitro derived pTh17 cells treated with dNP2-FLAG (control) or dNP2-FLAG-CARD (RCD) peptides. Data are representative of two to three independent experiments (A-D, F-G) or one independent experiment (E) (n=4–5). Statistical analyses: one-way ANOVA followed by Tukey’s post-hoc test (A), Student’s t -test (C, F-G) or paired Student’s t -test (B and E). * p

    Article Snippet: Purified cells were cultured in RPMI media containing 10% FBS and activated with anti-CD3 and anti-CD28 coated beads (Invitrogen) at a bead: cell ratio of 1:4 in media containing human rIL-2.

    Techniques: Cell Differentiation, Real-time Polymerase Chain Reaction, Expressing, In Vitro, Flow Cytometry, Mouse Assay, Infection, Derivative Assay, Transduction, Inhibition

    RIP2 deficient naïve CD4 + T cells skew towards pathogenic Th17 cells. Splenic naïve CD4 + T cells from WT and Rip2 −/− mice were differentiated in vitro into cTh17 cells, pTh17 cells, or Treg cells unless otherwise specified. (A) Flow cytometry plots of the percent of IL-17A expressing CD4 + T cells differentiated under cTh17 cell or pTh17 cell conditions. (B) Percent of IL-17A expression in CD4 + T cells differentiated under cTh17 cell or pTh17 cell conditions. Data are representative of four independent experiments (n=5–6). (C) IL-17A concentration in the culture supernatant of CD4 + T cells differentiated under cTh17 cell or pTh17 cell conditions. (D) IL-17A concentration in the culture supernatant when CD4 + T cells are co-cultured with WT or Rip2 −/− BMDC under pTh17 cell conditions. (E) Flow cytometry plots of CellTrace-labeled cells differentiated in vitro under pTh17 cell conditions. (F) Flow cytometry plots of CFSE-labeled T cell enriched splenocytes co-cultured with WT or Rip2 −/− regulatory T cells in the presence of anti-CD3 and anti-CD28 Ab. (G) Percent of FOXP3 expressing CD4 + T cells differentiated under Treg cell conditions. (H) IL-17A, IL-17F and IL-17A and F heterodimer concentrations in the culture supernatant of cells differentiated under pTh17 cell conditions. Data are representative of two to three independent experiments (n=3–6 mice/group). Statistical analyses: Student’s t -test. * p

    Journal: Immunity

    Article Title: T cell intrinsic Receptor Interacting Protein 2 regulates pathogenic T helper-17 cell differentiation

    doi: 10.1016/j.immuni.2018.08.022

    Figure Lengend Snippet: RIP2 deficient naïve CD4 + T cells skew towards pathogenic Th17 cells. Splenic naïve CD4 + T cells from WT and Rip2 −/− mice were differentiated in vitro into cTh17 cells, pTh17 cells, or Treg cells unless otherwise specified. (A) Flow cytometry plots of the percent of IL-17A expressing CD4 + T cells differentiated under cTh17 cell or pTh17 cell conditions. (B) Percent of IL-17A expression in CD4 + T cells differentiated under cTh17 cell or pTh17 cell conditions. Data are representative of four independent experiments (n=5–6). (C) IL-17A concentration in the culture supernatant of CD4 + T cells differentiated under cTh17 cell or pTh17 cell conditions. (D) IL-17A concentration in the culture supernatant when CD4 + T cells are co-cultured with WT or Rip2 −/− BMDC under pTh17 cell conditions. (E) Flow cytometry plots of CellTrace-labeled cells differentiated in vitro under pTh17 cell conditions. (F) Flow cytometry plots of CFSE-labeled T cell enriched splenocytes co-cultured with WT or Rip2 −/− regulatory T cells in the presence of anti-CD3 and anti-CD28 Ab. (G) Percent of FOXP3 expressing CD4 + T cells differentiated under Treg cell conditions. (H) IL-17A, IL-17F and IL-17A and F heterodimer concentrations in the culture supernatant of cells differentiated under pTh17 cell conditions. Data are representative of two to three independent experiments (n=3–6 mice/group). Statistical analyses: Student’s t -test. * p

    Article Snippet: Purified cells were cultured in RPMI media containing 10% FBS and activated with anti-CD3 and anti-CD28 coated beads (Invitrogen) at a bead: cell ratio of 1:4 in media containing human rIL-2.

    Techniques: Mouse Assay, In Vitro, Flow Cytometry, Expressing, Concentration Assay, Cell Culture, Labeling

    INCB057643 reduces the expression of FOXP3 in CD4 T cells and PD-L1 expression in macrophages. Immunohistochemistry for FOXP3 ( A ) or PD-L1 ( C ) in pancreas and liver of KPC mice treated with vehicle control or INCB057643 for 16 weeks. Scale bar = 30 µm. ( B ) CD4 T cells were isolated from a spleen of a wild type mouse using negative magnetic bead selection. CD4 T cells were plated with anti-CD3 (or PBS, a control for non-stimulated T cells), anti-CD28, IL-2 and TGF-β for 24 h prior to adding INCB057643 (100 nM) for 4 days. CD4 T cells were collected and levels of FOXP3 were determined by PCR. p

    Journal: Cancers

    Article Title: The Bromodomain Inhibitor, INCB057643, Targets Both Cancer Cells and the Tumor Microenvironment in Two Preclinical Models of Pancreatic Cancer

    doi: 10.3390/cancers13010096

    Figure Lengend Snippet: INCB057643 reduces the expression of FOXP3 in CD4 T cells and PD-L1 expression in macrophages. Immunohistochemistry for FOXP3 ( A ) or PD-L1 ( C ) in pancreas and liver of KPC mice treated with vehicle control or INCB057643 for 16 weeks. Scale bar = 30 µm. ( B ) CD4 T cells were isolated from a spleen of a wild type mouse using negative magnetic bead selection. CD4 T cells were plated with anti-CD3 (or PBS, a control for non-stimulated T cells), anti-CD28, IL-2 and TGF-β for 24 h prior to adding INCB057643 (100 nM) for 4 days. CD4 T cells were collected and levels of FOXP3 were determined by PCR. p

    Article Snippet: Isolated CD4 T cells were plated in a 24 well plate at 106 mL/well in RPMI media supplemented with 10% FBS, anti-mouse CD28 (clone 37.51, 3 μg/mL, Biolegend), recombinant IL-2 (5 ng/mL, Biolegend), and recombinant human TGFβ (5 ng/mL, R & D).

    Techniques: Expressing, Immunohistochemistry, Mouse Assay, Isolation, Selection, Polymerase Chain Reaction