anti-cd28 mab Search Results


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  • 99
    Thermo Fisher anti cd28
    Anti Cd28, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti cd28 mab
    The expression of CD40L on IL-21- expressing NKT cells PFMCs were stimulated for 6 hrs with or without PPD plus <t>anti-CD28</t> The cells were stained, gated on NKT cells and analyzed for the expression of CD40L on IL-21 + NKT cells. A. The expression of CD40L and production of IL-21 were detected by FACS. Data are representative of three independent experiments. B. Statistical results of the expression of CD40L on NKT cells. **, P
    Anti Cd28 Mab, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson anti cd28 mab
    2B4 −/− CD8 + T cells are resistant to the effects of selective <t>CD28</t> blockade. 10 6 Thy1.1 + OT-I or 10 6 Thy1.1 + 2B4 −/− OT-I and 10 6 Thy1.1 + OT-II T cells were adoptively transferred into naive B6 recipients, which were then challenged with an OVA-expressing skin graft in the presence of control Vκ dAb or anti-CD28 dAb, and then dosed on days 0, 2, 4, and 6 and three times per week continuously thereafter as described in the Materials and methods. Graft-draining LNs were harvested on day 10 after transplant and analyzed by flow cytometry. (A) Frequencies of donor-reactive CD8 + Thy1.1 + T cells WT and 2B4 −/− donor-reactive T cells in the presence or absence of anti-CD28 dAb. Data shown are gated on CD8 + T cells. (B) Summary data showing expansion of 2B4 −/− donor-reactive CD8 + T cells after treatment with anti-CD28 dAb as compared with expansion of WT OT-I T cells (P = 0.03). (C) IFN-γ and TNF production of donor-reactive CD8 + Thy1.1 + T cells in untreated animals that received WT or 2B4 −/− T cells. Data shown are gated on CD8 + Thy1.1 + T cells. (D) IFN-γ and TNF production of donor-reactive CD8 + Thy1.1 + T cells in anti-CD28 dAb–treated animals that received WT or 2B4 −/− T cells. Data shown are gated on CD8 + Thy1.1 + T cells. (E) Frequencies of IFN-γ + and TNF + donor-reactive CD8 + T cells in untreated recipients of WT versus 2B4 −/− OT-I T cells. (F) Frequencies of IFN-γ + (P = 0.0343) and TNF + (P = 0.0159) donor-reactive CD8 + T cells in anti-CD28 dAb–treated recipients of WT versus 2B4 −/− OT-I T cells. Flow plots are representative and graphs are summary data from two independent experiments with a total of 10 animals per group. (G and H) Recipients of WT or 2B4 −/− OT-I were left untreated (G) or treated with anti-CD28 dAb (H) and monitored for skin graft survival (P = 0.0299). *, P
    Anti Cd28 Mab, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 960 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher anti cd28 mabs
    Similar cytokine profiles in the primary cerebral lymphomas (PCL) and intrasplenic lymphoma (ISL) tumour microenvironments. phosphate-buffered saline (PBS) or A20.IIA-green fluorescent protein (GFP) cells were injected into the brain (black circles) or the spleen (white circles) of adult BALB/c mice. On day 21, cells isolated from the brain or splenocytes were cultivated for 36 h with <t>(aCD3/CD28)</t> or without (medium) <t>anti-CD3/CD28-coated</t> beads, and supernatants were analysed for interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-17 expression (two independent experiments, n = 10). The dashed horizontal line indicates the level of expression significant for each cytokine, as indicated by the manufacturer.
    Anti Cd28 Mabs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 103 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Becton Dickinson soluble anti cd28 mab
    PG490 inhibited both IKKα and IKKβ kinase activity. T cells were pretreated with various concentrations of PG490 for 2 h and then stimulated with TNF-α for 10 min (A) , PMA + ionomycin for 15 min (B) or <t>CD3/CD28</t> for 30 min (C) . Cell pellets were collected and total cell lysates were prepared and analyzed for the kinase activity of IKKα and IKKβ by immunoprecipitation kinase assays. The total IKKα and/or total IKKβ levels were determined by Western blotting. The analysis of PG490-mediated suppression of IKKα and IKKβ activities induced by TNF-α stimulation was performed on pooled data from T cells from 3 different donors (D) . *, P value of
    Soluble Anti Cd28 Mab, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 226 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher cd28 functional grade monoclonal antibody
    PG490 inhibited both IKKα and IKKβ kinase activity. T cells were pretreated with various concentrations of PG490 for 2 h and then stimulated with TNF-α for 10 min (A) , PMA + ionomycin for 15 min (B) or <t>CD3/CD28</t> for 30 min (C) . Cell pellets were collected and total cell lysates were prepared and analyzed for the kinase activity of IKKα and IKKβ by immunoprecipitation kinase assays. The total IKKα and/or total IKKβ levels were determined by Western blotting. The analysis of PG490-mediated suppression of IKKα and IKKβ activities induced by TNF-α stimulation was performed on pooled data from T cells from 3 different donors (D) . *, P value of
    Cd28 Functional Grade Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 245 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher anti cd28 antibodies
    Mitochondrial respiration, but not ATP production, promotes Akt/Foxo1 signaling in activated T cells ( A ) A schematic depicting electron transfer and disposition by cytosolic LDHA and mitochondrial electron transport chain (ETC) associated with NADH to NAD + conversion. ( B ) Naïve CD8 + T cells were isolated from Ldha fl/fl (wild-type, WT) and CD4 Cre Ldha fl/fl (knockout, KO) mice, and stimulated with 5 μg/ml anti-CD3, 2 μg/ml <t>anti-CD28,</t> and 100 U/ml IL-2 for 3 days. The amounts of NADH and NAD + were measured. The ratios of NAD + over NADH are plotted. ( C ) Measurements and quantifications of oxygen consumption rate (OCR) and spared respiratory capacity (SRC) of day-3 activated WT and KO CD8 + T cells. Sequential chemical treatments are indicated as shown in the graph. Oligo: oligomycin; Ant: antimycin; Rot: rotenone. (n=5 per genotype, mean ± SD) ( D ) A schematic of mitochondrial ETC, proton distribution, and ATP synthetase activity under the indicated treatment conditions. ( E ) Day-3 activated WT and KO CD8 + T cells were collected, and incubated in RPMI medium in the absence or presence of oligomycin and/or FCCP. T cells were subsequently re-stimulated with biotinylated anti-CD3 and anti-CD28 through streptavidin crosslinking, and immunoblotted for p-Akt (T308), Akt, p-Foxo1 (T24), p-Foxo1 (S256), Foxo1, LDHA, and β-Actin. Normalized expression of p-Akt (T308) to Akt, p-Foxo1 (T24) or p-Foxo1 (S256) to Foxo1 were marked. Unpaired t tests for the measurements between the two groups ( B and C ): **p
    Anti Cd28 Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1206 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher anti cd3 cd28 antibodies
    <t>Single-CD28</t> stimulated Treg reveal a highly demethylated FOXP3 gene and profound suppressor function. ( a ) FACS-sorted human Treg were stimulated with soluble CD28 mAb, plate bound <t>CD3</t> mAb or both (anti-CD3+CD28) in the presence of rhIL-2 for 7-days. Thereafter, cells were harvested and the demethylation status of FOXP3 gene was analyzed using bisulfate sequencing. n = 6–7. ( b ) CFSE-labeled FACS-sorted Treg were stimulated as described above. The divided cells (CFSE low population) were re-sorted (left panel) at day 7 of the cultures, and subsequently their suppressive function was analyzed in a co-culture suppression assay. Overlay histograms show the inhibition of responder T cells (Tresp) proliferation following the addition of graded doses of Treg. Numbers indicate the percentage of divided responder T cells. Grey line: stimulated Tresp, Black line: co-cultured with Treg of interest. The ratio of Treg:Tresp are indicated on the top. Representative experiment of n = 3 individual experiments conducted with cells obtained from different donors are shown. ( c ) Intracellular staining of cytokine IL-17A and IFNγ after additional stimulation with PMA, ionomycin, and Brefeldin-A for 4 hours. Numbers within the quadrant indicate the percentage of positive cells. Dotplots show a representative experiment of n = 6–10 individuals as shown in the cumulative data graph (right panel). Kruskal-Wallis followed by Dunns post-hoc test was used for statistical analysis ( a,c ). *P
    Anti Cd3 Cd28 Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 253 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher soluble anti cd28 monoclonal antibody
    p.L374P mutation in the C terminus of STIM 1 abolishes SOCE and causes CRAC channelopathy Pedigree of the patients P1 (II‐1) and P2 (II‐2) presenting with CRAC channelopathy. Filled circles (females) and squares (males) indicate homozygous patients; dotted symbols indicate heterozygous asymptomatic carriers; symbols with “?” indicate asymptomatic family members not available for DNA sequencing. Sanger sequencing of genomic DNA isolated from PBMC of the mother (I‐2), P1, and P2. STIM1 and STIM2 mRNA expression in PBMC of P1, P2, mother, and a healthy donor (HD) that were left unstimulated or stimulated with <t>anti‐CD3/CD28</t> for 24 h. mRNA levels of STIM1/2 normalized to 18S rRNA. Graph shows the mean ± SEM of duplicates from one experiment. STIM1 protein expression in CD4 + and CD8 + T cells from P1, P2, the mother, and a HD analyzed by flow cytometry. Shaded histograms: polyclonal rabbit IgG control antibody; open histograms: polyclonal anti‐STIM1 antibody. Bar graphs show the delta MFI calculated as MFI STIM1 – MFI IgG control that was normalized to HD T cells. Bar graphs are mean ± SEM of two independent repeat experiments. STIM1 protein expression in expanded human T cells analyzed by immunoblotting. One representative Western blot of 3 is shown. Bar graphs are the mean ± SEM of STIM1 expression normalized to actin from three independent experiments. Ca 2+ influx in Fura‐2 loaded PBMC of P1, P2, the mother, and a HD. T cells were stimulated with 1 μM thapsigargin (TG) in the absence of extracellular Ca 2+ followed by addition of 1 mM extracellular Ca 2+ . Bar graphs show the integrated Ca 2+ influx response (AUC, area under the curve) from 400 to 800 s and the peak Ca 2+ influx normalized to baseline Ca 2+ levels (F340/380) at 400 s. Data represent the mean ± SEM from 2 experiments. Statistical analysis by unpaired Student's t ‐test. ** P
    Soluble Anti Cd28 Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pharmingen anti cd28 mab
    Effect of exogenous IL-10 on LPMC IFN-γ production from WT and TGF-βRII DN mice. LPMC from uninfected WT (A and C) or TGF-βRII DN (A and B) mice were stimulated in vitro with <t>anti-CD3/CD28</t> (white bars) for 48 hr, in indicated cell numbers per well, with parallel cultures also containing 20 ng/ml (gray bars) or 100 ng/ml recombinant IL-10 (black bars). Supernatants were harvested and analyzed for IFN-γ by ELISA. Data show mean ± SD of (A) IFNγ secretion (two independent experiments with each experiment containing multiple determinations) or (B-C) the percentile ratio of LPMC IFNγ secretion in each condition to IFN-γ output of anti-CD3/28-stimulated culture (three independent experiments with each experiment containing multiple determinations). ( (A) WT Uninfected vs. Infected, ***p
    Anti Cd28 Mab, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Becton Dickinson anti mouse cd28 mab
    Intranasal immunization with MCS-pPES enhanced specific IFNγ-secreting CD4+ and CD8+ T response in the spleen. (A) ELISPOT analysis of the splenic TB-specific IFNγ-producing T response in pPES-, CS-DNA, MCS-DNA-, and BCG-immunized mice. Splenocytes were stimulated with mixed peptides, HSP65 protein or inactivated H37Rv plus <t>anti-CD28</t> for 36 h before IFN-γ ELISPOT assay was performed. Statistical results were from three independent experiments and presented as the mean ± SEM ( n = 6). (B) The frequency of CD4 and CD8 T cells in the lung producing IFN-γ, IL-2, or TNF-α in response to mixed peptides was measured. Cells were incubated with mixed peptides plus anti-CD28 for 10 h the percentages of IFN-γ, IL-2, and TNF-α-producing CD4+T and CD8+T cells were determined by FACS. Numbers in each quadrant represent percentages of positive cells in CD4+ or CD8+ T population. Results are represented as mean ± SEM ( n = 6) of three separate experiments. * p
    Anti Mouse Cd28 Mab, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher anti cd28 ebioscience antibody
    Intranasal immunization with MCS-pPES enhanced specific IFNγ-secreting CD4+ and CD8+ T response in the spleen. (A) ELISPOT analysis of the splenic TB-specific IFNγ-producing T response in pPES-, CS-DNA, MCS-DNA-, and BCG-immunized mice. Splenocytes were stimulated with mixed peptides, HSP65 protein or inactivated H37Rv plus <t>anti-CD28</t> for 36 h before IFN-γ ELISPOT assay was performed. Statistical results were from three independent experiments and presented as the mean ± SEM ( n = 6). (B) The frequency of CD4 and CD8 T cells in the lung producing IFN-γ, IL-2, or TNF-α in response to mixed peptides was measured. Cells were incubated with mixed peptides plus anti-CD28 for 10 h the percentages of IFN-γ, IL-2, and TNF-α-producing CD4+T and CD8+T cells were determined by FACS. Numbers in each quadrant represent percentages of positive cells in CD4+ or CD8+ T population. Results are represented as mean ± SEM ( n = 6) of three separate experiments. * p
    Anti Cd28 Ebioscience Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The expression of CD40L on IL-21- expressing NKT cells PFMCs were stimulated for 6 hrs with or without PPD plus anti-CD28 The cells were stained, gated on NKT cells and analyzed for the expression of CD40L on IL-21 + NKT cells. A. The expression of CD40L and production of IL-21 were detected by FACS. Data are representative of three independent experiments. B. Statistical results of the expression of CD40L on NKT cells. **, P

    Journal: Oncotarget

    Article Title: Antigen-specific human NKT cells from tuberculosis patients produce IL-21 to help B cells for the production of immunoglobulins

    doi:

    Figure Lengend Snippet: The expression of CD40L on IL-21- expressing NKT cells PFMCs were stimulated for 6 hrs with or without PPD plus anti-CD28 The cells were stained, gated on NKT cells and analyzed for the expression of CD40L on IL-21 + NKT cells. A. The expression of CD40L and production of IL-21 were detected by FACS. Data are representative of three independent experiments. B. Statistical results of the expression of CD40L on NKT cells. **, P

    Article Snippet: For the detection of intracellular cytokines, cells were incubated with PPD or Mtb-HAg plus anti-CD28 mAb for 6-8 hrs in the presence of brefeldin A (10 μg/mL; Sigma-Aldrich, St Louis, MO).

    Techniques: Expressing, Staining, FACS

    Antigen-induced expression of IL-21 with IFN-γ, TNF-α, IL-2 and IL-17 by CD3 + TCRvβ11 + NKT cells PFMCs were incubated for 6 hrs with or without PPD plus anti-CD28 NKT cells were gated and analyzed for the expression of cytokines. A. Statistical results of the expression of IL-21, IFN-γ, TNF-α, IL-2 and IL-17 after stimulation with PPD plus anti-CD28 are shown. *, P

    Journal: Oncotarget

    Article Title: Antigen-specific human NKT cells from tuberculosis patients produce IL-21 to help B cells for the production of immunoglobulins

    doi:

    Figure Lengend Snippet: Antigen-induced expression of IL-21 with IFN-γ, TNF-α, IL-2 and IL-17 by CD3 + TCRvβ11 + NKT cells PFMCs were incubated for 6 hrs with or without PPD plus anti-CD28 NKT cells were gated and analyzed for the expression of cytokines. A. Statistical results of the expression of IL-21, IFN-γ, TNF-α, IL-2 and IL-17 after stimulation with PPD plus anti-CD28 are shown. *, P

    Article Snippet: For the detection of intracellular cytokines, cells were incubated with PPD or Mtb-HAg plus anti-CD28 mAb for 6-8 hrs in the presence of brefeldin A (10 μg/mL; Sigma-Aldrich, St Louis, MO).

    Techniques: Expressing, Incubation

    Characterization of IL-21-expressing NKT cells PFMCs were stimulated for 6 hrs with PPD plus anti-CD28, the cells were stained for NKT cells and analyzed for phenotypes of NKT cells A. IL-21 + NKT and IL-21 − NKT cells were gated for the analysis of the expression of CD45RO, CD62L and CCR7. B. Statistical results of the percentages of CD45RO, CD62L and CCR7 on IL-21 + NKT and IL-21 − NKT cells. *, P

    Journal: Oncotarget

    Article Title: Antigen-specific human NKT cells from tuberculosis patients produce IL-21 to help B cells for the production of immunoglobulins

    doi:

    Figure Lengend Snippet: Characterization of IL-21-expressing NKT cells PFMCs were stimulated for 6 hrs with PPD plus anti-CD28, the cells were stained for NKT cells and analyzed for phenotypes of NKT cells A. IL-21 + NKT and IL-21 − NKT cells were gated for the analysis of the expression of CD45RO, CD62L and CCR7. B. Statistical results of the percentages of CD45RO, CD62L and CCR7 on IL-21 + NKT and IL-21 − NKT cells. *, P

    Article Snippet: For the detection of intracellular cytokines, cells were incubated with PPD or Mtb-HAg plus anti-CD28 mAb for 6-8 hrs in the presence of brefeldin A (10 μg/mL; Sigma-Aldrich, St Louis, MO).

    Techniques: Expressing, Staining

    IL-21-expressing NKT cells co-expressed CXCR5 PFMCs were stimulated for 6 hrs with or without PPD plus anti-CD28 The cells were stained, gated on NKT cells and analyzed for the expression of CXCR5 on IL-21 + NKT cells. A. The expression of CXCR5 and IL-21 were measured by FACS. Data are representative of three independent experiments. B. Statistical results of the expression of CXCR5 on NKT cells and the frequency of IL-21-expressing cells among CXCR5 + and CXCR5 − NKT cells. *, P

    Journal: Oncotarget

    Article Title: Antigen-specific human NKT cells from tuberculosis patients produce IL-21 to help B cells for the production of immunoglobulins

    doi:

    Figure Lengend Snippet: IL-21-expressing NKT cells co-expressed CXCR5 PFMCs were stimulated for 6 hrs with or without PPD plus anti-CD28 The cells were stained, gated on NKT cells and analyzed for the expression of CXCR5 on IL-21 + NKT cells. A. The expression of CXCR5 and IL-21 were measured by FACS. Data are representative of three independent experiments. B. Statistical results of the expression of CXCR5 on NKT cells and the frequency of IL-21-expressing cells among CXCR5 + and CXCR5 − NKT cells. *, P

    Article Snippet: For the detection of intracellular cytokines, cells were incubated with PPD or Mtb-HAg plus anti-CD28 mAb for 6-8 hrs in the presence of brefeldin A (10 μg/mL; Sigma-Aldrich, St Louis, MO).

    Techniques: Expressing, Staining, FACS

    The expression of BCL-6 and IL-21 by NKT cells PFMCs were stimulated for 6 hrs with or without PPD plus anti-CD28 The cells were stained, gated and analyzed for the expression of BCL-6 and IL-21 on NKT cells. A. The expression of BCL-6 on IL-21 + and IL-21 − NKT cells was measured by FACS. Data are representative of three independent experiments. B. Statistical results of the expression of BCL-6 on IL-21 + and IL-21 − NKT cells. *, P

    Journal: Oncotarget

    Article Title: Antigen-specific human NKT cells from tuberculosis patients produce IL-21 to help B cells for the production of immunoglobulins

    doi:

    Figure Lengend Snippet: The expression of BCL-6 and IL-21 by NKT cells PFMCs were stimulated for 6 hrs with or without PPD plus anti-CD28 The cells were stained, gated and analyzed for the expression of BCL-6 and IL-21 on NKT cells. A. The expression of BCL-6 on IL-21 + and IL-21 − NKT cells was measured by FACS. Data are representative of three independent experiments. B. Statistical results of the expression of BCL-6 on IL-21 + and IL-21 − NKT cells. *, P

    Article Snippet: For the detection of intracellular cytokines, cells were incubated with PPD or Mtb-HAg plus anti-CD28 mAb for 6-8 hrs in the presence of brefeldin A (10 μg/mL; Sigma-Aldrich, St Louis, MO).

    Techniques: Expressing, Staining, FACS

    2B4 −/− CD8 + T cells are resistant to the effects of selective CD28 blockade. 10 6 Thy1.1 + OT-I or 10 6 Thy1.1 + 2B4 −/− OT-I and 10 6 Thy1.1 + OT-II T cells were adoptively transferred into naive B6 recipients, which were then challenged with an OVA-expressing skin graft in the presence of control Vκ dAb or anti-CD28 dAb, and then dosed on days 0, 2, 4, and 6 and three times per week continuously thereafter as described in the Materials and methods. Graft-draining LNs were harvested on day 10 after transplant and analyzed by flow cytometry. (A) Frequencies of donor-reactive CD8 + Thy1.1 + T cells WT and 2B4 −/− donor-reactive T cells in the presence or absence of anti-CD28 dAb. Data shown are gated on CD8 + T cells. (B) Summary data showing expansion of 2B4 −/− donor-reactive CD8 + T cells after treatment with anti-CD28 dAb as compared with expansion of WT OT-I T cells (P = 0.03). (C) IFN-γ and TNF production of donor-reactive CD8 + Thy1.1 + T cells in untreated animals that received WT or 2B4 −/− T cells. Data shown are gated on CD8 + Thy1.1 + T cells. (D) IFN-γ and TNF production of donor-reactive CD8 + Thy1.1 + T cells in anti-CD28 dAb–treated animals that received WT or 2B4 −/− T cells. Data shown are gated on CD8 + Thy1.1 + T cells. (E) Frequencies of IFN-γ + and TNF + donor-reactive CD8 + T cells in untreated recipients of WT versus 2B4 −/− OT-I T cells. (F) Frequencies of IFN-γ + (P = 0.0343) and TNF + (P = 0.0159) donor-reactive CD8 + T cells in anti-CD28 dAb–treated recipients of WT versus 2B4 −/− OT-I T cells. Flow plots are representative and graphs are summary data from two independent experiments with a total of 10 animals per group. (G and H) Recipients of WT or 2B4 −/− OT-I were left untreated (G) or treated with anti-CD28 dAb (H) and monitored for skin graft survival (P = 0.0299). *, P

    Journal: The Journal of Experimental Medicine

    Article Title: 2B4 (CD244) induced by selective CD28 blockade functionally regulates allograft-specific CD8+ T cell responses

    doi: 10.1084/jem.20130902

    Figure Lengend Snippet: 2B4 −/− CD8 + T cells are resistant to the effects of selective CD28 blockade. 10 6 Thy1.1 + OT-I or 10 6 Thy1.1 + 2B4 −/− OT-I and 10 6 Thy1.1 + OT-II T cells were adoptively transferred into naive B6 recipients, which were then challenged with an OVA-expressing skin graft in the presence of control Vκ dAb or anti-CD28 dAb, and then dosed on days 0, 2, 4, and 6 and three times per week continuously thereafter as described in the Materials and methods. Graft-draining LNs were harvested on day 10 after transplant and analyzed by flow cytometry. (A) Frequencies of donor-reactive CD8 + Thy1.1 + T cells WT and 2B4 −/− donor-reactive T cells in the presence or absence of anti-CD28 dAb. Data shown are gated on CD8 + T cells. (B) Summary data showing expansion of 2B4 −/− donor-reactive CD8 + T cells after treatment with anti-CD28 dAb as compared with expansion of WT OT-I T cells (P = 0.03). (C) IFN-γ and TNF production of donor-reactive CD8 + Thy1.1 + T cells in untreated animals that received WT or 2B4 −/− T cells. Data shown are gated on CD8 + Thy1.1 + T cells. (D) IFN-γ and TNF production of donor-reactive CD8 + Thy1.1 + T cells in anti-CD28 dAb–treated animals that received WT or 2B4 −/− T cells. Data shown are gated on CD8 + Thy1.1 + T cells. (E) Frequencies of IFN-γ + and TNF + donor-reactive CD8 + T cells in untreated recipients of WT versus 2B4 −/− OT-I T cells. (F) Frequencies of IFN-γ + (P = 0.0343) and TNF + (P = 0.0159) donor-reactive CD8 + T cells in anti-CD28 dAb–treated recipients of WT versus 2B4 −/− OT-I T cells. Flow plots are representative and graphs are summary data from two independent experiments with a total of 10 animals per group. (G and H) Recipients of WT or 2B4 −/− OT-I were left untreated (G) or treated with anti-CD28 dAb (H) and monitored for skin graft survival (P = 0.0299). *, P

    Article Snippet: Splenocytes were harvested and stained with fluoresceinated anti-CD28 mAb clone E18 (BD) and analyzed by flow cytometry.

    Techniques: Expressing, Flow Cytometry, Cytometry

    Reduced up-regulation of ICOS after selective CD28 blockade is dependent on engagement of the 2B4 pathway. 10 6 Thy1.1 + OT-I or 10 6 Thy1.1 + 2B4 −/− OT-I and 10 6 Thy1.1 + OT-II T cells were adoptively transferred into naive B6 recipients, which were then challenged with an OVA-expressing skin graft in the presence of control dAb or anti-CD28 dAb, and then dosed on days 0, 2, 4, and 6 and three times per week continuously thereafter as described in the Materials and methods. Graft-draining LNs were harvested on day 10 after transplant and analyzed by flow cytometry. (A) ICOS expression on WT (left) and 2B4 −/− (right) donor-reactive CD8 + Thy1.1 + T cells in the presence and absence of anti-CD28 dAb. (B) Summary data from n = 5/group (representative of 2 independent experiments with a total of 10 mice per group) show the percent reduction in ICOS expression after anti-CD28 dAb treatment as compared with control dAb treatment in WT versus 2B4 −/− CD8 + T cells (P = 0.0018). Data shown are gated on CD8 + Thy1.1 + T cells. (C) Percent reduction in ICOS expression after anti-CD28 dAb treatment on CD4 + donor-reactive T cells in mice that received WT or 2B4 −/− OT-I T cells. Data shown are gated on CD4 + Thy1.1 + T cells. Summary data shown are from n = 5/group (representative of two independent experiments with a total of 10 mice per group). **, P

    Journal: The Journal of Experimental Medicine

    Article Title: 2B4 (CD244) induced by selective CD28 blockade functionally regulates allograft-specific CD8+ T cell responses

    doi: 10.1084/jem.20130902

    Figure Lengend Snippet: Reduced up-regulation of ICOS after selective CD28 blockade is dependent on engagement of the 2B4 pathway. 10 6 Thy1.1 + OT-I or 10 6 Thy1.1 + 2B4 −/− OT-I and 10 6 Thy1.1 + OT-II T cells were adoptively transferred into naive B6 recipients, which were then challenged with an OVA-expressing skin graft in the presence of control dAb or anti-CD28 dAb, and then dosed on days 0, 2, 4, and 6 and three times per week continuously thereafter as described in the Materials and methods. Graft-draining LNs were harvested on day 10 after transplant and analyzed by flow cytometry. (A) ICOS expression on WT (left) and 2B4 −/− (right) donor-reactive CD8 + Thy1.1 + T cells in the presence and absence of anti-CD28 dAb. (B) Summary data from n = 5/group (representative of 2 independent experiments with a total of 10 mice per group) show the percent reduction in ICOS expression after anti-CD28 dAb treatment as compared with control dAb treatment in WT versus 2B4 −/− CD8 + T cells (P = 0.0018). Data shown are gated on CD8 + Thy1.1 + T cells. (C) Percent reduction in ICOS expression after anti-CD28 dAb treatment on CD4 + donor-reactive T cells in mice that received WT or 2B4 −/− OT-I T cells. Data shown are gated on CD4 + Thy1.1 + T cells. Summary data shown are from n = 5/group (representative of two independent experiments with a total of 10 mice per group). **, P

    Article Snippet: Splenocytes were harvested and stained with fluoresceinated anti-CD28 mAb clone E18 (BD) and analyzed by flow cytometry.

    Techniques: Expressing, Flow Cytometry, Cytometry, Mouse Assay

    Donor-reactive CD4 + T cell accumulation and differentiation are more profoundly attenuated by selective CD28 blockade versus CTLA-4 Ig. 10 6 Thy1.1 + OT-I and 10 6 Thy1.1 + OT-II T cells were adoptively transferred into naive B6 recipients, which were then challenged with an OVA-expressing skin graft in the presence of control Vκ dAb, CTLA-4 Ig, or anti-CD28 dAb, and then dosed on days 0, 2, 4, and 6 and three times per week continuously thereafter, as described in the Materials and methods. (A) Assessment of frequencies of donor-reactive CD4 + Thy1.1 + T cells on day 10 after transplant in draining lymph nodes. Data shown are representative and gated on CD4 + T cells. (B and C) Summary data of 3 independent experiments with a total of 8–10 mice per group. Frequencies (B) and absolute numbers (C) of CD4 + Thy1.1 + T cells in anti-CD28 dAb-treated animals as compared with CTLA-4 Ig-treated animals (B, P = 0.0031; C, P = 0.00185). (D) Analysis of CD44 and CD62L expression on CD4 + Thy1.1 + T cells in DLN on day 10 after transplant. Data shown are representative. (E) Summary data of 3 independent experiments with a total of 8–10 mice per group (P = 0.0004). (F) IL-2 production by CD4 + Thy1.1 + T cells isolated from DLN on day 10 after transplant after ex vivo restimulation with OVA 323–339 (P

    Journal: The Journal of Experimental Medicine

    Article Title: 2B4 (CD244) induced by selective CD28 blockade functionally regulates allograft-specific CD8+ T cell responses

    doi: 10.1084/jem.20130902

    Figure Lengend Snippet: Donor-reactive CD4 + T cell accumulation and differentiation are more profoundly attenuated by selective CD28 blockade versus CTLA-4 Ig. 10 6 Thy1.1 + OT-I and 10 6 Thy1.1 + OT-II T cells were adoptively transferred into naive B6 recipients, which were then challenged with an OVA-expressing skin graft in the presence of control Vκ dAb, CTLA-4 Ig, or anti-CD28 dAb, and then dosed on days 0, 2, 4, and 6 and three times per week continuously thereafter, as described in the Materials and methods. (A) Assessment of frequencies of donor-reactive CD4 + Thy1.1 + T cells on day 10 after transplant in draining lymph nodes. Data shown are representative and gated on CD4 + T cells. (B and C) Summary data of 3 independent experiments with a total of 8–10 mice per group. Frequencies (B) and absolute numbers (C) of CD4 + Thy1.1 + T cells in anti-CD28 dAb-treated animals as compared with CTLA-4 Ig-treated animals (B, P = 0.0031; C, P = 0.00185). (D) Analysis of CD44 and CD62L expression on CD4 + Thy1.1 + T cells in DLN on day 10 after transplant. Data shown are representative. (E) Summary data of 3 independent experiments with a total of 8–10 mice per group (P = 0.0004). (F) IL-2 production by CD4 + Thy1.1 + T cells isolated from DLN on day 10 after transplant after ex vivo restimulation with OVA 323–339 (P

    Article Snippet: Splenocytes were harvested and stained with fluoresceinated anti-CD28 mAb clone E18 (BD) and analyzed by flow cytometry.

    Techniques: Expressing, Mouse Assay, Isolation, Ex Vivo

    Increased efficacy and ICOS down-regulation after selective CD28 blockade is independent of PD-L1–mediated signals. 10 6 Thy1.1 + OT-I and 10 6 Thy1.1 + OT-II T cells were adoptively transferred into naive B6 recipients, which were then challenged with an OVA-expressing skin graft in the presence of control Vκ dAb, anti-CD28 dAb, or anti-CD28 dAb + anti-PD-L1 mAb dosed on days 0, 2, 4, and 6 as described in Materials and methods. (A and B) Assessment of frequencies of donor-reactive CD4 + (A) and CD8 + (B) Thy1.1 + T cells on day 10 after transplant in draining lymph nodes. Data shown representative and are gated on CD4 + (A) or CD8 + (B) T cells. (C and D) Frequencies and absolute numbers of either CD4 + (C) or CD8 + (D) Thy1.1 + T cells in anti-CD28 dAb + anti-PD-L1 treated animals as compared with animals treated with anti-CD28 dAb alone are shown. (E) Cytokine production by CD4 + (IL-2) or CD8 + (IFN-γ + TNF + ) Thy1.1 + T cells isolated from DLN on day 10 after transplant after ex vivo restimulation with cognate antigen is shown. (F) ICOS expression on both CD4 + and CD8 + Thy1.1 + T cells isolated on day 10 after transplant is shown. All graphs are summary data of a total of 4–5 mice per group from two independent experiments. ***, P

    Journal: The Journal of Experimental Medicine

    Article Title: 2B4 (CD244) induced by selective CD28 blockade functionally regulates allograft-specific CD8+ T cell responses

    doi: 10.1084/jem.20130902

    Figure Lengend Snippet: Increased efficacy and ICOS down-regulation after selective CD28 blockade is independent of PD-L1–mediated signals. 10 6 Thy1.1 + OT-I and 10 6 Thy1.1 + OT-II T cells were adoptively transferred into naive B6 recipients, which were then challenged with an OVA-expressing skin graft in the presence of control Vκ dAb, anti-CD28 dAb, or anti-CD28 dAb + anti-PD-L1 mAb dosed on days 0, 2, 4, and 6 as described in Materials and methods. (A and B) Assessment of frequencies of donor-reactive CD4 + (A) and CD8 + (B) Thy1.1 + T cells on day 10 after transplant in draining lymph nodes. Data shown representative and are gated on CD4 + (A) or CD8 + (B) T cells. (C and D) Frequencies and absolute numbers of either CD4 + (C) or CD8 + (D) Thy1.1 + T cells in anti-CD28 dAb + anti-PD-L1 treated animals as compared with animals treated with anti-CD28 dAb alone are shown. (E) Cytokine production by CD4 + (IL-2) or CD8 + (IFN-γ + TNF + ) Thy1.1 + T cells isolated from DLN on day 10 after transplant after ex vivo restimulation with cognate antigen is shown. (F) ICOS expression on both CD4 + and CD8 + Thy1.1 + T cells isolated on day 10 after transplant is shown. All graphs are summary data of a total of 4–5 mice per group from two independent experiments. ***, P

    Article Snippet: Splenocytes were harvested and stained with fluoresceinated anti-CD28 mAb clone E18 (BD) and analyzed by flow cytometry.

    Techniques: Expressing, Isolation, Ex Vivo, Mouse Assay

    2B4 is up-regulated on endogenous, polyclonal alloreactive CD8 + T cells after treatment with anti-CD28 dAb. BALB/c skin grafts were placed onto B6 recipients that were treated with 100 µg anti-CD28 dAb on days 0, 2, 4, and 6 or left untreated. Animals were sacrificed at day 7 after transplant. (A) Splenocytes were restimulated for 4 h ex vivo with irradiated BALB/c stimulator cells, and intracellular IFN-γ was assessed. Results indicated significantly fewer CD8 + H-2K d− IFN-γ–secreting alloreactive T cells in CD28 dAb-treated recipients. (B) 2B4 expression on CD8 + T cells in grafted recipients was assessed. Data shown are representative and gated on CD8 + H-2K d− cells. (C), Percent 2B4 + of CD8 + T cells. (D) ICOS expression on CD8 + T cells in grafted recipients was assessed. Data shown are representative and gated on CD8 + H-2K d− cells. (E) Percent ICOS + of CD8 + T cells. All graphs are summary data from n = 5 animals per group (*, P

    Journal: The Journal of Experimental Medicine

    Article Title: 2B4 (CD244) induced by selective CD28 blockade functionally regulates allograft-specific CD8+ T cell responses

    doi: 10.1084/jem.20130902

    Figure Lengend Snippet: 2B4 is up-regulated on endogenous, polyclonal alloreactive CD8 + T cells after treatment with anti-CD28 dAb. BALB/c skin grafts were placed onto B6 recipients that were treated with 100 µg anti-CD28 dAb on days 0, 2, 4, and 6 or left untreated. Animals were sacrificed at day 7 after transplant. (A) Splenocytes were restimulated for 4 h ex vivo with irradiated BALB/c stimulator cells, and intracellular IFN-γ was assessed. Results indicated significantly fewer CD8 + H-2K d− IFN-γ–secreting alloreactive T cells in CD28 dAb-treated recipients. (B) 2B4 expression on CD8 + T cells in grafted recipients was assessed. Data shown are representative and gated on CD8 + H-2K d− cells. (C), Percent 2B4 + of CD8 + T cells. (D) ICOS expression on CD8 + T cells in grafted recipients was assessed. Data shown are representative and gated on CD8 + H-2K d− cells. (E) Percent ICOS + of CD8 + T cells. All graphs are summary data from n = 5 animals per group (*, P

    Article Snippet: Splenocytes were harvested and stained with fluoresceinated anti-CD28 mAb clone E18 (BD) and analyzed by flow cytometry.

    Techniques: Ex Vivo, Irradiation, Expressing

    Donor-reactive CD8 + T cell accumulation and differentiation are more profoundly attenuated by selective CD28 blockade versus CTLA-4 Ig. 10 6 Thy1.1 + OT-I and 10 6 Thy1.1 + OT-II T cells were adoptively transferred into naive B6 recipients, which were then challenged with an OVA-expressing skin graft in the presence of control Vκ dAb, CTLA-4 Ig, or anti-CD28 dAb, and then dosed on days 0, 2, 4, and 6 and three times per week continuously thereafter, as described in the Materials and methods. (A) Assessment of frequencies of donor-reactive CD8 + Thy1.1 + T cells on day 10 after transplant in draining lymph nodes. Data shown are representative and gated on CD8 + T cells. (B) Summary data of three independent experiments with a total of 8–10 mice per group. Frequencies (P = 0.0185) and absolute numbers (P = 0.0021) are shown. (C) Analysis of CD44 and CD62L expression on CD8 + Thy1.1 + T cells in DLN on day 10 after transplant. Data shown are representative. (D) Summary data of 3 independent experiments with a total of 8–10 mice per group. (P = 0.0004). (E) IFN-γ and TNF production by CD8 + Thy1.1 + T cells isolated from DLN on day 10 after transplant after ex vivo restimulation with SIINFEKL. (F) IFN-γ production in both CTLA-4 Ig– and anti–CD28 dAb-treated animals is shown relative to control Vκ dAb-treated animals (P

    Journal: The Journal of Experimental Medicine

    Article Title: 2B4 (CD244) induced by selective CD28 blockade functionally regulates allograft-specific CD8+ T cell responses

    doi: 10.1084/jem.20130902

    Figure Lengend Snippet: Donor-reactive CD8 + T cell accumulation and differentiation are more profoundly attenuated by selective CD28 blockade versus CTLA-4 Ig. 10 6 Thy1.1 + OT-I and 10 6 Thy1.1 + OT-II T cells were adoptively transferred into naive B6 recipients, which were then challenged with an OVA-expressing skin graft in the presence of control Vκ dAb, CTLA-4 Ig, or anti-CD28 dAb, and then dosed on days 0, 2, 4, and 6 and three times per week continuously thereafter, as described in the Materials and methods. (A) Assessment of frequencies of donor-reactive CD8 + Thy1.1 + T cells on day 10 after transplant in draining lymph nodes. Data shown are representative and gated on CD8 + T cells. (B) Summary data of three independent experiments with a total of 8–10 mice per group. Frequencies (P = 0.0185) and absolute numbers (P = 0.0021) are shown. (C) Analysis of CD44 and CD62L expression on CD8 + Thy1.1 + T cells in DLN on day 10 after transplant. Data shown are representative. (D) Summary data of 3 independent experiments with a total of 8–10 mice per group. (P = 0.0004). (E) IFN-γ and TNF production by CD8 + Thy1.1 + T cells isolated from DLN on day 10 after transplant after ex vivo restimulation with SIINFEKL. (F) IFN-γ production in both CTLA-4 Ig– and anti–CD28 dAb-treated animals is shown relative to control Vκ dAb-treated animals (P

    Article Snippet: Splenocytes were harvested and stained with fluoresceinated anti-CD28 mAb clone E18 (BD) and analyzed by flow cytometry.

    Techniques: Expressing, Mouse Assay, Isolation, Ex Vivo

    CD28 co-stimulatory blockade in the presence of CTLA-4 signals results in reduced up-regulation of ICOS and increased expression of the co-inhibitor 2B4. 10 6 Thy1.1 + OT-I and 10 6 Thy1.1 + OT-II T cells were adoptively transferred into naive B6 recipients, which were then challenged with an OVA-expressing skin graft in the presence of control dAb, CTLA-4 Ig, or anti-CD28 dAb, and then dosed on days 0, 2, 4, and 6 and three times per week continuously thereafter, as described in the Materials and methods. Graft-draining LNs were harvested on day 10 after transplant and analyzed by flow cytometry. (A) ICOS expression on naive donor-reactive T cells (gray) or donor-reactive CD4 + and CD8 + T cells isolated from control Vκ dAb dAb-treated animals (red), CTLA-4 Ig-treated animals (green), and anti-CD28 dAb-treated animals (blue). Data shown are representative and gated on CD4 + Thy1.1 + cells (top) and CD8 + Thy1.1 + cells (bottom). (B) Summary data from 3 independent experiments with 8–10 animals per group. CD4 + T cells, P = 0.0079; CD8 + T cells, P = 0.0317. (C) Up-regulation of 2B4 on donor-reactive CD8 + T cells isolated from untreated animals (red), CTLA-4 Ig-treated animals (green), or anti-CD28 dAb-treated animals (blue). Naive CD8 + T cells are shown in black. Data shown are representative and gated on CD8 + Thy1.1 + cells. (D) Summary data from 3 independent experiments with 8–10 animals per group. 2B4 expression on CD8 + T cells isolated from animals treated with anti-CD28 dAb as compared with control Vκ dAb or CTLA-4 Ig (P

    Journal: The Journal of Experimental Medicine

    Article Title: 2B4 (CD244) induced by selective CD28 blockade functionally regulates allograft-specific CD8+ T cell responses

    doi: 10.1084/jem.20130902

    Figure Lengend Snippet: CD28 co-stimulatory blockade in the presence of CTLA-4 signals results in reduced up-regulation of ICOS and increased expression of the co-inhibitor 2B4. 10 6 Thy1.1 + OT-I and 10 6 Thy1.1 + OT-II T cells were adoptively transferred into naive B6 recipients, which were then challenged with an OVA-expressing skin graft in the presence of control dAb, CTLA-4 Ig, or anti-CD28 dAb, and then dosed on days 0, 2, 4, and 6 and three times per week continuously thereafter, as described in the Materials and methods. Graft-draining LNs were harvested on day 10 after transplant and analyzed by flow cytometry. (A) ICOS expression on naive donor-reactive T cells (gray) or donor-reactive CD4 + and CD8 + T cells isolated from control Vκ dAb dAb-treated animals (red), CTLA-4 Ig-treated animals (green), and anti-CD28 dAb-treated animals (blue). Data shown are representative and gated on CD4 + Thy1.1 + cells (top) and CD8 + Thy1.1 + cells (bottom). (B) Summary data from 3 independent experiments with 8–10 animals per group. CD4 + T cells, P = 0.0079; CD8 + T cells, P = 0.0317. (C) Up-regulation of 2B4 on donor-reactive CD8 + T cells isolated from untreated animals (red), CTLA-4 Ig-treated animals (green), or anti-CD28 dAb-treated animals (blue). Naive CD8 + T cells are shown in black. Data shown are representative and gated on CD8 + Thy1.1 + cells. (D) Summary data from 3 independent experiments with 8–10 animals per group. 2B4 expression on CD8 + T cells isolated from animals treated with anti-CD28 dAb as compared with control Vκ dAb or CTLA-4 Ig (P

    Article Snippet: Splenocytes were harvested and stained with fluoresceinated anti-CD28 mAb clone E18 (BD) and analyzed by flow cytometry.

    Techniques: Expressing, Flow Cytometry, Cytometry, Isolation

    Selective CD28 blockade results in superior graft survival as compared with CTLA-4 Ig, where both CD28 co-stimulatory and CTLA-4 co-inhibitory signals are blocked. (A) B6 recipients of BALB/c skin grafts were treated with CTLA-4 Ig or anti-CD28 dAb in the presence of anti-CD154 mAb on days 0, 2, 4, and 6, and then three times per week continuously thereafter until day 50, as described in the Materials and methods. Anti-CD154 alone–treated animals (red line, control) served as negative controls. MST of control-treated animals was 14 d and MST of animals treated with CTLA-4 Ig and anti-CD154 was 32 d. MST of animals treated with anti-CD28 dAbs and anti-CD154 > 50 d (P = 0.0013 as compared with CTLA-4 Ig/anti-CD154; n = 5/group). (B) Experimental design of TCR transgenic model of minor antigen disparity wherein 10 6 Thy1.1 + OT-I and 10 6 Thy1.1 + OT-II T cells were adoptively transferred into naive B6 recipients, which were then challenged with an OVA-expressing skin graft in the presence of either control dAb, CTLA-4 Ig, or anti-CD28 dAb, which were dosed on days 0, 2, 4, and 6 and then three times per week continuously thereafter as described in Materials and methods. (C) Graft survival data from the experimental design depicted in B. Control dAb and CTLA-4 Ig-treated animals rejected their grafts with MSTs of 19 and 34 d, respectively, and anti-CD28 dAb-treated animals exhibited an MST of > 100 d (P

    Journal: The Journal of Experimental Medicine

    Article Title: 2B4 (CD244) induced by selective CD28 blockade functionally regulates allograft-specific CD8+ T cell responses

    doi: 10.1084/jem.20130902

    Figure Lengend Snippet: Selective CD28 blockade results in superior graft survival as compared with CTLA-4 Ig, where both CD28 co-stimulatory and CTLA-4 co-inhibitory signals are blocked. (A) B6 recipients of BALB/c skin grafts were treated with CTLA-4 Ig or anti-CD28 dAb in the presence of anti-CD154 mAb on days 0, 2, 4, and 6, and then three times per week continuously thereafter until day 50, as described in the Materials and methods. Anti-CD154 alone–treated animals (red line, control) served as negative controls. MST of control-treated animals was 14 d and MST of animals treated with CTLA-4 Ig and anti-CD154 was 32 d. MST of animals treated with anti-CD28 dAbs and anti-CD154 > 50 d (P = 0.0013 as compared with CTLA-4 Ig/anti-CD154; n = 5/group). (B) Experimental design of TCR transgenic model of minor antigen disparity wherein 10 6 Thy1.1 + OT-I and 10 6 Thy1.1 + OT-II T cells were adoptively transferred into naive B6 recipients, which were then challenged with an OVA-expressing skin graft in the presence of either control dAb, CTLA-4 Ig, or anti-CD28 dAb, which were dosed on days 0, 2, 4, and 6 and then three times per week continuously thereafter as described in Materials and methods. (C) Graft survival data from the experimental design depicted in B. Control dAb and CTLA-4 Ig-treated animals rejected their grafts with MSTs of 19 and 34 d, respectively, and anti-CD28 dAb-treated animals exhibited an MST of > 100 d (P

    Article Snippet: Splenocytes were harvested and stained with fluoresceinated anti-CD28 mAb clone E18 (BD) and analyzed by flow cytometry.

    Techniques: Microscale Thermophoresis, Transgenic Assay, Expressing

    Increased efficacy and 2B4 up-regulation after selective CD28 blockade is dependent on CTLA-4–mediated signals. 10 6 Thy1.1 + OT-I and 10 6 Thy1.1 + OT-II T cells were adoptively transferred into naive B6 recipients, which were then challenged with an OVA-expressing skin graft in the presence of control Vκ dAb, anti-CD28 dAb, or anti-CD28 dAb + anti–CTLA-4 mAb dosed on days 0, 2, 4, and 6 as described in the Materials and methods. (A and B) Assessment of frequencies of donor-reactive CD4 + (A) and CD8 + (B) Thy1.1 + T cells on day 10 after transplant in draining lymph nodes. Data shown are representative and gated on CD4 + (A) or CD8 + (B) T cells. (C and D) Frequencies and absolute numbers of both CD4 + (C) and CD8 + (D) Thy1.1 + T cells in anti-CD28 dAb + anti–CTLA-4–treated animals as compared with animals treated with anti-CD28 dAb alone (P = 0.0079 for CD28 dAb versus CD28 dAb + anti–CTLA-4 groups). (E and F) IL-2 production by CD4 + Thy1.1 + T cells isolated from DLN on day 10 after transplant after ex vivo restimulation with OVA 323–339 (P = 0.0079 for CD28 dAb versus CD28 dAb + anti–CTLA-4 groups). (F) IFN-γ and TNF production by CD8 + Thy1.1 + T cells isolated from DLN on day 10 after transplant after ex vivo restimulation with SIINFEKL (P = 0.0079 for CD28 dAb vs. CD28 dAb + anti–CTLA-4 groups). (G) ICOS expression is shown on both CD4 + and CD8 + Thy1.1 + T cells isolated on day 10 after transplant from anti-CD28 dAb + anti–CTLA-4 treated animals relative to animals treated with anti-CD28 dAb alone (P = 0.0079). (H and I) 2B4 expression is shown on CD8 + Thy1.1 + T cells isolated on day 10 after transplant from anti-CD28 dAb + anti–CTLA-4–treated animals relative to animals treated with anti-CD28 dAb alone (P = 0.0159). All graphs are summary data of 2 independent experiments with a total of 8–10 mice per group. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: 2B4 (CD244) induced by selective CD28 blockade functionally regulates allograft-specific CD8+ T cell responses

    doi: 10.1084/jem.20130902

    Figure Lengend Snippet: Increased efficacy and 2B4 up-regulation after selective CD28 blockade is dependent on CTLA-4–mediated signals. 10 6 Thy1.1 + OT-I and 10 6 Thy1.1 + OT-II T cells were adoptively transferred into naive B6 recipients, which were then challenged with an OVA-expressing skin graft in the presence of control Vκ dAb, anti-CD28 dAb, or anti-CD28 dAb + anti–CTLA-4 mAb dosed on days 0, 2, 4, and 6 as described in the Materials and methods. (A and B) Assessment of frequencies of donor-reactive CD4 + (A) and CD8 + (B) Thy1.1 + T cells on day 10 after transplant in draining lymph nodes. Data shown are representative and gated on CD4 + (A) or CD8 + (B) T cells. (C and D) Frequencies and absolute numbers of both CD4 + (C) and CD8 + (D) Thy1.1 + T cells in anti-CD28 dAb + anti–CTLA-4–treated animals as compared with animals treated with anti-CD28 dAb alone (P = 0.0079 for CD28 dAb versus CD28 dAb + anti–CTLA-4 groups). (E and F) IL-2 production by CD4 + Thy1.1 + T cells isolated from DLN on day 10 after transplant after ex vivo restimulation with OVA 323–339 (P = 0.0079 for CD28 dAb versus CD28 dAb + anti–CTLA-4 groups). (F) IFN-γ and TNF production by CD8 + Thy1.1 + T cells isolated from DLN on day 10 after transplant after ex vivo restimulation with SIINFEKL (P = 0.0079 for CD28 dAb vs. CD28 dAb + anti–CTLA-4 groups). (G) ICOS expression is shown on both CD4 + and CD8 + Thy1.1 + T cells isolated on day 10 after transplant from anti-CD28 dAb + anti–CTLA-4 treated animals relative to animals treated with anti-CD28 dAb alone (P = 0.0079). (H and I) 2B4 expression is shown on CD8 + Thy1.1 + T cells isolated on day 10 after transplant from anti-CD28 dAb + anti–CTLA-4–treated animals relative to animals treated with anti-CD28 dAb alone (P = 0.0159). All graphs are summary data of 2 independent experiments with a total of 8–10 mice per group. *, P

    Article Snippet: Splenocytes were harvested and stained with fluoresceinated anti-CD28 mAb clone E18 (BD) and analyzed by flow cytometry.

    Techniques: Expressing, Isolation, Ex Vivo, Mouse Assay

    Similar cytokine profiles in the primary cerebral lymphomas (PCL) and intrasplenic lymphoma (ISL) tumour microenvironments. phosphate-buffered saline (PBS) or A20.IIA-green fluorescent protein (GFP) cells were injected into the brain (black circles) or the spleen (white circles) of adult BALB/c mice. On day 21, cells isolated from the brain or splenocytes were cultivated for 36 h with (aCD3/CD28) or without (medium) anti-CD3/CD28-coated beads, and supernatants were analysed for interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-17 expression (two independent experiments, n = 10). The dashed horizontal line indicates the level of expression significant for each cytokine, as indicated by the manufacturer.

    Journal: Clinical and Experimental Immunology

    Article Title: Immune adaptive microenvironment profiles in intracerebral and intrasplenic lymphomas share common characteristics

    doi: 10.1111/j.1365-2249.2011.04416.x

    Figure Lengend Snippet: Similar cytokine profiles in the primary cerebral lymphomas (PCL) and intrasplenic lymphoma (ISL) tumour microenvironments. phosphate-buffered saline (PBS) or A20.IIA-green fluorescent protein (GFP) cells were injected into the brain (black circles) or the spleen (white circles) of adult BALB/c mice. On day 21, cells isolated from the brain or splenocytes were cultivated for 36 h with (aCD3/CD28) or without (medium) anti-CD3/CD28-coated beads, and supernatants were analysed for interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-17 expression (two independent experiments, n = 10). The dashed horizontal line indicates the level of expression significant for each cytokine, as indicated by the manufacturer.

    Article Snippet: Brain or spleen cells (105 ) were stimulated with anti-CD3/CD28 monoclonal antibody coated beads (Dynabeads, Dynal Biotech, Compiègne, France), as recommended by the manufacturer.

    Techniques: Injection, Mouse Assay, Isolation, Expressing

    PG490 inhibited both IKKα and IKKβ kinase activity. T cells were pretreated with various concentrations of PG490 for 2 h and then stimulated with TNF-α for 10 min (A) , PMA + ionomycin for 15 min (B) or CD3/CD28 for 30 min (C) . Cell pellets were collected and total cell lysates were prepared and analyzed for the kinase activity of IKKα and IKKβ by immunoprecipitation kinase assays. The total IKKα and/or total IKKβ levels were determined by Western blotting. The analysis of PG490-mediated suppression of IKKα and IKKβ activities induced by TNF-α stimulation was performed on pooled data from T cells from 3 different donors (D) . *, P value of

    Journal: Journal of Translational Medicine

    Article Title: Differential immunomodulatory effects by Tripterygium wilfordii Hook f-derived refined extract PG27 and its purified component PG490 (triptolide) in human peripheral blood T cells: potential therapeutics for arthritis and possible mechanisms explaining in part Chinese herbal theory “Junn-Chenn-Zuou-SS”

    doi: 10.1186/1479-5876-11-294

    Figure Lengend Snippet: PG490 inhibited both IKKα and IKKβ kinase activity. T cells were pretreated with various concentrations of PG490 for 2 h and then stimulated with TNF-α for 10 min (A) , PMA + ionomycin for 15 min (B) or CD3/CD28 for 30 min (C) . Cell pellets were collected and total cell lysates were prepared and analyzed for the kinase activity of IKKα and IKKβ by immunoprecipitation kinase assays. The total IKKα and/or total IKKβ levels were determined by Western blotting. The analysis of PG490-mediated suppression of IKKα and IKKβ activities induced by TNF-α stimulation was performed on pooled data from T cells from 3 different donors (D) . *, P value of

    Article Snippet: Cell stimulation, cytokine determination, and cell survival measurement To activate T cells, the following stimuli were used: phorbol 12-myristate 13-acetate (PMA; Sigma) at 10 ng/mL, ionomycin (Sigma) at 1 μM, immobilized monoclonal antibody (mAb) anti-CD3 (OKT3; ATCC) at 10 μg/mL, soluble anti-CD28 mAb (Beckton Dickinson) at 1 μg/mL, and TNF-α at 10 ng/mL.

    Techniques: Activity Assay, Immunoprecipitation, Western Blot

    Side-by-side comparisons of PG27 and PG490 for immunosuppressive potency and drug cytotoxicity. T cells from a single donor were pretreated in parallel with DMSO (Ctl) or various concentrations (in serial dilutions) of PG490 or PG27 that contained an equal amount of PG490, and then stimulated with PMA + ionomycin (A) or 3 different stimuli (PMA + ionomycin, CD3/CD28 and TNF-α) as indicated (B) for 24 h. In (A) , the concentrations of PG27 and PG490 at 1× (the highest concentration) were 200 ng/mL and 2.56 ng/mL, respectively. In (B) , the concentrations of PG27 and PG490 at 1× (the highest concentration) were 6400 ng/mL and 81.28 ng/mL, respectively. The supernatants were collected for the measurement of IL-2 (A) and the cells were collected for the measurement of cytotoxicity by MTT colorimetric assays (B) . The results are representative of T cells from at least 3 different donors.

    Journal: Journal of Translational Medicine

    Article Title: Differential immunomodulatory effects by Tripterygium wilfordii Hook f-derived refined extract PG27 and its purified component PG490 (triptolide) in human peripheral blood T cells: potential therapeutics for arthritis and possible mechanisms explaining in part Chinese herbal theory “Junn-Chenn-Zuou-SS”

    doi: 10.1186/1479-5876-11-294

    Figure Lengend Snippet: Side-by-side comparisons of PG27 and PG490 for immunosuppressive potency and drug cytotoxicity. T cells from a single donor were pretreated in parallel with DMSO (Ctl) or various concentrations (in serial dilutions) of PG490 or PG27 that contained an equal amount of PG490, and then stimulated with PMA + ionomycin (A) or 3 different stimuli (PMA + ionomycin, CD3/CD28 and TNF-α) as indicated (B) for 24 h. In (A) , the concentrations of PG27 and PG490 at 1× (the highest concentration) were 200 ng/mL and 2.56 ng/mL, respectively. In (B) , the concentrations of PG27 and PG490 at 1× (the highest concentration) were 6400 ng/mL and 81.28 ng/mL, respectively. The supernatants were collected for the measurement of IL-2 (A) and the cells were collected for the measurement of cytotoxicity by MTT colorimetric assays (B) . The results are representative of T cells from at least 3 different donors.

    Article Snippet: Cell stimulation, cytokine determination, and cell survival measurement To activate T cells, the following stimuli were used: phorbol 12-myristate 13-acetate (PMA; Sigma) at 10 ng/mL, ionomycin (Sigma) at 1 μM, immobilized monoclonal antibody (mAb) anti-CD3 (OKT3; ATCC) at 10 μg/mL, soluble anti-CD28 mAb (Beckton Dickinson) at 1 μg/mL, and TNF-α at 10 ng/mL.

    Techniques: CTL Assay, Concentration Assay, MTT Assay

    PG27 inhibited NF-κB and AP-1 activation induced by various stimuli. Human peripheral blood T cells at a concentration of 2 × 10 6 /mL were pretreated with various concentrations of PG27 for 2 h and then stimulated with PMA + ionomycin for 2 h (A) , CD3/CD28 for 6 h (B) or TNF-α for 6 h (C) . The nuclear extracts were prepared and analyzed for both NF-κB and AP-1 DNA-binding activity by EMSA. As a control, the DNA-binding activity of Oct-1 was measured (C) . In (D) , before adding the radiolabeled oligonucleotides to the reaction mixture, the nuclear extracts were preincubated with 5 μl of the indicated mAb for 30 min. The asterisk indicates the supershifted bands. In (E) , T cells at a concentration of 1 × 10 6 /mL were mixed with pNF-κB-Luc or pAP-1-Luc reporter plasmids and transfection reagents. The transfection was performed using an Amaxa Nucleofector according to the manufacturer’s instructions. After transfection for 48 h, the cells were aliquoted equally for the individual conditions and pretreated with various concentrations of PG27 for 2 h. After stimulation with TNF-α for another 18 h, cells were collected and total cell lysates were analyzed for luciferase activity. Cell survival was determined by trypan blue exclusion assays. Representative data of at least 3 independent experiments are shown.

    Journal: Journal of Translational Medicine

    Article Title: Differential immunomodulatory effects by Tripterygium wilfordii Hook f-derived refined extract PG27 and its purified component PG490 (triptolide) in human peripheral blood T cells: potential therapeutics for arthritis and possible mechanisms explaining in part Chinese herbal theory “Junn-Chenn-Zuou-SS”

    doi: 10.1186/1479-5876-11-294

    Figure Lengend Snippet: PG27 inhibited NF-κB and AP-1 activation induced by various stimuli. Human peripheral blood T cells at a concentration of 2 × 10 6 /mL were pretreated with various concentrations of PG27 for 2 h and then stimulated with PMA + ionomycin for 2 h (A) , CD3/CD28 for 6 h (B) or TNF-α for 6 h (C) . The nuclear extracts were prepared and analyzed for both NF-κB and AP-1 DNA-binding activity by EMSA. As a control, the DNA-binding activity of Oct-1 was measured (C) . In (D) , before adding the radiolabeled oligonucleotides to the reaction mixture, the nuclear extracts were preincubated with 5 μl of the indicated mAb for 30 min. The asterisk indicates the supershifted bands. In (E) , T cells at a concentration of 1 × 10 6 /mL were mixed with pNF-κB-Luc or pAP-1-Luc reporter plasmids and transfection reagents. The transfection was performed using an Amaxa Nucleofector according to the manufacturer’s instructions. After transfection for 48 h, the cells were aliquoted equally for the individual conditions and pretreated with various concentrations of PG27 for 2 h. After stimulation with TNF-α for another 18 h, cells were collected and total cell lysates were analyzed for luciferase activity. Cell survival was determined by trypan blue exclusion assays. Representative data of at least 3 independent experiments are shown.

    Article Snippet: Cell stimulation, cytokine determination, and cell survival measurement To activate T cells, the following stimuli were used: phorbol 12-myristate 13-acetate (PMA; Sigma) at 10 ng/mL, ionomycin (Sigma) at 1 μM, immobilized monoclonal antibody (mAb) anti-CD3 (OKT3; ATCC) at 10 μg/mL, soluble anti-CD28 mAb (Beckton Dickinson) at 1 μg/mL, and TNF-α at 10 ng/mL.

    Techniques: Activation Assay, Concentration Assay, Binding Assay, Activity Assay, Transfection, Luciferase

    PG490 inhibited both NF-κB and AP-1 DNA-binding and transcriptional activity. T cells were pretreated with various concentrations of PG490 (A) for 2 h and then stimulated with TNF-α (B) or CD3/CD28 (C) for 6 h. The nuclear extracts were analyzed for both NF-κB and AP-1 DNA-binding activity by EMSA. In (D) , T cells were pretreated with PG490 for 2 h or left untreated, and then stimulated with TNF-α for various lengths of time as indicated. After washing, cell pellets were collected and cytoplasmic extracts were analyzed for the protein levels of IκBα and β-actin by Western blotting. In (E) , T cells were mixed together with pNF-κB-Luc or pAP-1-Luc reporter plasmids and the transfection procedures were performed as described for Figure 2 . After electroporation for 48 h, the cells were aliquoted equally and pretreated with various concentrations of PG490 for 2 h. After stimulation with TNF-α for another 18 h, cells were collected and analyzed for luciferase activity. Representative data of at least 3 independent experiments are shown.

    Journal: Journal of Translational Medicine

    Article Title: Differential immunomodulatory effects by Tripterygium wilfordii Hook f-derived refined extract PG27 and its purified component PG490 (triptolide) in human peripheral blood T cells: potential therapeutics for arthritis and possible mechanisms explaining in part Chinese herbal theory “Junn-Chenn-Zuou-SS”

    doi: 10.1186/1479-5876-11-294

    Figure Lengend Snippet: PG490 inhibited both NF-κB and AP-1 DNA-binding and transcriptional activity. T cells were pretreated with various concentrations of PG490 (A) for 2 h and then stimulated with TNF-α (B) or CD3/CD28 (C) for 6 h. The nuclear extracts were analyzed for both NF-κB and AP-1 DNA-binding activity by EMSA. In (D) , T cells were pretreated with PG490 for 2 h or left untreated, and then stimulated with TNF-α for various lengths of time as indicated. After washing, cell pellets were collected and cytoplasmic extracts were analyzed for the protein levels of IκBα and β-actin by Western blotting. In (E) , T cells were mixed together with pNF-κB-Luc or pAP-1-Luc reporter plasmids and the transfection procedures were performed as described for Figure 2 . After electroporation for 48 h, the cells were aliquoted equally and pretreated with various concentrations of PG490 for 2 h. After stimulation with TNF-α for another 18 h, cells were collected and analyzed for luciferase activity. Representative data of at least 3 independent experiments are shown.

    Article Snippet: Cell stimulation, cytokine determination, and cell survival measurement To activate T cells, the following stimuli were used: phorbol 12-myristate 13-acetate (PMA; Sigma) at 10 ng/mL, ionomycin (Sigma) at 1 μM, immobilized monoclonal antibody (mAb) anti-CD3 (OKT3; ATCC) at 10 μg/mL, soluble anti-CD28 mAb (Beckton Dickinson) at 1 μg/mL, and TNF-α at 10 ng/mL.

    Techniques: Binding Assay, Activity Assay, Western Blot, Transfection, Electroporation, Luciferase

    PG27 and PG490 down regulated MAPK activity. T cells at a concentration of 5 × 10 6 /mL were pretreated with various concentrations of PG27 (A and B) or PG490 (C and D) for 2 h and then stimulated with PMA + ionomycin for 30 min (A) , TNF-α for 10 min (B and C) or CD3/CD28 for 30 min (D) . Cell pellets were collected and total cell lysates were immunoprecipitated with anti-JNK, anti-p38 or anti-ERK antibodies. After sequential washes, the substrates (GST-c-Jun for JNK and MBP for both p38 and ERK) and [γ- 32 P]ATP were added individually. After the kinase reaction, the mixture was analyzed by SDS-PAGE. Representative data of at least 3 independent experiments are shown.

    Journal: Journal of Translational Medicine

    Article Title: Differential immunomodulatory effects by Tripterygium wilfordii Hook f-derived refined extract PG27 and its purified component PG490 (triptolide) in human peripheral blood T cells: potential therapeutics for arthritis and possible mechanisms explaining in part Chinese herbal theory “Junn-Chenn-Zuou-SS”

    doi: 10.1186/1479-5876-11-294

    Figure Lengend Snippet: PG27 and PG490 down regulated MAPK activity. T cells at a concentration of 5 × 10 6 /mL were pretreated with various concentrations of PG27 (A and B) or PG490 (C and D) for 2 h and then stimulated with PMA + ionomycin for 30 min (A) , TNF-α for 10 min (B and C) or CD3/CD28 for 30 min (D) . Cell pellets were collected and total cell lysates were immunoprecipitated with anti-JNK, anti-p38 or anti-ERK antibodies. After sequential washes, the substrates (GST-c-Jun for JNK and MBP for both p38 and ERK) and [γ- 32 P]ATP were added individually. After the kinase reaction, the mixture was analyzed by SDS-PAGE. Representative data of at least 3 independent experiments are shown.

    Article Snippet: Cell stimulation, cytokine determination, and cell survival measurement To activate T cells, the following stimuli were used: phorbol 12-myristate 13-acetate (PMA; Sigma) at 10 ng/mL, ionomycin (Sigma) at 1 μM, immobilized monoclonal antibody (mAb) anti-CD3 (OKT3; ATCC) at 10 μg/mL, soluble anti-CD28 mAb (Beckton Dickinson) at 1 μg/mL, and TNF-α at 10 ng/mL.

    Techniques: Activity Assay, Concentration Assay, Immunoprecipitation, SDS Page

    PG27 inhibited IL-2 production from activated T cells in a concentration-dependent manner. Human peripheral blood T cells at a concentration of 1 × 10 6 /mL were pretreated with various concentrations of PG27 for 2 h and then stimulated with PMA + ionomycin (shown as P + I) (A) or CD3/CD28 (B) for 24 h. The supernatants were collected for the determination of IL-2 concentrations. In parallel, cell survival in the various conditions was determined by trypan blue exclusion assays (C) . The results shown are from independent experiments. Each experiment was performed in triplicate using T cells from at least 3 different donors.

    Journal: Journal of Translational Medicine

    Article Title: Differential immunomodulatory effects by Tripterygium wilfordii Hook f-derived refined extract PG27 and its purified component PG490 (triptolide) in human peripheral blood T cells: potential therapeutics for arthritis and possible mechanisms explaining in part Chinese herbal theory “Junn-Chenn-Zuou-SS”

    doi: 10.1186/1479-5876-11-294

    Figure Lengend Snippet: PG27 inhibited IL-2 production from activated T cells in a concentration-dependent manner. Human peripheral blood T cells at a concentration of 1 × 10 6 /mL were pretreated with various concentrations of PG27 for 2 h and then stimulated with PMA + ionomycin (shown as P + I) (A) or CD3/CD28 (B) for 24 h. The supernatants were collected for the determination of IL-2 concentrations. In parallel, cell survival in the various conditions was determined by trypan blue exclusion assays (C) . The results shown are from independent experiments. Each experiment was performed in triplicate using T cells from at least 3 different donors.

    Article Snippet: Cell stimulation, cytokine determination, and cell survival measurement To activate T cells, the following stimuli were used: phorbol 12-myristate 13-acetate (PMA; Sigma) at 10 ng/mL, ionomycin (Sigma) at 1 μM, immobilized monoclonal antibody (mAb) anti-CD3 (OKT3; ATCC) at 10 μg/mL, soluble anti-CD28 mAb (Beckton Dickinson) at 1 μg/mL, and TNF-α at 10 ng/mL.

    Techniques: Concentration Assay

    Mitochondrial respiration, but not ATP production, promotes Akt/Foxo1 signaling in activated T cells ( A ) A schematic depicting electron transfer and disposition by cytosolic LDHA and mitochondrial electron transport chain (ETC) associated with NADH to NAD + conversion. ( B ) Naïve CD8 + T cells were isolated from Ldha fl/fl (wild-type, WT) and CD4 Cre Ldha fl/fl (knockout, KO) mice, and stimulated with 5 μg/ml anti-CD3, 2 μg/ml anti-CD28, and 100 U/ml IL-2 for 3 days. The amounts of NADH and NAD + were measured. The ratios of NAD + over NADH are plotted. ( C ) Measurements and quantifications of oxygen consumption rate (OCR) and spared respiratory capacity (SRC) of day-3 activated WT and KO CD8 + T cells. Sequential chemical treatments are indicated as shown in the graph. Oligo: oligomycin; Ant: antimycin; Rot: rotenone. (n=5 per genotype, mean ± SD) ( D ) A schematic of mitochondrial ETC, proton distribution, and ATP synthetase activity under the indicated treatment conditions. ( E ) Day-3 activated WT and KO CD8 + T cells were collected, and incubated in RPMI medium in the absence or presence of oligomycin and/or FCCP. T cells were subsequently re-stimulated with biotinylated anti-CD3 and anti-CD28 through streptavidin crosslinking, and immunoblotted for p-Akt (T308), Akt, p-Foxo1 (T24), p-Foxo1 (S256), Foxo1, LDHA, and β-Actin. Normalized expression of p-Akt (T308) to Akt, p-Foxo1 (T24) or p-Foxo1 (S256) to Foxo1 were marked. Unpaired t tests for the measurements between the two groups ( B and C ): **p

    Journal: bioRxiv

    Article Title: Glycolysis Fuels Phosphoinositide 3-Kinase Signaling to Bolster T Cell Immunity

    doi: 10.1101/2020.03.12.989707

    Figure Lengend Snippet: Mitochondrial respiration, but not ATP production, promotes Akt/Foxo1 signaling in activated T cells ( A ) A schematic depicting electron transfer and disposition by cytosolic LDHA and mitochondrial electron transport chain (ETC) associated with NADH to NAD + conversion. ( B ) Naïve CD8 + T cells were isolated from Ldha fl/fl (wild-type, WT) and CD4 Cre Ldha fl/fl (knockout, KO) mice, and stimulated with 5 μg/ml anti-CD3, 2 μg/ml anti-CD28, and 100 U/ml IL-2 for 3 days. The amounts of NADH and NAD + were measured. The ratios of NAD + over NADH are plotted. ( C ) Measurements and quantifications of oxygen consumption rate (OCR) and spared respiratory capacity (SRC) of day-3 activated WT and KO CD8 + T cells. Sequential chemical treatments are indicated as shown in the graph. Oligo: oligomycin; Ant: antimycin; Rot: rotenone. (n=5 per genotype, mean ± SD) ( D ) A schematic of mitochondrial ETC, proton distribution, and ATP synthetase activity under the indicated treatment conditions. ( E ) Day-3 activated WT and KO CD8 + T cells were collected, and incubated in RPMI medium in the absence or presence of oligomycin and/or FCCP. T cells were subsequently re-stimulated with biotinylated anti-CD3 and anti-CD28 through streptavidin crosslinking, and immunoblotted for p-Akt (T308), Akt, p-Foxo1 (T24), p-Foxo1 (S256), Foxo1, LDHA, and β-Actin. Normalized expression of p-Akt (T308) to Akt, p-Foxo1 (T24) or p-Foxo1 (S256) to Foxo1 were marked. Unpaired t tests for the measurements between the two groups ( B and C ): **p

    Article Snippet: Antibodies for in vitro T cell culture: Monoclonal anti-mouse CD28-biotin (Clone 37.51; Cat. # 13-0281-81), monoclonal anti-mouse and CD3ε-biotin (Clone 145-2C11; Functional Grade; Cat. # 36-0031-85) were purchased from Invitrogen.

    Techniques: Isolation, Knock-Out, Mouse Assay, Activity Assay, Incubation, Expressing

    Schematics of cell sorting and cell culture, and characterization of anti-CD3-induced TCR proximal signaling in the absence or presence of PI3K inhibitor ( A ) A schematic of flow cytometry sorting strategy for naïve and H-2K b -OVA + CD8 + T cells from wild-type mice infected with LM-OVA for 7 days. ( B ) A schematic of in vitro CD8 + T cell culture with various doses of anti-CD3 and the PI3K inhibitor, CAL-101 in the presence of 2 μg/ml anti-CD28, and 100 U/ml IL-2. ( C ) Naïve CD8 + T cells were stimulated with increasing doses of anti-CD3 in the presence of 2 μg/ml anti-CD28, and 100 U/ml IL-2 for 24 hours. Cellular extracts were immunoblotted for p-Zap70 (T319), Zap70, p-LAT (T191), LAT, and β-Actin. Normalized expression of p-Zap70 (T319) to Zap70 and p-LAT (T191) to LAT were marked. ( D ) Naïve CD8 + T cells were stimulated with 5 μg/ml anti-CD3, 2 μg/ml anti-CD28, and 100 U/ml IL-2 in the absence or presence of increasing doses of the PI3K inhibitor, CAL-101, for 24 hours. Cellular extracts were immunoblotted for p-Zap70 (T319), Zap70, p-LAT (T191), LAT, and β-Actin. Normalized expression of p-Zap70 (T319) to Zap70 and p-LAT (T191) to LAT were marked.

    Journal: bioRxiv

    Article Title: Glycolysis Fuels Phosphoinositide 3-Kinase Signaling to Bolster T Cell Immunity

    doi: 10.1101/2020.03.12.989707

    Figure Lengend Snippet: Schematics of cell sorting and cell culture, and characterization of anti-CD3-induced TCR proximal signaling in the absence or presence of PI3K inhibitor ( A ) A schematic of flow cytometry sorting strategy for naïve and H-2K b -OVA + CD8 + T cells from wild-type mice infected with LM-OVA for 7 days. ( B ) A schematic of in vitro CD8 + T cell culture with various doses of anti-CD3 and the PI3K inhibitor, CAL-101 in the presence of 2 μg/ml anti-CD28, and 100 U/ml IL-2. ( C ) Naïve CD8 + T cells were stimulated with increasing doses of anti-CD3 in the presence of 2 μg/ml anti-CD28, and 100 U/ml IL-2 for 24 hours. Cellular extracts were immunoblotted for p-Zap70 (T319), Zap70, p-LAT (T191), LAT, and β-Actin. Normalized expression of p-Zap70 (T319) to Zap70 and p-LAT (T191) to LAT were marked. ( D ) Naïve CD8 + T cells were stimulated with 5 μg/ml anti-CD3, 2 μg/ml anti-CD28, and 100 U/ml IL-2 in the absence or presence of increasing doses of the PI3K inhibitor, CAL-101, for 24 hours. Cellular extracts were immunoblotted for p-Zap70 (T319), Zap70, p-LAT (T191), LAT, and β-Actin. Normalized expression of p-Zap70 (T319) to Zap70 and p-LAT (T191) to LAT were marked.

    Article Snippet: Antibodies for in vitro T cell culture: Monoclonal anti-mouse CD28-biotin (Clone 37.51; Cat. # 13-0281-81), monoclonal anti-mouse and CD3ε-biotin (Clone 145-2C11; Functional Grade; Cat. # 36-0031-85) were purchased from Invitrogen.

    Techniques: FACS, Cell Culture, Flow Cytometry, Mouse Assay, Infection, In Vitro, Expressing

    Mitochondrion electron transport chain (ETC)-mediated redox control promotes glycolysis and Akt/Foxo1 signaling ( A ) Naïve CD8 + T cells were isolated from Ldha fl/fl (wild-type, WT) and CD4 Cre Ldha fl/fl (knockout, KO) mice, and stimulated with 5 μg/ml anti-CD3, 2 μg/ml anti-CD28, and 100 U/ml IL-2 for 3 days before subject to measurements of extracellular acidification rate (ECAR) with a Seahorse Glycolysis stress test kit. Sequential chemical treatments are indicated as shown in the graph. Glu: glucose; Oligo: oligomycin; 2-DG: 2-deoxyglucose. (n=5 per genotype, mean ± SD). ( B ) OCR over ECAR ratios in WT and KO T cells (n=5 per genotype, mean ± SD). ( C ) A schematic of glycolysis-associated ATP generation via substrate level phosphorylation and mitochondrial ATP generation via oxidative phosphorylation as well as the redox regulatory mechanisms under the indicated treatment conditions: T cell medium (TCM), Oligomycin, FCCP, and Oligomycin + FCCP. ( D ) Day-3 activated WT T cells were collected, and incubated in RPMI medium in the absence or presence of oligomycin (Oligo) and/or FCCP and/or Rotenon (Rot) plus Antimycin (Ant). T cells were subsequently re-stimulated with biotinylated anti-CD3 and anti-CD28 through streptavidin crosslinking, and immunoblotted for p-Akt (T308), Akt, p-Foxo1 (T24), p-Foxo1 (S256), Foxo1, LDHA, and β-Actin. Normalized expression of p-Akt (T308) to Akt, p-Foxo1 (T24) or p-Foxo1 (S256) to Foxo1 were marked. ( E ) ECAR measurement for day-3 activated WT T cells treated with the indicated drugs in Seahorse Mitostress tests (n=5 per genotype, mean ± SD). Unpaired t tests for the measurements between the two groups ( A and B ): ****p

    Journal: bioRxiv

    Article Title: Glycolysis Fuels Phosphoinositide 3-Kinase Signaling to Bolster T Cell Immunity

    doi: 10.1101/2020.03.12.989707

    Figure Lengend Snippet: Mitochondrion electron transport chain (ETC)-mediated redox control promotes glycolysis and Akt/Foxo1 signaling ( A ) Naïve CD8 + T cells were isolated from Ldha fl/fl (wild-type, WT) and CD4 Cre Ldha fl/fl (knockout, KO) mice, and stimulated with 5 μg/ml anti-CD3, 2 μg/ml anti-CD28, and 100 U/ml IL-2 for 3 days before subject to measurements of extracellular acidification rate (ECAR) with a Seahorse Glycolysis stress test kit. Sequential chemical treatments are indicated as shown in the graph. Glu: glucose; Oligo: oligomycin; 2-DG: 2-deoxyglucose. (n=5 per genotype, mean ± SD). ( B ) OCR over ECAR ratios in WT and KO T cells (n=5 per genotype, mean ± SD). ( C ) A schematic of glycolysis-associated ATP generation via substrate level phosphorylation and mitochondrial ATP generation via oxidative phosphorylation as well as the redox regulatory mechanisms under the indicated treatment conditions: T cell medium (TCM), Oligomycin, FCCP, and Oligomycin + FCCP. ( D ) Day-3 activated WT T cells were collected, and incubated in RPMI medium in the absence or presence of oligomycin (Oligo) and/or FCCP and/or Rotenon (Rot) plus Antimycin (Ant). T cells were subsequently re-stimulated with biotinylated anti-CD3 and anti-CD28 through streptavidin crosslinking, and immunoblotted for p-Akt (T308), Akt, p-Foxo1 (T24), p-Foxo1 (S256), Foxo1, LDHA, and β-Actin. Normalized expression of p-Akt (T308) to Akt, p-Foxo1 (T24) or p-Foxo1 (S256) to Foxo1 were marked. ( E ) ECAR measurement for day-3 activated WT T cells treated with the indicated drugs in Seahorse Mitostress tests (n=5 per genotype, mean ± SD). Unpaired t tests for the measurements between the two groups ( A and B ): ****p

    Article Snippet: Antibodies for in vitro T cell culture: Monoclonal anti-mouse CD28-biotin (Clone 37.51; Cat. # 13-0281-81), monoclonal anti-mouse and CD3ε-biotin (Clone 145-2C11; Functional Grade; Cat. # 36-0031-85) were purchased from Invitrogen.

    Techniques: Isolation, Knock-Out, Mouse Assay, Incubation, Expressing

    LDHA deficiency causes reduced ATP production ( A ) Naïve CD8 + T cells were isolated from Ldha fl/fl (wild-type, WT) and CD4 Cre Ldha fl/fl (knockout, KO) mice, and stimulated with 5 μg/ml anti-CD3, 2 μg/ml anti-CD28, and 100 U/ml IL-2 for 3 days. Cellular ATP levels were measured (n=3 per genotype, mean ± SD). ( B ) A schematic depicting a streptolysin-O (SLO)-based ATP delivery system. Unpaired t test for the measurement between the two groups ( A ): ***p

    Journal: bioRxiv

    Article Title: Glycolysis Fuels Phosphoinositide 3-Kinase Signaling to Bolster T Cell Immunity

    doi: 10.1101/2020.03.12.989707

    Figure Lengend Snippet: LDHA deficiency causes reduced ATP production ( A ) Naïve CD8 + T cells were isolated from Ldha fl/fl (wild-type, WT) and CD4 Cre Ldha fl/fl (knockout, KO) mice, and stimulated with 5 μg/ml anti-CD3, 2 μg/ml anti-CD28, and 100 U/ml IL-2 for 3 days. Cellular ATP levels were measured (n=3 per genotype, mean ± SD). ( B ) A schematic depicting a streptolysin-O (SLO)-based ATP delivery system. Unpaired t test for the measurement between the two groups ( A ): ***p

    Article Snippet: Antibodies for in vitro T cell culture: Monoclonal anti-mouse CD28-biotin (Clone 37.51; Cat. # 13-0281-81), monoclonal anti-mouse and CD3ε-biotin (Clone 145-2C11; Functional Grade; Cat. # 36-0031-85) were purchased from Invitrogen.

    Techniques: Isolation, Knock-Out, Mouse Assay

    Mitochondrial ATP generation supports Akt/Foxo1 signaling in naïve T cells ( A ) Naïve Ldha fl/fl (wild-type, WT) and CD4 Cre Ldha fl/fl (knockout, KO) CD8 + T cells were harvested for measurements of extracellular acidification rate (ECAR) with a Seahorse Glycolysis stress test kit. Sequential chemical treatments are indicated as shown in the graph. Glu: glucose; Oligo: oligomycin; 2-DG: 2-deoxyglucose. (n=4 per genotype, mean ± SD). ( B ) Naïve WT and KO T cells were measured for oxygen consumption rate (OCR) and spared respiratory capacity (SRC) with a Seahorse Mitostress test kit. Sequential chemical treatments are indicated as shown in the graph. Oligo: oligomycin; Ant: antimycin; Rot: rotenone. (n=4 per genotype, mean ± SD). ( C ) OCR over ECAR ratios in WT and KO T cells (n=4 per genotype, mean ± SD). ( D ) Naïve WT and KO T cells were incubated in RPMI medium in the absence or presence of oligomycin and/or FCCP. T cells were subsequently re-stimulated with biotinylated anti-CD3 and anti-CD28 through streptavidin crosslinking, and immunoblotted for p-Akt (T308), Akt, p-Foxo1 (T24), p-Foxo1 (S256), Foxo1, LDHA, and b-Actin. Normalized expression of p-Akt (T308) to Akt, p-Foxo1 (T24) or p-Foxo1 (S256) to Foxo1 were marked. Unpaired t tests for the measurements between the two groups ( A to C ), ns: not-significant.

    Journal: bioRxiv

    Article Title: Glycolysis Fuels Phosphoinositide 3-Kinase Signaling to Bolster T Cell Immunity

    doi: 10.1101/2020.03.12.989707

    Figure Lengend Snippet: Mitochondrial ATP generation supports Akt/Foxo1 signaling in naïve T cells ( A ) Naïve Ldha fl/fl (wild-type, WT) and CD4 Cre Ldha fl/fl (knockout, KO) CD8 + T cells were harvested for measurements of extracellular acidification rate (ECAR) with a Seahorse Glycolysis stress test kit. Sequential chemical treatments are indicated as shown in the graph. Glu: glucose; Oligo: oligomycin; 2-DG: 2-deoxyglucose. (n=4 per genotype, mean ± SD). ( B ) Naïve WT and KO T cells were measured for oxygen consumption rate (OCR) and spared respiratory capacity (SRC) with a Seahorse Mitostress test kit. Sequential chemical treatments are indicated as shown in the graph. Oligo: oligomycin; Ant: antimycin; Rot: rotenone. (n=4 per genotype, mean ± SD). ( C ) OCR over ECAR ratios in WT and KO T cells (n=4 per genotype, mean ± SD). ( D ) Naïve WT and KO T cells were incubated in RPMI medium in the absence or presence of oligomycin and/or FCCP. T cells were subsequently re-stimulated with biotinylated anti-CD3 and anti-CD28 through streptavidin crosslinking, and immunoblotted for p-Akt (T308), Akt, p-Foxo1 (T24), p-Foxo1 (S256), Foxo1, LDHA, and b-Actin. Normalized expression of p-Akt (T308) to Akt, p-Foxo1 (T24) or p-Foxo1 (S256) to Foxo1 were marked. Unpaired t tests for the measurements between the two groups ( A to C ), ns: not-significant.

    Article Snippet: Antibodies for in vitro T cell culture: Monoclonal anti-mouse CD28-biotin (Clone 37.51; Cat. # 13-0281-81), monoclonal anti-mouse and CD3ε-biotin (Clone 145-2C11; Functional Grade; Cat. # 36-0031-85) were purchased from Invitrogen.

    Techniques: Knock-Out, Incubation, Expressing

    Reduced glycolytic ATP production accounts for defective PI3K/Akt/Foxo1 signaling in LDHA-deficient T cells following antigen stimulation ( A ) A schematic depicting how naïve CD8 + T cells were treated for assessment of kinase signaling and PIP3 generation. ( B ) Naïve CD8 + T cells were isolated from Ldha fl/fl (wild-type, WT) and CD4 Cre Ldha fl/fl (knockout, KO) mice, and stimulated with 5 μg/ml anti-CD3, 2 μg/ml anti-CD28, and 100 U/ml IL-2 for 3 days. Day-3 activated T cells and freshly isolated naïve WT and KO CD8 + T cells were left untreated, or labeled with biotinylated anti-CD3 and anti-CD28 and re-stimulated by streptavidin crosslinking. Cell lysates were prepared and immunoblotted for p-Akt (T308), Akt, p-Foxo1 (T24), p-Foxo1 (S256), Foxo1, LDHA, p-Zap70 (T319), Zap70, p-LAT (T191), LAT, p-AMPKα (T172), AMPKα, and β-Actin. Normalized expression of p-Akt (T308) to Akt, p-Foxo1 (T24) or p-Foxo1 (S256) to Foxo1, p-Zap70 (T319) to Zap70, p-LAT (T191) to LAT, and p-AMPKα (T172) to AMPKα were marked. ( C ) Day-3 activated WT and KO CD8 + T cells were collected, rested in RPMI medium for 3 hours, and incubated in the absence or presence of 10 mM ATP and/or 250 U/ml Streptolysin O (SLO). T cells were subsequently re-stimulated with biotinylated anti-CD3 and anti-CD28 through streptavidin crosslinking, and immunoblotted for p-Akt (T308), Akt, p-Foxo1 (T24), p-Foxo1 (S256), Foxo1, LDHA, and β-Actin. Normalized expression of p-Akt (T308) to Akt, p-Foxo1 (T24) or p-Foxo1 (S256) to Foxo1 were marked. ( D ) Representative immunofluorescent images of PIP3 and its quantification in WT and KO CD8 + T cells after receiving the indicated treatments as described in ( C ). Unpaired t tests for the measurements between the two groups ( D ): **p

    Journal: bioRxiv

    Article Title: Glycolysis Fuels Phosphoinositide 3-Kinase Signaling to Bolster T Cell Immunity

    doi: 10.1101/2020.03.12.989707

    Figure Lengend Snippet: Reduced glycolytic ATP production accounts for defective PI3K/Akt/Foxo1 signaling in LDHA-deficient T cells following antigen stimulation ( A ) A schematic depicting how naïve CD8 + T cells were treated for assessment of kinase signaling and PIP3 generation. ( B ) Naïve CD8 + T cells were isolated from Ldha fl/fl (wild-type, WT) and CD4 Cre Ldha fl/fl (knockout, KO) mice, and stimulated with 5 μg/ml anti-CD3, 2 μg/ml anti-CD28, and 100 U/ml IL-2 for 3 days. Day-3 activated T cells and freshly isolated naïve WT and KO CD8 + T cells were left untreated, or labeled with biotinylated anti-CD3 and anti-CD28 and re-stimulated by streptavidin crosslinking. Cell lysates were prepared and immunoblotted for p-Akt (T308), Akt, p-Foxo1 (T24), p-Foxo1 (S256), Foxo1, LDHA, p-Zap70 (T319), Zap70, p-LAT (T191), LAT, p-AMPKα (T172), AMPKα, and β-Actin. Normalized expression of p-Akt (T308) to Akt, p-Foxo1 (T24) or p-Foxo1 (S256) to Foxo1, p-Zap70 (T319) to Zap70, p-LAT (T191) to LAT, and p-AMPKα (T172) to AMPKα were marked. ( C ) Day-3 activated WT and KO CD8 + T cells were collected, rested in RPMI medium for 3 hours, and incubated in the absence or presence of 10 mM ATP and/or 250 U/ml Streptolysin O (SLO). T cells were subsequently re-stimulated with biotinylated anti-CD3 and anti-CD28 through streptavidin crosslinking, and immunoblotted for p-Akt (T308), Akt, p-Foxo1 (T24), p-Foxo1 (S256), Foxo1, LDHA, and β-Actin. Normalized expression of p-Akt (T308) to Akt, p-Foxo1 (T24) or p-Foxo1 (S256) to Foxo1 were marked. ( D ) Representative immunofluorescent images of PIP3 and its quantification in WT and KO CD8 + T cells after receiving the indicated treatments as described in ( C ). Unpaired t tests for the measurements between the two groups ( D ): **p

    Article Snippet: Antibodies for in vitro T cell culture: Monoclonal anti-mouse CD28-biotin (Clone 37.51; Cat. # 13-0281-81), monoclonal anti-mouse and CD3ε-biotin (Clone 145-2C11; Functional Grade; Cat. # 36-0031-85) were purchased from Invitrogen.

    Techniques: Isolation, Knock-Out, Mouse Assay, Labeling, Expressing, Incubation

    Single-CD28 stimulated Treg reveal a highly demethylated FOXP3 gene and profound suppressor function. ( a ) FACS-sorted human Treg were stimulated with soluble CD28 mAb, plate bound CD3 mAb or both (anti-CD3+CD28) in the presence of rhIL-2 for 7-days. Thereafter, cells were harvested and the demethylation status of FOXP3 gene was analyzed using bisulfate sequencing. n = 6–7. ( b ) CFSE-labeled FACS-sorted Treg were stimulated as described above. The divided cells (CFSE low population) were re-sorted (left panel) at day 7 of the cultures, and subsequently their suppressive function was analyzed in a co-culture suppression assay. Overlay histograms show the inhibition of responder T cells (Tresp) proliferation following the addition of graded doses of Treg. Numbers indicate the percentage of divided responder T cells. Grey line: stimulated Tresp, Black line: co-cultured with Treg of interest. The ratio of Treg:Tresp are indicated on the top. Representative experiment of n = 3 individual experiments conducted with cells obtained from different donors are shown. ( c ) Intracellular staining of cytokine IL-17A and IFNγ after additional stimulation with PMA, ionomycin, and Brefeldin-A for 4 hours. Numbers within the quadrant indicate the percentage of positive cells. Dotplots show a representative experiment of n = 6–10 individuals as shown in the cumulative data graph (right panel). Kruskal-Wallis followed by Dunns post-hoc test was used for statistical analysis ( a,c ). *P

    Journal: Scientific Reports

    Article Title: Single CD28 stimulation induces stable and polyclonal expansion of human regulatory T cells

    doi: 10.1038/srep43003

    Figure Lengend Snippet: Single-CD28 stimulated Treg reveal a highly demethylated FOXP3 gene and profound suppressor function. ( a ) FACS-sorted human Treg were stimulated with soluble CD28 mAb, plate bound CD3 mAb or both (anti-CD3+CD28) in the presence of rhIL-2 for 7-days. Thereafter, cells were harvested and the demethylation status of FOXP3 gene was analyzed using bisulfate sequencing. n = 6–7. ( b ) CFSE-labeled FACS-sorted Treg were stimulated as described above. The divided cells (CFSE low population) were re-sorted (left panel) at day 7 of the cultures, and subsequently their suppressive function was analyzed in a co-culture suppression assay. Overlay histograms show the inhibition of responder T cells (Tresp) proliferation following the addition of graded doses of Treg. Numbers indicate the percentage of divided responder T cells. Grey line: stimulated Tresp, Black line: co-cultured with Treg of interest. The ratio of Treg:Tresp are indicated on the top. Representative experiment of n = 3 individual experiments conducted with cells obtained from different donors are shown. ( c ) Intracellular staining of cytokine IL-17A and IFNγ after additional stimulation with PMA, ionomycin, and Brefeldin-A for 4 hours. Numbers within the quadrant indicate the percentage of positive cells. Dotplots show a representative experiment of n = 6–10 individuals as shown in the cumulative data graph (right panel). Kruskal-Wallis followed by Dunns post-hoc test was used for statistical analysis ( a,c ). *P

    Article Snippet: Cell culture Treg were stimulated with rhIL-2 (25 U/mL) alone as control, or together with either soluble CD28 agonist mAb (1 μg/mL, Clone L293, Cat# 348040; BD Bioscience), plate-bound CD3 mAb (5 μg/mL, Clone UCHT1, BD Bioscience), plate-bound CD3 (5 μg/mL) plus soluble CD28 mAb (1μg/mL), CD28-superagonist ANC28.1 (1 μg/mL, Clone ANC28.1/5D10, Cat# 177–820, preservative free; Ancell, Bayport, USA), or anti-CD3/anti-CD28 mAb-coated microbeads (Invitrogen by Life technologies, Bleiswijk, The Netherlands) used in a 1:2 bead-to-cell ratio.

    Techniques: FACS, Sequencing, Labeling, Co-Culture Assay, Suppression Assay, Inhibition, Cell Culture, Staining

    Single-CD28 stimulation induces Treg proliferation and promotes high level FOXP3 expression. Flow cytometry of FACS-sorted human Treg (CD4+CD25 high ) that were labeled with CFSE and stimulated with soluble CD28 mAb, plate bound CD3 mAb or both (Anti-CD3+CD28 ) in the presence of rhIL-2. As a control Treg cultured in the presence of rhIL-2 only were included. Cell division indicated by the dilution of CFSE ( a ), intracellular expression of FOXP3 ( b ) and active caspase 3 ( c ) were determined at day 7 of the cultures. Numbers within the histograms indicate the percentage of divided cells ( a ) and numbers within the quadrant show the percentage of positive cells ( b,c ). Cumulative data of Treg proliferation ( a , right panel), the median fluorescence intensity (MFI) of FOXP3 ( b , right panel), and the percentage of apoptotic cells ( c ) right panel) are also shown. Bar in cumulative data indicates the mean value. Kruskal-Wallis followed by Dunns post-hoc test ( a ) n = 3; b, n = 3) and Wilcoxon signed-rank test ( c , n = 5) were used for statistical analysis. *P

    Journal: Scientific Reports

    Article Title: Single CD28 stimulation induces stable and polyclonal expansion of human regulatory T cells

    doi: 10.1038/srep43003

    Figure Lengend Snippet: Single-CD28 stimulation induces Treg proliferation and promotes high level FOXP3 expression. Flow cytometry of FACS-sorted human Treg (CD4+CD25 high ) that were labeled with CFSE and stimulated with soluble CD28 mAb, plate bound CD3 mAb or both (Anti-CD3+CD28 ) in the presence of rhIL-2. As a control Treg cultured in the presence of rhIL-2 only were included. Cell division indicated by the dilution of CFSE ( a ), intracellular expression of FOXP3 ( b ) and active caspase 3 ( c ) were determined at day 7 of the cultures. Numbers within the histograms indicate the percentage of divided cells ( a ) and numbers within the quadrant show the percentage of positive cells ( b,c ). Cumulative data of Treg proliferation ( a , right panel), the median fluorescence intensity (MFI) of FOXP3 ( b , right panel), and the percentage of apoptotic cells ( c ) right panel) are also shown. Bar in cumulative data indicates the mean value. Kruskal-Wallis followed by Dunns post-hoc test ( a ) n = 3; b, n = 3) and Wilcoxon signed-rank test ( c , n = 5) were used for statistical analysis. *P

    Article Snippet: Cell culture Treg were stimulated with rhIL-2 (25 U/mL) alone as control, or together with either soluble CD28 agonist mAb (1 μg/mL, Clone L293, Cat# 348040; BD Bioscience), plate-bound CD3 mAb (5 μg/mL, Clone UCHT1, BD Bioscience), plate-bound CD3 (5 μg/mL) plus soluble CD28 mAb (1μg/mL), CD28-superagonist ANC28.1 (1 μg/mL, Clone ANC28.1/5D10, Cat# 177–820, preservative free; Ancell, Bayport, USA), or anti-CD3/anti-CD28 mAb-coated microbeads (Invitrogen by Life technologies, Bleiswijk, The Netherlands) used in a 1:2 bead-to-cell ratio.

    Techniques: Expressing, Flow Cytometry, Cytometry, FACS, Labeling, Cell Culture, Fluorescence

    CD28-superagonist drives polyclonal Treg proliferation and enhances expression of FOXP3, Helios and CTLA-4. Flow cytometry of FACS-sorted human Treg that were stimulated with soluble CD28-superagonist(CD28 SA) or anti-CD3/CD28 mAb coated microbeads (anti-CD3/CD28 beads) in the presence of rhIL-2. As a control Treg cultured in the presence of rhIL-2 only were included. ( a ) Fold expansion of Treg during 7 days of culture. n = 6–9. ( b ) TcR Vβ repertoire analysis following the indicated stimuli (see legend). One experiment of two similar ones conducted with cells obtained from different donors is shown. ( c ) Intracellular expression of FOXP3, Helios and CTLA-4 by Treg after 7 days stimulation (X-axis). Numbers within the histogram show the median fluorescence intensity (MFI). n = 6. ( d ) CFSE-based co-culture suppression assays showing a representative experiment of two independent ones. The ratio of Treg:Tresp are indicated at the top. Numbers indicate the percentage of divided responder T cells. Grey line: stimulated Tresp; Black line: co-cultured with CD28-superagonist or anti-CD3/CD28 beads expanded Treg. ( e ) Demethylation status of FOXP3 gene was analyzed using bisulfate sequencing. n = 3. ( f ) Intracellular staining of IL-17A and IFNγ at day 7 of the cultures. Numbers within the quadrant indicate the percentage of positive cells. Dot plots show a representative experiment of five individual ones conducted with cells obtained from different donors as shown in the cumulative data graphs; n = 5. ( g ) Measurement of IL-17A and IFNγ production in culture supernatants using luminex at day 7 of the cultures. n = 11 (for IL-17A) and n = 7 (for IFNγ). Bar in cumulative data indicated the mean value. Kruskal-Wallis followed by Dunns post-hoc test was used for statistical analysis ( a,c,e–g ). *P

    Journal: Scientific Reports

    Article Title: Single CD28 stimulation induces stable and polyclonal expansion of human regulatory T cells

    doi: 10.1038/srep43003

    Figure Lengend Snippet: CD28-superagonist drives polyclonal Treg proliferation and enhances expression of FOXP3, Helios and CTLA-4. Flow cytometry of FACS-sorted human Treg that were stimulated with soluble CD28-superagonist(CD28 SA) or anti-CD3/CD28 mAb coated microbeads (anti-CD3/CD28 beads) in the presence of rhIL-2. As a control Treg cultured in the presence of rhIL-2 only were included. ( a ) Fold expansion of Treg during 7 days of culture. n = 6–9. ( b ) TcR Vβ repertoire analysis following the indicated stimuli (see legend). One experiment of two similar ones conducted with cells obtained from different donors is shown. ( c ) Intracellular expression of FOXP3, Helios and CTLA-4 by Treg after 7 days stimulation (X-axis). Numbers within the histogram show the median fluorescence intensity (MFI). n = 6. ( d ) CFSE-based co-culture suppression assays showing a representative experiment of two independent ones. The ratio of Treg:Tresp are indicated at the top. Numbers indicate the percentage of divided responder T cells. Grey line: stimulated Tresp; Black line: co-cultured with CD28-superagonist or anti-CD3/CD28 beads expanded Treg. ( e ) Demethylation status of FOXP3 gene was analyzed using bisulfate sequencing. n = 3. ( f ) Intracellular staining of IL-17A and IFNγ at day 7 of the cultures. Numbers within the quadrant indicate the percentage of positive cells. Dot plots show a representative experiment of five individual ones conducted with cells obtained from different donors as shown in the cumulative data graphs; n = 5. ( g ) Measurement of IL-17A and IFNγ production in culture supernatants using luminex at day 7 of the cultures. n = 11 (for IL-17A) and n = 7 (for IFNγ). Bar in cumulative data indicated the mean value. Kruskal-Wallis followed by Dunns post-hoc test was used for statistical analysis ( a,c,e–g ). *P

    Article Snippet: Cell culture Treg were stimulated with rhIL-2 (25 U/mL) alone as control, or together with either soluble CD28 agonist mAb (1 μg/mL, Clone L293, Cat# 348040; BD Bioscience), plate-bound CD3 mAb (5 μg/mL, Clone UCHT1, BD Bioscience), plate-bound CD3 (5 μg/mL) plus soluble CD28 mAb (1μg/mL), CD28-superagonist ANC28.1 (1 μg/mL, Clone ANC28.1/5D10, Cat# 177–820, preservative free; Ancell, Bayport, USA), or anti-CD3/anti-CD28 mAb-coated microbeads (Invitrogen by Life technologies, Bleiswijk, The Netherlands) used in a 1:2 bead-to-cell ratio.

    Techniques: Expressing, Flow Cytometry, Cytometry, FACS, Cell Culture, Fluorescence, Co-Culture Assay, Sequencing, Staining, Luminex

    CD28-superagonist stimulation drives both naïve and memory Treg proliferation. Flow cytometry of FACS-sorted human naïve (CD4+CD45RA+CD25+) and memory (CD4+CD45RA-CD25 high ) Treg that were stimulated with CD28-superagonist(CD28 SA) or anti-CD3/CD28 mAb coated-microbeads in the presence of rhIL-2. As a control Treg cultured in the presence of rhIL-2 only were included. Dotplots show intracellular staining of KI67 ( a ), isoform switch of CD45RA and CD45RO ( b ), and intracellular expression of FOXP3 and Helios ( c ). Numbers within ( a ) and ( b ) indicate the percentage of positive cells, and numbers within the histogram ( c ) indicate the MFI values. A representative example of n = 4 individual experiments conducted with cells obtained from different donors is shown. Aggregate data are shown in the figures at right side of the flow cytometry plots. Kruskal-Wallis followed by Dunns post-hoc test ( a,b ) and Wilcoxon signed-rank test ( c ) were used for statistical analysis. *P

    Journal: Scientific Reports

    Article Title: Single CD28 stimulation induces stable and polyclonal expansion of human regulatory T cells

    doi: 10.1038/srep43003

    Figure Lengend Snippet: CD28-superagonist stimulation drives both naïve and memory Treg proliferation. Flow cytometry of FACS-sorted human naïve (CD4+CD45RA+CD25+) and memory (CD4+CD45RA-CD25 high ) Treg that were stimulated with CD28-superagonist(CD28 SA) or anti-CD3/CD28 mAb coated-microbeads in the presence of rhIL-2. As a control Treg cultured in the presence of rhIL-2 only were included. Dotplots show intracellular staining of KI67 ( a ), isoform switch of CD45RA and CD45RO ( b ), and intracellular expression of FOXP3 and Helios ( c ). Numbers within ( a ) and ( b ) indicate the percentage of positive cells, and numbers within the histogram ( c ) indicate the MFI values. A representative example of n = 4 individual experiments conducted with cells obtained from different donors is shown. Aggregate data are shown in the figures at right side of the flow cytometry plots. Kruskal-Wallis followed by Dunns post-hoc test ( a,b ) and Wilcoxon signed-rank test ( c ) were used for statistical analysis. *P

    Article Snippet: Cell culture Treg were stimulated with rhIL-2 (25 U/mL) alone as control, or together with either soluble CD28 agonist mAb (1 μg/mL, Clone L293, Cat# 348040; BD Bioscience), plate-bound CD3 mAb (5 μg/mL, Clone UCHT1, BD Bioscience), plate-bound CD3 (5 μg/mL) plus soluble CD28 mAb (1μg/mL), CD28-superagonist ANC28.1 (1 μg/mL, Clone ANC28.1/5D10, Cat# 177–820, preservative free; Ancell, Bayport, USA), or anti-CD3/anti-CD28 mAb-coated microbeads (Invitrogen by Life technologies, Bleiswijk, The Netherlands) used in a 1:2 bead-to-cell ratio.

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

    PI3K and mTOR differentially regulate CD28-superagonist mediated Treg stability. Flow cytometry of FACS-sorted human CD4+CD25 high Treg that were stimulated with CD28-superagonist(CD28 SA) or anti-CD3/CD28 mAb coated-microbeads and exogenously added rhIL-2. The cells were pretreated for 30 minutes with the PI3K inhibitor wortmannin, or mTOR inhibitor rapamycin as indicated on the top. ( a ) Intracellular FOXP3 expression and ( b ) intracellular IL-17A and IFNγ staining at day 7 of the cultures. Numbers within the histogram indicate the MFI of FOXP3 and the percentage of positive cells, respectively ( a ), and numbers within the quadrants indicate the percentage of cytokine producing cells. A representative example of n = 5 ( a,b ) individual experiments conducted with cells obtained from different donors is shown. Aggregate data are shown in the figures bellow the flow cytometry plots. Wilcoxon signed-rank test was used for the comparison between medium group and inhibitor-treated group. *P

    Journal: Scientific Reports

    Article Title: Single CD28 stimulation induces stable and polyclonal expansion of human regulatory T cells

    doi: 10.1038/srep43003

    Figure Lengend Snippet: PI3K and mTOR differentially regulate CD28-superagonist mediated Treg stability. Flow cytometry of FACS-sorted human CD4+CD25 high Treg that were stimulated with CD28-superagonist(CD28 SA) or anti-CD3/CD28 mAb coated-microbeads and exogenously added rhIL-2. The cells were pretreated for 30 minutes with the PI3K inhibitor wortmannin, or mTOR inhibitor rapamycin as indicated on the top. ( a ) Intracellular FOXP3 expression and ( b ) intracellular IL-17A and IFNγ staining at day 7 of the cultures. Numbers within the histogram indicate the MFI of FOXP3 and the percentage of positive cells, respectively ( a ), and numbers within the quadrants indicate the percentage of cytokine producing cells. A representative example of n = 5 ( a,b ) individual experiments conducted with cells obtained from different donors is shown. Aggregate data are shown in the figures bellow the flow cytometry plots. Wilcoxon signed-rank test was used for the comparison between medium group and inhibitor-treated group. *P

    Article Snippet: Cell culture Treg were stimulated with rhIL-2 (25 U/mL) alone as control, or together with either soluble CD28 agonist mAb (1 μg/mL, Clone L293, Cat# 348040; BD Bioscience), plate-bound CD3 mAb (5 μg/mL, Clone UCHT1, BD Bioscience), plate-bound CD3 (5 μg/mL) plus soluble CD28 mAb (1μg/mL), CD28-superagonist ANC28.1 (1 μg/mL, Clone ANC28.1/5D10, Cat# 177–820, preservative free; Ancell, Bayport, USA), or anti-CD3/anti-CD28 mAb-coated microbeads (Invitrogen by Life technologies, Bleiswijk, The Netherlands) used in a 1:2 bead-to-cell ratio.

    Techniques: Flow Cytometry, Cytometry, FACS, Expressing, Staining

    p.L374P mutation in the C terminus of STIM 1 abolishes SOCE and causes CRAC channelopathy Pedigree of the patients P1 (II‐1) and P2 (II‐2) presenting with CRAC channelopathy. Filled circles (females) and squares (males) indicate homozygous patients; dotted symbols indicate heterozygous asymptomatic carriers; symbols with “?” indicate asymptomatic family members not available for DNA sequencing. Sanger sequencing of genomic DNA isolated from PBMC of the mother (I‐2), P1, and P2. STIM1 and STIM2 mRNA expression in PBMC of P1, P2, mother, and a healthy donor (HD) that were left unstimulated or stimulated with anti‐CD3/CD28 for 24 h. mRNA levels of STIM1/2 normalized to 18S rRNA. Graph shows the mean ± SEM of duplicates from one experiment. STIM1 protein expression in CD4 + and CD8 + T cells from P1, P2, the mother, and a HD analyzed by flow cytometry. Shaded histograms: polyclonal rabbit IgG control antibody; open histograms: polyclonal anti‐STIM1 antibody. Bar graphs show the delta MFI calculated as MFI STIM1 – MFI IgG control that was normalized to HD T cells. Bar graphs are mean ± SEM of two independent repeat experiments. STIM1 protein expression in expanded human T cells analyzed by immunoblotting. One representative Western blot of 3 is shown. Bar graphs are the mean ± SEM of STIM1 expression normalized to actin from three independent experiments. Ca 2+ influx in Fura‐2 loaded PBMC of P1, P2, the mother, and a HD. T cells were stimulated with 1 μM thapsigargin (TG) in the absence of extracellular Ca 2+ followed by addition of 1 mM extracellular Ca 2+ . Bar graphs show the integrated Ca 2+ influx response (AUC, area under the curve) from 400 to 800 s and the peak Ca 2+ influx normalized to baseline Ca 2+ levels (F340/380) at 400 s. Data represent the mean ± SEM from 2 experiments. Statistical analysis by unpaired Student's t ‐test. ** P

    Journal: EMBO Molecular Medicine

    Article Title: STIM1‐mediated calcium influx controls antifungal immunity and the metabolic function of non‐pathogenic Th17 cells

    doi: 10.15252/emmm.201911592

    Figure Lengend Snippet: p.L374P mutation in the C terminus of STIM 1 abolishes SOCE and causes CRAC channelopathy Pedigree of the patients P1 (II‐1) and P2 (II‐2) presenting with CRAC channelopathy. Filled circles (females) and squares (males) indicate homozygous patients; dotted symbols indicate heterozygous asymptomatic carriers; symbols with “?” indicate asymptomatic family members not available for DNA sequencing. Sanger sequencing of genomic DNA isolated from PBMC of the mother (I‐2), P1, and P2. STIM1 and STIM2 mRNA expression in PBMC of P1, P2, mother, and a healthy donor (HD) that were left unstimulated or stimulated with anti‐CD3/CD28 for 24 h. mRNA levels of STIM1/2 normalized to 18S rRNA. Graph shows the mean ± SEM of duplicates from one experiment. STIM1 protein expression in CD4 + and CD8 + T cells from P1, P2, the mother, and a HD analyzed by flow cytometry. Shaded histograms: polyclonal rabbit IgG control antibody; open histograms: polyclonal anti‐STIM1 antibody. Bar graphs show the delta MFI calculated as MFI STIM1 – MFI IgG control that was normalized to HD T cells. Bar graphs are mean ± SEM of two independent repeat experiments. STIM1 protein expression in expanded human T cells analyzed by immunoblotting. One representative Western blot of 3 is shown. Bar graphs are the mean ± SEM of STIM1 expression normalized to actin from three independent experiments. Ca 2+ influx in Fura‐2 loaded PBMC of P1, P2, the mother, and a HD. T cells were stimulated with 1 μM thapsigargin (TG) in the absence of extracellular Ca 2+ followed by addition of 1 mM extracellular Ca 2+ . Bar graphs show the integrated Ca 2+ influx response (AUC, area under the curve) from 400 to 800 s and the peak Ca 2+ influx normalized to baseline Ca 2+ levels (F340/380) at 400 s. Data represent the mean ± SEM from 2 experiments. Statistical analysis by unpaired Student's t ‐test. ** P

    Article Snippet: PBMCs or purified CD4+ T cells were stimulated with 5 μg/ml plate‐bound anti‐CD3 (clone OKT3) and 10 μg/ml soluble anti‐CD28 (clone CD28.2, both eBioscience) monoclonal antibodies in the presence or absence of 1 μM FK506 (Sigma‐Aldrich) in RPMI 1640 medium supplemented with 10% FCS, 1% l ‐glutamine, and 1% penicillin/streptomycin (Mediatech Inc., VA).

    Techniques: Mutagenesis, DNA Sequencing, Sequencing, Isolation, Expressing, Flow Cytometry, Western Blot

    Reduced mTOR and S6 phosphorylation in T cells of STIM 1‐deficient patients isolated ex vivo Analysis of mTOR and p70S6 phosphorylation in CD4 + and CD8 + T cells freshly isolated from PBMC of P1 (red), P2 (blue), their mother (gray), and a HD (black) and stimulated with anti‐CD3/CD28 for 24 h or left unstimulated. HD T cells were stimulated in the presence or absence of 1 μM FK506. Histograms represent intracellular staining of cells with anti‐phospho‐mTOR (Ser2448) (A) and anti‐phospho‐p70S6 (Ser235/236) (B) antibodies. Filled gray histograms represent unstimulated cells. Numbers above gates in histograms and in bar graphs indicate the frequencies of CD4 + and CD8 + T cells positive for phospho‐mTOR and phospho‐p70S6 staining.

    Journal: EMBO Molecular Medicine

    Article Title: STIM1‐mediated calcium influx controls antifungal immunity and the metabolic function of non‐pathogenic Th17 cells

    doi: 10.15252/emmm.201911592

    Figure Lengend Snippet: Reduced mTOR and S6 phosphorylation in T cells of STIM 1‐deficient patients isolated ex vivo Analysis of mTOR and p70S6 phosphorylation in CD4 + and CD8 + T cells freshly isolated from PBMC of P1 (red), P2 (blue), their mother (gray), and a HD (black) and stimulated with anti‐CD3/CD28 for 24 h or left unstimulated. HD T cells were stimulated in the presence or absence of 1 μM FK506. Histograms represent intracellular staining of cells with anti‐phospho‐mTOR (Ser2448) (A) and anti‐phospho‐p70S6 (Ser235/236) (B) antibodies. Filled gray histograms represent unstimulated cells. Numbers above gates in histograms and in bar graphs indicate the frequencies of CD4 + and CD8 + T cells positive for phospho‐mTOR and phospho‐p70S6 staining.

    Article Snippet: PBMCs or purified CD4+ T cells were stimulated with 5 μg/ml plate‐bound anti‐CD3 (clone OKT3) and 10 μg/ml soluble anti‐CD28 (clone CD28.2, both eBioscience) monoclonal antibodies in the presence or absence of 1 μM FK506 (Sigma‐Aldrich) in RPMI 1640 medium supplemented with 10% FCS, 1% l ‐glutamine, and 1% penicillin/streptomycin (Mediatech Inc., VA).

    Techniques: Isolation, Ex Vivo, Staining

    STIM 1 p.L374P mutation causes defect in T‐cell proliferation and cytokine production Cell size (A) and proliferation (B) of CD4 + T cells from P1 (red), P2 (blue), their mother (gray), and a HD (black) stimulated with anti‐CD3 (5 μg/ml) and anti‐CD28 (10 μg/ml) in the presence or absence of 1 μM FK506 for 24 h. (A) Representative histograms of FSC (left panel) and percentages of T‐cell blasts (defined as cells to the right of the dotted vertical line) analyzed by flow cytometry (right panel). (B) Representative histograms of CFSE dilution (left panel) and percentages of proliferating cells (defined as cells to the left of the dotted vertical line) (right panel). Bar graphs in A and B are the mean ± SEM from two independent experiments. Cytokine production by PBMC from P1, P2, the mother, and an unrelated HD after stimulation with PMA (40 ng/ml) and ionomycin (500 ng/ml) for 4 h. Cytokines were analyzed by flow cytometry following surface staining with antibodies against CD3, CD4, and CD45RO, permeabilization and intracellular cytokine staining for GM‐CSF, IL‐22, and IL‐17A. Representative flow cytometry plots (C) and quantification of Th17 (GM‐CSF, IL‐22, IL‐17A), Th1 (TNF‐α, IFN‐γ), and Th2 (IL‐4) cytokines (D). Data represent the mean ± SEM from two independent experiments. Data information: Statistical analysis by unpaired Student's t ‐test. * P

    Journal: EMBO Molecular Medicine

    Article Title: STIM1‐mediated calcium influx controls antifungal immunity and the metabolic function of non‐pathogenic Th17 cells

    doi: 10.15252/emmm.201911592

    Figure Lengend Snippet: STIM 1 p.L374P mutation causes defect in T‐cell proliferation and cytokine production Cell size (A) and proliferation (B) of CD4 + T cells from P1 (red), P2 (blue), their mother (gray), and a HD (black) stimulated with anti‐CD3 (5 μg/ml) and anti‐CD28 (10 μg/ml) in the presence or absence of 1 μM FK506 for 24 h. (A) Representative histograms of FSC (left panel) and percentages of T‐cell blasts (defined as cells to the right of the dotted vertical line) analyzed by flow cytometry (right panel). (B) Representative histograms of CFSE dilution (left panel) and percentages of proliferating cells (defined as cells to the left of the dotted vertical line) (right panel). Bar graphs in A and B are the mean ± SEM from two independent experiments. Cytokine production by PBMC from P1, P2, the mother, and an unrelated HD after stimulation with PMA (40 ng/ml) and ionomycin (500 ng/ml) for 4 h. Cytokines were analyzed by flow cytometry following surface staining with antibodies against CD3, CD4, and CD45RO, permeabilization and intracellular cytokine staining for GM‐CSF, IL‐22, and IL‐17A. Representative flow cytometry plots (C) and quantification of Th17 (GM‐CSF, IL‐22, IL‐17A), Th1 (TNF‐α, IFN‐γ), and Th2 (IL‐4) cytokines (D). Data represent the mean ± SEM from two independent experiments. Data information: Statistical analysis by unpaired Student's t ‐test. * P

    Article Snippet: PBMCs or purified CD4+ T cells were stimulated with 5 μg/ml plate‐bound anti‐CD3 (clone OKT3) and 10 μg/ml soluble anti‐CD28 (clone CD28.2, both eBioscience) monoclonal antibodies in the presence or absence of 1 μM FK506 (Sigma‐Aldrich) in RPMI 1640 medium supplemented with 10% FCS, 1% l ‐glutamine, and 1% penicillin/streptomycin (Mediatech Inc., VA).

    Techniques: Mutagenesis, Flow Cytometry, Staining

    Effect of exogenous IL-10 on LPMC IFN-γ production from WT and TGF-βRII DN mice. LPMC from uninfected WT (A and C) or TGF-βRII DN (A and B) mice were stimulated in vitro with anti-CD3/CD28 (white bars) for 48 hr, in indicated cell numbers per well, with parallel cultures also containing 20 ng/ml (gray bars) or 100 ng/ml recombinant IL-10 (black bars). Supernatants were harvested and analyzed for IFN-γ by ELISA. Data show mean ± SD of (A) IFNγ secretion (two independent experiments with each experiment containing multiple determinations) or (B-C) the percentile ratio of LPMC IFNγ secretion in each condition to IFN-γ output of anti-CD3/28-stimulated culture (three independent experiments with each experiment containing multiple determinations). ( (A) WT Uninfected vs. Infected, ***p

    Journal: European journal of immunology

    Article Title: Role of T Cell TGF-? Signaling in Intestinal Cytokine Responses and Helminthic Immune Modulation

    doi: 10.1002/eji.200838956

    Figure Lengend Snippet: Effect of exogenous IL-10 on LPMC IFN-γ production from WT and TGF-βRII DN mice. LPMC from uninfected WT (A and C) or TGF-βRII DN (A and B) mice were stimulated in vitro with anti-CD3/CD28 (white bars) for 48 hr, in indicated cell numbers per well, with parallel cultures also containing 20 ng/ml (gray bars) or 100 ng/ml recombinant IL-10 (black bars). Supernatants were harvested and analyzed for IFN-γ by ELISA. Data show mean ± SD of (A) IFNγ secretion (two independent experiments with each experiment containing multiple determinations) or (B-C) the percentile ratio of LPMC IFNγ secretion in each condition to IFN-γ output of anti-CD3/28-stimulated culture (three independent experiments with each experiment containing multiple determinations). ( (A) WT Uninfected vs. Infected, ***p

    Article Snippet: For most experiments, the cells were cultured alone or with anti-CD3 (2C11, ATCC) and anti-CD28 mAb (Pharmingen, San Diego, CA) (each at 1 μg/ml).

    Techniques: Mouse Assay, In Vitro, Recombinant, Enzyme-linked Immunosorbent Assay, Infection

    Helminths cannot modulate the Th1 response and only weakly promote the Th2 response in LPMC from TGF-βRII DN mice. LPMC from uninfected or helminth-infected WT or TGF-βRII DN mice were stimulated in vitro with anti-CD3/CD28 mAb for 48 mAb. Supernatants were harvested and analyzed for IFN-γ, IL-4 and IL-5 by ELISA. Data show mean ± SD from at least three independent experiments with each experiment containing multiple determinations. (IFN-γ: WT Uninfected vs. Infected, ***p

    Journal: European journal of immunology

    Article Title: Role of T Cell TGF-? Signaling in Intestinal Cytokine Responses and Helminthic Immune Modulation

    doi: 10.1002/eji.200838956

    Figure Lengend Snippet: Helminths cannot modulate the Th1 response and only weakly promote the Th2 response in LPMC from TGF-βRII DN mice. LPMC from uninfected or helminth-infected WT or TGF-βRII DN mice were stimulated in vitro with anti-CD3/CD28 mAb for 48 mAb. Supernatants were harvested and analyzed for IFN-γ, IL-4 and IL-5 by ELISA. Data show mean ± SD from at least three independent experiments with each experiment containing multiple determinations. (IFN-γ: WT Uninfected vs. Infected, ***p

    Article Snippet: For most experiments, the cells were cultured alone or with anti-CD3 (2C11, ATCC) and anti-CD28 mAb (Pharmingen, San Diego, CA) (each at 1 μg/ml).

    Techniques: Mouse Assay, Infection, In Vitro, Enzyme-linked Immunosorbent Assay

    Helminthic induction of IL-10 in LPMC is dependent on T cell TGF-β signaling. LPMC from uninfected or helminth-infected WT or TGF-βR DN mice were stimulated in vitro with anti-CD3/CD28 mAb for 48 hr. Supernatants were harvested and analyzed for IL-10 by ELISA. Data show mean ± SD from three independent experiments with each experiment containing multiple determinations. (WT Uninfected vs. Infected, ***p

    Journal: European journal of immunology

    Article Title: Role of T Cell TGF-? Signaling in Intestinal Cytokine Responses and Helminthic Immune Modulation

    doi: 10.1002/eji.200838956

    Figure Lengend Snippet: Helminthic induction of IL-10 in LPMC is dependent on T cell TGF-β signaling. LPMC from uninfected or helminth-infected WT or TGF-βR DN mice were stimulated in vitro with anti-CD3/CD28 mAb for 48 hr. Supernatants were harvested and analyzed for IL-10 by ELISA. Data show mean ± SD from three independent experiments with each experiment containing multiple determinations. (WT Uninfected vs. Infected, ***p

    Article Snippet: For most experiments, the cells were cultured alone or with anti-CD3 (2C11, ATCC) and anti-CD28 mAb (Pharmingen, San Diego, CA) (each at 1 μg/ml).

    Techniques: Infection, Mouse Assay, In Vitro, Enzyme-linked Immunosorbent Assay

    TCR-triggered TGF-β production in LPMC from uninfected control or H. polygyrus -infected WT and TGF-βRII DN mice. LPMC from uninfected or helminth-infected WT or TGF-βRII DN mice were cultured in vitro alone (Cells) or with anti-CD3/CD28 mAb (Cells + αCD3/28) for 48 h in TGF-β culture medium. Supernatants then were analyzed for TGF-β by ELISA. Data show mean ± SD from three independent experiments with each experiment containing multiple determinations. (WT: unstimulated LPMC Uninfected vs. Infected, p > 0.05, NS; anti-CD3/CD28 stimulated Uninfected vs. Infected, **p

    Journal: European journal of immunology

    Article Title: Role of T Cell TGF-? Signaling in Intestinal Cytokine Responses and Helminthic Immune Modulation

    doi: 10.1002/eji.200838956

    Figure Lengend Snippet: TCR-triggered TGF-β production in LPMC from uninfected control or H. polygyrus -infected WT and TGF-βRII DN mice. LPMC from uninfected or helminth-infected WT or TGF-βRII DN mice were cultured in vitro alone (Cells) or with anti-CD3/CD28 mAb (Cells + αCD3/28) for 48 h in TGF-β culture medium. Supernatants then were analyzed for TGF-β by ELISA. Data show mean ± SD from three independent experiments with each experiment containing multiple determinations. (WT: unstimulated LPMC Uninfected vs. Infected, p > 0.05, NS; anti-CD3/CD28 stimulated Uninfected vs. Infected, **p

    Article Snippet: For most experiments, the cells were cultured alone or with anti-CD3 (2C11, ATCC) and anti-CD28 mAb (Pharmingen, San Diego, CA) (each at 1 μg/ml).

    Techniques: Infection, Mouse Assay, Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay

    Intranasal immunization with MCS-pPES enhanced specific IFNγ-secreting CD4+ and CD8+ T response in the spleen. (A) ELISPOT analysis of the splenic TB-specific IFNγ-producing T response in pPES-, CS-DNA, MCS-DNA-, and BCG-immunized mice. Splenocytes were stimulated with mixed peptides, HSP65 protein or inactivated H37Rv plus anti-CD28 for 36 h before IFN-γ ELISPOT assay was performed. Statistical results were from three independent experiments and presented as the mean ± SEM ( n = 6). (B) The frequency of CD4 and CD8 T cells in the lung producing IFN-γ, IL-2, or TNF-α in response to mixed peptides was measured. Cells were incubated with mixed peptides plus anti-CD28 for 10 h the percentages of IFN-γ, IL-2, and TNF-α-producing CD4+T and CD8+T cells were determined by FACS. Numbers in each quadrant represent percentages of positive cells in CD4+ or CD8+ T population. Results are represented as mean ± SEM ( n = 6) of three separate experiments. * p

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Intranasal Vaccination with Mannosylated Chitosan Formulated DNA Vaccine Enables Robust IgA and Cellular Response Induction in the Lungs of Mice and Improves Protection against Pulmonary Mycobacterial Challenge

    doi: 10.3389/fcimb.2017.00445

    Figure Lengend Snippet: Intranasal immunization with MCS-pPES enhanced specific IFNγ-secreting CD4+ and CD8+ T response in the spleen. (A) ELISPOT analysis of the splenic TB-specific IFNγ-producing T response in pPES-, CS-DNA, MCS-DNA-, and BCG-immunized mice. Splenocytes were stimulated with mixed peptides, HSP65 protein or inactivated H37Rv plus anti-CD28 for 36 h before IFN-γ ELISPOT assay was performed. Statistical results were from three independent experiments and presented as the mean ± SEM ( n = 6). (B) The frequency of CD4 and CD8 T cells in the lung producing IFN-γ, IL-2, or TNF-α in response to mixed peptides was measured. Cells were incubated with mixed peptides plus anti-CD28 for 10 h the percentages of IFN-γ, IL-2, and TNF-α-producing CD4+T and CD8+T cells were determined by FACS. Numbers in each quadrant represent percentages of positive cells in CD4+ or CD8+ T population. Results are represented as mean ± SEM ( n = 6) of three separate experiments. * p

    Article Snippet: Splenocytes or pulmonary lymphocytes (5 × 105 /well) from individual mice were stimulated for 36 h at 37°C with RPMI 1640 alone, 10 μM single peptide or mixed M. tb peptides (ESAT-61–20 , MTB10.43–11 , Ag85B241–255 , PPE25241–255 , and PE194–18 ), 10 μg/ml HSP65 protein, 5 μg/ml inactivated H37Rv or concanavalin A (5 μg/ml; Sigma-Aldrich) plus anti-mouse CD28 mAb (1 μg/ml; BD Pharmingen).

    Techniques: Enzyme-linked Immunospot, Mouse Assay, Incubation, FACS

    MCS-pPES intranasal immunization enhanced a poly-functional Th1 response in the lung. (A) 2 weeks after final immunization, pulmonic lymphocytes were stimulated with mixed peptides plus anti-CD28 for 36 h, and specific IFNγ-producing T cell spots were determined by ELISPOT assay. A representative result was shown. Statistical results were from three independent experiments and presented as the mean ± SEM ( n = 6). (B) The frequency of CD4 and CD8 T cells in the lung producing IFN-γ, IL-2, or TNF-α in response to mixed peptides was measured by FACS. Numbers in each quadrant indicate percentages of positive cells in CD4+ or CD8+ T population. Results are represented as mean ± SEM ( n = 6) of three separate experiments. (C) CD4+TNF-α+ and CD4+TNF-α- cells within CD4+ T cells were further gated, and the expression of IFNγ and IL-2 was shown. (D) The total peptide-specific Th1 response within CD4+ T cells was defined as the number of cells expressing any combination of IFN-γ, TNF-α or IL-2. The average percentages for each subset are shown. Results are represented as mean ± SEM (n = 6) of three separate experiments. (E) The quality of the T cell responses in (C,D) . Pie charts showed the fraction of total cytokine-response in the lung comprising any combination of IFN-γ, IL-2, or TNF-α. The percentages of cells producing three cytokines (triple positive), two cytokines (double positive) or one cytokine (single positive) within the total CD4+T cell response were shown from three independent experiments. * p

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Intranasal Vaccination with Mannosylated Chitosan Formulated DNA Vaccine Enables Robust IgA and Cellular Response Induction in the Lungs of Mice and Improves Protection against Pulmonary Mycobacterial Challenge

    doi: 10.3389/fcimb.2017.00445

    Figure Lengend Snippet: MCS-pPES intranasal immunization enhanced a poly-functional Th1 response in the lung. (A) 2 weeks after final immunization, pulmonic lymphocytes were stimulated with mixed peptides plus anti-CD28 for 36 h, and specific IFNγ-producing T cell spots were determined by ELISPOT assay. A representative result was shown. Statistical results were from three independent experiments and presented as the mean ± SEM ( n = 6). (B) The frequency of CD4 and CD8 T cells in the lung producing IFN-γ, IL-2, or TNF-α in response to mixed peptides was measured by FACS. Numbers in each quadrant indicate percentages of positive cells in CD4+ or CD8+ T population. Results are represented as mean ± SEM ( n = 6) of three separate experiments. (C) CD4+TNF-α+ and CD4+TNF-α- cells within CD4+ T cells were further gated, and the expression of IFNγ and IL-2 was shown. (D) The total peptide-specific Th1 response within CD4+ T cells was defined as the number of cells expressing any combination of IFN-γ, TNF-α or IL-2. The average percentages for each subset are shown. Results are represented as mean ± SEM (n = 6) of three separate experiments. (E) The quality of the T cell responses in (C,D) . Pie charts showed the fraction of total cytokine-response in the lung comprising any combination of IFN-γ, IL-2, or TNF-α. The percentages of cells producing three cytokines (triple positive), two cytokines (double positive) or one cytokine (single positive) within the total CD4+T cell response were shown from three independent experiments. * p

    Article Snippet: Splenocytes or pulmonary lymphocytes (5 × 105 /well) from individual mice were stimulated for 36 h at 37°C with RPMI 1640 alone, 10 μM single peptide or mixed M. tb peptides (ESAT-61–20 , MTB10.43–11 , Ag85B241–255 , PPE25241–255 , and PE194–18 ), 10 μg/ml HSP65 protein, 5 μg/ml inactivated H37Rv or concanavalin A (5 μg/ml; Sigma-Aldrich) plus anti-mouse CD28 mAb (1 μg/ml; BD Pharmingen).

    Techniques: Functional Assay, Enzyme-linked Immunospot, FACS, Expressing