ifn-γ Search Results


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  • 99
    Thermo Fisher ifn γ
    Cytokine production from human whole blood stimulated by liposomes containing GLA, 3M-052, both TLR ligands, or neither (empty). Stimulated whole blood was analyzed for production of Th1-associated cytokines a IL-12p70 and b <t>IFN-γ,</t> as well as chemokines c Mip-1β and d MCP-1. The x -axes represent serial dilutions with starting concentrations of 4 µg/ml for GLA and 1.5 µg/ml 3M-052. The y -axes represent concentrations of target analytes secreted post whole blood stimulation with error bars representing the standard error of the mean from three blood donors with each individual value averaged from duplicate wells
    Ifn γ, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18942 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore ifn γ
    Cytokine production from human whole blood stimulated by liposomes containing GLA, 3M-052, both TLR ligands, or neither (empty). Stimulated whole blood was analyzed for production of Th1-associated cytokines a IL-12p70 and b <t>IFN-γ,</t> as well as chemokines c Mip-1β and d MCP-1. The x -axes represent serial dilutions with starting concentrations of 4 µg/ml for GLA and 1.5 µg/ml 3M-052. The y -axes represent concentrations of target analytes secreted post whole blood stimulation with error bars representing the standard error of the mean from three blood donors with each individual value averaged from duplicate wells
    Ifn γ, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6048 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti ifn γ
    M exp reduced the generation of IL-17-producing cells due to their lower CD86 expression. (A) Representative plots showing the percentages of IL-17, <t>IL-17/IFN-γ,</t> and IFN-γ-producing CD4 + cells after co-culture with M exp , M 25+ , and M 25− . (B) Cumulative data of nine independent experiments showing the percentages of IL-17, IL-17/IFN-γ, and IFN-γ and IL-4 producing CD4 + cells. Representative plots (C) and cumulative data (D) of four independent experiments showing the percentages of memory IL-17-producing CD4 + cells after co-culture with M 25− alone or in the presence of infliximab, tociluzmab, and abatacept. (E) Cell count of CD4 + cells ( n = 4) co-cultured with M 25− alone or in the presence of Abatacept. (F) Cumulative data of four independent experiments showing the percentages of memory IL-17-producing CD4 + stimulated with anti-CD3/28 beads in medium alone or M 25− conditioned medium. In all the experiments, data, presented as mean ± SEM, were analyzed using one-way ANOVA followed by Tukey with * p
    Anti Ifn γ, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2696 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PeproTech ifn γ
    IL-22BP protein and mRNA levels in monocyte-derived DCs. (A) (Upper left) Diagram showing exons with the primer-binding positions for IL22RA2 variant discrimination strategy. (Lower left) Expression of IL22RA2v1, v2 , and v3 in moDC after 6 days of culture in differentiation medium (DM) ± AM580, a retinoic acid receptor agonist, by RT-PCR. IL22RA2v2 expression vector was used as positive control for IL22RA2v2 expression. (Upper right) Expression of IL22RA2 variants in moDC following 6 days of culture in DM ± AM580 by RT-qPCR using the isoform-specific primers indicated in the left diagram (mean ± SEM; n = 3) relative to the housekeeping gene GAPDH . (B) Effect of different maturation stimuli (CpG and LPS + <t>IFN-γ</t> or Poly I:C) on IL22RA2, IL12B , and IL6 expression in moDC after 6 days of culture in DM by RT-qPCR using Taqman probes relative to the housekeeping gene ACTB . Extent of maturation is represented as percentage of CD83 + fluorescence by flow cytometry. Data showing representative experiment of 3 performed. (C) IL-22BP secretion by moDC cultured in DM detected by ELISA corresponds to IL22RA2 mRNA expression levels analyzed by RT-qPCR using Taqman probes and both increase over the cultivation period (mean ± SEM, n = 2). (D) Cell lysates (CL) of moDCs and conditioned media (CM) were harvested after 6 days in DM ± AM580 and immunoblotted for detection of IL-22BP (IL-22BP antibody 1) using tubulin as a loading control, IL22RA2 mRNA expression relative to the mean of the housekeeping genes GAPDH and ACTB .
    Ifn γ, supplied by PeproTech, used in various techniques. Bioz Stars score: 99/100, based on 4737 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ifnγ
    Systemic inflammatory toxicity mainly derived from IL-2-Fc rather than anti-CD137. Groups of C57Bl/6 mice ( n = 4/group) were inoculated with 5 × 10 5 B16F10 tumor cells s.c. on day 0, then received i.v. injections of αCD137 and IL-2-Fc (αCD137/IL-2-Fc), Lipo-IL-2-Fc mixed with αCD137 (Lipo-IL-2-Fc/αCD137), or Lipo-αCD137 mixed with IL-2-Fc (Lipo- αCD137/IL-2-Fc) on day 8. One day later, sera were collected from peripheral blood and levels of <t>IFNγ</t> ( a ), IL-6 ( b ), MCP-1 ( c ) and TNFα ( d ) were assayed by luminex assays. All measurements shown are mean ± s.e.m.
    Ifnγ, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4844 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson ifn γ
    Quality characterization of the specific T-cell anti-VACV immune response. ( A ) Groups of four C57BL/6 mice were i.m immunized with 10 7 pfu of MVAwt (white bars) or MVAΔC12L (black bars), and seven dpi the magnitude of the T-cell response against B8R peptide in the spleen was measured by <t>IFN-γ</t> and IL-2 Elispot assays and ( B ) degranulation of specific-CD8 + T-cells was assessed with CD107a/b staining simultaneously with IFN-γ and TNF-α production by ICS, after 5 hr of stimulation with B8R peptide. Analysis of the total B8R specific-CD8 + T-cells expressing each of the functions (left panel) and polyfunctionality of the specific-CD8 + T-cells were performed (right panel). Bars represent the frequency of CD8 + T-cells expressing the particular combination of functions indicated on the x axis. Results are expressed as mean % CD8 + anti-B8R T-cells ± SD. Each pie chart represents the mean contribution of each effector function to the total response. Green sectors stand for CD8 + cells positive for only one function (SP), yellow sectors stand for bifunctional CD8 + cells (DP), and red sectors stand for trifunctional cells (TP). ( C ) Groups of four C57BL/6 mice were i.m immunized with 10 7 pfu of MVAwt or MVAΔC12L and 7 (acute) or 35 (memory) dpi, specific cytokine production in splenocyte-culture supernatants was evaluated by ELISA after 72 hr stimulation with the indicated peptides. ( D ) Splenocytes from BALB/c mice i.m immunized with 10 7 pfu of MVAwt (green) or MVAΔC12L (red) were assayed by IFN-γ Elispot against serial dilutions of VACV E3 peptide. T-cell functional avidity is defined as the concentration required to achieve half-maximal of the response (EC 50 ). Data represents the percentage of the maximal response (net number of SFU per 10 6 cells stimulated with a peptide concentration of 20 µg/ml). Statistically significant differences: *p
    Ifn γ, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 25355 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pharmingen ifn γ
    Isolation of non-MAIT MR1-restricted T cell clones after stimulation with A375-MR1 cells in the absence of microbial antigens. ( A ) FACS analysis of purified T cells previously expanded with irradiated A375-MR1 cells following overnight co-culture with A375-MR1 cells. Left dot plot shows CD3 and CellTrace violet (CTV) staining in live cells. Upper right and bottom right dot plots show CD69 and CD137 expression on CD3 + CTV − and CD3 + CTV + gated cells, respectively. Arrows indicate gating hierarchy. Numbers indicate the percentages of cells within the gates. Cells from Donor A are illustrated as a representative donor. ( B, D ) Cumulative results of T cell clones screening from Donors A and B. T cell clones were generated from CD3 + CTV - CD137 high sorted T cells as depicted in A. Graphs show the individual clones (x axis) and their <t>IFN-γ</t> release (y axis), expressed as ratio between the amount of cytokine secreted in response to A375-MR1 cells vs. A375 WT cells. Each dot represents a single T cell clone, tested at the same time in the indicated experimental conditions. The horizontal red line marks the arbitrary IFN-γ ratio cut-off of two, above which MR1-dependent T cell clone reactivity was set. The intercept of the vertical red line indicates the number of MR1-restricted T cell clones in each donor. Red boxes highlight T cell clones whose reactivity was MR1-dependent. Results are representative of two independent experiments. ( C, E ) IFN-γ release by 14 representative clones from Donor A and 11 clones from Donor B after stimulation with A375 WT, A375-MR1 and A375-MR1 in the presence of blocking anti-MR1 mAbs (α-MR1). Dots represent the IFN-γ release (mean ± SD of duplicate cultures) by each clone. Results are representative of three independent experiments. *p
    Ifn γ, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 3055 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems recombinant human ifn γ
    GBP1 is required for <t>IFN-γ-induced</t> processing of IDO, IL-6 and IL-8. ( A , B ) Expression of GBP1 was dramatically upregulated by treatment with IFN-γ at 10 ng/ml, as determined by RT-PCR ( A ) and western blot ( B . ( C ) Knockdown of GBP1 impaired the mRNA expression of indoleamine 2,3 dioxygenase (IDO) induced by treatment with IFN-γ (#p
    Recombinant Human Ifn γ, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 716 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher interferon gamma ifn γ
    GBP1 is required for <t>IFN-γ-induced</t> processing of IDO, IL-6 and IL-8. ( A , B ) Expression of GBP1 was dramatically upregulated by treatment with IFN-γ at 10 ng/ml, as determined by RT-PCR ( A ) and western blot ( B . ( C ) Knockdown of GBP1 impaired the mRNA expression of indoleamine 2,3 dioxygenase (IDO) induced by treatment with IFN-γ (#p
    Interferon Gamma Ifn γ, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 353 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti ifn γ
    Antigen targeting to DCs via DC-ASGPR favors the generation of antigen-specific IL-10–producing CD4 + T cells. IFNDCs (5 × 10 3 ) were loaded with 1 µg/ml α-DC-ASGPR-HA1 or α-LOX-1-HA1, and then co-cultured with CFSE-labeled autologous total CD4 + T cells (1–2 × 10 5 ) for 7 d. (A) CD4 + T cells were restimulated with peptide HA1 250-266 for 48h, and cytokine levels in the culture supernatants were measured. Each line represents the data from a single experiment. P-values were tested by Student’s t test. (B) CD4 + T cells were restimulated with indicated peptides and stained for intracellular IL-10. HA1 280-296 is a negative control. Peptides tested in B had been selected in Fig. S2 (F and G) . Four independent experiments showed similar results. (C) Naive CD4 + T cells (1–2 × 10 5 ) were co-cultured with IFNDCs (5 × 10 3 ) loaded with 1 µg/ml recombinant fusion proteins for 7 d. CD4 + T cells were restimulated with peptides indicated for 48 h. IL-10 and <t>IFN-γ</t> levels in culture supernatants were assessed. Error bars represent mean ± SEM of triplicate assay. Three independent experiments with 59 PSA-derived peptides showed similar results. (D) Frequency of PSA-specific IL-10–producing CD4 + T cells elicited by recombinant fusion proteins. Four independent experiments showed similar results. (E) Experiments in C were performed with blood mDCs (Lin − HLA-DR + CD11c + CD123 − ). (F) Experiments in D were performed with blood mDCs. In both E and F, two independent experiments showed similar results. (G) Expression levels of Foxp3, PD-1, and CTLA-4 on HA1 250-266 -specific (left) and PSA 30-44 -specific CD4 + T cells producing IL-10 (right). (H) After 7 d of co-culture of purified naive CD4 + T cells and DCs loaded with either α-LOX-1-PSA or α-DC-ASGPR-PSA, CD4 + T cells were stained for intracellular IFN-γ and IL-10 during restimulation with 50 ng/ml PMA and 1 µg/ml ionomycin. Three independent experiments showed similar results (G and H).
    Anti Ifn γ, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 4124 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mab Technologies ifn γ
    AEC sup induces IL-1β expression and secretion without an effect of intracellular killing of BCG. BMM and PuM were infected with GFP-BCG. After infection, cells were treated with either <t>IFN-γ</t> or AEC sup or left untreated. a) Total RNA was extracted from both cell-types after 4 and 24 h of treatment and the mean-fold accumulation of IL-1β transcripts shown. Data shows the average of 3 independent experiments. b) Cell culture supernatants were collected 24 h after infection and IL-1β measured with ELISA. Data shows the average of 6 independent experiments. c) The effect of incubating cells with IL-1β (1000 pg/ml) on intracellular growth of BCG. Data representative of 3 independent experiments d) The effect of AECs up on intracellular growth of BCG in BMM from caspase1 -/- mice. Bacterial growth was evaluated by determining RLU. Values are expressed as means ± SD from 5 wells. The data is representative of 2 independent experiments. The differences between groups of BMM were analyzed using a one-way ANOVA followed by Bonferroni‘s Multiple Comparison Test. * significantly different from medium control, P
    Ifn γ, supplied by Mab Technologies, used in various techniques. Bioz Stars score: 92/100, based on 1704 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PeproTech ifnγ
    AEC sup induces IL-1β expression and secretion without an effect of intracellular killing of BCG. BMM and PuM were infected with GFP-BCG. After infection, cells were treated with either <t>IFN-γ</t> or AEC sup or left untreated. a) Total RNA was extracted from both cell-types after 4 and 24 h of treatment and the mean-fold accumulation of IL-1β transcripts shown. Data shows the average of 3 independent experiments. b) Cell culture supernatants were collected 24 h after infection and IL-1β measured with ELISA. Data shows the average of 6 independent experiments. c) The effect of incubating cells with IL-1β (1000 pg/ml) on intracellular growth of BCG. Data representative of 3 independent experiments d) The effect of AECs up on intracellular growth of BCG in BMM from caspase1 -/- mice. Bacterial growth was evaluated by determining RLU. Values are expressed as means ± SD from 5 wells. The data is representative of 2 independent experiments. The differences between groups of BMM were analyzed using a one-way ANOVA followed by Bonferroni‘s Multiple Comparison Test. * significantly different from medium control, P
    Ifnγ, supplied by PeproTech, used in various techniques. Bioz Stars score: 99/100, based on 1672 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genzyme ifn γ
    Surface analysis of <t>IFN-γ–stimulated</t> Mφ reveals simultaneous increase in TRAIL expression with downregulation of TRAIL-R2 expression. (A) Flow cytometric analysis of TRAIL-R1, -R2, -R3, and -R4 expression on unstimulated Mφ. Filled histograms represent staining by M271 (anti–TRAIL-R1 mAb), M413 (anti–TRAIL-R2 mAb), M430 (anti–TRAIL-R3 mAb), or M444 (anti–TRAIL-R4 mAb), and open histograms represent staining with isotype control mAb. (B) RT-PCR analysis of TRAIL receptor mRNA expression in normal Mφ. (C) TRAIL, TRAIL-R2, and TRAIL-R3 expression after various times of IFN-γ stimulation. Filled histograms represent staining by M413 (anti–TRAIL-R2 mAb), M430 (anti–TRAIL-R3 mAb), or M181 (anti-TRAIL mAb), and open histograms represent staining with isotype control mAb. For comparison, Mφ cultured in the absence or presence of GM-CSF for 8 h were also stained for TRAIL, TRAIL-R2, and TRAIL-R3. (D) RT-PCR analysis of TRAIL, TRAIL-R2, and TRAIL-R3 mRNA levels after Mφ culture in the absence or presence of IFN-γ and GM-CSF. β-actin was used as a control over the same time course. Similar results were observed with Mφ from two other donors.
    Ifn γ, supplied by Genzyme, used in various techniques. Bioz Stars score: 92/100, based on 795 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems mouse ifn γ
    Effects of ATP on ConA-induced production of pro-inflammatory cytokines by splenic lymphocytes isolated from BALB/c mice. (A–E) Lymphocytes were pre-incubated with various concentrations of ATP (100, 250, or 500 μM), and then stimulated with 50 μg/mL ConA for 24 h. (F–J) Lymphocytes were pre-incubated with 250 μM ATP, ADP, AMP, or adenosine, and then stimulated with 50 μg/mL ConA for 24 h. Concentrations of IL-6 (A,F) , TNF-α (B,G) , IL-17 (C,H) , <t>IFN-γ</t> (D,I) , or IL-4 (E,J) in the culture medium were measured by ELISA. Each value represents the mean ± SE ( n = 4). Significant difference between the vehicle control group and the ConA-treated group in the absence of ATP or other ligand is indicated by ### P
    Mouse Ifn γ, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 408 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PeproTech recombinant murine ifn γ
    Interferon signaling mediates resistance to cholesterol-dependent cytolysins. a, Percentage of PI-positive BMDMs treated with TLR1/2 agonist (Pam3CSK4; 50 ng/mL), TLR3 agonist (Poly(I:C); 1 μg/mL), TLR4 agonist (LPS; 50 ng/mL), TLR7 agonist (CL307; 100 nM), TLR9 (ODN1668; 100 nM) agonist, or unstimulated (NT) for 24 h and then challenged with PFO for up to 60 min in the presence of PI. Cells were imaged every 10 min to assess changes in PI incorporation. b, Percentage of PI-positive BMDMs treated with the various concentrations of TLR4 agonist for 24 h and then challenged with PFO for up to 60 min in the presence of PI. Cells were imaged every 7 min to assess changes in PI incorporation. c, Percentage of PI-positive BMDMs treated with IFN-β or <t>IFN-γ</t> (20 ng/mL) for 24 h and then challenged with PFO for 60 min in the presence of PI. d, Percentage of PI-positive BMDMs treated with NOD2 agonist (N-Glycolyl-MDP; 20 μg/ml), STING agonist (2’,3’cGAMP and c-di-GMP; both 2 μg/mL) for 24 h and then challenged with PFO for 60 min in the presence of PI. e, Percentage of PI-positive BMDMs treated with STING ligand for 24 h and then challenged with Streptolysin O (SLO) for 4 h in the presence of PI. f, Percentage of PI-positive BMDMs treated with IFN-β or IFN-γ (20 ng/mL) for 24 h and then challenged with SLO for 4 h in the presence of PI. g, Percentage of PI-positive BMDMs treated with IFN-β or IFN-γ (20 ng/mL) for 24 h and then challenged with ALO for 60 min in the presence of PI. h, Percentage of PI-positive BMDMs treated with IFN-β (100 ng/mL) or TLR1/2 agonist, or IFN-β (100 ng/mL) together with TLR1/2 agonist for 24 h and then challenged with PFO for 60 min in the presence of PI. i, Percentage of PI-positive BMDMs treated with IFN-β (100 ng/mL) or TLR1/2 agonist, or IFN-β (100 ng/mL) together with TLR1/2 agonist for 24 h and then challenged with SLO for 4 h in the presence of PI. Data are representatives of three independent experiments, and are shown as mean ± s.e.m. (n = 3). Statistical significance was determined using an RM one-way ANOVA with Dunnett’s correction (a-g) or a two-way ANOVA with Dunnett’s correction (h, i). ***P
    Recombinant Murine Ifn γ, supplied by PeproTech, used in various techniques. Bioz Stars score: 92/100, based on 428 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems recombinant mouse ifn γ
    NLRP6 -/- neutrophils exhibit enhanced intracellular killing through increased <t>IFN-γ</t> and NADPH oxidase-dependent ROS production. (A) BMDNs from WT and KO mice were infected with S . aureus (MOI 10) and the intracellular killing ability of neutrophils was measured at the indicated time points. (B) ROS production by BMDNs from WT and KO mice were compared after infection with S . aureus (MOI 1). (C) WT and KO mice (N = 6-8/group) were infected with a sublethal dose of S . aureus (5 X 10 7 CFU/mouse). At designated time points, lungs were collected and processed for immunoblotting to compare the expression of NADPH oxidase enzyme components between WT and KO mice. (D) IFN-γ in BALF obtained from mice in 3C was measured. (E) Lungs obtained from S . aureus -infected WT and KO mice were homogenized and expressions of phospho-38, phospho-JNK, and phospho-STAT3 were measured through immunoblotting. (F) BMDNs from WT and KO mice were isolated and pre-treated with either IFN-γ or PBS for 30 minutes before infection with S . aureus (MOI 10). Two-hours post-infection, WT neutrophils were lysed and immunoblotted to measure the expression of NADPH oxidase enzyme components. (G) ROS production by neutrophils in 3F were measured as described in the methods. All figures are representative of three independent experiments. ROS: reactive oxygen species, RFU: relative florescence unit. WT: Wild-type, KO: NLRP6 KO. *, p
    Recombinant Mouse Ifn γ, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 380 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mouse ifn γ
    PD-1: PD-L1 pathway inhibits CD8 + T-cell <t>IFN-γ</t> and IL-2 expression in the post-encephalitic brain
    Mouse Ifn γ, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 462 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems interferon γ
    Blockade of TIM-4 improves hepatic acute reaction response injury and reduces the secretion of inflammatory cytokines. (A) KCs were isolated from mice in the sham, control mAb and TIM-4 mAb groups on day 7 post-surgery. Cells were stained with specific secondary antibodies labeled with tetramethylrhodamine (red) and DAPI (blue) and then examined using laser confocal microscopy (magnification ×800). (B) KCs were isolated from each group and stained with FITC-TIM-4 mAb for flow cytometry. (C) Blood samples were obtained (0.5 ml/mouse) from murine abdominal aortas in each group on day 7 post-surgery. Levels of AST, ALT and TBIL were determined in a clinical biochemical laboratory. (D-F) Levels of inflammatory cytokines, including TNF-α, <t>IFN-γ,</t> CCL2, CXCL2 and TGF-β were detected using ELISA and western blotting. (G) Representative micrographs of the pathological damage observed in each group post-transplantation following staining with hematoxylin and eosin (magnification ×400). (H) Donor mice received CL treatment to destroy KCs prior to surgery. orthotopic liver transplantation or sham surgery was subsequently performed and mice were treated either with or without TIM-4 mAb. All mice underwent analysis to determine the RAI score on day 7 following surgery. (I) The expression of key phosphoproteins involved in the NF-κB and p38 MAPK signaling pathways, including p65 and p38, respectively, were analyzed using western blotting. Data are presented as the mean ± standard deviation. a P
    Interferon γ, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 1042 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems human ifn γ
    Blockade of TIM-4 improves hepatic acute reaction response injury and reduces the secretion of inflammatory cytokines. (A) KCs were isolated from mice in the sham, control mAb and TIM-4 mAb groups on day 7 post-surgery. Cells were stained with specific secondary antibodies labeled with tetramethylrhodamine (red) and DAPI (blue) and then examined using laser confocal microscopy (magnification ×800). (B) KCs were isolated from each group and stained with FITC-TIM-4 mAb for flow cytometry. (C) Blood samples were obtained (0.5 ml/mouse) from murine abdominal aortas in each group on day 7 post-surgery. Levels of AST, ALT and TBIL were determined in a clinical biochemical laboratory. (D-F) Levels of inflammatory cytokines, including TNF-α, <t>IFN-γ,</t> CCL2, CXCL2 and TGF-β were detected using ELISA and western blotting. (G) Representative micrographs of the pathological damage observed in each group post-transplantation following staining with hematoxylin and eosin (magnification ×400). (H) Donor mice received CL treatment to destroy KCs prior to surgery. orthotopic liver transplantation or sham surgery was subsequently performed and mice were treated either with or without TIM-4 mAb. All mice underwent analysis to determine the RAI score on day 7 following surgery. (I) The expression of key phosphoproteins involved in the NF-κB and p38 MAPK signaling pathways, including p65 and p38, respectively, were analyzed using western blotting. Data are presented as the mean ± standard deviation. a P
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    Thermo Fisher interferon ifn γ
    Intradermal vaccination of C57BL/6 wild-type mice with peptide antigens and photochemical internalization (PCI) effectively induces antigen-specific CD8+ effector T cell responses. Groups of five C57BL/6 wild-type mice were intradermally immunized with TRP-2 180–188 or HPV 43–78 peptide ± PCI treatment. Mice received three identical vaccination treatments with 14-day intervals. (A) 10 days after the last vaccination, frequencies of activated, antigen-specific CD8+ CTLs were analyses from blood samples with HPV 49–57 or TRP-2 180–188 peptide-loaded MHC class I pentamers (CD8+ CD44+ pentamer+ cells). Plots show representative examples of flow cytometric analysis for HPV 43–78 (upper panels) or TRP-2 180–188 (lower panels) vaccinated mice without (left) or with PCI treatment (right). (B) Quantification of activated HPV 43–48 -specific (CD8+ CD44+ H-2Kb/HPV 49–57 -pentamer+ cells, left) and TRP-2 180–188 -specific (CD8+ CD44+ H-2Kb/TRP-2 180–188 -pentamer+ cells, right) activated CD8+ T cells from all five mice in each group of the experiment. (C) Frequencies of interferon <t>(IFN)-γ</t> effector cytokine producing CD8+ CTLs were analyzed from spleen cells after overnight stimulation with TRP-2 180–188 or HPV 43–78 peptide, respectively (CD8+ CD44+ IFN-γ+ cells). Plots show representative examples of flow cytometric analysis for HPV 43–78 (upper panels) or TRP-2 180–188 (lower panels) vaccinated mice without (left) or with PCI treatment (right). (D) Quantification of IFN-γ effector cytokine production from CD8+ T cells (CD8+ CD44+ IFN-γ+ T cells) from HPV 43–48 -vaccinated (left) and TRP-2 180–188 -vaccinated mice for all five mice in each group of the experiments. Horizontal lines in scatter graphs indicate median values; P values were calculated using an unpaired two-tailed t -test to compare pairs of datasets (* P
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    Mab Technologies anti ifn γ mab
    Intradermal vaccination of C57BL/6 wild-type mice with peptide antigens and photochemical internalization (PCI) effectively induces antigen-specific CD8+ effector T cell responses. Groups of five C57BL/6 wild-type mice were intradermally immunized with TRP-2 180–188 or HPV 43–78 peptide ± PCI treatment. Mice received three identical vaccination treatments with 14-day intervals. (A) 10 days after the last vaccination, frequencies of activated, antigen-specific CD8+ CTLs were analyses from blood samples with HPV 49–57 or TRP-2 180–188 peptide-loaded MHC class I pentamers (CD8+ CD44+ pentamer+ cells). Plots show representative examples of flow cytometric analysis for HPV 43–78 (upper panels) or TRP-2 180–188 (lower panels) vaccinated mice without (left) or with PCI treatment (right). (B) Quantification of activated HPV 43–48 -specific (CD8+ CD44+ H-2Kb/HPV 49–57 -pentamer+ cells, left) and TRP-2 180–188 -specific (CD8+ CD44+ H-2Kb/TRP-2 180–188 -pentamer+ cells, right) activated CD8+ T cells from all five mice in each group of the experiment. (C) Frequencies of interferon <t>(IFN)-γ</t> effector cytokine producing CD8+ CTLs were analyzed from spleen cells after overnight stimulation with TRP-2 180–188 or HPV 43–78 peptide, respectively (CD8+ CD44+ IFN-γ+ cells). Plots show representative examples of flow cytometric analysis for HPV 43–78 (upper panels) or TRP-2 180–188 (lower panels) vaccinated mice without (left) or with PCI treatment (right). (D) Quantification of IFN-γ effector cytokine production from CD8+ T cells (CD8+ CD44+ IFN-γ+ T cells) from HPV 43–48 -vaccinated (left) and TRP-2 180–188 -vaccinated mice for all five mice in each group of the experiments. Horizontal lines in scatter graphs indicate median values; P values were calculated using an unpaired two-tailed t -test to compare pairs of datasets (* P
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    Pharmingen anti ifn γ
    Effects of cytokine-specific antibodies on LPS-induced <t>IFN-γ</t> production. Isolated splenocytes were incubated with LPS (1 μg/ml) or 10 8 CFU of heat-killed bacteria per ml for 24 h in the presence of nonspecific goat IgG (10 μg/ml), anti-IL-10 (10 μg/ml), anti-IL-12 (1 μg/ml), anti-IL-15 (10 μg/ml), or anti-IL-18 (10 μg/ml). The IFN-γ levels in the conditioned media were determined by ELISA ( n = 8 to 12 wells/group). †, significantly ( P
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    Image Search Results


    Cytokine production from human whole blood stimulated by liposomes containing GLA, 3M-052, both TLR ligands, or neither (empty). Stimulated whole blood was analyzed for production of Th1-associated cytokines a IL-12p70 and b IFN-γ, as well as chemokines c Mip-1β and d MCP-1. The x -axes represent serial dilutions with starting concentrations of 4 µg/ml for GLA and 1.5 µg/ml 3M-052. The y -axes represent concentrations of target analytes secreted post whole blood stimulation with error bars representing the standard error of the mean from three blood donors with each individual value averaged from duplicate wells

    Journal: NPJ Vaccines

    Article Title: Adjuvant composition and delivery route shape immune response quality and protective efficacy of a recombinant vaccine for Entamoeba histolytica

    doi: 10.1038/s41541-018-0060-x

    Figure Lengend Snippet: Cytokine production from human whole blood stimulated by liposomes containing GLA, 3M-052, both TLR ligands, or neither (empty). Stimulated whole blood was analyzed for production of Th1-associated cytokines a IL-12p70 and b IFN-γ, as well as chemokines c Mip-1β and d MCP-1. The x -axes represent serial dilutions with starting concentrations of 4 µg/ml for GLA and 1.5 µg/ml 3M-052. The y -axes represent concentrations of target analytes secreted post whole blood stimulation with error bars representing the standard error of the mean from three blood donors with each individual value averaged from duplicate wells

    Article Snippet: Samples were incubated for 24 h at 37 °C/5% CO2 in a humidified incubator and plasma supernatants assayed by ELISA for the selected cytokines (Mip-1β [R & D Systems, catalog #DY271]; IL-12p70 [eBioscience, catalog #88-7126-86]; IFN-γ [eBioscience, catalog #88-7316-86]; MCP-1 [eBioscience, catalog #88-7399-88]).

    Techniques:

    Biological activity of adjuvant formulation: importance of PEG length and complementary roles for GLA and 3M-052. Five mice per group were immunized three times with a 2-week interval between immunizations using a mixed intranasal/subcutaneous regimen. Mice were euthanized a week after third immunization and samples collected. a Stool supernatants were diluted 250-fold and anti-LecA IgA titer was determined by ELISA. b Plasma samples were diluted 100,000-fold and titers of anti-LecA IgG1 (black circles) and IgG2a (red circles) subtypes were determined by ELISA. c Intracellular IFN-γ levels were measured using flow cytometry as described (negative values obtained after subtracting values from unstimulated cells were considered zero; the outlier test described below was conducted prior to this data transformation). Error bars represent standard error of the mean. For the analysis of the data in plot a , data were analyzed by one-way ANOVA with Sidak’s correction for selected comparisons. For the analysis of the data in plot b , data were log-transformed with a small offset if necessary and Sidak’s correction for selected comparisons was employed. All formulations in plot b elicited statistically increased ( p

    Journal: NPJ Vaccines

    Article Title: Adjuvant composition and delivery route shape immune response quality and protective efficacy of a recombinant vaccine for Entamoeba histolytica

    doi: 10.1038/s41541-018-0060-x

    Figure Lengend Snippet: Biological activity of adjuvant formulation: importance of PEG length and complementary roles for GLA and 3M-052. Five mice per group were immunized three times with a 2-week interval between immunizations using a mixed intranasal/subcutaneous regimen. Mice were euthanized a week after third immunization and samples collected. a Stool supernatants were diluted 250-fold and anti-LecA IgA titer was determined by ELISA. b Plasma samples were diluted 100,000-fold and titers of anti-LecA IgG1 (black circles) and IgG2a (red circles) subtypes were determined by ELISA. c Intracellular IFN-γ levels were measured using flow cytometry as described (negative values obtained after subtracting values from unstimulated cells were considered zero; the outlier test described below was conducted prior to this data transformation). Error bars represent standard error of the mean. For the analysis of the data in plot a , data were analyzed by one-way ANOVA with Sidak’s correction for selected comparisons. For the analysis of the data in plot b , data were log-transformed with a small offset if necessary and Sidak’s correction for selected comparisons was employed. All formulations in plot b elicited statistically increased ( p

    Article Snippet: Samples were incubated for 24 h at 37 °C/5% CO2 in a humidified incubator and plasma supernatants assayed by ELISA for the selected cytokines (Mip-1β [R & D Systems, catalog #DY271]; IL-12p70 [eBioscience, catalog #88-7126-86]; IFN-γ [eBioscience, catalog #88-7316-86]; MCP-1 [eBioscience, catalog #88-7399-88]).

    Techniques: Activity Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Transformation Assay

    M exp reduced the generation of IL-17-producing cells due to their lower CD86 expression. (A) Representative plots showing the percentages of IL-17, IL-17/IFN-γ, and IFN-γ-producing CD4 + cells after co-culture with M exp , M 25+ , and M 25− . (B) Cumulative data of nine independent experiments showing the percentages of IL-17, IL-17/IFN-γ, and IFN-γ and IL-4 producing CD4 + cells. Representative plots (C) and cumulative data (D) of four independent experiments showing the percentages of memory IL-17-producing CD4 + cells after co-culture with M 25− alone or in the presence of infliximab, tociluzmab, and abatacept. (E) Cell count of CD4 + cells ( n = 4) co-cultured with M 25− alone or in the presence of Abatacept. (F) Cumulative data of four independent experiments showing the percentages of memory IL-17-producing CD4 + stimulated with anti-CD3/28 beads in medium alone or M 25− conditioned medium. In all the experiments, data, presented as mean ± SEM, were analyzed using one-way ANOVA followed by Tukey with * p

    Journal: Frontiers in Immunology

    Article Title: Expanded Regulatory T Cells Induce Alternatively Activated Monocytes With a Reduced Capacity to Expand T Helper-17 Cells

    doi: 10.3389/fimmu.2018.01625

    Figure Lengend Snippet: M exp reduced the generation of IL-17-producing cells due to their lower CD86 expression. (A) Representative plots showing the percentages of IL-17, IL-17/IFN-γ, and IFN-γ-producing CD4 + cells after co-culture with M exp , M 25+ , and M 25− . (B) Cumulative data of nine independent experiments showing the percentages of IL-17, IL-17/IFN-γ, and IFN-γ and IL-4 producing CD4 + cells. Representative plots (C) and cumulative data (D) of four independent experiments showing the percentages of memory IL-17-producing CD4 + cells after co-culture with M 25− alone or in the presence of infliximab, tociluzmab, and abatacept. (E) Cell count of CD4 + cells ( n = 4) co-cultured with M 25− alone or in the presence of Abatacept. (F) Cumulative data of four independent experiments showing the percentages of memory IL-17-producing CD4 + stimulated with anti-CD3/28 beads in medium alone or M 25− conditioned medium. In all the experiments, data, presented as mean ± SEM, were analyzed using one-way ANOVA followed by Tukey with * p

    Article Snippet: Cells were then stained with, anti-HLA-A2 and CD45RO followed by cell permeabilization and intracellular staining using anti-IL-17 (BL168, BioLegend, USA), anti-IL-4 (8D4-8, Thermo Fisher Scientific, UK), and anti-IFN-γ (B27, Thermo Fisher Scientific, UK).

    Techniques: Expressing, Co-Culture Assay, Cell Counting, Cell Culture

    Experimental protocol. Co-culture experiments have been settled in 48-well plates pre-coated with anti-CD3 monoclonal antibody (50 ng/mL). 0.5 × 10 6 monocytes (HLA-A2 − ) have been cultured in the presence of 0.25 × 10 6 T cells (HLA-A2 + ) for 6 days. The resultant monocyte populations have been identified as M 25+ [monocytes co-cultured with freshly isolated CD4 + CD25 + regulatory T cells (Tregs)], M 25− [monocytes co-cultured with freshly isolated CD4 + CD25 − conventional T cell (Tconv)], and M exp (monocytes co-cultured with expanded Tregs). Cells were then detached and labeled with Live/Dead dying and HLA-A2 antibody for cell sorting following the gate strategy reported in Figure S1 in Supplementary Material. At the same time, the expression of CD86, CD14, CD80 CD40, CD206, CD163, and HLA-DR has been evaluated by flow cytometry. After sorting, 150 × 10 5 cells have been lysed for WB analysis and 50 × 10 3 cells per condition were placed in a new 48-well plate and left at 37°C overnight to ensure complete adhesion. Adhering cells have been stimulated with LPS for 24 h and then co-cultured, for other 6 days, with allogeneic CD4 T cells (10 5 per condition, HLA-A2 + coming from the same donor of the Tconv/Treg) in presence of soluble anti-CD3 (50 ng/mL). Intracellular staining to evaluate IL-4, IL-17, and IFN-γ-producing CD4 T cells has been executed at the end of the co-culture. Supernatant after LPS stimulation (50 ng/mL) has been used for cytokine evaluation and for the experiment using the M 25− conditioned medium.

    Journal: Frontiers in Immunology

    Article Title: Expanded Regulatory T Cells Induce Alternatively Activated Monocytes With a Reduced Capacity to Expand T Helper-17 Cells

    doi: 10.3389/fimmu.2018.01625

    Figure Lengend Snippet: Experimental protocol. Co-culture experiments have been settled in 48-well plates pre-coated with anti-CD3 monoclonal antibody (50 ng/mL). 0.5 × 10 6 monocytes (HLA-A2 − ) have been cultured in the presence of 0.25 × 10 6 T cells (HLA-A2 + ) for 6 days. The resultant monocyte populations have been identified as M 25+ [monocytes co-cultured with freshly isolated CD4 + CD25 + regulatory T cells (Tregs)], M 25− [monocytes co-cultured with freshly isolated CD4 + CD25 − conventional T cell (Tconv)], and M exp (monocytes co-cultured with expanded Tregs). Cells were then detached and labeled with Live/Dead dying and HLA-A2 antibody for cell sorting following the gate strategy reported in Figure S1 in Supplementary Material. At the same time, the expression of CD86, CD14, CD80 CD40, CD206, CD163, and HLA-DR has been evaluated by flow cytometry. After sorting, 150 × 10 5 cells have been lysed for WB analysis and 50 × 10 3 cells per condition were placed in a new 48-well plate and left at 37°C overnight to ensure complete adhesion. Adhering cells have been stimulated with LPS for 24 h and then co-cultured, for other 6 days, with allogeneic CD4 T cells (10 5 per condition, HLA-A2 + coming from the same donor of the Tconv/Treg) in presence of soluble anti-CD3 (50 ng/mL). Intracellular staining to evaluate IL-4, IL-17, and IFN-γ-producing CD4 T cells has been executed at the end of the co-culture. Supernatant after LPS stimulation (50 ng/mL) has been used for cytokine evaluation and for the experiment using the M 25− conditioned medium.

    Article Snippet: Cells were then stained with, anti-HLA-A2 and CD45RO followed by cell permeabilization and intracellular staining using anti-IL-17 (BL168, BioLegend, USA), anti-IL-4 (8D4-8, Thermo Fisher Scientific, UK), and anti-IFN-γ (B27, Thermo Fisher Scientific, UK).

    Techniques: Co-Culture Assay, Cell Culture, Isolation, Labeling, FACS, Expressing, Flow Cytometry, Cytometry, Western Blot, Staining

    IL-22BP protein and mRNA levels in monocyte-derived DCs. (A) (Upper left) Diagram showing exons with the primer-binding positions for IL22RA2 variant discrimination strategy. (Lower left) Expression of IL22RA2v1, v2 , and v3 in moDC after 6 days of culture in differentiation medium (DM) ± AM580, a retinoic acid receptor agonist, by RT-PCR. IL22RA2v2 expression vector was used as positive control for IL22RA2v2 expression. (Upper right) Expression of IL22RA2 variants in moDC following 6 days of culture in DM ± AM580 by RT-qPCR using the isoform-specific primers indicated in the left diagram (mean ± SEM; n = 3) relative to the housekeeping gene GAPDH . (B) Effect of different maturation stimuli (CpG and LPS + IFN-γ or Poly I:C) on IL22RA2, IL12B , and IL6 expression in moDC after 6 days of culture in DM by RT-qPCR using Taqman probes relative to the housekeeping gene ACTB . Extent of maturation is represented as percentage of CD83 + fluorescence by flow cytometry. Data showing representative experiment of 3 performed. (C) IL-22BP secretion by moDC cultured in DM detected by ELISA corresponds to IL22RA2 mRNA expression levels analyzed by RT-qPCR using Taqman probes and both increase over the cultivation period (mean ± SEM, n = 2). (D) Cell lysates (CL) of moDCs and conditioned media (CM) were harvested after 6 days in DM ± AM580 and immunoblotted for detection of IL-22BP (IL-22BP antibody 1) using tubulin as a loading control, IL22RA2 mRNA expression relative to the mean of the housekeeping genes GAPDH and ACTB .

    Journal: Frontiers in Immunology

    Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

    doi: 10.3389/fimmu.2018.02934

    Figure Lengend Snippet: IL-22BP protein and mRNA levels in monocyte-derived DCs. (A) (Upper left) Diagram showing exons with the primer-binding positions for IL22RA2 variant discrimination strategy. (Lower left) Expression of IL22RA2v1, v2 , and v3 in moDC after 6 days of culture in differentiation medium (DM) ± AM580, a retinoic acid receptor agonist, by RT-PCR. IL22RA2v2 expression vector was used as positive control for IL22RA2v2 expression. (Upper right) Expression of IL22RA2 variants in moDC following 6 days of culture in DM ± AM580 by RT-qPCR using the isoform-specific primers indicated in the left diagram (mean ± SEM; n = 3) relative to the housekeeping gene GAPDH . (B) Effect of different maturation stimuli (CpG and LPS + IFN-γ or Poly I:C) on IL22RA2, IL12B , and IL6 expression in moDC after 6 days of culture in DM by RT-qPCR using Taqman probes relative to the housekeeping gene ACTB . Extent of maturation is represented as percentage of CD83 + fluorescence by flow cytometry. Data showing representative experiment of 3 performed. (C) IL-22BP secretion by moDC cultured in DM detected by ELISA corresponds to IL22RA2 mRNA expression levels analyzed by RT-qPCR using Taqman probes and both increase over the cultivation period (mean ± SEM, n = 2). (D) Cell lysates (CL) of moDCs and conditioned media (CM) were harvested after 6 days in DM ± AM580 and immunoblotted for detection of IL-22BP (IL-22BP antibody 1) using tubulin as a loading control, IL22RA2 mRNA expression relative to the mean of the housekeeping genes GAPDH and ACTB .

    Article Snippet: Maturation of moDCs was induced by adding IFN-γ (Peprotech, AF-300-02; 500 pg/ ml) for overnight priming (16 h) followed by late addition of LPS for 6 hrs (Sigma, L6143; 2 μg/ml) to the culture or, alternatively by addition of CpG (Miltenyi, 130-100-243; 1 μg/ml) or LPS/Poly(I:C) (Sigma, P1530; 1 μg/ml) for 6 h. HEK293, HeLa and A549 were cultured in DMEM supplemented with 10% FBS and 2 mM L-glutamine; U937 cells were cultured in RPMI supplemented with 0.05 mM β-mercaptoethanol, 10 % FBS and 2 mM L-glutamine.

    Techniques: Derivative Assay, Binding Assay, Variant Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Positive Control, Quantitative RT-PCR, Fluorescence, Flow Cytometry, Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay

    CIK Cell Culture, Identification, and Adenovirus-Binding Ability Detection (A–C) CIK cells were isolated from human peripheral blood and differentiated by adding IFN-γ, anti-CD3 mAb, IL-1α, and IL-2 in different culture stages. CIK cells were observed under a phase contrast microscope at 200× after culturing for 5 (A), 10 (B), and 14 (C) days. Microscope observation showed that the cell number increased gradually and reached its peak and formed a cell colony on day 14, which is a defining characteristic of CIK cells. (D–I) CIK cells were harvested on day 14 and embedded in wax blocks. (D) CIK cells were sectioned and stained with H E, and they were observed under a microscope at 400× magnification. Microscope observation showed morphologically regular cells, with larger nuclei and less cytoplasm. (E and F) CIK cell markers (E, CD3 and F, CD56) were detected by immunohistochemistry and found to be expressed on CIK cell membranes at 400× magnification. (G) CD46 protein (the receptor for KGHV500 and KGHV400 adenoviruses) was also detected on CIK cell surfaces at 400× magnification. (H and I) KGHV500 (H) and KGHV400 (I) adenovirus hexon was detected on CIK cell membranes at 400× magnification.

    Journal: Molecular Therapy Oncolytics

    Article Title: Recombinant Adenovirus KGHV500 and CIK Cells Codeliver Anti-p21-Ras scFv for the Treatment of Gastric Cancer with Wild-Type Ras Overexpression

    doi: 10.1016/j.omto.2018.10.003

    Figure Lengend Snippet: CIK Cell Culture, Identification, and Adenovirus-Binding Ability Detection (A–C) CIK cells were isolated from human peripheral blood and differentiated by adding IFN-γ, anti-CD3 mAb, IL-1α, and IL-2 in different culture stages. CIK cells were observed under a phase contrast microscope at 200× after culturing for 5 (A), 10 (B), and 14 (C) days. Microscope observation showed that the cell number increased gradually and reached its peak and formed a cell colony on day 14, which is a defining characteristic of CIK cells. (D–I) CIK cells were harvested on day 14 and embedded in wax blocks. (D) CIK cells were sectioned and stained with H E, and they were observed under a microscope at 400× magnification. Microscope observation showed morphologically regular cells, with larger nuclei and less cytoplasm. (E and F) CIK cell markers (E, CD3 and F, CD56) were detected by immunohistochemistry and found to be expressed on CIK cell membranes at 400× magnification. (G) CD46 protein (the receptor for KGHV500 and KGHV400 adenoviruses) was also detected on CIK cell surfaces at 400× magnification. (H and I) KGHV500 (H) and KGHV400 (I) adenovirus hexon was detected on CIK cell membranes at 400× magnification.

    Article Snippet: Then, 1,000 U/mL recombinant human IFN-γ (PeproTech, 300-02, USA) was added to the culture on day 0.

    Techniques: Cell Culture, Binding Assay, Isolation, Microscopy, Staining, Immunohistochemistry

    Systemic inflammatory toxicity mainly derived from IL-2-Fc rather than anti-CD137. Groups of C57Bl/6 mice ( n = 4/group) were inoculated with 5 × 10 5 B16F10 tumor cells s.c. on day 0, then received i.v. injections of αCD137 and IL-2-Fc (αCD137/IL-2-Fc), Lipo-IL-2-Fc mixed with αCD137 (Lipo-IL-2-Fc/αCD137), or Lipo-αCD137 mixed with IL-2-Fc (Lipo- αCD137/IL-2-Fc) on day 8. One day later, sera were collected from peripheral blood and levels of IFNγ ( a ), IL-6 ( b ), MCP-1 ( c ) and TNFα ( d ) were assayed by luminex assays. All measurements shown are mean ± s.e.m.

    Journal: Nature Communications

    Article Title: Nanoparticle anchoring targets immune agonists to tumors enabling anti-cancer immunity without systemic toxicity

    doi: 10.1038/s41467-017-02251-3

    Figure Lengend Snippet: Systemic inflammatory toxicity mainly derived from IL-2-Fc rather than anti-CD137. Groups of C57Bl/6 mice ( n = 4/group) were inoculated with 5 × 10 5 B16F10 tumor cells s.c. on day 0, then received i.v. injections of αCD137 and IL-2-Fc (αCD137/IL-2-Fc), Lipo-IL-2-Fc mixed with αCD137 (Lipo-IL-2-Fc/αCD137), or Lipo-αCD137 mixed with IL-2-Fc (Lipo- αCD137/IL-2-Fc) on day 8. One day later, sera were collected from peripheral blood and levels of IFNγ ( a ), IL-6 ( b ), MCP-1 ( c ) and TNFα ( d ) were assayed by luminex assays. All measurements shown are mean ± s.e.m.

    Article Snippet: Fluorescent antibodies against mouse CD45 (1:100 dilution, cat# 17–0451–82), CD3 (1:100 dilution, cat# 48–0032–82), CD4 (1:100 dilution, cat# 45–0042–82), NK1.1 (1:100 dilution, cat# 25–5941–81), IFNγ (1:50 dilution, cat# 12–7311–41), CD16/32 (1:100 dilution, cat# 14–0161–82), CD25 (1:100 dilution, cat# 45–0251–82) and Foxp3 (1:50 dilution, cat# 17–5773–82) were from eBioscience.

    Techniques: Derivative Assay, Mouse Assay, Luminex

    Quality characterization of the specific T-cell anti-VACV immune response. ( A ) Groups of four C57BL/6 mice were i.m immunized with 10 7 pfu of MVAwt (white bars) or MVAΔC12L (black bars), and seven dpi the magnitude of the T-cell response against B8R peptide in the spleen was measured by IFN-γ and IL-2 Elispot assays and ( B ) degranulation of specific-CD8 + T-cells was assessed with CD107a/b staining simultaneously with IFN-γ and TNF-α production by ICS, after 5 hr of stimulation with B8R peptide. Analysis of the total B8R specific-CD8 + T-cells expressing each of the functions (left panel) and polyfunctionality of the specific-CD8 + T-cells were performed (right panel). Bars represent the frequency of CD8 + T-cells expressing the particular combination of functions indicated on the x axis. Results are expressed as mean % CD8 + anti-B8R T-cells ± SD. Each pie chart represents the mean contribution of each effector function to the total response. Green sectors stand for CD8 + cells positive for only one function (SP), yellow sectors stand for bifunctional CD8 + cells (DP), and red sectors stand for trifunctional cells (TP). ( C ) Groups of four C57BL/6 mice were i.m immunized with 10 7 pfu of MVAwt or MVAΔC12L and 7 (acute) or 35 (memory) dpi, specific cytokine production in splenocyte-culture supernatants was evaluated by ELISA after 72 hr stimulation with the indicated peptides. ( D ) Splenocytes from BALB/c mice i.m immunized with 10 7 pfu of MVAwt (green) or MVAΔC12L (red) were assayed by IFN-γ Elispot against serial dilutions of VACV E3 peptide. T-cell functional avidity is defined as the concentration required to achieve half-maximal of the response (EC 50 ). Data represents the percentage of the maximal response (net number of SFU per 10 6 cells stimulated with a peptide concentration of 20 µg/ml). Statistically significant differences: *p

    Journal: PLoS ONE

    Article Title: Improving the MVA Vaccine Potential by Deleting the Viral Gene Coding for the IL-18 Binding Protein

    doi: 10.1371/journal.pone.0032220

    Figure Lengend Snippet: Quality characterization of the specific T-cell anti-VACV immune response. ( A ) Groups of four C57BL/6 mice were i.m immunized with 10 7 pfu of MVAwt (white bars) or MVAΔC12L (black bars), and seven dpi the magnitude of the T-cell response against B8R peptide in the spleen was measured by IFN-γ and IL-2 Elispot assays and ( B ) degranulation of specific-CD8 + T-cells was assessed with CD107a/b staining simultaneously with IFN-γ and TNF-α production by ICS, after 5 hr of stimulation with B8R peptide. Analysis of the total B8R specific-CD8 + T-cells expressing each of the functions (left panel) and polyfunctionality of the specific-CD8 + T-cells were performed (right panel). Bars represent the frequency of CD8 + T-cells expressing the particular combination of functions indicated on the x axis. Results are expressed as mean % CD8 + anti-B8R T-cells ± SD. Each pie chart represents the mean contribution of each effector function to the total response. Green sectors stand for CD8 + cells positive for only one function (SP), yellow sectors stand for bifunctional CD8 + cells (DP), and red sectors stand for trifunctional cells (TP). ( C ) Groups of four C57BL/6 mice were i.m immunized with 10 7 pfu of MVAwt or MVAΔC12L and 7 (acute) or 35 (memory) dpi, specific cytokine production in splenocyte-culture supernatants was evaluated by ELISA after 72 hr stimulation with the indicated peptides. ( D ) Splenocytes from BALB/c mice i.m immunized with 10 7 pfu of MVAwt (green) or MVAΔC12L (red) were assayed by IFN-γ Elispot against serial dilutions of VACV E3 peptide. T-cell functional avidity is defined as the concentration required to achieve half-maximal of the response (EC 50 ). Data represents the percentage of the maximal response (net number of SFU per 10 6 cells stimulated with a peptide concentration of 20 µg/ml). Statistically significant differences: *p

    Article Snippet: After 72 hr incubation at 37°C in 5% CO2 , culture supernatants were harvested at −80°C and analyzed by ELISA for IFN-γ (BD OptEIA Set Mouse IFN-γ) and TNF-α (BD OptEIA Set Mouse TNF mono/poly) following the manufacturer's instructions.

    Techniques: Mouse Assay, Enzyme-linked Immunospot, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Functional Assay, Concentration Assay

    Immunogenicity of MVAΔC12L in BALB/c mice. Groups of four BALB/c mice were i.p immunized with 5×10 7 pfu of MVAwt (white bars) or MVAΔC12L (black bars), and seven dpi specific-CD8 + T-cell responses against the E3 and F2(G) VACV peptides were evaluated in the spleen. ( A ) The magnitude of the specific responses was measured by IFN-γ (left) and IL-2 (right) Elispot assays. Background (RPMI negative control) subtracted results are depicted as mean spot forming units (SFU) per 10 6 splenocytes ± SD. ( B ) Quality of the response analyzed by ICS. Degranulation of specific-CD8 + T-cells was assessed with CD107a/b mAb (cytotoxicity marker) simultaneously with IFN-γ production, after 5 hr of stimulation with VACV peptides Results are expressed as mean % CD8 + T-cells ± SD. Statistically significant differences: *p

    Journal: PLoS ONE

    Article Title: Improving the MVA Vaccine Potential by Deleting the Viral Gene Coding for the IL-18 Binding Protein

    doi: 10.1371/journal.pone.0032220

    Figure Lengend Snippet: Immunogenicity of MVAΔC12L in BALB/c mice. Groups of four BALB/c mice were i.p immunized with 5×10 7 pfu of MVAwt (white bars) or MVAΔC12L (black bars), and seven dpi specific-CD8 + T-cell responses against the E3 and F2(G) VACV peptides were evaluated in the spleen. ( A ) The magnitude of the specific responses was measured by IFN-γ (left) and IL-2 (right) Elispot assays. Background (RPMI negative control) subtracted results are depicted as mean spot forming units (SFU) per 10 6 splenocytes ± SD. ( B ) Quality of the response analyzed by ICS. Degranulation of specific-CD8 + T-cells was assessed with CD107a/b mAb (cytotoxicity marker) simultaneously with IFN-γ production, after 5 hr of stimulation with VACV peptides Results are expressed as mean % CD8 + T-cells ± SD. Statistically significant differences: *p

    Article Snippet: After 72 hr incubation at 37°C in 5% CO2 , culture supernatants were harvested at −80°C and analyzed by ELISA for IFN-γ (BD OptEIA Set Mouse IFN-γ) and TNF-α (BD OptEIA Set Mouse TNF mono/poly) following the manufacturer's instructions.

    Techniques: Mouse Assay, Enzyme-linked Immunospot, Negative Control, Marker

    MVAΔC12L elicits higher cellular responses at lower doses of immunization and by different immunization routes. Groups of four BALB/c mice were immunized by intraperitoneal (IP) intramuscular (IM) or intranasal (IN) route with 10 7 pfu of MVAwt (white bars) or MVAΔC12L (black bars) and seven dpi specific-CD8 + T-cell responses against E3 and F2(G) peptides were evaluated in the spleen. The magnitude of the response was measured by IFN-γ Elispot assay after 24 hr stimulation. Background (RPMI control) subtracted results are depicted as mean IFN-γ spot forming units (SFU) per 10 6 splenocytes ± SD. Statistically significant differences: *p

    Journal: PLoS ONE

    Article Title: Improving the MVA Vaccine Potential by Deleting the Viral Gene Coding for the IL-18 Binding Protein

    doi: 10.1371/journal.pone.0032220

    Figure Lengend Snippet: MVAΔC12L elicits higher cellular responses at lower doses of immunization and by different immunization routes. Groups of four BALB/c mice were immunized by intraperitoneal (IP) intramuscular (IM) or intranasal (IN) route with 10 7 pfu of MVAwt (white bars) or MVAΔC12L (black bars) and seven dpi specific-CD8 + T-cell responses against E3 and F2(G) peptides were evaluated in the spleen. The magnitude of the response was measured by IFN-γ Elispot assay after 24 hr stimulation. Background (RPMI control) subtracted results are depicted as mean IFN-γ spot forming units (SFU) per 10 6 splenocytes ± SD. Statistically significant differences: *p

    Article Snippet: After 72 hr incubation at 37°C in 5% CO2 , culture supernatants were harvested at −80°C and analyzed by ELISA for IFN-γ (BD OptEIA Set Mouse IFN-γ) and TNF-α (BD OptEIA Set Mouse TNF mono/poly) following the manufacturer's instructions.

    Techniques: Mouse Assay, Enzyme-linked Immunospot

    In vitro characterization of MVAΔC12L. ( A ) Corroboration of correct C12L gene deletion and abrogation of IL-18 bp expression. DNA and total RNA were extracted from CEFs infected with MVAΔC12L, MVAwt or mock-infected cells (lanes 1, 2 and 3 respectively). C12L (left) and HA (control gene, middle) sequences were amplified by PCR using specific primers. Absence of C12L mRNA (right) was assessed by RT-PCR using specific primers. M: molecular weight marker (1 kb plus DNA ladder, Invitrogen). ( B ) Analysis of virus growth in CEFs after infection at low moi (0.01 pfu/cell), with MVAΔC12L (white triangles) or MVAwt (black squares). Quantification of extra (upper panel) and intracellular (lower panel) virus yields at the different indicated time points was performed as described in Materials and Methods . ( C ) Inhibition of mouse IL-18 biological activity. To evaluate the inhibition of the IL-18-induced IFN-γ production, mice naïve splenocytes were treated with Con A (200 ng/ml) and rIL-18 (5 ng/ml) in the presence of supernatants (SN) from 10 5 CEFs that had been mock-infected or infected with the indicated viruses. The level of IFN-γ in the culture SN was determined by a standard ELISA assay 24 hs later. Statistically significant differences between MVAwt and MVAΔC12L: **p

    Journal: PLoS ONE

    Article Title: Improving the MVA Vaccine Potential by Deleting the Viral Gene Coding for the IL-18 Binding Protein

    doi: 10.1371/journal.pone.0032220

    Figure Lengend Snippet: In vitro characterization of MVAΔC12L. ( A ) Corroboration of correct C12L gene deletion and abrogation of IL-18 bp expression. DNA and total RNA were extracted from CEFs infected with MVAΔC12L, MVAwt or mock-infected cells (lanes 1, 2 and 3 respectively). C12L (left) and HA (control gene, middle) sequences were amplified by PCR using specific primers. Absence of C12L mRNA (right) was assessed by RT-PCR using specific primers. M: molecular weight marker (1 kb plus DNA ladder, Invitrogen). ( B ) Analysis of virus growth in CEFs after infection at low moi (0.01 pfu/cell), with MVAΔC12L (white triangles) or MVAwt (black squares). Quantification of extra (upper panel) and intracellular (lower panel) virus yields at the different indicated time points was performed as described in Materials and Methods . ( C ) Inhibition of mouse IL-18 biological activity. To evaluate the inhibition of the IL-18-induced IFN-γ production, mice naïve splenocytes were treated with Con A (200 ng/ml) and rIL-18 (5 ng/ml) in the presence of supernatants (SN) from 10 5 CEFs that had been mock-infected or infected with the indicated viruses. The level of IFN-γ in the culture SN was determined by a standard ELISA assay 24 hs later. Statistically significant differences between MVAwt and MVAΔC12L: **p

    Article Snippet: After 72 hr incubation at 37°C in 5% CO2 , culture supernatants were harvested at −80°C and analyzed by ELISA for IFN-γ (BD OptEIA Set Mouse IFN-γ) and TNF-α (BD OptEIA Set Mouse TNF mono/poly) following the manufacturer's instructions.

    Techniques: In Vitro, Expressing, Infection, Amplification, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Marker, Inhibition, Activity Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Analysis of the immune response generated in local draining lymph nodes (LNs) to the site of immunization. Groups of four mice were immunized as indicated in the bar charts with 10 7 pfu of MVAwt (white bars) or MVAΔC12L (black bars) and seven dpi specific T-cell responses against the indicated peptides were evaluated in the regional draining LNs to the different immunization routes as depicted in the Figure. The magnitude of the response was measured by IFN-γ Elispot assay after 24 hr stimulation. Background (RPMI control) subtracted data are depicted as mean IFN-γ spot forming units (SFU) per 10 6 cells ± SD. SC: subcutaneous; IN: intranasal; IM: intramuscular; ILN: inguinal lymph nodes; CLN: cervical lymph nodes. Statistically significant differences: *p

    Journal: PLoS ONE

    Article Title: Improving the MVA Vaccine Potential by Deleting the Viral Gene Coding for the IL-18 Binding Protein

    doi: 10.1371/journal.pone.0032220

    Figure Lengend Snippet: Analysis of the immune response generated in local draining lymph nodes (LNs) to the site of immunization. Groups of four mice were immunized as indicated in the bar charts with 10 7 pfu of MVAwt (white bars) or MVAΔC12L (black bars) and seven dpi specific T-cell responses against the indicated peptides were evaluated in the regional draining LNs to the different immunization routes as depicted in the Figure. The magnitude of the response was measured by IFN-γ Elispot assay after 24 hr stimulation. Background (RPMI control) subtracted data are depicted as mean IFN-γ spot forming units (SFU) per 10 6 cells ± SD. SC: subcutaneous; IN: intranasal; IM: intramuscular; ILN: inguinal lymph nodes; CLN: cervical lymph nodes. Statistically significant differences: *p

    Article Snippet: After 72 hr incubation at 37°C in 5% CO2 , culture supernatants were harvested at −80°C and analyzed by ELISA for IFN-γ (BD OptEIA Set Mouse IFN-γ) and TNF-α (BD OptEIA Set Mouse TNF mono/poly) following the manufacturer's instructions.

    Techniques: Generated, Mouse Assay, Enzyme-linked Immunospot

    MVAΔC12L improves T-cell memory responses conferring a higher grade of protection against a VACV challenge. ( A ) Groups of four BALB/c mice were i.p immunized with 10 7 pfu of MVAwt or MVAΔC12L. At 40 dpi the magnitude of the T-cell response in the spleen was measured by a more sensitive IFN-γ Elispot assay as described in Materials and Methods . ( B ) Groups of four C57BL/6 mice were i.m inoculated with 10 7 pfu of the indicated vectors and at 40 dpi the magnitude of the response was evaluated by IFN-γ Elispot assay against the CD8 + (B8R) and CD4 + (E9L) VACV peptides in the spleen and ILNs. ( C ) Groups of 11 BALB/c mice were i.p vaccinated with 10 6 pfu of MVAwt, MVAΔC12L or mock immunized and 45 dpi all animals were intranasally challenged with 2×10 6 pfu of the VACV WR strain. Mice were daily weighed during 12 days and the mean % of accumulative weight loss for each group was calculated (see Materials and Methods ). ( D ) Detailed % of accumulative weight loss of individual animals within each indicated group at 5, 6, 7 and 8 days post-challenge, median values are shown. ( E ) Groups of 4 BALB/c mice were i.p immunized with 2×10 6 pfu of the vectors and 40 dpi the response against E3 peptide and/or P815-MVA infected cells was evaluated by IFN-γ and IL-2 Elispot assays. Background (RPMI control) subtracted results are depicted as mean spot forming units (SFU) per 10 6 cells ± SD. Statistically significant differences: *p

    Journal: PLoS ONE

    Article Title: Improving the MVA Vaccine Potential by Deleting the Viral Gene Coding for the IL-18 Binding Protein

    doi: 10.1371/journal.pone.0032220

    Figure Lengend Snippet: MVAΔC12L improves T-cell memory responses conferring a higher grade of protection against a VACV challenge. ( A ) Groups of four BALB/c mice were i.p immunized with 10 7 pfu of MVAwt or MVAΔC12L. At 40 dpi the magnitude of the T-cell response in the spleen was measured by a more sensitive IFN-γ Elispot assay as described in Materials and Methods . ( B ) Groups of four C57BL/6 mice were i.m inoculated with 10 7 pfu of the indicated vectors and at 40 dpi the magnitude of the response was evaluated by IFN-γ Elispot assay against the CD8 + (B8R) and CD4 + (E9L) VACV peptides in the spleen and ILNs. ( C ) Groups of 11 BALB/c mice were i.p vaccinated with 10 6 pfu of MVAwt, MVAΔC12L or mock immunized and 45 dpi all animals were intranasally challenged with 2×10 6 pfu of the VACV WR strain. Mice were daily weighed during 12 days and the mean % of accumulative weight loss for each group was calculated (see Materials and Methods ). ( D ) Detailed % of accumulative weight loss of individual animals within each indicated group at 5, 6, 7 and 8 days post-challenge, median values are shown. ( E ) Groups of 4 BALB/c mice were i.p immunized with 2×10 6 pfu of the vectors and 40 dpi the response against E3 peptide and/or P815-MVA infected cells was evaluated by IFN-γ and IL-2 Elispot assays. Background (RPMI control) subtracted results are depicted as mean spot forming units (SFU) per 10 6 cells ± SD. Statistically significant differences: *p

    Article Snippet: After 72 hr incubation at 37°C in 5% CO2 , culture supernatants were harvested at −80°C and analyzed by ELISA for IFN-γ (BD OptEIA Set Mouse IFN-γ) and TNF-α (BD OptEIA Set Mouse TNF mono/poly) following the manufacturer's instructions.

    Techniques: Mouse Assay, Enzyme-linked Immunospot, Infection

    Immunogenicity of MVAΔC12L in C57BL/6 mice. Groups of four C57BL/6 mice were i.p vaccinated with 5×10 7 pfu of MVAwt (white bars) or MVAΔC12L (black bars) and seven dpi T-cell responses against VACV peptides B8R (CD8 + -specific), E9L, H3L and L4R (CD4 + -specific) were evaluated in the spleen. ( A ) The magnitude of the response was measured by IFN-γ Elispot assay after 24 hr stimulation. Background (RPMI negative control) subtracted results are depicted as mean IFN-γ spot forming units (SFU) per 10 6 splenocytes ± SD. ( B ) IFN-γ production in splenocyte-culture supernatants was evaluated by ELISA after 72 hr stimulation. ( C ) Quality of the response was analyzed by ICS. Degranulation of specific-CD8 + T-cells was assessed with CD107a/b mAb together with IFN-γ production, after 5 hr stimulation with the indicated peptides. Results are expressed as mean % of specific CD8 + T-cells ± SD. Statistically significant differences: *p

    Journal: PLoS ONE

    Article Title: Improving the MVA Vaccine Potential by Deleting the Viral Gene Coding for the IL-18 Binding Protein

    doi: 10.1371/journal.pone.0032220

    Figure Lengend Snippet: Immunogenicity of MVAΔC12L in C57BL/6 mice. Groups of four C57BL/6 mice were i.p vaccinated with 5×10 7 pfu of MVAwt (white bars) or MVAΔC12L (black bars) and seven dpi T-cell responses against VACV peptides B8R (CD8 + -specific), E9L, H3L and L4R (CD4 + -specific) were evaluated in the spleen. ( A ) The magnitude of the response was measured by IFN-γ Elispot assay after 24 hr stimulation. Background (RPMI negative control) subtracted results are depicted as mean IFN-γ spot forming units (SFU) per 10 6 splenocytes ± SD. ( B ) IFN-γ production in splenocyte-culture supernatants was evaluated by ELISA after 72 hr stimulation. ( C ) Quality of the response was analyzed by ICS. Degranulation of specific-CD8 + T-cells was assessed with CD107a/b mAb together with IFN-γ production, after 5 hr stimulation with the indicated peptides. Results are expressed as mean % of specific CD8 + T-cells ± SD. Statistically significant differences: *p

    Article Snippet: After 72 hr incubation at 37°C in 5% CO2 , culture supernatants were harvested at −80°C and analyzed by ELISA for IFN-γ (BD OptEIA Set Mouse IFN-γ) and TNF-α (BD OptEIA Set Mouse TNF mono/poly) following the manufacturer's instructions.

    Techniques: Mouse Assay, Enzyme-linked Immunospot, Negative Control, Enzyme-linked Immunosorbent Assay

    MVAΔC12L induced strengthened cellular responses against recombinant HIV antigens compared to MVA after DNA/MVA immunizations. ( A ) Description of the different vectors employed in the DNA/MVA immunization schemes applied, each group consisted of three to four BALB/c mice. ( B ) Seven days after the boost, specific-CD8 + T-cell responses against NefBF (homologous) or NefB (heterologous) peptides were evaluated in the spleen. ( C ) Ten days after the boost specific responses against HIV-1 subtype C antigens were evaluated in the spleen. To the left it is depicted the responses found against the total pool of peptides analyzed, in the right panel it can be seen the detailed analysis showing the pool of peptides targeted comprising the different HIV antigens that are expressed from the vectors. The magnitude of the response was measured by IFN-γ Elispot assay after 24 hr stimulation. Background (RPMI control) subtracted results are depicted as mean IFN-γ spot forming units (SFU) per 10 6 splenocytes ± SD. Statistically significant differences: *p

    Journal: PLoS ONE

    Article Title: Improving the MVA Vaccine Potential by Deleting the Viral Gene Coding for the IL-18 Binding Protein

    doi: 10.1371/journal.pone.0032220

    Figure Lengend Snippet: MVAΔC12L induced strengthened cellular responses against recombinant HIV antigens compared to MVA after DNA/MVA immunizations. ( A ) Description of the different vectors employed in the DNA/MVA immunization schemes applied, each group consisted of three to four BALB/c mice. ( B ) Seven days after the boost, specific-CD8 + T-cell responses against NefBF (homologous) or NefB (heterologous) peptides were evaluated in the spleen. ( C ) Ten days after the boost specific responses against HIV-1 subtype C antigens were evaluated in the spleen. To the left it is depicted the responses found against the total pool of peptides analyzed, in the right panel it can be seen the detailed analysis showing the pool of peptides targeted comprising the different HIV antigens that are expressed from the vectors. The magnitude of the response was measured by IFN-γ Elispot assay after 24 hr stimulation. Background (RPMI control) subtracted results are depicted as mean IFN-γ spot forming units (SFU) per 10 6 splenocytes ± SD. Statistically significant differences: *p

    Article Snippet: After 72 hr incubation at 37°C in 5% CO2 , culture supernatants were harvested at −80°C and analyzed by ELISA for IFN-γ (BD OptEIA Set Mouse IFN-γ) and TNF-α (BD OptEIA Set Mouse TNF mono/poly) following the manufacturer's instructions.

    Techniques: Recombinant, Mouse Assay, Enzyme-linked Immunospot

    Isolation of non-MAIT MR1-restricted T cell clones after stimulation with A375-MR1 cells in the absence of microbial antigens. ( A ) FACS analysis of purified T cells previously expanded with irradiated A375-MR1 cells following overnight co-culture with A375-MR1 cells. Left dot plot shows CD3 and CellTrace violet (CTV) staining in live cells. Upper right and bottom right dot plots show CD69 and CD137 expression on CD3 + CTV − and CD3 + CTV + gated cells, respectively. Arrows indicate gating hierarchy. Numbers indicate the percentages of cells within the gates. Cells from Donor A are illustrated as a representative donor. ( B, D ) Cumulative results of T cell clones screening from Donors A and B. T cell clones were generated from CD3 + CTV - CD137 high sorted T cells as depicted in A. Graphs show the individual clones (x axis) and their IFN-γ release (y axis), expressed as ratio between the amount of cytokine secreted in response to A375-MR1 cells vs. A375 WT cells. Each dot represents a single T cell clone, tested at the same time in the indicated experimental conditions. The horizontal red line marks the arbitrary IFN-γ ratio cut-off of two, above which MR1-dependent T cell clone reactivity was set. The intercept of the vertical red line indicates the number of MR1-restricted T cell clones in each donor. Red boxes highlight T cell clones whose reactivity was MR1-dependent. Results are representative of two independent experiments. ( C, E ) IFN-γ release by 14 representative clones from Donor A and 11 clones from Donor B after stimulation with A375 WT, A375-MR1 and A375-MR1 in the presence of blocking anti-MR1 mAbs (α-MR1). Dots represent the IFN-γ release (mean ± SD of duplicate cultures) by each clone. Results are representative of three independent experiments. *p

    Journal: eLife

    Article Title: Functionally diverse human T cells recognize non-microbial antigens presented by MR1

    doi: 10.7554/eLife.24476

    Figure Lengend Snippet: Isolation of non-MAIT MR1-restricted T cell clones after stimulation with A375-MR1 cells in the absence of microbial antigens. ( A ) FACS analysis of purified T cells previously expanded with irradiated A375-MR1 cells following overnight co-culture with A375-MR1 cells. Left dot plot shows CD3 and CellTrace violet (CTV) staining in live cells. Upper right and bottom right dot plots show CD69 and CD137 expression on CD3 + CTV − and CD3 + CTV + gated cells, respectively. Arrows indicate gating hierarchy. Numbers indicate the percentages of cells within the gates. Cells from Donor A are illustrated as a representative donor. ( B, D ) Cumulative results of T cell clones screening from Donors A and B. T cell clones were generated from CD3 + CTV - CD137 high sorted T cells as depicted in A. Graphs show the individual clones (x axis) and their IFN-γ release (y axis), expressed as ratio between the amount of cytokine secreted in response to A375-MR1 cells vs. A375 WT cells. Each dot represents a single T cell clone, tested at the same time in the indicated experimental conditions. The horizontal red line marks the arbitrary IFN-γ ratio cut-off of two, above which MR1-dependent T cell clone reactivity was set. The intercept of the vertical red line indicates the number of MR1-restricted T cell clones in each donor. Red boxes highlight T cell clones whose reactivity was MR1-dependent. Results are representative of two independent experiments. ( C, E ) IFN-γ release by 14 representative clones from Donor A and 11 clones from Donor B after stimulation with A375 WT, A375-MR1 and A375-MR1 in the presence of blocking anti-MR1 mAbs (α-MR1). Dots represent the IFN-γ release (mean ± SD of duplicate cultures) by each clone. Results are representative of three independent experiments. *p

    Article Snippet: MR1T cell activation was evaluated by measuring IFN-γ, IL-8 (R & D) and IL-13 (Pharmingen) in the supernatants by ELISA.

    Techniques: Isolation, Clone Assay, FACS, Purification, Irradiation, Co-Culture Assay, Staining, Expressing, Generated, Blocking Assay

    MR1 surface expression by monocyte-derived dendritic cells and LS 174 T cells. ( A ) Flow cytometry detection of MR1 surface protein expression on monocyte-derived dendritic cells (Mo-DCs) generated from blood of three healthy donors. Grey histograms represent staining with isotype-matched control mAbs. ( B ) Recognition of Mo-DCs from the three donors in A by DGB129 MR1T cell clones in the absence or presence of anti-MR1 (α-MR1) mAbs. IFN-γ release in the supernatants is shown and expressed as mean ± SD. Results are representative of three independent experiments. *p

    Journal: eLife

    Article Title: Functionally diverse human T cells recognize non-microbial antigens presented by MR1

    doi: 10.7554/eLife.24476

    Figure Lengend Snippet: MR1 surface expression by monocyte-derived dendritic cells and LS 174 T cells. ( A ) Flow cytometry detection of MR1 surface protein expression on monocyte-derived dendritic cells (Mo-DCs) generated from blood of three healthy donors. Grey histograms represent staining with isotype-matched control mAbs. ( B ) Recognition of Mo-DCs from the three donors in A by DGB129 MR1T cell clones in the absence or presence of anti-MR1 (α-MR1) mAbs. IFN-γ release in the supernatants is shown and expressed as mean ± SD. Results are representative of three independent experiments. *p

    Article Snippet: MR1T cell activation was evaluated by measuring IFN-γ, IL-8 (R & D) and IL-13 (Pharmingen) in the supernatants by ELISA.

    Techniques: Expressing, Derivative Assay, Flow Cytometry, Cytometry, Generated, Staining, Clone Assay

    MR1T cells recognize diverse antigens not derived from RPMI 1640 medium. Stimulation of the DGB129 MR1T cell clone by MR1-overexpressing ( A ) A375 cells (A375-MR1) and ( B ) THP-1 cells (THP1-MR1) grown for 4 days in RPMI 1640 or in PBS both supplemented with 5% human serum. Inhibition of T cell clone reactivity by anti-MR1 blocking mAbs (α-MR1) is shown. DGB129 cells recognize APCs loaded with fractions isolated from ( C ) THP-1 cell lysate or from ( D ) in vivo grown mouse breast tumor EMT6. Fractions E1 and E2 contain hydrophobic molecules; fractions N1-N4 contain hydrophilic molecules. ( E ) DGB70 MR1T cells react to N3 fraction of THP-1 lysate. ( F ) Stimulation of DGB129 and DGB70 T cells by THP-1-derived fractions N3 and N4 loaded onto plastic-bound recombinant MR1. Shown is T cell release of IFN-γ or GM-CSF mean ± SD of duplicate cultures (representative of three independent experiments). Total cytokine release is shown in panels A, B, F; fold increase over background is shown in panels C, D, E. *p

    Journal: eLife

    Article Title: Functionally diverse human T cells recognize non-microbial antigens presented by MR1

    doi: 10.7554/eLife.24476

    Figure Lengend Snippet: MR1T cells recognize diverse antigens not derived from RPMI 1640 medium. Stimulation of the DGB129 MR1T cell clone by MR1-overexpressing ( A ) A375 cells (A375-MR1) and ( B ) THP-1 cells (THP1-MR1) grown for 4 days in RPMI 1640 or in PBS both supplemented with 5% human serum. Inhibition of T cell clone reactivity by anti-MR1 blocking mAbs (α-MR1) is shown. DGB129 cells recognize APCs loaded with fractions isolated from ( C ) THP-1 cell lysate or from ( D ) in vivo grown mouse breast tumor EMT6. Fractions E1 and E2 contain hydrophobic molecules; fractions N1-N4 contain hydrophilic molecules. ( E ) DGB70 MR1T cells react to N3 fraction of THP-1 lysate. ( F ) Stimulation of DGB129 and DGB70 T cells by THP-1-derived fractions N3 and N4 loaded onto plastic-bound recombinant MR1. Shown is T cell release of IFN-γ or GM-CSF mean ± SD of duplicate cultures (representative of three independent experiments). Total cytokine release is shown in panels A, B, F; fold increase over background is shown in panels C, D, E. *p

    Article Snippet: MR1T cell activation was evaluated by measuring IFN-γ, IL-8 (R & D) and IL-13 (Pharmingen) in the supernatants by ELISA.

    Techniques: Derivative Assay, Inhibition, Blocking Assay, Isolation, In Vivo, Recombinant

    Non-MAIT MR1-restricted T cells are readily detectable in the blood of healthy individuals. ( A ) Flow cytometry analysis of purified T cells from a representative donor (Donor C) after overnight co-culture with A375 WT or A375-MR1 cells. Dot plots show CD69 and CD137 expression on live CD3 + cells. Numbers indicate the percentage of cells in the gates. ( B ) Frequency of CD69 + CD137 + T cells from five different donors after overnight co-culture with A375 WT or A375-MR1 cells. ( C ) Cumulative results of T cell clone stimulation assays from Donor C. T cell clones were generated from CD3 + CD69 + CD137 + sorted T cells as depicted in A, right dot plot. The graph shows the number of tested clones (x axis) and IFN-γ release (y axis) expressed as ratio between the amount of cytokine secreted in response to A375-MR1 cells vs. A375-WT cells. Each dot represents a single T cell clone, tested at the same time in the indicated experimental conditions. The horizontal red line marks the arbitrary IFN-γ ratio threshold of two above which MR1-dependent T cell clone reactivity was set. The intercept of the vertical red line indicates the number of MR1-restricted T cell clones. Red box highlights T cell clones whose reactivity was MR1-dependent. Results are representative of two independent experiments. ( D ) Recognition of A375-MR1 but not A375 WT cells in the absence of exogenous antigens by eight representative MR1-restricted T cell clones from Donor C. Inhibition of T cell clone reactivity to A375-MR1 cells by blocking anti-MR1 mAbs (α-MR1). Dots represent the IFN-γ release (mean ± SD of duplicate cultures) by each clone tested in the three experimental conditions. Results are representative of three independent experiments. *p

    Journal: eLife

    Article Title: Functionally diverse human T cells recognize non-microbial antigens presented by MR1

    doi: 10.7554/eLife.24476

    Figure Lengend Snippet: Non-MAIT MR1-restricted T cells are readily detectable in the blood of healthy individuals. ( A ) Flow cytometry analysis of purified T cells from a representative donor (Donor C) after overnight co-culture with A375 WT or A375-MR1 cells. Dot plots show CD69 and CD137 expression on live CD3 + cells. Numbers indicate the percentage of cells in the gates. ( B ) Frequency of CD69 + CD137 + T cells from five different donors after overnight co-culture with A375 WT or A375-MR1 cells. ( C ) Cumulative results of T cell clone stimulation assays from Donor C. T cell clones were generated from CD3 + CD69 + CD137 + sorted T cells as depicted in A, right dot plot. The graph shows the number of tested clones (x axis) and IFN-γ release (y axis) expressed as ratio between the amount of cytokine secreted in response to A375-MR1 cells vs. A375-WT cells. Each dot represents a single T cell clone, tested at the same time in the indicated experimental conditions. The horizontal red line marks the arbitrary IFN-γ ratio threshold of two above which MR1-dependent T cell clone reactivity was set. The intercept of the vertical red line indicates the number of MR1-restricted T cell clones. Red box highlights T cell clones whose reactivity was MR1-dependent. Results are representative of two independent experiments. ( D ) Recognition of A375-MR1 but not A375 WT cells in the absence of exogenous antigens by eight representative MR1-restricted T cell clones from Donor C. Inhibition of T cell clone reactivity to A375-MR1 cells by blocking anti-MR1 mAbs (α-MR1). Dots represent the IFN-γ release (mean ± SD of duplicate cultures) by each clone tested in the three experimental conditions. Results are representative of three independent experiments. *p

    Article Snippet: MR1T cell activation was evaluated by measuring IFN-γ, IL-8 (R & D) and IL-13 (Pharmingen) in the supernatants by ELISA.

    Techniques: Flow Cytometry, Cytometry, Purification, Co-Culture Assay, Expressing, Clone Assay, Generated, Inhibition, Blocking Assay

    Recognition of non-microbial antigens by MR1-restricted T cells. ( A ) Surface expression of MR1 by CCRFSB, THP-1 and A375-MR1 cells. Grey histograms indicate staining with isotype-matched control mAbs. Stimulation of ( B ) T cell clone DGB129 or ( C ) MAIT cell clone SMC3 by the three cell lines in A in the absence (no Ag) or presence of E. coli lysate ( E. coli ) and/or anti-MR1 blocking mAbs (α-MR1). Columns indicate IFN-γ release (mean + SD). Stimulation of ( D ) DGB129 MR1T or ( E ) SMC3 MAIT cells by THP-1 cells, constitutively expressing surface MR1, loaded with synthetic 6,7-dimethyl-8-D-ribityllumazine (RL-6,7-diMe) with or without anti-MR1 mAbs. Columns indicate mean IFN-γ release + SD. Stimulation of ( F ) SKW-3 cells expressing the DGB129 TCR (SKW3-DGB129) or ( G ) J.RT3-T3.5 cells expressing the MAIT MRC25 clone TCR (J.RT3-MAIT) with A375 cells that expressed (A375-MR1) or lacked (A375-WT) MR1, with or without E. coli lysate and/or anti-MR1 mAbs. CD69 median fluorescence intensity (MFI) ± SD of duplicate cultures of transduced T cells are shown. The CD69 MFI of transduced T cells cultured in the absence of APCs is also shown. Data are representative of four ( A, B and C ), two ( D and E ), and three ( F and G ) independent experiments. *p

    Journal: eLife

    Article Title: Functionally diverse human T cells recognize non-microbial antigens presented by MR1

    doi: 10.7554/eLife.24476

    Figure Lengend Snippet: Recognition of non-microbial antigens by MR1-restricted T cells. ( A ) Surface expression of MR1 by CCRFSB, THP-1 and A375-MR1 cells. Grey histograms indicate staining with isotype-matched control mAbs. Stimulation of ( B ) T cell clone DGB129 or ( C ) MAIT cell clone SMC3 by the three cell lines in A in the absence (no Ag) or presence of E. coli lysate ( E. coli ) and/or anti-MR1 blocking mAbs (α-MR1). Columns indicate IFN-γ release (mean + SD). Stimulation of ( D ) DGB129 MR1T or ( E ) SMC3 MAIT cells by THP-1 cells, constitutively expressing surface MR1, loaded with synthetic 6,7-dimethyl-8-D-ribityllumazine (RL-6,7-diMe) with or without anti-MR1 mAbs. Columns indicate mean IFN-γ release + SD. Stimulation of ( F ) SKW-3 cells expressing the DGB129 TCR (SKW3-DGB129) or ( G ) J.RT3-T3.5 cells expressing the MAIT MRC25 clone TCR (J.RT3-MAIT) with A375 cells that expressed (A375-MR1) or lacked (A375-WT) MR1, with or without E. coli lysate and/or anti-MR1 mAbs. CD69 median fluorescence intensity (MFI) ± SD of duplicate cultures of transduced T cells are shown. The CD69 MFI of transduced T cells cultured in the absence of APCs is also shown. Data are representative of four ( A, B and C ), two ( D and E ), and three ( F and G ) independent experiments. *p

    Article Snippet: MR1T cell activation was evaluated by measuring IFN-γ, IL-8 (R & D) and IL-13 (Pharmingen) in the supernatants by ELISA.

    Techniques: Expressing, Staining, Blocking Assay, Fluorescence, Cell Culture

    Cytokines released by antigen-stimulated MR1T and MAIT cell clones. ( A ) IFN-γ released by 7 MR1T cell clones stimulated with A375-MR1 cells and 2 MAIT cell clones (MRC25 and SMC3) stimulated with A375-MR1 cells pulsed with E. coli lysate. ELISA results are expressed as mean ± SD of IFN-γ release measured in duplicate cultures. ( B ) Analysis of 16 additional cytokines by multiplex cytokine assay performed on the same supernatants for which IFN-γ is shown in A. Results are representative of two independent experiments. DOI: http://dx.doi.org/10.7554/eLife.24476.013

    Journal: eLife

    Article Title: Functionally diverse human T cells recognize non-microbial antigens presented by MR1

    doi: 10.7554/eLife.24476

    Figure Lengend Snippet: Cytokines released by antigen-stimulated MR1T and MAIT cell clones. ( A ) IFN-γ released by 7 MR1T cell clones stimulated with A375-MR1 cells and 2 MAIT cell clones (MRC25 and SMC3) stimulated with A375-MR1 cells pulsed with E. coli lysate. ELISA results are expressed as mean ± SD of IFN-γ release measured in duplicate cultures. ( B ) Analysis of 16 additional cytokines by multiplex cytokine assay performed on the same supernatants for which IFN-γ is shown in A. Results are representative of two independent experiments. DOI: http://dx.doi.org/10.7554/eLife.24476.013

    Article Snippet: MR1T cell activation was evaluated by measuring IFN-γ, IL-8 (R & D) and IL-13 (Pharmingen) in the supernatants by ELISA.

    Techniques: Clone Assay, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Cytokine Assay

    MR1T cell clones do not recognize Ac-6-FP. ( A ) Stimulation of three representative MR1T cell clones by A375-MR1 cells in the absence or presence of acetyl-6-formyl pterin (Ac-6-FP). ( B ) Stimulation of two MAIT cell clones (MRC25 and SMC3) by A375-MR1 cells pulsed with E. coli lysate in the absence or presence of Ac-6-FP. ( C ) A375-MR1 cells were treated with zoledronate (Zol) in the absence or presence of Ac-6-FP (25 µg/ml) and used to stimulate a TCR Vγ9-Vδ2 cell clone (G2B9). ( D ) A375 cells expressing K43A mutant MR1 molecules (A375-MR1 K43A) were used to stimulate the three MR1T cell clones shown in A, in the absence or presence of Ac-6-FP (25 µg/ml). ( E ) Stimulation of the two MAIT cell clones used in B by A375-MR1 K43A cells pulsed with E. coli lysate in the absence or presence of Ac-6-FP (25 µg/ml). Results are expressed as mean ± SD of IFN-γ release assessed in duplicate cultures and are representative of three independent experiments. *p

    Journal: eLife

    Article Title: Functionally diverse human T cells recognize non-microbial antigens presented by MR1

    doi: 10.7554/eLife.24476

    Figure Lengend Snippet: MR1T cell clones do not recognize Ac-6-FP. ( A ) Stimulation of three representative MR1T cell clones by A375-MR1 cells in the absence or presence of acetyl-6-formyl pterin (Ac-6-FP). ( B ) Stimulation of two MAIT cell clones (MRC25 and SMC3) by A375-MR1 cells pulsed with E. coli lysate in the absence or presence of Ac-6-FP. ( C ) A375-MR1 cells were treated with zoledronate (Zol) in the absence or presence of Ac-6-FP (25 µg/ml) and used to stimulate a TCR Vγ9-Vδ2 cell clone (G2B9). ( D ) A375 cells expressing K43A mutant MR1 molecules (A375-MR1 K43A) were used to stimulate the three MR1T cell clones shown in A, in the absence or presence of Ac-6-FP (25 µg/ml). ( E ) Stimulation of the two MAIT cell clones used in B by A375-MR1 K43A cells pulsed with E. coli lysate in the absence or presence of Ac-6-FP (25 µg/ml). Results are expressed as mean ± SD of IFN-γ release assessed in duplicate cultures and are representative of three independent experiments. *p

    Article Snippet: MR1T cell activation was evaluated by measuring IFN-γ, IL-8 (R & D) and IL-13 (Pharmingen) in the supernatants by ELISA.

    Techniques: Clone Assay, Expressing, Mutagenesis

    GBP1 is required for IFN-γ-induced processing of IDO, IL-6 and IL-8. ( A , B ) Expression of GBP1 was dramatically upregulated by treatment with IFN-γ at 10 ng/ml, as determined by RT-PCR ( A ) and western blot ( B . ( C ) Knockdown of GBP1 impaired the mRNA expression of indoleamine 2,3 dioxygenase (IDO) induced by treatment with IFN-γ (#p

    Journal: Scientific Reports

    Article Title: Guanylate Binding Protein 1 Inhibits Osteogenic Differentiation of Human Mesenchymal Stromal Cells Derived from Bone Marrow

    doi: 10.1038/s41598-018-19401-2

    Figure Lengend Snippet: GBP1 is required for IFN-γ-induced processing of IDO, IL-6 and IL-8. ( A , B ) Expression of GBP1 was dramatically upregulated by treatment with IFN-γ at 10 ng/ml, as determined by RT-PCR ( A ) and western blot ( B . ( C ) Knockdown of GBP1 impaired the mRNA expression of indoleamine 2,3 dioxygenase (IDO) induced by treatment with IFN-γ (#p

    Article Snippet: 10 ng/ml recombinant human IFN-γ (R & D systems, Minneapolis, MN, USA) was used to treat hBM-MSCs as indicated.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Depletion of GBP1 partially rescued the inhibited osteogenesis by IFN-γ treatment in hBM-MSCs. ( A , B ) Knockdown of GBP1 partially restored the inhibited ALP activity by IFN-γ treatment, as determined by ALP staining ( A ) and quantitative ALP activity assay ( B ). ( C ) Knockdown of GBP1 partially rescued the reduced ECM mineralization by IFN-γ treatment. ( D ) Quantification of ARS staining in ( D ). *p

    Journal: Scientific Reports

    Article Title: Guanylate Binding Protein 1 Inhibits Osteogenic Differentiation of Human Mesenchymal Stromal Cells Derived from Bone Marrow

    doi: 10.1038/s41598-018-19401-2

    Figure Lengend Snippet: Depletion of GBP1 partially rescued the inhibited osteogenesis by IFN-γ treatment in hBM-MSCs. ( A , B ) Knockdown of GBP1 partially restored the inhibited ALP activity by IFN-γ treatment, as determined by ALP staining ( A ) and quantitative ALP activity assay ( B ). ( C ) Knockdown of GBP1 partially rescued the reduced ECM mineralization by IFN-γ treatment. ( D ) Quantification of ARS staining in ( D ). *p

    Article Snippet: 10 ng/ml recombinant human IFN-γ (R & D systems, Minneapolis, MN, USA) was used to treat hBM-MSCs as indicated.

    Techniques: ALP Assay, Activity Assay, Staining

    Antigen targeting to DCs via DC-ASGPR favors the generation of antigen-specific IL-10–producing CD4 + T cells. IFNDCs (5 × 10 3 ) were loaded with 1 µg/ml α-DC-ASGPR-HA1 or α-LOX-1-HA1, and then co-cultured with CFSE-labeled autologous total CD4 + T cells (1–2 × 10 5 ) for 7 d. (A) CD4 + T cells were restimulated with peptide HA1 250-266 for 48h, and cytokine levels in the culture supernatants were measured. Each line represents the data from a single experiment. P-values were tested by Student’s t test. (B) CD4 + T cells were restimulated with indicated peptides and stained for intracellular IL-10. HA1 280-296 is a negative control. Peptides tested in B had been selected in Fig. S2 (F and G) . Four independent experiments showed similar results. (C) Naive CD4 + T cells (1–2 × 10 5 ) were co-cultured with IFNDCs (5 × 10 3 ) loaded with 1 µg/ml recombinant fusion proteins for 7 d. CD4 + T cells were restimulated with peptides indicated for 48 h. IL-10 and IFN-γ levels in culture supernatants were assessed. Error bars represent mean ± SEM of triplicate assay. Three independent experiments with 59 PSA-derived peptides showed similar results. (D) Frequency of PSA-specific IL-10–producing CD4 + T cells elicited by recombinant fusion proteins. Four independent experiments showed similar results. (E) Experiments in C were performed with blood mDCs (Lin − HLA-DR + CD11c + CD123 − ). (F) Experiments in D were performed with blood mDCs. In both E and F, two independent experiments showed similar results. (G) Expression levels of Foxp3, PD-1, and CTLA-4 on HA1 250-266 -specific (left) and PSA 30-44 -specific CD4 + T cells producing IL-10 (right). (H) After 7 d of co-culture of purified naive CD4 + T cells and DCs loaded with either α-LOX-1-PSA or α-DC-ASGPR-PSA, CD4 + T cells were stained for intracellular IFN-γ and IL-10 during restimulation with 50 ng/ml PMA and 1 µg/ml ionomycin. Three independent experiments showed similar results (G and H).

    Journal: The Journal of Experimental Medicine

    Article Title: Targeting self- and foreign antigens to dendritic cells via DC-ASGPR generates IL-10-producing suppressive CD4+ T cells

    doi: 10.1084/jem.20110399

    Figure Lengend Snippet: Antigen targeting to DCs via DC-ASGPR favors the generation of antigen-specific IL-10–producing CD4 + T cells. IFNDCs (5 × 10 3 ) were loaded with 1 µg/ml α-DC-ASGPR-HA1 or α-LOX-1-HA1, and then co-cultured with CFSE-labeled autologous total CD4 + T cells (1–2 × 10 5 ) for 7 d. (A) CD4 + T cells were restimulated with peptide HA1 250-266 for 48h, and cytokine levels in the culture supernatants were measured. Each line represents the data from a single experiment. P-values were tested by Student’s t test. (B) CD4 + T cells were restimulated with indicated peptides and stained for intracellular IL-10. HA1 280-296 is a negative control. Peptides tested in B had been selected in Fig. S2 (F and G) . Four independent experiments showed similar results. (C) Naive CD4 + T cells (1–2 × 10 5 ) were co-cultured with IFNDCs (5 × 10 3 ) loaded with 1 µg/ml recombinant fusion proteins for 7 d. CD4 + T cells were restimulated with peptides indicated for 48 h. IL-10 and IFN-γ levels in culture supernatants were assessed. Error bars represent mean ± SEM of triplicate assay. Three independent experiments with 59 PSA-derived peptides showed similar results. (D) Frequency of PSA-specific IL-10–producing CD4 + T cells elicited by recombinant fusion proteins. Four independent experiments showed similar results. (E) Experiments in C were performed with blood mDCs (Lin − HLA-DR + CD11c + CD123 − ). (F) Experiments in D were performed with blood mDCs. In both E and F, two independent experiments showed similar results. (G) Expression levels of Foxp3, PD-1, and CTLA-4 on HA1 250-266 -specific (left) and PSA 30-44 -specific CD4 + T cells producing IL-10 (right). (H) After 7 d of co-culture of purified naive CD4 + T cells and DCs loaded with either α-LOX-1-PSA or α-DC-ASGPR-PSA, CD4 + T cells were stained for intracellular IFN-γ and IL-10 during restimulation with 50 ng/ml PMA and 1 µg/ml ionomycin. Three independent experiments showed similar results (G and H).

    Article Snippet: Anti-IFN-γ and anti-IL-10 mAbs were purchased from BD.

    Techniques: Cell Culture, Labeling, Staining, Negative Control, Recombinant, Derivative Assay, Expressing, Co-Culture Assay, Purification

    Immunization with α-DC-ASGPR-HA1 and α-DC-ASGPR-PSA generates antigen-specific IL-10–producing T cell responses in nonhuman primates. (A) Expression levels of LOX-1 and DC-ASGPR in PBMCs of cynomolgus macaques. (B and C) 12 animals primed and boosted i.d. with live influenza viruses (H1N1, A/PR8/38) were divided into two groups (6 animals/group). One group of animals was immunized i.d. with 250 µg α-LOX-1-HA1 (right arm) and 250 µg α-LOX-1-PSA (left arm). The other group was immunized i.d. with 250 µg α-DC-ASGPR-HA1 (right arm) and 250 µg α-DC-ASGPR-PSA (left arm). After the second boosting with the recombinant fusion proteins, PBMCs (2 × 10 5 cells/200 µl of medium/well in 96-well plates) were restimulated with 25 µM peptide pools of HA1 (B) or PSA (C), respectively. Cytokines in the culture supernatants were measured by ELISA. Peptide pools of HIVgag were controls. Values presented in B and D are after substraction of control values. Statistical significance was tested by ANOVA. (D) 2 × 10 5 /200 µl PBMCs were stimulated with 0.1 µg/ml Staphylococcal enterotoxin B for 48h. IL-10 and IFN-γ levels in culture supernatants were measured.

    Journal: The Journal of Experimental Medicine

    Article Title: Targeting self- and foreign antigens to dendritic cells via DC-ASGPR generates IL-10-producing suppressive CD4+ T cells

    doi: 10.1084/jem.20110399

    Figure Lengend Snippet: Immunization with α-DC-ASGPR-HA1 and α-DC-ASGPR-PSA generates antigen-specific IL-10–producing T cell responses in nonhuman primates. (A) Expression levels of LOX-1 and DC-ASGPR in PBMCs of cynomolgus macaques. (B and C) 12 animals primed and boosted i.d. with live influenza viruses (H1N1, A/PR8/38) were divided into two groups (6 animals/group). One group of animals was immunized i.d. with 250 µg α-LOX-1-HA1 (right arm) and 250 µg α-LOX-1-PSA (left arm). The other group was immunized i.d. with 250 µg α-DC-ASGPR-HA1 (right arm) and 250 µg α-DC-ASGPR-PSA (left arm). After the second boosting with the recombinant fusion proteins, PBMCs (2 × 10 5 cells/200 µl of medium/well in 96-well plates) were restimulated with 25 µM peptide pools of HA1 (B) or PSA (C), respectively. Cytokines in the culture supernatants were measured by ELISA. Peptide pools of HIVgag were controls. Values presented in B and D are after substraction of control values. Statistical significance was tested by ANOVA. (D) 2 × 10 5 /200 µl PBMCs were stimulated with 0.1 µg/ml Staphylococcal enterotoxin B for 48h. IL-10 and IFN-γ levels in culture supernatants were measured.

    Article Snippet: Anti-IFN-γ and anti-IL-10 mAbs were purchased from BD.

    Techniques: Expressing, Recombinant, Enzyme-linked Immunosorbent Assay

    CD4 + T cells generated with α-DC-ASGPR-HA1 suppress HA1-specific IFN-γ–, TNF-, and IL-2–producing CD4 + T cell responses. (A) Experimental scheme for B and C. (B) FACS-sorted CFSE low effector cells generated with α-DC-ASGPR-HA1 (top) or α–LOX-1-HA1 (bottom) were co-cultured with IFNDCs (5 × 10 3 ) loaded with HA1 250-266 in the upper wells. Newly purified and CFSE-labeled CD4 + T cells (1–2 × 10 5 ) and IFNDCs (5 × 10 3 ) loaded with α–LOX-1-HA1 were co-cultured in the lower wells of trans-well plates. On day 6, proliferation of CD4 + T cells in the lower wells were assessed by measuring CFSE dilution. α–IL-10/IL-10R or control IgG was added. Statistical significance was tested by ANOVA. Three independent experiments showed similar results. Error bars represent SD. (C) Frequency of IFN-γ–, TNF-, and IL-2–expressing CD4 + T cells was measured. Each dot in lower panels represents data from independent experiments. P-values were acquired by Student’s t test.

    Journal: The Journal of Experimental Medicine

    Article Title: Targeting self- and foreign antigens to dendritic cells via DC-ASGPR generates IL-10-producing suppressive CD4+ T cells

    doi: 10.1084/jem.20110399

    Figure Lengend Snippet: CD4 + T cells generated with α-DC-ASGPR-HA1 suppress HA1-specific IFN-γ–, TNF-, and IL-2–producing CD4 + T cell responses. (A) Experimental scheme for B and C. (B) FACS-sorted CFSE low effector cells generated with α-DC-ASGPR-HA1 (top) or α–LOX-1-HA1 (bottom) were co-cultured with IFNDCs (5 × 10 3 ) loaded with HA1 250-266 in the upper wells. Newly purified and CFSE-labeled CD4 + T cells (1–2 × 10 5 ) and IFNDCs (5 × 10 3 ) loaded with α–LOX-1-HA1 were co-cultured in the lower wells of trans-well plates. On day 6, proliferation of CD4 + T cells in the lower wells were assessed by measuring CFSE dilution. α–IL-10/IL-10R or control IgG was added. Statistical significance was tested by ANOVA. Three independent experiments showed similar results. Error bars represent SD. (C) Frequency of IFN-γ–, TNF-, and IL-2–expressing CD4 + T cells was measured. Each dot in lower panels represents data from independent experiments. P-values were acquired by Student’s t test.

    Article Snippet: Anti-IFN-γ and anti-IL-10 mAbs were purchased from BD.

    Techniques: Generated, FACS, Cell Culture, Purification, Labeling, Expressing

    DC-ASGPR ligation generates antigen-specific IL-10–producing CD4 + T cells. IFNDCs (5 × 10 3 ) were loaded with 1 µg/ml recombinant fusion proteins of PSA, pooled peptides (10 µM), or none overnight. CFSE-labeled autologous naive CD4 + T cells (1–2 × 10 5 ) were co-cultured with primed DCs for 7 d. CD4 + T cells were restimulated with 1 µM PSA 102-116 . After 48 h, IL-10, IFN-γ, TNF, and IL-2 in the culture supernatants were assessed. Each dot represents the data from independent experiments. P-values were calculated by Student’s t test.

    Journal: The Journal of Experimental Medicine

    Article Title: Targeting self- and foreign antigens to dendritic cells via DC-ASGPR generates IL-10-producing suppressive CD4+ T cells

    doi: 10.1084/jem.20110399

    Figure Lengend Snippet: DC-ASGPR ligation generates antigen-specific IL-10–producing CD4 + T cells. IFNDCs (5 × 10 3 ) were loaded with 1 µg/ml recombinant fusion proteins of PSA, pooled peptides (10 µM), or none overnight. CFSE-labeled autologous naive CD4 + T cells (1–2 × 10 5 ) were co-cultured with primed DCs for 7 d. CD4 + T cells were restimulated with 1 µM PSA 102-116 . After 48 h, IL-10, IFN-γ, TNF, and IL-2 in the culture supernatants were assessed. Each dot represents the data from independent experiments. P-values were calculated by Student’s t test.

    Article Snippet: Anti-IFN-γ and anti-IL-10 mAbs were purchased from BD.

    Techniques: Ligation, Recombinant, Labeling, Cell Culture

    DC-ASGPR ligation induces IL-10 from DCs dependent on ERK/p38 contributing to the generation of antigen-specific IL-10–producing CD4 + T cells. (A) IFNDCs (10 5 ) were incubated with indicated inhibitors for 1 h, washed, and loaded with 1 µg/ml IgG4-PSA, α-LOX-1-PSA, or α-DC-ASGPR-PSA. After 24 h, culture supernatants were harvested and IL-10 production was assessed. Error bars indicate the mean ± SEM of two independent experiments with triplicate assay. (B) After 15 min of loading IFNDCs with 1 µg/ml recombinant proteins, α-DC-ASGPR-PSA, or α-LOX-1-PSA, cells were stained with indicated antibodies. Representative data from four independent experiments are presented. (C) 5 × 10 3 IFNDCs were treated with 2.5 µM PD0325901 (MEK inhibitor) or SB203580 (p38 inhibitor) for 1 h and washed thoroughly. DCs were loaded with 1 µg/ml α-DC-ASGPR-PSA or α-LOX-1-PSA. CFSE-labeled autologous naive CD4 + T cells (1–2 × 10 5 ) were co-cultured for 7 d. CD4 + T cells were then restimulated with 1 µM PSA 30-44 for 48 h. IL-10, IFN-γ, and IL-2 in culture supernatants were assessed. Each dot represents the data generated with a single experiment. Background values acquired with control peptide (PSA 82-96 ) were substracted. (D) Purified naive CD4 + T cells (1–2 × 10 5 ) were co-cultured with α-DC-ASGPR-PSA–loaded IFNDCs in the presence of control IgG or α–IL-10/IL-10R antibodies for 7 d. Cells were then restimulated with indicated peptides (1 µM) and stained for intracellular IL-10. Summary of the data from four independent experiments are presented on the right. (E) Naive CD4 + T cells (1–2 × 10 5 ) were co-cultured for 7 d with IFNDCs (5 × 10 3 ) loaded with 1 µg/ml α-LOX-1-PSA in the presence or absence of 20 pg/ml IL-10. Cells were then restimulated with indicated peptides (1 µM) and stained for intracellular IL-10. Summary of the data from five independent experiments are presented in right panel. P-values were calculated with Student’s t test.

    Journal: The Journal of Experimental Medicine

    Article Title: Targeting self- and foreign antigens to dendritic cells via DC-ASGPR generates IL-10-producing suppressive CD4+ T cells

    doi: 10.1084/jem.20110399

    Figure Lengend Snippet: DC-ASGPR ligation induces IL-10 from DCs dependent on ERK/p38 contributing to the generation of antigen-specific IL-10–producing CD4 + T cells. (A) IFNDCs (10 5 ) were incubated with indicated inhibitors for 1 h, washed, and loaded with 1 µg/ml IgG4-PSA, α-LOX-1-PSA, or α-DC-ASGPR-PSA. After 24 h, culture supernatants were harvested and IL-10 production was assessed. Error bars indicate the mean ± SEM of two independent experiments with triplicate assay. (B) After 15 min of loading IFNDCs with 1 µg/ml recombinant proteins, α-DC-ASGPR-PSA, or α-LOX-1-PSA, cells were stained with indicated antibodies. Representative data from four independent experiments are presented. (C) 5 × 10 3 IFNDCs were treated with 2.5 µM PD0325901 (MEK inhibitor) or SB203580 (p38 inhibitor) for 1 h and washed thoroughly. DCs were loaded with 1 µg/ml α-DC-ASGPR-PSA or α-LOX-1-PSA. CFSE-labeled autologous naive CD4 + T cells (1–2 × 10 5 ) were co-cultured for 7 d. CD4 + T cells were then restimulated with 1 µM PSA 30-44 for 48 h. IL-10, IFN-γ, and IL-2 in culture supernatants were assessed. Each dot represents the data generated with a single experiment. Background values acquired with control peptide (PSA 82-96 ) were substracted. (D) Purified naive CD4 + T cells (1–2 × 10 5 ) were co-cultured with α-DC-ASGPR-PSA–loaded IFNDCs in the presence of control IgG or α–IL-10/IL-10R antibodies for 7 d. Cells were then restimulated with indicated peptides (1 µM) and stained for intracellular IL-10. Summary of the data from four independent experiments are presented on the right. (E) Naive CD4 + T cells (1–2 × 10 5 ) were co-cultured for 7 d with IFNDCs (5 × 10 3 ) loaded with 1 µg/ml α-LOX-1-PSA in the presence or absence of 20 pg/ml IL-10. Cells were then restimulated with indicated peptides (1 µM) and stained for intracellular IL-10. Summary of the data from five independent experiments are presented in right panel. P-values were calculated with Student’s t test.

    Article Snippet: Anti-IFN-γ and anti-IL-10 mAbs were purchased from BD.

    Techniques: Ligation, Incubation, Recombinant, Staining, Labeling, Cell Culture, Generated, Purification

    AEC sup induces IL-1β expression and secretion without an effect of intracellular killing of BCG. BMM and PuM were infected with GFP-BCG. After infection, cells were treated with either IFN-γ or AEC sup or left untreated. a) Total RNA was extracted from both cell-types after 4 and 24 h of treatment and the mean-fold accumulation of IL-1β transcripts shown. Data shows the average of 3 independent experiments. b) Cell culture supernatants were collected 24 h after infection and IL-1β measured with ELISA. Data shows the average of 6 independent experiments. c) The effect of incubating cells with IL-1β (1000 pg/ml) on intracellular growth of BCG. Data representative of 3 independent experiments d) The effect of AECs up on intracellular growth of BCG in BMM from caspase1 -/- mice. Bacterial growth was evaluated by determining RLU. Values are expressed as means ± SD from 5 wells. The data is representative of 2 independent experiments. The differences between groups of BMM were analyzed using a one-way ANOVA followed by Bonferroni‘s Multiple Comparison Test. * significantly different from medium control, P

    Journal: PLoS ONE

    Article Title: Macrophage Control of Phagocytosed Mycobacteria Is Increased by Factors Secreted by Alveolar Epithelial Cells through Nitric Oxide Independent Mechanisms

    doi: 10.1371/journal.pone.0103411

    Figure Lengend Snippet: AEC sup induces IL-1β expression and secretion without an effect of intracellular killing of BCG. BMM and PuM were infected with GFP-BCG. After infection, cells were treated with either IFN-γ or AEC sup or left untreated. a) Total RNA was extracted from both cell-types after 4 and 24 h of treatment and the mean-fold accumulation of IL-1β transcripts shown. Data shows the average of 3 independent experiments. b) Cell culture supernatants were collected 24 h after infection and IL-1β measured with ELISA. Data shows the average of 6 independent experiments. c) The effect of incubating cells with IL-1β (1000 pg/ml) on intracellular growth of BCG. Data representative of 3 independent experiments d) The effect of AECs up on intracellular growth of BCG in BMM from caspase1 -/- mice. Bacterial growth was evaluated by determining RLU. Values are expressed as means ± SD from 5 wells. The data is representative of 2 independent experiments. The differences between groups of BMM were analyzed using a one-way ANOVA followed by Bonferroni‘s Multiple Comparison Test. * significantly different from medium control, P

    Article Snippet: After further washing, cells were either kept untreated or treated with 20 ng/ml of IFN-γ (Mabtech, Sweden), or IL-1β (1 or 10 ng/ml) or AECsup (diluted 1∶2 in cell culture media).

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

    IFN-γ and AEC sup induce an oxidative burst following infection with BCG. BMM were treated with IFN-γ (20 ng/ml) or AEC sup 24 h prior to infection and then infected with GFP-BCG. a) ROS production was monitored in the cultures for 16 h by measuring luminescence with luminol as substrate. b) The effect of incubating cells with superoxide dismutase (SOD) and catalase on intracellular killing of BCG is shown. Values are expressed as means ± SD from 5 wells. Differences between treatments with or without SOD/catalase were analyzed using a paired t-test. * significant effect of SOD/catalase treatment, P

    Journal: PLoS ONE

    Article Title: Macrophage Control of Phagocytosed Mycobacteria Is Increased by Factors Secreted by Alveolar Epithelial Cells through Nitric Oxide Independent Mechanisms

    doi: 10.1371/journal.pone.0103411

    Figure Lengend Snippet: IFN-γ and AEC sup induce an oxidative burst following infection with BCG. BMM were treated with IFN-γ (20 ng/ml) or AEC sup 24 h prior to infection and then infected with GFP-BCG. a) ROS production was monitored in the cultures for 16 h by measuring luminescence with luminol as substrate. b) The effect of incubating cells with superoxide dismutase (SOD) and catalase on intracellular killing of BCG is shown. Values are expressed as means ± SD from 5 wells. Differences between treatments with or without SOD/catalase were analyzed using a paired t-test. * significant effect of SOD/catalase treatment, P

    Article Snippet: After further washing, cells were either kept untreated or treated with 20 ng/ml of IFN-γ (Mabtech, Sweden), or IL-1β (1 or 10 ng/ml) or AECsup (diluted 1∶2 in cell culture media).

    Techniques: Infection

    Inhibiting phagosomal acidification does not affect intracellular killing of BCG. BMM and PuM were infected with GFP-BCG. After infection, cells were treated with either IFN-γ or AEC sup with or without chloroquine (10 µM). Bacterial growth was evaluated by determining RLU. Values are expressed as means ± SD from 5 wells. The data is representative of 2 independent experiments. Differences between treatments analyzed using a one-way ANOVA followed by Bonferroni's Multiple Comparison Test. * significantly different from medium control, P

    Journal: PLoS ONE

    Article Title: Macrophage Control of Phagocytosed Mycobacteria Is Increased by Factors Secreted by Alveolar Epithelial Cells through Nitric Oxide Independent Mechanisms

    doi: 10.1371/journal.pone.0103411

    Figure Lengend Snippet: Inhibiting phagosomal acidification does not affect intracellular killing of BCG. BMM and PuM were infected with GFP-BCG. After infection, cells were treated with either IFN-γ or AEC sup with or without chloroquine (10 µM). Bacterial growth was evaluated by determining RLU. Values are expressed as means ± SD from 5 wells. The data is representative of 2 independent experiments. Differences between treatments analyzed using a one-way ANOVA followed by Bonferroni's Multiple Comparison Test. * significantly different from medium control, P

    Article Snippet: After further washing, cells were either kept untreated or treated with 20 ng/ml of IFN-γ (Mabtech, Sweden), or IL-1β (1 or 10 ng/ml) or AECsup (diluted 1∶2 in cell culture media).

    Techniques: Infection

    Impaired intracellular control of BCG growth by PuM upon IFN-γ treatment. BMM and PuM were infected with GFP-BCG. After infection, cells were left untreated or treated with IFN-γ (20, 40 and 80 ng/ml) for 48 h. Bacterial growth was evaluated by determining RLU. Data are shown as % reduction of phagocytosed bacteria evaluated as RLU. Values are means ± SD of the mean of a representative experiment from 3 independent experiments with 4 replicates each. The differences between groups of BMM and PuM were analyzed using a one-way ANOVA followed by Bonferroni‘s Multiple Comparison Test, * significantly different from medium control, P

    Journal: PLoS ONE

    Article Title: Macrophage Control of Phagocytosed Mycobacteria Is Increased by Factors Secreted by Alveolar Epithelial Cells through Nitric Oxide Independent Mechanisms

    doi: 10.1371/journal.pone.0103411

    Figure Lengend Snippet: Impaired intracellular control of BCG growth by PuM upon IFN-γ treatment. BMM and PuM were infected with GFP-BCG. After infection, cells were left untreated or treated with IFN-γ (20, 40 and 80 ng/ml) for 48 h. Bacterial growth was evaluated by determining RLU. Data are shown as % reduction of phagocytosed bacteria evaluated as RLU. Values are means ± SD of the mean of a representative experiment from 3 independent experiments with 4 replicates each. The differences between groups of BMM and PuM were analyzed using a one-way ANOVA followed by Bonferroni‘s Multiple Comparison Test, * significantly different from medium control, P

    Article Snippet: After further washing, cells were either kept untreated or treated with 20 ng/ml of IFN-γ (Mabtech, Sweden), or IL-1β (1 or 10 ng/ml) or AECsup (diluted 1∶2 in cell culture media).

    Techniques: Infection

    IFN-γ and AECsup induce different cytokine profiles in PuM and BMM. BMM and PuM were infected with GFP-BCG. After infection, cells were left untreated or treated with either IFN-γ or AEC sup . Cell culture supernatants were collected at 4 and 24 h after treatments. IL-12, IP-10 and IL-6, levels were measured using ELISA. Values are expressed as means ± SD, from 3 independent experiments. The differences between groups of PuM and BMM were analyzed using a one-way ANOVA followed by Bonferroni‘s Multiple Comparison Test, * Significantly different from medium control, p

    Journal: PLoS ONE

    Article Title: Macrophage Control of Phagocytosed Mycobacteria Is Increased by Factors Secreted by Alveolar Epithelial Cells through Nitric Oxide Independent Mechanisms

    doi: 10.1371/journal.pone.0103411

    Figure Lengend Snippet: IFN-γ and AECsup induce different cytokine profiles in PuM and BMM. BMM and PuM were infected with GFP-BCG. After infection, cells were left untreated or treated with either IFN-γ or AEC sup . Cell culture supernatants were collected at 4 and 24 h after treatments. IL-12, IP-10 and IL-6, levels were measured using ELISA. Values are expressed as means ± SD, from 3 independent experiments. The differences between groups of PuM and BMM were analyzed using a one-way ANOVA followed by Bonferroni‘s Multiple Comparison Test, * Significantly different from medium control, p

    Article Snippet: After further washing, cells were either kept untreated or treated with 20 ng/ml of IFN-γ (Mabtech, Sweden), or IL-1β (1 or 10 ng/ml) or AECsup (diluted 1∶2 in cell culture media).

    Techniques: Infection, Cell Culture, Enzyme-linked Immunosorbent Assay

    PuM are specifically unresponsive to IFN-γ in the control of intracellular bacteria. PuM, AM and PEM were infected with GFP-BCG. After infection, cells were treated with either IFN-γ or AEC sup for 48 h or left untreated. Bacterial growth was evaluated by determining RLU. Data are shown as % reduction of phagocytosed bacteria evaluated as RLU. Values are means ± SD of the mean of 2 independent experiments with 4 replicates each. The differences between groups of PuM, AM and PEM were analyzed using a one-way ANOVA followed by Bonferroni‘s Multiple Comparison Test, * significantly different from medium control, P

    Journal: PLoS ONE

    Article Title: Macrophage Control of Phagocytosed Mycobacteria Is Increased by Factors Secreted by Alveolar Epithelial Cells through Nitric Oxide Independent Mechanisms

    doi: 10.1371/journal.pone.0103411

    Figure Lengend Snippet: PuM are specifically unresponsive to IFN-γ in the control of intracellular bacteria. PuM, AM and PEM were infected with GFP-BCG. After infection, cells were treated with either IFN-γ or AEC sup for 48 h or left untreated. Bacterial growth was evaluated by determining RLU. Data are shown as % reduction of phagocytosed bacteria evaluated as RLU. Values are means ± SD of the mean of 2 independent experiments with 4 replicates each. The differences between groups of PuM, AM and PEM were analyzed using a one-way ANOVA followed by Bonferroni‘s Multiple Comparison Test, * significantly different from medium control, P

    Article Snippet: After further washing, cells were either kept untreated or treated with 20 ng/ml of IFN-γ (Mabtech, Sweden), or IL-1β (1 or 10 ng/ml) or AECsup (diluted 1∶2 in cell culture media).

    Techniques: End-sequence Profiling, Infection

    AEC sup has opposite effects to IFN-γ on iNOS, and Arg-1 expression in BMM and PuM and mediates killing through an iNOS independent mechanism. BMM and PuM were infected with GFP-BCG. After infection, cells were treated with either IFN-γ or AEC sup or 1 mM of NG-monomethyl-L-arginine (NMMLA) together with either IFN-γ or AEC sup or left untreated. Total RNA was extracted from both cell-types after 4 and 24 h of treatment and the mean-fold accumulation of a) iNOS, b) Arg-1 ± SD from 3-4 experiments. In c) the effect of the iNOS inhibitor NMMLA on intracellular growth of BCG is shown. Bacterial growth was evaluated by determining RLU. Data are expressed as mean ± SD. The differences between groups of BMM and PuM were analyzed using non-parametric, one-way ANOVA (Kruskal-Wallis) with Dunn‘s post-test. * significantly different from medium control, P

    Journal: PLoS ONE

    Article Title: Macrophage Control of Phagocytosed Mycobacteria Is Increased by Factors Secreted by Alveolar Epithelial Cells through Nitric Oxide Independent Mechanisms

    doi: 10.1371/journal.pone.0103411

    Figure Lengend Snippet: AEC sup has opposite effects to IFN-γ on iNOS, and Arg-1 expression in BMM and PuM and mediates killing through an iNOS independent mechanism. BMM and PuM were infected with GFP-BCG. After infection, cells were treated with either IFN-γ or AEC sup or 1 mM of NG-monomethyl-L-arginine (NMMLA) together with either IFN-γ or AEC sup or left untreated. Total RNA was extracted from both cell-types after 4 and 24 h of treatment and the mean-fold accumulation of a) iNOS, b) Arg-1 ± SD from 3-4 experiments. In c) the effect of the iNOS inhibitor NMMLA on intracellular growth of BCG is shown. Bacterial growth was evaluated by determining RLU. Data are expressed as mean ± SD. The differences between groups of BMM and PuM were analyzed using non-parametric, one-way ANOVA (Kruskal-Wallis) with Dunn‘s post-test. * significantly different from medium control, P

    Article Snippet: After further washing, cells were either kept untreated or treated with 20 ng/ml of IFN-γ (Mabtech, Sweden), or IL-1β (1 or 10 ng/ml) or AECsup (diluted 1∶2 in cell culture media).

    Techniques: Expressing, Infection

    Impaired intracellular control of BCG growth by PuM upon IFN-γ treatment is regulated by SOCS1. PuM from IFN-γ -/- SOCS1 -/- mice were infected with GFP-BCG. After infection, cells were left untreated or treated with IFN-γ, AEC sup and IFN-γ + AEC sup for 48 h. Bacterial growth was evaluated by determining RLU. Data are shown as % reduction of phagocytosed bacteria evaluated as RLU. Values are means ± SD of the mean of a representative experiment from 2 independent experiments with 4 replicates. The differences between groups of IFN-γ -/- SOCS1 -/- PuM were analyzed using a one-way ANOVA followed by Bonferroni‘s Multiple Comparison Test. * significantly different from medium control, P

    Journal: PLoS ONE

    Article Title: Macrophage Control of Phagocytosed Mycobacteria Is Increased by Factors Secreted by Alveolar Epithelial Cells through Nitric Oxide Independent Mechanisms

    doi: 10.1371/journal.pone.0103411

    Figure Lengend Snippet: Impaired intracellular control of BCG growth by PuM upon IFN-γ treatment is regulated by SOCS1. PuM from IFN-γ -/- SOCS1 -/- mice were infected with GFP-BCG. After infection, cells were left untreated or treated with IFN-γ, AEC sup and IFN-γ + AEC sup for 48 h. Bacterial growth was evaluated by determining RLU. Data are shown as % reduction of phagocytosed bacteria evaluated as RLU. Values are means ± SD of the mean of a representative experiment from 2 independent experiments with 4 replicates. The differences between groups of IFN-γ -/- SOCS1 -/- PuM were analyzed using a one-way ANOVA followed by Bonferroni‘s Multiple Comparison Test. * significantly different from medium control, P

    Article Snippet: After further washing, cells were either kept untreated or treated with 20 ng/ml of IFN-γ (Mabtech, Sweden), or IL-1β (1 or 10 ng/ml) or AECsup (diluted 1∶2 in cell culture media).

    Techniques: Mouse Assay, Infection

    Surface analysis of IFN-γ–stimulated Mφ reveals simultaneous increase in TRAIL expression with downregulation of TRAIL-R2 expression. (A) Flow cytometric analysis of TRAIL-R1, -R2, -R3, and -R4 expression on unstimulated Mφ. Filled histograms represent staining by M271 (anti–TRAIL-R1 mAb), M413 (anti–TRAIL-R2 mAb), M430 (anti–TRAIL-R3 mAb), or M444 (anti–TRAIL-R4 mAb), and open histograms represent staining with isotype control mAb. (B) RT-PCR analysis of TRAIL receptor mRNA expression in normal Mφ. (C) TRAIL, TRAIL-R2, and TRAIL-R3 expression after various times of IFN-γ stimulation. Filled histograms represent staining by M413 (anti–TRAIL-R2 mAb), M430 (anti–TRAIL-R3 mAb), or M181 (anti-TRAIL mAb), and open histograms represent staining with isotype control mAb. For comparison, Mφ cultured in the absence or presence of GM-CSF for 8 h were also stained for TRAIL, TRAIL-R2, and TRAIL-R3. (D) RT-PCR analysis of TRAIL, TRAIL-R2, and TRAIL-R3 mRNA levels after Mφ culture in the absence or presence of IFN-γ and GM-CSF. β-actin was used as a control over the same time course. Similar results were observed with Mφ from two other donors.

    Journal: The Journal of Experimental Medicine

    Article Title: Monocyte-mediated Tumoricidal Activity via the Tumor Necrosis Factor-related Cytokine, TRAIL

    doi:

    Figure Lengend Snippet: Surface analysis of IFN-γ–stimulated Mφ reveals simultaneous increase in TRAIL expression with downregulation of TRAIL-R2 expression. (A) Flow cytometric analysis of TRAIL-R1, -R2, -R3, and -R4 expression on unstimulated Mφ. Filled histograms represent staining by M271 (anti–TRAIL-R1 mAb), M413 (anti–TRAIL-R2 mAb), M430 (anti–TRAIL-R3 mAb), or M444 (anti–TRAIL-R4 mAb), and open histograms represent staining with isotype control mAb. (B) RT-PCR analysis of TRAIL receptor mRNA expression in normal Mφ. (C) TRAIL, TRAIL-R2, and TRAIL-R3 expression after various times of IFN-γ stimulation. Filled histograms represent staining by M413 (anti–TRAIL-R2 mAb), M430 (anti–TRAIL-R3 mAb), or M181 (anti-TRAIL mAb), and open histograms represent staining with isotype control mAb. For comparison, Mφ cultured in the absence or presence of GM-CSF for 8 h were also stained for TRAIL, TRAIL-R2, and TRAIL-R3. (D) RT-PCR analysis of TRAIL, TRAIL-R2, and TRAIL-R3 mRNA levels after Mφ culture in the absence or presence of IFN-γ and GM-CSF. β-actin was used as a control over the same time course. Similar results were observed with Mφ from two other donors.

    Article Snippet: Reagents and sources were as follows: IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-15, GM-CSF (100 ng/ml; Immunex ); IFN-α, IFN-γ, IL-10, IL-12 (100 ng/ml; Genzyme Corp. ); LPS (5 ng/ml; DIFCO); MOPC-21, nonspecific IgG1 isotype control; MOPC-173, nonspecific IgG2a isotype control; M181, IgG1 anti-TRAIL ( Immunex ); mAb11, IgG1 anti-TNF; NOK-1, IgG1 anti-FasL ( PharMingen ).

    Techniques: Expressing, Flow Cytometry, Staining, Reverse Transcription Polymerase Chain Reaction, Cell Culture

    TRAIL-mediated tumoricidal activity by human Mφ occurs after stimulation with IFN. (A–D) Mφ were incubated for 12 h in the absence or presence of either GM-CSF, IFN-γ, or IFN-α and then cultured for 8 h with 51 Cr-labeled (A) OVCAR3, (B) WM 164, or (C) WM 793 target cells at the indicated E/T ratios. As a positive control, soluble LZ-TRAIL (LZT) was added to target cells at the indicated concentrations. (D) Inclusion of the fusion protein TRAIL-R2:Fc (20 μg/ml) to 12-h IFN-γ–stimulated Mφ inhibited killing of WM 793 target cells, whereas addition of Fas:Fc (20 μg/ml) did not. (E and F) Addition of the NO inhibitor L-NMMA (300 μM) did not alter the antitumor activity of (E) IFN-γ– or (F) IFN-α–stimulated Mφ against WM 793 target cells. Data points represent the mean of triplicate wells, and experiments were repeated at least three times with similar results. For clarity, SD bars were omitted from the graphs, but were

    Journal: The Journal of Experimental Medicine

    Article Title: Monocyte-mediated Tumoricidal Activity via the Tumor Necrosis Factor-related Cytokine, TRAIL

    doi:

    Figure Lengend Snippet: TRAIL-mediated tumoricidal activity by human Mφ occurs after stimulation with IFN. (A–D) Mφ were incubated for 12 h in the absence or presence of either GM-CSF, IFN-γ, or IFN-α and then cultured for 8 h with 51 Cr-labeled (A) OVCAR3, (B) WM 164, or (C) WM 793 target cells at the indicated E/T ratios. As a positive control, soluble LZ-TRAIL (LZT) was added to target cells at the indicated concentrations. (D) Inclusion of the fusion protein TRAIL-R2:Fc (20 μg/ml) to 12-h IFN-γ–stimulated Mφ inhibited killing of WM 793 target cells, whereas addition of Fas:Fc (20 μg/ml) did not. (E and F) Addition of the NO inhibitor L-NMMA (300 μM) did not alter the antitumor activity of (E) IFN-γ– or (F) IFN-α–stimulated Mφ against WM 793 target cells. Data points represent the mean of triplicate wells, and experiments were repeated at least three times with similar results. For clarity, SD bars were omitted from the graphs, but were

    Article Snippet: Reagents and sources were as follows: IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-15, GM-CSF (100 ng/ml; Immunex ); IFN-α, IFN-γ, IL-10, IL-12 (100 ng/ml; Genzyme Corp. ); LPS (5 ng/ml; DIFCO); MOPC-21, nonspecific IgG1 isotype control; MOPC-173, nonspecific IgG2a isotype control; M181, IgG1 anti-TRAIL ( Immunex ); mAb11, IgG1 anti-TNF; NOK-1, IgG1 anti-FasL ( PharMingen ).

    Techniques: Activity Assay, Incubation, Cell Culture, Labeling, Positive Control

    Phosphatidylserine externalization on OVCAR3 tumor cells during apoptosis induced by IFN-stimulated, TRAIL- expressing Mφ. OVCAR3 tumor cells were cultured for 6 h in medium alone or in the presence of LZ-TRAIL (1 μg/ml), unstimulated, or cytokine (GM-CSF, IFN-γ, IFN-α [100 ng/ml for 12 h])–stimulated Mφ (E/T ratio 2:1). Cells were then stained with FITC–annexin V and analyzed by flow cytometry. The percent of FITC–annexin V positive tumor cells is indicated for each condition. Histograms represent 10 4 gated tumor cells. Similar results were seen with Mφ from three other donors.

    Journal: The Journal of Experimental Medicine

    Article Title: Monocyte-mediated Tumoricidal Activity via the Tumor Necrosis Factor-related Cytokine, TRAIL

    doi:

    Figure Lengend Snippet: Phosphatidylserine externalization on OVCAR3 tumor cells during apoptosis induced by IFN-stimulated, TRAIL- expressing Mφ. OVCAR3 tumor cells were cultured for 6 h in medium alone or in the presence of LZ-TRAIL (1 μg/ml), unstimulated, or cytokine (GM-CSF, IFN-γ, IFN-α [100 ng/ml for 12 h])–stimulated Mφ (E/T ratio 2:1). Cells were then stained with FITC–annexin V and analyzed by flow cytometry. The percent of FITC–annexin V positive tumor cells is indicated for each condition. Histograms represent 10 4 gated tumor cells. Similar results were seen with Mφ from three other donors.

    Article Snippet: Reagents and sources were as follows: IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-15, GM-CSF (100 ng/ml; Immunex ); IFN-α, IFN-γ, IL-10, IL-12 (100 ng/ml; Genzyme Corp. ); LPS (5 ng/ml; DIFCO); MOPC-21, nonspecific IgG1 isotype control; MOPC-173, nonspecific IgG2a isotype control; M181, IgG1 anti-TRAIL ( Immunex ); mAb11, IgG1 anti-TNF; NOK-1, IgG1 anti-FasL ( PharMingen ).

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

    TNF-mediated apoptosis by human Mφ occurs after stimulation with LPS but not IFN-γ. (A) Mφ were incubated in the absence or presence of LPS for 2 or 12 h and then cultured for 8 h with 51 Cr-labeled L929 target cells at the indicated E/T ratios. As a positive control, soluble (s)TNF was added to targets cells at the indicated concentrations. (B) Inclusion of TNFR: Fc (20 μg/ml) to 2-h LPS-stimulated Mφ inhibited the killing of L929 target cells, whereas addition of TRAIL-R2:Fc (20 μg/ml) did not. (C) Killing of WM 793 tumor cells by Mφ stimulated with IFN-γ for 12 h can be inhibited by TRAIL-R2:Fc (20 μg/ml), but not TNFR:Fc (20 μg/ml). Data points represent the mean of triplicate wells, and the experiments were repeated at least three times with similar results. For clarity, SD bars were omitted from the graphs, but were

    Journal: The Journal of Experimental Medicine

    Article Title: Monocyte-mediated Tumoricidal Activity via the Tumor Necrosis Factor-related Cytokine, TRAIL

    doi:

    Figure Lengend Snippet: TNF-mediated apoptosis by human Mφ occurs after stimulation with LPS but not IFN-γ. (A) Mφ were incubated in the absence or presence of LPS for 2 or 12 h and then cultured for 8 h with 51 Cr-labeled L929 target cells at the indicated E/T ratios. As a positive control, soluble (s)TNF was added to targets cells at the indicated concentrations. (B) Inclusion of TNFR: Fc (20 μg/ml) to 2-h LPS-stimulated Mφ inhibited the killing of L929 target cells, whereas addition of TRAIL-R2:Fc (20 μg/ml) did not. (C) Killing of WM 793 tumor cells by Mφ stimulated with IFN-γ for 12 h can be inhibited by TRAIL-R2:Fc (20 μg/ml), but not TNFR:Fc (20 μg/ml). Data points represent the mean of triplicate wells, and the experiments were repeated at least three times with similar results. For clarity, SD bars were omitted from the graphs, but were

    Article Snippet: Reagents and sources were as follows: IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-15, GM-CSF (100 ng/ml; Immunex ); IFN-α, IFN-γ, IL-10, IL-12 (100 ng/ml; Genzyme Corp. ); LPS (5 ng/ml; DIFCO); MOPC-21, nonspecific IgG1 isotype control; MOPC-173, nonspecific IgG2a isotype control; M181, IgG1 anti-TRAIL ( Immunex ); mAb11, IgG1 anti-TNF; NOK-1, IgG1 anti-FasL ( PharMingen ).

    Techniques: Incubation, Cell Culture, Labeling, Positive Control

    IFN-γ stimulation renders Mφ resistant to TRAIL-induced death, but not tumor cell targets. (A) Peripheral blood Mφ were incubated for 12 h in the absence or presence of GM-CSF and IFN-γ, then tested for sensitivity to LZ-TRAIL. (B) OVCAR3 tumor cells were incubated for 12 h in the absence or presence of IFN-γ, then tested for sensitivity to LZ-TRAIL. (C) OVCAR3 tumor cells were cultured for 12 h in the absence or presence of IFN-γ, then incubated with unstimulated or IFN-γ–stimulated Mφ. Percent specific lysis was measured by 51 Cr release after 8 h, and each data point represents the mean of triplicate wells. For clarity, SD bars were omitted from the graphs, but were

    Journal: The Journal of Experimental Medicine

    Article Title: Monocyte-mediated Tumoricidal Activity via the Tumor Necrosis Factor-related Cytokine, TRAIL

    doi:

    Figure Lengend Snippet: IFN-γ stimulation renders Mφ resistant to TRAIL-induced death, but not tumor cell targets. (A) Peripheral blood Mφ were incubated for 12 h in the absence or presence of GM-CSF and IFN-γ, then tested for sensitivity to LZ-TRAIL. (B) OVCAR3 tumor cells were incubated for 12 h in the absence or presence of IFN-γ, then tested for sensitivity to LZ-TRAIL. (C) OVCAR3 tumor cells were cultured for 12 h in the absence or presence of IFN-γ, then incubated with unstimulated or IFN-γ–stimulated Mφ. Percent specific lysis was measured by 51 Cr release after 8 h, and each data point represents the mean of triplicate wells. For clarity, SD bars were omitted from the graphs, but were

    Article Snippet: Reagents and sources were as follows: IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-15, GM-CSF (100 ng/ml; Immunex ); IFN-α, IFN-γ, IL-10, IL-12 (100 ng/ml; Genzyme Corp. ); LPS (5 ng/ml; DIFCO); MOPC-21, nonspecific IgG1 isotype control; MOPC-173, nonspecific IgG2a isotype control; M181, IgG1 anti-TRAIL ( Immunex ); mAb11, IgG1 anti-TNF; NOK-1, IgG1 anti-FasL ( PharMingen ).

    Techniques: Incubation, Cell Culture, Lysis

    TRAIL, FasL, and TNF expression on human Mφ. (A) Mφ were incubated for 2 or 12 h in the absence or presence of IFN-γ, IFN-α, GM-CSF, or LPS and then analyzed for TRAIL or FasL surface expression. Filled histograms represent staining at 12 h by either M181 (anti-TRAIL mAb) or NOK-1 (anti-FasL mAb). Open histograms represent staining at 2 h with the same mAb, whereas dotted histograms represent staining with isotype control mAb. (B) Mφ were incubated as in A and then analyzed for TNF surface expression. Filled histograms represent staining at 2 h by mAb11 (anti-TNF mAb). Open histograms represent staining at 12 h with the same mAb, whereas dotted histograms represent staining with isotype control mAb. Histograms represent 10 4 gated Mφ in all conditions, and viability was > 95% as assessed by propidium iodide exclusion. These observations were reproduced using Mφ from at least five different donors.

    Journal: The Journal of Experimental Medicine

    Article Title: Monocyte-mediated Tumoricidal Activity via the Tumor Necrosis Factor-related Cytokine, TRAIL

    doi:

    Figure Lengend Snippet: TRAIL, FasL, and TNF expression on human Mφ. (A) Mφ were incubated for 2 or 12 h in the absence or presence of IFN-γ, IFN-α, GM-CSF, or LPS and then analyzed for TRAIL or FasL surface expression. Filled histograms represent staining at 12 h by either M181 (anti-TRAIL mAb) or NOK-1 (anti-FasL mAb). Open histograms represent staining at 2 h with the same mAb, whereas dotted histograms represent staining with isotype control mAb. (B) Mφ were incubated as in A and then analyzed for TNF surface expression. Filled histograms represent staining at 2 h by mAb11 (anti-TNF mAb). Open histograms represent staining at 12 h with the same mAb, whereas dotted histograms represent staining with isotype control mAb. Histograms represent 10 4 gated Mφ in all conditions, and viability was > 95% as assessed by propidium iodide exclusion. These observations were reproduced using Mφ from at least five different donors.

    Article Snippet: Reagents and sources were as follows: IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-15, GM-CSF (100 ng/ml; Immunex ); IFN-α, IFN-γ, IL-10, IL-12 (100 ng/ml; Genzyme Corp. ); LPS (5 ng/ml; DIFCO); MOPC-21, nonspecific IgG1 isotype control; MOPC-173, nonspecific IgG2a isotype control; M181, IgG1 anti-TRAIL ( Immunex ); mAb11, IgG1 anti-TNF; NOK-1, IgG1 anti-FasL ( PharMingen ).

    Techniques: Expressing, Incubation, Staining

    Effects of ATP on ConA-induced production of pro-inflammatory cytokines by splenic lymphocytes isolated from BALB/c mice. (A–E) Lymphocytes were pre-incubated with various concentrations of ATP (100, 250, or 500 μM), and then stimulated with 50 μg/mL ConA for 24 h. (F–J) Lymphocytes were pre-incubated with 250 μM ATP, ADP, AMP, or adenosine, and then stimulated with 50 μg/mL ConA for 24 h. Concentrations of IL-6 (A,F) , TNF-α (B,G) , IL-17 (C,H) , IFN-γ (D,I) , or IL-4 (E,J) in the culture medium were measured by ELISA. Each value represents the mean ± SE ( n = 4). Significant difference between the vehicle control group and the ConA-treated group in the absence of ATP or other ligand is indicated by ### P

    Journal: Frontiers in Pharmacology

    Article Title: Adenine Nucleotides Attenuate Murine T Cell Activation Induced by Concanavalin A or T Cell Receptor Stimulation

    doi: 10.3389/fphar.2017.00986

    Figure Lengend Snippet: Effects of ATP on ConA-induced production of pro-inflammatory cytokines by splenic lymphocytes isolated from BALB/c mice. (A–E) Lymphocytes were pre-incubated with various concentrations of ATP (100, 250, or 500 μM), and then stimulated with 50 μg/mL ConA for 24 h. (F–J) Lymphocytes were pre-incubated with 250 μM ATP, ADP, AMP, or adenosine, and then stimulated with 50 μg/mL ConA for 24 h. Concentrations of IL-6 (A,F) , TNF-α (B,G) , IL-17 (C,H) , IFN-γ (D,I) , or IL-4 (E,J) in the culture medium were measured by ELISA. Each value represents the mean ± SE ( n = 4). Significant difference between the vehicle control group and the ConA-treated group in the absence of ATP or other ligand is indicated by ### P

    Article Snippet: IFN-γ, IL-4, TNF-α production was determined with mouse IFN-γ, IL-4, TNF-α Quantikine ELISA kits (R & D Systems) according to the manufacturer’s instructions.

    Techniques: Isolation, Mouse Assay, Incubation, Enzyme-linked Immunosorbent Assay

    Interferon signaling mediates resistance to cholesterol-dependent cytolysins. a, Percentage of PI-positive BMDMs treated with TLR1/2 agonist (Pam3CSK4; 50 ng/mL), TLR3 agonist (Poly(I:C); 1 μg/mL), TLR4 agonist (LPS; 50 ng/mL), TLR7 agonist (CL307; 100 nM), TLR9 (ODN1668; 100 nM) agonist, or unstimulated (NT) for 24 h and then challenged with PFO for up to 60 min in the presence of PI. Cells were imaged every 10 min to assess changes in PI incorporation. b, Percentage of PI-positive BMDMs treated with the various concentrations of TLR4 agonist for 24 h and then challenged with PFO for up to 60 min in the presence of PI. Cells were imaged every 7 min to assess changes in PI incorporation. c, Percentage of PI-positive BMDMs treated with IFN-β or IFN-γ (20 ng/mL) for 24 h and then challenged with PFO for 60 min in the presence of PI. d, Percentage of PI-positive BMDMs treated with NOD2 agonist (N-Glycolyl-MDP; 20 μg/ml), STING agonist (2’,3’cGAMP and c-di-GMP; both 2 μg/mL) for 24 h and then challenged with PFO for 60 min in the presence of PI. e, Percentage of PI-positive BMDMs treated with STING ligand for 24 h and then challenged with Streptolysin O (SLO) for 4 h in the presence of PI. f, Percentage of PI-positive BMDMs treated with IFN-β or IFN-γ (20 ng/mL) for 24 h and then challenged with SLO for 4 h in the presence of PI. g, Percentage of PI-positive BMDMs treated with IFN-β or IFN-γ (20 ng/mL) for 24 h and then challenged with ALO for 60 min in the presence of PI. h, Percentage of PI-positive BMDMs treated with IFN-β (100 ng/mL) or TLR1/2 agonist, or IFN-β (100 ng/mL) together with TLR1/2 agonist for 24 h and then challenged with PFO for 60 min in the presence of PI. i, Percentage of PI-positive BMDMs treated with IFN-β (100 ng/mL) or TLR1/2 agonist, or IFN-β (100 ng/mL) together with TLR1/2 agonist for 24 h and then challenged with SLO for 4 h in the presence of PI. Data are representatives of three independent experiments, and are shown as mean ± s.e.m. (n = 3). Statistical significance was determined using an RM one-way ANOVA with Dunnett’s correction (a-g) or a two-way ANOVA with Dunnett’s correction (h, i). ***P

    Journal: Nature immunology

    Article Title: Interferon-mediated reprogramming of membrane cholesterol to evade bacterial toxins

    doi: 10.1038/s41590-020-0695-4

    Figure Lengend Snippet: Interferon signaling mediates resistance to cholesterol-dependent cytolysins. a, Percentage of PI-positive BMDMs treated with TLR1/2 agonist (Pam3CSK4; 50 ng/mL), TLR3 agonist (Poly(I:C); 1 μg/mL), TLR4 agonist (LPS; 50 ng/mL), TLR7 agonist (CL307; 100 nM), TLR9 (ODN1668; 100 nM) agonist, or unstimulated (NT) for 24 h and then challenged with PFO for up to 60 min in the presence of PI. Cells were imaged every 10 min to assess changes in PI incorporation. b, Percentage of PI-positive BMDMs treated with the various concentrations of TLR4 agonist for 24 h and then challenged with PFO for up to 60 min in the presence of PI. Cells were imaged every 7 min to assess changes in PI incorporation. c, Percentage of PI-positive BMDMs treated with IFN-β or IFN-γ (20 ng/mL) for 24 h and then challenged with PFO for 60 min in the presence of PI. d, Percentage of PI-positive BMDMs treated with NOD2 agonist (N-Glycolyl-MDP; 20 μg/ml), STING agonist (2’,3’cGAMP and c-di-GMP; both 2 μg/mL) for 24 h and then challenged with PFO for 60 min in the presence of PI. e, Percentage of PI-positive BMDMs treated with STING ligand for 24 h and then challenged with Streptolysin O (SLO) for 4 h in the presence of PI. f, Percentage of PI-positive BMDMs treated with IFN-β or IFN-γ (20 ng/mL) for 24 h and then challenged with SLO for 4 h in the presence of PI. g, Percentage of PI-positive BMDMs treated with IFN-β or IFN-γ (20 ng/mL) for 24 h and then challenged with ALO for 60 min in the presence of PI. h, Percentage of PI-positive BMDMs treated with IFN-β (100 ng/mL) or TLR1/2 agonist, or IFN-β (100 ng/mL) together with TLR1/2 agonist for 24 h and then challenged with PFO for 60 min in the presence of PI. i, Percentage of PI-positive BMDMs treated with IFN-β (100 ng/mL) or TLR1/2 agonist, or IFN-β (100 ng/mL) together with TLR1/2 agonist for 24 h and then challenged with SLO for 4 h in the presence of PI. Data are representatives of three independent experiments, and are shown as mean ± s.e.m. (n = 3). Statistical significance was determined using an RM one-way ANOVA with Dunnett’s correction (a-g) or a two-way ANOVA with Dunnett’s correction (h, i). ***P

    Article Snippet: Cytokines: recombinant Murine IFN-γ (Peprotech, 315–05), recombinant Mouse IFN-β1 (carrier-free, BioLegend, 581302), recombinant human IFN-β (Peprotech, 300–02BC), IFN-α is a kind gift from R. Modlin lab (UCLA).

    Techniques:

    IFN signals reprogram cholesterol metabolism to decrease the pool of cholesterol targeted by CDCs a. Total cholesterol (nmol/10 7 cells) from C57BL/6 bone marrow–derived macrophages (BMDMs) stimulated with IFN-β (20 ng/mL), IFN-γ (20 ng/mL), or unstimulated (NT) for 48 h. Synthesized cholesterol was determined by GC-MS and isotopomer spectral analysis modeling (n = 4). b. Total plasma membrane cholesterol (nmol/10 7 cells) from C57BL/6 bone marrow–derived macrophages (BMDMs) stimulated with IFN-γ (40 ng/mL) or unstimulated (NT) for 24 h (n = 4). c. Quantification of [ 15 N]ALO-D4 or [ 15 N]OlyA binding on untreated or IFN-β (100 ng/mL, 16 h)-stimulated BMDMs determined by NanoSIMS. Quantification based on average 15 N/ 14 N ratio by cell. Data are mean ± s.e.m (n = 18, 14 for ALO and n = 8, 10 for OlyA). d. Confocal images of WT BMDM cultures stimulated with IFNs (20 ng/mL) for 24 h, treated with Sphingomyelinase (SMase; 200 mU/mL) for 30 min at 37 ˚C, and then stained with fluorescent ALO-D4 and DAPI. Scale bars represent 50 μm. e. Violin plots of cellular fluorescent intensity quantified from d (n = 6868, 6811, 3404, 3838, 2930, 2642). Data are representative of three ( a , d , e ) independent experiments, three independent samples ( c ) or from 4 biological replicates ( b ). Data in a - c are shown as mean ± s.e.m., violin plot in e is shown with median (solid lines) and 25% and 75% percentiles (dashed lines). Statistical significance was determined using a one-way ANOVA with Dunn’s correction ( a ), an unpaired two-tailed Student’s t test ( b ), or a two-tailed Mann-Whitney test ( c , e ). *** P

    Journal: Nature immunology

    Article Title: Interferon-mediated reprogramming of membrane cholesterol to evade bacterial toxins

    doi: 10.1038/s41590-020-0695-4

    Figure Lengend Snippet: IFN signals reprogram cholesterol metabolism to decrease the pool of cholesterol targeted by CDCs a. Total cholesterol (nmol/10 7 cells) from C57BL/6 bone marrow–derived macrophages (BMDMs) stimulated with IFN-β (20 ng/mL), IFN-γ (20 ng/mL), or unstimulated (NT) for 48 h. Synthesized cholesterol was determined by GC-MS and isotopomer spectral analysis modeling (n = 4). b. Total plasma membrane cholesterol (nmol/10 7 cells) from C57BL/6 bone marrow–derived macrophages (BMDMs) stimulated with IFN-γ (40 ng/mL) or unstimulated (NT) for 24 h (n = 4). c. Quantification of [ 15 N]ALO-D4 or [ 15 N]OlyA binding on untreated or IFN-β (100 ng/mL, 16 h)-stimulated BMDMs determined by NanoSIMS. Quantification based on average 15 N/ 14 N ratio by cell. Data are mean ± s.e.m (n = 18, 14 for ALO and n = 8, 10 for OlyA). d. Confocal images of WT BMDM cultures stimulated with IFNs (20 ng/mL) for 24 h, treated with Sphingomyelinase (SMase; 200 mU/mL) for 30 min at 37 ˚C, and then stained with fluorescent ALO-D4 and DAPI. Scale bars represent 50 μm. e. Violin plots of cellular fluorescent intensity quantified from d (n = 6868, 6811, 3404, 3838, 2930, 2642). Data are representative of three ( a , d , e ) independent experiments, three independent samples ( c ) or from 4 biological replicates ( b ). Data in a - c are shown as mean ± s.e.m., violin plot in e is shown with median (solid lines) and 25% and 75% percentiles (dashed lines). Statistical significance was determined using a one-way ANOVA with Dunn’s correction ( a ), an unpaired two-tailed Student’s t test ( b ), or a two-tailed Mann-Whitney test ( c , e ). *** P

    Article Snippet: Cytokines: recombinant Murine IFN-γ (Peprotech, 315–05), recombinant Mouse IFN-β1 (carrier-free, BioLegend, 581302), recombinant human IFN-β (Peprotech, 300–02BC), IFN-α is a kind gift from R. Modlin lab (UCLA).

    Techniques: Derivative Assay, Synthesized, Gas Chromatography-Mass Spectrometry, Binding Assay, Staining, Two Tailed Test, MANN-WHITNEY

    IFN signals decrease plasma membrane binding to ALO-D4 protein. a, Confocal images of neutrophils stimulated with IFN-β or IFN-γ (20 ng/mL) for 6 h, and then stained with fluorescent ALO-D4 and DAPI. b, Violin plots of cellular fluorescent intensity quantified from a (n = 20334, 18546, 16290). c, Confocal images of WT or type I interferon receptor–deficient (IFNAR KO) BMDMs stimulated with TLR3 agonist (1 μg/mL) or IFN-β (20 ng/mL) for 24 h, and then stained with fluorescent ALO-D4 and DAPI. d, Violin plots of cellular fluorescent intensity quantified from c (n = 5543, 6682, 4673, 8231, 5201, 7906). Data are representatives of three independent experiments. Violin plots are shown with median (solid lines in b, d) and 25% and 75% percentiles (dashed lines in b), and statistical significance was determined using a Kruskal-Wallis test with Dunn’s correction. ***P

    Journal: Nature immunology

    Article Title: Interferon-mediated reprogramming of membrane cholesterol to evade bacterial toxins

    doi: 10.1038/s41590-020-0695-4

    Figure Lengend Snippet: IFN signals decrease plasma membrane binding to ALO-D4 protein. a, Confocal images of neutrophils stimulated with IFN-β or IFN-γ (20 ng/mL) for 6 h, and then stained with fluorescent ALO-D4 and DAPI. b, Violin plots of cellular fluorescent intensity quantified from a (n = 20334, 18546, 16290). c, Confocal images of WT or type I interferon receptor–deficient (IFNAR KO) BMDMs stimulated with TLR3 agonist (1 μg/mL) or IFN-β (20 ng/mL) for 24 h, and then stained with fluorescent ALO-D4 and DAPI. d, Violin plots of cellular fluorescent intensity quantified from c (n = 5543, 6682, 4673, 8231, 5201, 7906). Data are representatives of three independent experiments. Violin plots are shown with median (solid lines in b, d) and 25% and 75% percentiles (dashed lines in b), and statistical significance was determined using a Kruskal-Wallis test with Dunn’s correction. ***P

    Article Snippet: Cytokines: recombinant Murine IFN-γ (Peprotech, 315–05), recombinant Mouse IFN-β1 (carrier-free, BioLegend, 581302), recombinant human IFN-β (Peprotech, 300–02BC), IFN-α is a kind gift from R. Modlin lab (UCLA).

    Techniques: Binding Assay, Staining

    IFN signals reprogram cholesterol metabolism to decrease the pool of cholesterol targeted by CDCs. a, Total cholesterol (nmol/107 cells) from C57BL/6 bone marrow–derived macrophages (BMDMs) stimulated with cGAMP (2 μg/mL), TLR3 agonist (Poly(I:C); 1 μg/mL), or unstimulated (NT) for 48 h. Total cholesterol was determined by GC-MS (n = 4). b, Total plasma membrane cholesterol (normalized to total FAMEs) from C57BL/6 bone marrow–derived macrophages (BMDMs) stimulated with IFN-γ (40 ng/mL) or unstimulated (NT) for 24 h (n = 4). c, Relative Fillipin III fluorescence intensity of plasma membranes of untreated macrophages or macrophages stimulated with IFN-β (20 ng/mL) or IFN-γ (20 ng/mL) for 24 h (n = 32, 38, 34, 42). MβCD-Cholesterol loaded macrophages indicate dynamic range of Fillipin III fluorescence and are included as a positive control. d, Confocal images of BMDM stimulated with IFN-β (20 ng/mL) for 24 h, and then stained with fluorescent ALO-D4 or OlyA and DAPI. Scale bar, 50 μm. e, Violin plots of cellular fluorescent intensity quantified from d (n = 2225, 2021, 2225, 2021). f, Cholera Toxin B staining of BMDM stimulated with IFN-β or IFN-γ (20 ng/mL) for 24 h. Median fluorescence intensity (MFI) are indicated on the left. Data are representative of three (a, d, e, f) independent experiments, three independent samples (c) or from 4 biological replicates (b). Data in a-c are shown as mean ± s.e.m., violin plots in e are shown with median (solid lines). Statistical significance was determined using an unpaired two-tailed Student’s t-test (a, b), a one-way ANOVA with Dunnett’s correction (c), or a two-tailed Mann–Whitney test (e) ***P

    Journal: Nature immunology

    Article Title: Interferon-mediated reprogramming of membrane cholesterol to evade bacterial toxins

    doi: 10.1038/s41590-020-0695-4

    Figure Lengend Snippet: IFN signals reprogram cholesterol metabolism to decrease the pool of cholesterol targeted by CDCs. a, Total cholesterol (nmol/107 cells) from C57BL/6 bone marrow–derived macrophages (BMDMs) stimulated with cGAMP (2 μg/mL), TLR3 agonist (Poly(I:C); 1 μg/mL), or unstimulated (NT) for 48 h. Total cholesterol was determined by GC-MS (n = 4). b, Total plasma membrane cholesterol (normalized to total FAMEs) from C57BL/6 bone marrow–derived macrophages (BMDMs) stimulated with IFN-γ (40 ng/mL) or unstimulated (NT) for 24 h (n = 4). c, Relative Fillipin III fluorescence intensity of plasma membranes of untreated macrophages or macrophages stimulated with IFN-β (20 ng/mL) or IFN-γ (20 ng/mL) for 24 h (n = 32, 38, 34, 42). MβCD-Cholesterol loaded macrophages indicate dynamic range of Fillipin III fluorescence and are included as a positive control. d, Confocal images of BMDM stimulated with IFN-β (20 ng/mL) for 24 h, and then stained with fluorescent ALO-D4 or OlyA and DAPI. Scale bar, 50 μm. e, Violin plots of cellular fluorescent intensity quantified from d (n = 2225, 2021, 2225, 2021). f, Cholera Toxin B staining of BMDM stimulated with IFN-β or IFN-γ (20 ng/mL) for 24 h. Median fluorescence intensity (MFI) are indicated on the left. Data are representative of three (a, d, e, f) independent experiments, three independent samples (c) or from 4 biological replicates (b). Data in a-c are shown as mean ± s.e.m., violin plots in e are shown with median (solid lines). Statistical significance was determined using an unpaired two-tailed Student’s t-test (a, b), a one-way ANOVA with Dunnett’s correction (c), or a two-tailed Mann–Whitney test (e) ***P

    Article Snippet: Cytokines: recombinant Murine IFN-γ (Peprotech, 315–05), recombinant Mouse IFN-β1 (carrier-free, BioLegend, 581302), recombinant human IFN-β (Peprotech, 300–02BC), IFN-α is a kind gift from R. Modlin lab (UCLA).

    Techniques: Derivative Assay, Gas Chromatography-Mass Spectrometry, Fluorescence, Positive Control, Staining, Two Tailed Test, MANN-WHITNEY

    Production of 25-hydroxycholesterol is required to maintain changes in plasma membrane cholesterol and mediates resistance to CDCs a. Confocal images of control or cholesterol 25-hydroxylase-deficient (CH25H KO) BMDMs cultures stimulated with IFN-β (20 ng/mL), or IFN-γ (20 ng/mL) for 24 h, and then stained with fluorescent ALO-D4 and DAPI. b. Violin plots of cellular fluorescent intensity quantified from a (from left to right: n = 5294, 5505, 4564, 4066, 5370, 4962). c. Percentage of PI-positive control or CH25H KO BMDMs stimulated with IFNs (20 ng/mL) for 24 h and then challenged with PFO for 60 minutes in the presence of PI. d. Percentage of PI-positive control or CH25H KO BMDMs stimulated with IFNs (20 ng/mL) for 24 h and then challenged with SLO for 2 h in the presence of PI. e. Flow cytometry plots of S. aureus phagocytosed by control or CH25H KO BMDMs. Macrophage cultures were treated with IFNs (100 ng/mL) or 25HC (3 μM). After 24 h, BMDMs were washed and then incubated with PFO for 15 min. PFO containing media was then replaced with fresh media containing pHrodo-red-labeled S. aureus . Median fluorescence intensity (MFI) are indicated on the right. f. Percentage of PI-positive control or CH25H KO BMDMs incubated with 25HC (1 μM) overnight and then challenged with PFO for 60 minutes in the presence of PI. g. Net synthesized and total cholesterol (nmol/10 7 cells) from CH25H-deficient or control bone marrow–derived macrophages (BMDMs) stimulated with IFN-β (20 ng/mL) or unstimulated (NT) for 48 h. Synthesized cholesterol was determined by GC-MS and isotopomer spectral analysis modeling. h. Confocal images of CH25H KO BMDMs treated with Simvastatin (5 μM) for 24 h and then stained with fluorescent ALO-D4 and DAPI. i. Violin plots of cellular fluorescent intensity quantified from h (n = 6666, 2867). Data are representative of three independent experiments. Data in c , d , f , and g are shown as mean ± s.e.m. (n = 3 in c , d , f and n = 4 in g ). Violin plots are show with median (solid lines in b , i ) and 25% and 75% percentiles (dashed lines in i ). Statistical significance was determined using a Kruskal-Wallis test with Dunn’s correction ( b ), a two-way ANOVA with Tukey’s correction ( c , d , f , g ), or a two-tailed Mann-Whitney test ( i ). *** P

    Journal: Nature immunology

    Article Title: Interferon-mediated reprogramming of membrane cholesterol to evade bacterial toxins

    doi: 10.1038/s41590-020-0695-4

    Figure Lengend Snippet: Production of 25-hydroxycholesterol is required to maintain changes in plasma membrane cholesterol and mediates resistance to CDCs a. Confocal images of control or cholesterol 25-hydroxylase-deficient (CH25H KO) BMDMs cultures stimulated with IFN-β (20 ng/mL), or IFN-γ (20 ng/mL) for 24 h, and then stained with fluorescent ALO-D4 and DAPI. b. Violin plots of cellular fluorescent intensity quantified from a (from left to right: n = 5294, 5505, 4564, 4066, 5370, 4962). c. Percentage of PI-positive control or CH25H KO BMDMs stimulated with IFNs (20 ng/mL) for 24 h and then challenged with PFO for 60 minutes in the presence of PI. d. Percentage of PI-positive control or CH25H KO BMDMs stimulated with IFNs (20 ng/mL) for 24 h and then challenged with SLO for 2 h in the presence of PI. e. Flow cytometry plots of S. aureus phagocytosed by control or CH25H KO BMDMs. Macrophage cultures were treated with IFNs (100 ng/mL) or 25HC (3 μM). After 24 h, BMDMs were washed and then incubated with PFO for 15 min. PFO containing media was then replaced with fresh media containing pHrodo-red-labeled S. aureus . Median fluorescence intensity (MFI) are indicated on the right. f. Percentage of PI-positive control or CH25H KO BMDMs incubated with 25HC (1 μM) overnight and then challenged with PFO for 60 minutes in the presence of PI. g. Net synthesized and total cholesterol (nmol/10 7 cells) from CH25H-deficient or control bone marrow–derived macrophages (BMDMs) stimulated with IFN-β (20 ng/mL) or unstimulated (NT) for 48 h. Synthesized cholesterol was determined by GC-MS and isotopomer spectral analysis modeling. h. Confocal images of CH25H KO BMDMs treated with Simvastatin (5 μM) for 24 h and then stained with fluorescent ALO-D4 and DAPI. i. Violin plots of cellular fluorescent intensity quantified from h (n = 6666, 2867). Data are representative of three independent experiments. Data in c , d , f , and g are shown as mean ± s.e.m. (n = 3 in c , d , f and n = 4 in g ). Violin plots are show with median (solid lines in b , i ) and 25% and 75% percentiles (dashed lines in i ). Statistical significance was determined using a Kruskal-Wallis test with Dunn’s correction ( b ), a two-way ANOVA with Tukey’s correction ( c , d , f , g ), or a two-tailed Mann-Whitney test ( i ). *** P

    Article Snippet: Cytokines: recombinant Murine IFN-γ (Peprotech, 315–05), recombinant Mouse IFN-β1 (carrier-free, BioLegend, 581302), recombinant human IFN-β (Peprotech, 300–02BC), IFN-α is a kind gift from R. Modlin lab (UCLA).

    Techniques: Staining, Positive Control, Flow Cytometry, Incubation, Labeling, Fluorescence, Synthesized, Derivative Assay, Gas Chromatography-Mass Spectrometry, Two Tailed Test, MANN-WHITNEY

    Cholesterol synthesis is linked to CDC susceptibility a. Net synthesized cholesterol (nmol/10 7 cells) from C57BL/6 bone marrow–derived macrophages (BMDMs) stimulated with IFN-β (20 ng/mL), IFN-γ (20 ng/mL), or unstimulated (NT) for 48 h. Synthesized cholesterol was determined by GC-MS and isotopomer spectral analysis modeling (n = 4). b. Confocal images of WT BMDM treated with Simvastatin (1 μM-5 μM) for 24 h and then stained with fluorescent ALO-D4 and DAPI. c. Violin plots of cellular fluorescent intensity quantified from b (n = 5713, 2252, 2808, 5556). d. Violin plots of cellular fluorescent intensity quantified from control or SCAP KO BMDMs stained with fluorescent ALO-D4 and DAPI (n = 3306, 2927). e. Percentage of PI–positive control or SCAP KO BMDMs challenged with PFO for 60 min in the presence of PI (n = 3). f. Confocal images of WT BMDMs cultures incubated with 25HC (3 μM) for 4 h, and then stained with fluorescent ALO-D4 and DAPI. g. Violin plots of cellular fluorescent intensity quantified from f (n = 5861, 5769). Data are representatives of three independent experiments. Data in a , and e are shown as mean ± s.e.m. Violin plots in c , d, and g are shown with median (solid lines) and 25% and 75% percentiles (dashed lines). Statistical significance was determined using a Kruskal-Wallis test with Dunn’s correction ( c ), a two-tailed Mann-Whitney test ( d, g ), or an RM one-way ANOVA ( e ). *** P

    Journal: Nature immunology

    Article Title: Interferon-mediated reprogramming of membrane cholesterol to evade bacterial toxins

    doi: 10.1038/s41590-020-0695-4

    Figure Lengend Snippet: Cholesterol synthesis is linked to CDC susceptibility a. Net synthesized cholesterol (nmol/10 7 cells) from C57BL/6 bone marrow–derived macrophages (BMDMs) stimulated with IFN-β (20 ng/mL), IFN-γ (20 ng/mL), or unstimulated (NT) for 48 h. Synthesized cholesterol was determined by GC-MS and isotopomer spectral analysis modeling (n = 4). b. Confocal images of WT BMDM treated with Simvastatin (1 μM-5 μM) for 24 h and then stained with fluorescent ALO-D4 and DAPI. c. Violin plots of cellular fluorescent intensity quantified from b (n = 5713, 2252, 2808, 5556). d. Violin plots of cellular fluorescent intensity quantified from control or SCAP KO BMDMs stained with fluorescent ALO-D4 and DAPI (n = 3306, 2927). e. Percentage of PI–positive control or SCAP KO BMDMs challenged with PFO for 60 min in the presence of PI (n = 3). f. Confocal images of WT BMDMs cultures incubated with 25HC (3 μM) for 4 h, and then stained with fluorescent ALO-D4 and DAPI. g. Violin plots of cellular fluorescent intensity quantified from f (n = 5861, 5769). Data are representatives of three independent experiments. Data in a , and e are shown as mean ± s.e.m. Violin plots in c , d, and g are shown with median (solid lines) and 25% and 75% percentiles (dashed lines). Statistical significance was determined using a Kruskal-Wallis test with Dunn’s correction ( c ), a two-tailed Mann-Whitney test ( d, g ), or an RM one-way ANOVA ( e ). *** P

    Article Snippet: Cytokines: recombinant Murine IFN-γ (Peprotech, 315–05), recombinant Mouse IFN-β1 (carrier-free, BioLegend, 581302), recombinant human IFN-β (Peprotech, 300–02BC), IFN-α is a kind gift from R. Modlin lab (UCLA).

    Techniques: Synthesized, Derivative Assay, Gas Chromatography-Mass Spectrometry, Staining, Positive Control, Incubation, Two Tailed Test, MANN-WHITNEY

    Cholesterol esterification contributes to CDC resistance of macrophages a. qPCR analysis of genes that regulate cholesterol homeostasis in WT BMDM treated with IFNs (20 ng/mL) for 24 h. b. Quantification (nmol/10 7 cells) of total cholesterol ester (CE) in WT BMDMs stimulated with IFN-β (20 ng/mL) or IFN-γ (20 ng/mL) for 48 h. CE pool sizes were determined by direct infusion mass spectrometry (left panel: n = 3, right panel: n = 4). c. Percentage of PI–positive BMDMs treated with IFN-β (20 ng/mL) and IFN-γ (20 ng/mL) for 24 h in the presence of the ACAT inhibitor 58–035 (4.3 μM) and then challenged with PFO for 60 min in the presence of PI. d. Violin plots of cellular fluorescent intensity quantified from control or CH25H KO BMDMs treated with ACAT inhibitor 58–035 (4.3 μM) for 24 h and then stained with fluorescent ALO-D4 and DAPI (n = 6666, 9997). e. Percentage of PI–positive control or ABCA1 KO BMDMs stimulated with IFNs (20 ng/mL) for 24 h and then challenged with PFO for 60 minutes in the presence of PI. f. Violin plots of cellular fluorescent intensity quantified from control or CH25H KO BMDMs treated with LXR agonist GW3965 (1 μM) for 24 h and then stained with fluorescent ALO-D4 and DAPI (n= 2304, 3954, 2712, 2322). g. Percentage of PI–positive control or CH25H KO BMDMs treated with LXR agonist GW3965 (1 μM) for 24 h and then challenged with PFO for 60 min in the presence of PI. Data are representative of three independent experiments. Data in a, b, c, e, and g are shown as mean ± s.e.m. (n = 3) unless otherwise specified. Violin plots in d , and f are shown with median (solid lines) and 25% and 75% percentiles (dashed lines). Statistical significance was determined using a one-way ANOVA with Dunnett’s correction ( a ), an unpaired two-tailed Student’s t test ( b ), a two-way ANOVA with Tukey’s correction ( c , e , g ), a two-tailed Mann-Whitney test ( d ), or a Kruskal-Wallis test with Dunn’s correction ( f ). *** P

    Journal: Nature immunology

    Article Title: Interferon-mediated reprogramming of membrane cholesterol to evade bacterial toxins

    doi: 10.1038/s41590-020-0695-4

    Figure Lengend Snippet: Cholesterol esterification contributes to CDC resistance of macrophages a. qPCR analysis of genes that regulate cholesterol homeostasis in WT BMDM treated with IFNs (20 ng/mL) for 24 h. b. Quantification (nmol/10 7 cells) of total cholesterol ester (CE) in WT BMDMs stimulated with IFN-β (20 ng/mL) or IFN-γ (20 ng/mL) for 48 h. CE pool sizes were determined by direct infusion mass spectrometry (left panel: n = 3, right panel: n = 4). c. Percentage of PI–positive BMDMs treated with IFN-β (20 ng/mL) and IFN-γ (20 ng/mL) for 24 h in the presence of the ACAT inhibitor 58–035 (4.3 μM) and then challenged with PFO for 60 min in the presence of PI. d. Violin plots of cellular fluorescent intensity quantified from control or CH25H KO BMDMs treated with ACAT inhibitor 58–035 (4.3 μM) for 24 h and then stained with fluorescent ALO-D4 and DAPI (n = 6666, 9997). e. Percentage of PI–positive control or ABCA1 KO BMDMs stimulated with IFNs (20 ng/mL) for 24 h and then challenged with PFO for 60 minutes in the presence of PI. f. Violin plots of cellular fluorescent intensity quantified from control or CH25H KO BMDMs treated with LXR agonist GW3965 (1 μM) for 24 h and then stained with fluorescent ALO-D4 and DAPI (n= 2304, 3954, 2712, 2322). g. Percentage of PI–positive control or CH25H KO BMDMs treated with LXR agonist GW3965 (1 μM) for 24 h and then challenged with PFO for 60 min in the presence of PI. Data are representative of three independent experiments. Data in a, b, c, e, and g are shown as mean ± s.e.m. (n = 3) unless otherwise specified. Violin plots in d , and f are shown with median (solid lines) and 25% and 75% percentiles (dashed lines). Statistical significance was determined using a one-way ANOVA with Dunnett’s correction ( a ), an unpaired two-tailed Student’s t test ( b ), a two-way ANOVA with Tukey’s correction ( c , e , g ), a two-tailed Mann-Whitney test ( d ), or a Kruskal-Wallis test with Dunn’s correction ( f ). *** P

    Article Snippet: Cytokines: recombinant Murine IFN-γ (Peprotech, 315–05), recombinant Mouse IFN-β1 (carrier-free, BioLegend, 581302), recombinant human IFN-β (Peprotech, 300–02BC), IFN-α is a kind gift from R. Modlin lab (UCLA).

    Techniques: Real-time Polymerase Chain Reaction, Mass Spectrometry, Staining, Positive Control, Two Tailed Test, MANN-WHITNEY

    Cholesterol esterification contributes to CDC resistance of macrophages. a, Quantification (nmol/107 cells) of cholesterol ester species (16:0. 18:1, 20:4) in BMDMs stimulated with TLR3 agonist (1 μg/mL) in FBS or LPDS for 48 h. CE species pool sizes were determined by direct infusion MS. b, Percentage of PI–positive WT BMDMs treated with IFN-β, or IFN-γ (20 ng/mL), or in combination with ACATi 58–035 (4.3 μM) for 24 h and then challenged with PFO for 60 min in the presence of PI. c, Quantification (nmol/107 cells) of total cholesterol ester (CE) in control or CH25H KO BMDMs stimulated with IFN-β (20 ng/mL) or IFN-γ (20 ng/mL) for 48 h. CE pool sizes were determined by direct infusion mass spectrometry. d, Percentage of PI-positive CH25H KO BMDMs treated with ACATi 58–035 (4.3 μM) for 24 h and then challenged PFO for 60 min in the presence of PI. e, Violin plots of cellular fluorescent intensity quantified from control or ABCA1 KO or ABCG1 KO BMDMs stimulated with IFNs (20 ng/mL) for 24 h and then stained with fluorescent ALO-D4 and DAPI (n = 5943, 4126, 5727, 6914, 5740, 7898; n=7201, 7532, 7563, 7417). f, Percentage of PI-positive control or ABCA1 KO BMDMs treated with IFNs (20 ng/mL) for 24 h and then challenged PFO for 60 min in the presence of PI. g, Percentage of PI-positive control or CH25H KO BMDMs treated with LXR agonist GW3965 (1 μM) for 24 h and then challenged PFO for 60 min in the presence of PI. Data are representatives of two (a, c) or three (b, d, e, f, g) independent experiments. Data in a, b, c, d, f and g are shown as mean + s.e.m. (n = 3 in a, b, d, g; n = 4 in c). Violin plots in e are shown with median (solid lines) and 25% and 75% percentiles (dashed lines). Statistical significance was determined using an unpaired two-tailed Student’s t-test (a), a two-way ANOVA with Tukey’s correction (b, c, f, g), a paired two-tailed Student’s t-test (d), or a Kruskal–Wallis test with Dunn’s correction (e). ***P

    Journal: Nature immunology

    Article Title: Interferon-mediated reprogramming of membrane cholesterol to evade bacterial toxins

    doi: 10.1038/s41590-020-0695-4

    Figure Lengend Snippet: Cholesterol esterification contributes to CDC resistance of macrophages. a, Quantification (nmol/107 cells) of cholesterol ester species (16:0. 18:1, 20:4) in BMDMs stimulated with TLR3 agonist (1 μg/mL) in FBS or LPDS for 48 h. CE species pool sizes were determined by direct infusion MS. b, Percentage of PI–positive WT BMDMs treated with IFN-β, or IFN-γ (20 ng/mL), or in combination with ACATi 58–035 (4.3 μM) for 24 h and then challenged with PFO for 60 min in the presence of PI. c, Quantification (nmol/107 cells) of total cholesterol ester (CE) in control or CH25H KO BMDMs stimulated with IFN-β (20 ng/mL) or IFN-γ (20 ng/mL) for 48 h. CE pool sizes were determined by direct infusion mass spectrometry. d, Percentage of PI-positive CH25H KO BMDMs treated with ACATi 58–035 (4.3 μM) for 24 h and then challenged PFO for 60 min in the presence of PI. e, Violin plots of cellular fluorescent intensity quantified from control or ABCA1 KO or ABCG1 KO BMDMs stimulated with IFNs (20 ng/mL) for 24 h and then stained with fluorescent ALO-D4 and DAPI (n = 5943, 4126, 5727, 6914, 5740, 7898; n=7201, 7532, 7563, 7417). f, Percentage of PI-positive control or ABCA1 KO BMDMs treated with IFNs (20 ng/mL) for 24 h and then challenged PFO for 60 min in the presence of PI. g, Percentage of PI-positive control or CH25H KO BMDMs treated with LXR agonist GW3965 (1 μM) for 24 h and then challenged PFO for 60 min in the presence of PI. Data are representatives of two (a, c) or three (b, d, e, f, g) independent experiments. Data in a, b, c, d, f and g are shown as mean + s.e.m. (n = 3 in a, b, d, g; n = 4 in c). Violin plots in e are shown with median (solid lines) and 25% and 75% percentiles (dashed lines). Statistical significance was determined using an unpaired two-tailed Student’s t-test (a), a two-way ANOVA with Tukey’s correction (b, c, f, g), a paired two-tailed Student’s t-test (d), or a Kruskal–Wallis test with Dunn’s correction (e). ***P

    Article Snippet: Cytokines: recombinant Murine IFN-γ (Peprotech, 315–05), recombinant Mouse IFN-β1 (carrier-free, BioLegend, 581302), recombinant human IFN-β (Peprotech, 300–02BC), IFN-α is a kind gift from R. Modlin lab (UCLA).

    Techniques: Mass Spectrometry, Staining, Positive Control, Two Tailed Test

    IFN signals decrease plasma membrane binding to ALO-D4 protein a. Super resolution confocal images of macrophage cultures stimulated with the indicated IFNs (20 ng/mL) for 24 h and then stained with fluorescent ALO-D4 and DAPI. b. Confocal images of macrophage cultures stimulated with the indicated IFNs (20 ng/mL) for 24 h and then stained with fluorescent ALO-D4 and DAPI. c. Violin plots of cellular fluorescent intensity quantified from b (from left to right: n = 6539, 4561, 5367). d. Confocal images of macrophage cultures stimulated with the indicated PRR ligands for 24 h and then stained with fluorescent ALO-D4 and DAPI. e. Violin plots of cellular fluorescent intensity quantified from d (n = 5931, 4766, 5660). f. Confocal images of macrophage cultures stimulated with the indicated TLR agonists for 24 h and then stained with fluorescent ALO-D4 and DAPI. g. Violin plots of cellular fluorescent intensity quantified from f (n = 4358, 3270). h. Confocal images of human peripheral blood monocyte (hPBMC)–derived macrophages stimulated with human IFN-α or IFN-β (10 ng/mL) for 24 h and then stained with fluorescent ALO-D4 and DAPI. i. Violin plots of cellular fluorescent intensity quantified from h (n = 2084, 1818, 1728). j. Confocal images of macrophage cultures stimulated with IFN-β (20 ng/mL) for 2 h and then stained with fluorescent ALO-D4 and DAPI. k. Violin plots of cellular fluorescent intensity quantified from j (n = 6550, 7012). l. Confocal images of WT or type I interferon receptor–deficient (IFNAR KO) BMDMs stimulated with IFN-γ (20 ng/mL) for 24 h and then stained with fluorescent ALO-D4 and DAPI. m. Violin plots of cellular fluorescent intensity quantified from l (n = 4358, 2853, 7089, 5458). Data in a are representatives of three independent samples. Data in b - m are representatives of three ( b - k ) or two ( l , m ) independent experiments. Violin plots in c , e , g , i , k , and m are shown with median (solid line) and 25% and 75% percentiles (dashed lines), and statistical significance was determined using a Kruskal-Wallis test with Dunn’s correction ( c, e, i ) or a two-tailed Mann-Whitney test ( g , k , m ). *** P

    Journal: Nature immunology

    Article Title: Interferon-mediated reprogramming of membrane cholesterol to evade bacterial toxins

    doi: 10.1038/s41590-020-0695-4

    Figure Lengend Snippet: IFN signals decrease plasma membrane binding to ALO-D4 protein a. Super resolution confocal images of macrophage cultures stimulated with the indicated IFNs (20 ng/mL) for 24 h and then stained with fluorescent ALO-D4 and DAPI. b. Confocal images of macrophage cultures stimulated with the indicated IFNs (20 ng/mL) for 24 h and then stained with fluorescent ALO-D4 and DAPI. c. Violin plots of cellular fluorescent intensity quantified from b (from left to right: n = 6539, 4561, 5367). d. Confocal images of macrophage cultures stimulated with the indicated PRR ligands for 24 h and then stained with fluorescent ALO-D4 and DAPI. e. Violin plots of cellular fluorescent intensity quantified from d (n = 5931, 4766, 5660). f. Confocal images of macrophage cultures stimulated with the indicated TLR agonists for 24 h and then stained with fluorescent ALO-D4 and DAPI. g. Violin plots of cellular fluorescent intensity quantified from f (n = 4358, 3270). h. Confocal images of human peripheral blood monocyte (hPBMC)–derived macrophages stimulated with human IFN-α or IFN-β (10 ng/mL) for 24 h and then stained with fluorescent ALO-D4 and DAPI. i. Violin plots of cellular fluorescent intensity quantified from h (n = 2084, 1818, 1728). j. Confocal images of macrophage cultures stimulated with IFN-β (20 ng/mL) for 2 h and then stained with fluorescent ALO-D4 and DAPI. k. Violin plots of cellular fluorescent intensity quantified from j (n = 6550, 7012). l. Confocal images of WT or type I interferon receptor–deficient (IFNAR KO) BMDMs stimulated with IFN-γ (20 ng/mL) for 24 h and then stained with fluorescent ALO-D4 and DAPI. m. Violin plots of cellular fluorescent intensity quantified from l (n = 4358, 2853, 7089, 5458). Data in a are representatives of three independent samples. Data in b - m are representatives of three ( b - k ) or two ( l , m ) independent experiments. Violin plots in c , e , g , i , k , and m are shown with median (solid line) and 25% and 75% percentiles (dashed lines), and statistical significance was determined using a Kruskal-Wallis test with Dunn’s correction ( c, e, i ) or a two-tailed Mann-Whitney test ( g , k , m ). *** P

    Article Snippet: Cytokines: recombinant Murine IFN-γ (Peprotech, 315–05), recombinant Mouse IFN-β1 (carrier-free, BioLegend, 581302), recombinant human IFN-β (Peprotech, 300–02BC), IFN-α is a kind gift from R. Modlin lab (UCLA).

    Techniques: Binding Assay, Staining, Derivative Assay, Two Tailed Test, MANN-WHITNEY

    Interferon signaling mediates resistance to cholesterol-dependent cytolysins a. Percentage of propidium iodide (PI)–positive BMDMs treated with the TLR1/2 agonist (Pam3CSK4; 50 ng/mL), TLR3 agonist (Poly(I:C); 1 μg/mL), TLR4 agonist (LPS; 50 ng/mL), TLR7 agonist (CL307; 100 nM), TLR9 (ODN1668; 100 nM) agonist, or unstimulated (NT) for 24 h and then challenged with perfringolysin O (PFO) for 60 min in the presence of PI. b. Percentage of PI–positive BMDMs treated with IFN-β (20 ng/mL) and IFN-γ (20 ng/mL) for 24 h and then challenged with PFO for 60 min in the presence of PI. c. Percentage of PI–positive BMDMs treated with NOD2 agonist (N-Glycolyl-MDP; 20 μg/ml), STING agonist (2’,3’cGAMP and c-di-GMP; both 2 μg/mL) for 24 h and then challenged with PFO for 60 min in the presence of PI. d. Percentage of PI–positive BMDMs treated with the indicated IFNs (20 ng/mL) for 24 h and then challenged with Streptolysin-O (SLO) for 2 h in the presence of PI. e. Percentage of PI–positive BMDMs treated with the indicated IFNs (20 ng/mL) for 24 h and then challenged with anthrolysin-O (ALO) for 2 h in the presence of PI. f. Percentage of PI–positive BMDMs treated with TLR1/2 (50 ng/mL) together with IFNs (100 ng/mL) for 24 h and then challenged with PFO for 60 min in the presence of PI. g. Percentage of PI–positive BMDMs treated with TLR1/2 (50 ng/mL) together with IFNs (100 ng/mL) for 24 h and then challenged with SLO for 2 h in the presence of PI. h. Percentage of PI–positive neutrophils treated with IFN-β (20 ng/mL) and IFN-γ (20 ng/mL) for 4 h and then challenged with PFO for 60 min in the presence of PI (n = 3). i. Flow cytometry plots of S. aureus phagocytosed by control or IFNs-stimulated BMDMs. Macrophage cultures were stimulated with IFNs (100 ng/mL) for 24 h. BMDMs were then washed and then incubated with PFO for 15 min. PFO containing media was then replaced with fresh media containing pHrodo-red-labeled S. aureus . Median fluorescence intensity (MFI) are indicated on the right. Data are representatives of three independent experiments. Data in a - h are shown as mean ± s.e.m.(n=3). Statistical significance was determined using a one-way ANOVA with Dunnett’s correction ( a - e ), a two-way ANOVA with Tukey’s correction ( f, g ), or an RM one-way ANOVA with Dunnett’s correction ( h ). *** P

    Journal: Nature immunology

    Article Title: Interferon-mediated reprogramming of membrane cholesterol to evade bacterial toxins

    doi: 10.1038/s41590-020-0695-4

    Figure Lengend Snippet: Interferon signaling mediates resistance to cholesterol-dependent cytolysins a. Percentage of propidium iodide (PI)–positive BMDMs treated with the TLR1/2 agonist (Pam3CSK4; 50 ng/mL), TLR3 agonist (Poly(I:C); 1 μg/mL), TLR4 agonist (LPS; 50 ng/mL), TLR7 agonist (CL307; 100 nM), TLR9 (ODN1668; 100 nM) agonist, or unstimulated (NT) for 24 h and then challenged with perfringolysin O (PFO) for 60 min in the presence of PI. b. Percentage of PI–positive BMDMs treated with IFN-β (20 ng/mL) and IFN-γ (20 ng/mL) for 24 h and then challenged with PFO for 60 min in the presence of PI. c. Percentage of PI–positive BMDMs treated with NOD2 agonist (N-Glycolyl-MDP; 20 μg/ml), STING agonist (2’,3’cGAMP and c-di-GMP; both 2 μg/mL) for 24 h and then challenged with PFO for 60 min in the presence of PI. d. Percentage of PI–positive BMDMs treated with the indicated IFNs (20 ng/mL) for 24 h and then challenged with Streptolysin-O (SLO) for 2 h in the presence of PI. e. Percentage of PI–positive BMDMs treated with the indicated IFNs (20 ng/mL) for 24 h and then challenged with anthrolysin-O (ALO) for 2 h in the presence of PI. f. Percentage of PI–positive BMDMs treated with TLR1/2 (50 ng/mL) together with IFNs (100 ng/mL) for 24 h and then challenged with PFO for 60 min in the presence of PI. g. Percentage of PI–positive BMDMs treated with TLR1/2 (50 ng/mL) together with IFNs (100 ng/mL) for 24 h and then challenged with SLO for 2 h in the presence of PI. h. Percentage of PI–positive neutrophils treated with IFN-β (20 ng/mL) and IFN-γ (20 ng/mL) for 4 h and then challenged with PFO for 60 min in the presence of PI (n = 3). i. Flow cytometry plots of S. aureus phagocytosed by control or IFNs-stimulated BMDMs. Macrophage cultures were stimulated with IFNs (100 ng/mL) for 24 h. BMDMs were then washed and then incubated with PFO for 15 min. PFO containing media was then replaced with fresh media containing pHrodo-red-labeled S. aureus . Median fluorescence intensity (MFI) are indicated on the right. Data are representatives of three independent experiments. Data in a - h are shown as mean ± s.e.m.(n=3). Statistical significance was determined using a one-way ANOVA with Dunnett’s correction ( a - e ), a two-way ANOVA with Tukey’s correction ( f, g ), or an RM one-way ANOVA with Dunnett’s correction ( h ). *** P

    Article Snippet: Cytokines: recombinant Murine IFN-γ (Peprotech, 315–05), recombinant Mouse IFN-β1 (carrier-free, BioLegend, 581302), recombinant human IFN-β (Peprotech, 300–02BC), IFN-α is a kind gift from R. Modlin lab (UCLA).

    Techniques: Flow Cytometry, Incubation, Labeling, Fluorescence

    NLRP6 -/- neutrophils exhibit enhanced intracellular killing through increased IFN-γ and NADPH oxidase-dependent ROS production. (A) BMDNs from WT and KO mice were infected with S . aureus (MOI 10) and the intracellular killing ability of neutrophils was measured at the indicated time points. (B) ROS production by BMDNs from WT and KO mice were compared after infection with S . aureus (MOI 1). (C) WT and KO mice (N = 6-8/group) were infected with a sublethal dose of S . aureus (5 X 10 7 CFU/mouse). At designated time points, lungs were collected and processed for immunoblotting to compare the expression of NADPH oxidase enzyme components between WT and KO mice. (D) IFN-γ in BALF obtained from mice in 3C was measured. (E) Lungs obtained from S . aureus -infected WT and KO mice were homogenized and expressions of phospho-38, phospho-JNK, and phospho-STAT3 were measured through immunoblotting. (F) BMDNs from WT and KO mice were isolated and pre-treated with either IFN-γ or PBS for 30 minutes before infection with S . aureus (MOI 10). Two-hours post-infection, WT neutrophils were lysed and immunoblotted to measure the expression of NADPH oxidase enzyme components. (G) ROS production by neutrophils in 3F were measured as described in the methods. All figures are representative of three independent experiments. ROS: reactive oxygen species, RFU: relative florescence unit. WT: Wild-type, KO: NLRP6 KO. *, p

    Journal: PLoS Pathogens

    Article Title: NLRP6 negatively regulates pulmonary host defense in Gram-positive bacterial infection through modulating neutrophil recruitment and function

    doi: 10.1371/journal.ppat.1007308

    Figure Lengend Snippet: NLRP6 -/- neutrophils exhibit enhanced intracellular killing through increased IFN-γ and NADPH oxidase-dependent ROS production. (A) BMDNs from WT and KO mice were infected with S . aureus (MOI 10) and the intracellular killing ability of neutrophils was measured at the indicated time points. (B) ROS production by BMDNs from WT and KO mice were compared after infection with S . aureus (MOI 1). (C) WT and KO mice (N = 6-8/group) were infected with a sublethal dose of S . aureus (5 X 10 7 CFU/mouse). At designated time points, lungs were collected and processed for immunoblotting to compare the expression of NADPH oxidase enzyme components between WT and KO mice. (D) IFN-γ in BALF obtained from mice in 3C was measured. (E) Lungs obtained from S . aureus -infected WT and KO mice were homogenized and expressions of phospho-38, phospho-JNK, and phospho-STAT3 were measured through immunoblotting. (F) BMDNs from WT and KO mice were isolated and pre-treated with either IFN-γ or PBS for 30 minutes before infection with S . aureus (MOI 10). Two-hours post-infection, WT neutrophils were lysed and immunoblotted to measure the expression of NADPH oxidase enzyme components. (G) ROS production by neutrophils in 3F were measured as described in the methods. All figures are representative of three independent experiments. ROS: reactive oxygen species, RFU: relative florescence unit. WT: Wild-type, KO: NLRP6 KO. *, p

    Article Snippet: The effect of IFN-γ on ROS production was determined by pretreating neutrophils with either 20 ng/ml of recombinant mouse IFN-γ (R & D Systems, MN) or an equal volume of PBS for 30 minutes before infection with S . aureus .

    Techniques: Mouse Assay, Infection, Expressing, Isolation

    NK cells and CD4 + T cells are the major sources of IFN-γ production during pulmonary S . aureus infection. WT and KO mice (N = 6-8/group) were infected with S . aureus (5 X 10 7 CFU/mouse). Twelve-hours post-infection, mice were euthanized to harvest lungs. Single cell suspensions obtained through digestion of lungs were treated with PMA/Ionomycin and GolgiStop for 5 hours and then stained for flow cytometry. (A) Representative zebra plot showing CD3 - IFN-γ + NK 1.1 + cells. (B) Quantification of A . (C) Representative zebra plot showing CD3 + IFN-γ + CD4 + T cells. (D) Quantification of C . (E) WT and KO mice (N = 6-8/group) were infected with S . aureus (5 X 10 7 CFU/mouse). Twenty-four hours post-infection, mice were euthanized to collect lungs tissues. Single cell suspensions obtained after lung digestion were stained to estimate NK and CD4 T cells using flowcytometry. The figures shown above are representatives of three independent experiments. *, p

    Journal: PLoS Pathogens

    Article Title: NLRP6 negatively regulates pulmonary host defense in Gram-positive bacterial infection through modulating neutrophil recruitment and function

    doi: 10.1371/journal.ppat.1007308

    Figure Lengend Snippet: NK cells and CD4 + T cells are the major sources of IFN-γ production during pulmonary S . aureus infection. WT and KO mice (N = 6-8/group) were infected with S . aureus (5 X 10 7 CFU/mouse). Twelve-hours post-infection, mice were euthanized to harvest lungs. Single cell suspensions obtained through digestion of lungs were treated with PMA/Ionomycin and GolgiStop for 5 hours and then stained for flow cytometry. (A) Representative zebra plot showing CD3 - IFN-γ + NK 1.1 + cells. (B) Quantification of A . (C) Representative zebra plot showing CD3 + IFN-γ + CD4 + T cells. (D) Quantification of C . (E) WT and KO mice (N = 6-8/group) were infected with S . aureus (5 X 10 7 CFU/mouse). Twenty-four hours post-infection, mice were euthanized to collect lungs tissues. Single cell suspensions obtained after lung digestion were stained to estimate NK and CD4 T cells using flowcytometry. The figures shown above are representatives of three independent experiments. *, p

    Article Snippet: The effect of IFN-γ on ROS production was determined by pretreating neutrophils with either 20 ng/ml of recombinant mouse IFN-γ (R & D Systems, MN) or an equal volume of PBS for 30 minutes before infection with S . aureus .

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

    IFN-γ mediates bacterial clearance in NLRP6 KO mice during S . aureus -induced pneumonia. KO mice (n = 6-8/group) were treated with either anti-IFN-γ antibody (100 μg/mouse, i.p.) or isotype control antibody 12 hours prior to infection with S . aureus (5 X 10 7 CFU/mouse). WT mice were treated with equal volume of isotype control antibody 12 hours prior to infection with S . aureus (5 X 10 7 CFU/mouse). Twelve-hours post-infection, mice were euthanized to collect lungs and BALF. Bacterial burden in the (A) lungs, and (B) BALF were compared. All figures are representative of three independent experiments. *, p

    Journal: PLoS Pathogens

    Article Title: NLRP6 negatively regulates pulmonary host defense in Gram-positive bacterial infection through modulating neutrophil recruitment and function

    doi: 10.1371/journal.ppat.1007308

    Figure Lengend Snippet: IFN-γ mediates bacterial clearance in NLRP6 KO mice during S . aureus -induced pneumonia. KO mice (n = 6-8/group) were treated with either anti-IFN-γ antibody (100 μg/mouse, i.p.) or isotype control antibody 12 hours prior to infection with S . aureus (5 X 10 7 CFU/mouse). WT mice were treated with equal volume of isotype control antibody 12 hours prior to infection with S . aureus (5 X 10 7 CFU/mouse). Twelve-hours post-infection, mice were euthanized to collect lungs and BALF. Bacterial burden in the (A) lungs, and (B) BALF were compared. All figures are representative of three independent experiments. *, p

    Article Snippet: The effect of IFN-γ on ROS production was determined by pretreating neutrophils with either 20 ng/ml of recombinant mouse IFN-γ (R & D Systems, MN) or an equal volume of PBS for 30 minutes before infection with S . aureus .

    Techniques: Mouse Assay, Infection

    PD-1: PD-L1 pathway inhibits CD8 + T-cell IFN-γ and IL-2 expression in the post-encephalitic brain

    Journal: Glia

    Article Title: Glial cells suppress post-encephalitic CD8+ T lymphocytes through PD-L1

    doi: 10.1002/glia.22701

    Figure Lengend Snippet: PD-1: PD-L1 pathway inhibits CD8 + T-cell IFN-γ and IL-2 expression in the post-encephalitic brain

    Article Snippet: IFN-γ and IL-2 concentration were measured using a mouse IFN-γ or IL-2 ELISA kit (eBioscience) according to manufacturers instructions.

    Techniques: Expressing

    Blockade of TIM-4 improves hepatic acute reaction response injury and reduces the secretion of inflammatory cytokines. (A) KCs were isolated from mice in the sham, control mAb and TIM-4 mAb groups on day 7 post-surgery. Cells were stained with specific secondary antibodies labeled with tetramethylrhodamine (red) and DAPI (blue) and then examined using laser confocal microscopy (magnification ×800). (B) KCs were isolated from each group and stained with FITC-TIM-4 mAb for flow cytometry. (C) Blood samples were obtained (0.5 ml/mouse) from murine abdominal aortas in each group on day 7 post-surgery. Levels of AST, ALT and TBIL were determined in a clinical biochemical laboratory. (D-F) Levels of inflammatory cytokines, including TNF-α, IFN-γ, CCL2, CXCL2 and TGF-β were detected using ELISA and western blotting. (G) Representative micrographs of the pathological damage observed in each group post-transplantation following staining with hematoxylin and eosin (magnification ×400). (H) Donor mice received CL treatment to destroy KCs prior to surgery. orthotopic liver transplantation or sham surgery was subsequently performed and mice were treated either with or without TIM-4 mAb. All mice underwent analysis to determine the RAI score on day 7 following surgery. (I) The expression of key phosphoproteins involved in the NF-κB and p38 MAPK signaling pathways, including p65 and p38, respectively, were analyzed using western blotting. Data are presented as the mean ± standard deviation. a P

    Journal: International Journal of Molecular Medicine

    Article Title: TIM-4 blockade of KCs combined with exogenous TGF-β injection helps to reverse acute rejection and prolong the survival rate of mice receiving liver allografts

    doi: 10.3892/ijmm.2018.3606

    Figure Lengend Snippet: Blockade of TIM-4 improves hepatic acute reaction response injury and reduces the secretion of inflammatory cytokines. (A) KCs were isolated from mice in the sham, control mAb and TIM-4 mAb groups on day 7 post-surgery. Cells were stained with specific secondary antibodies labeled with tetramethylrhodamine (red) and DAPI (blue) and then examined using laser confocal microscopy (magnification ×800). (B) KCs were isolated from each group and stained with FITC-TIM-4 mAb for flow cytometry. (C) Blood samples were obtained (0.5 ml/mouse) from murine abdominal aortas in each group on day 7 post-surgery. Levels of AST, ALT and TBIL were determined in a clinical biochemical laboratory. (D-F) Levels of inflammatory cytokines, including TNF-α, IFN-γ, CCL2, CXCL2 and TGF-β were detected using ELISA and western blotting. (G) Representative micrographs of the pathological damage observed in each group post-transplantation following staining with hematoxylin and eosin (magnification ×400). (H) Donor mice received CL treatment to destroy KCs prior to surgery. orthotopic liver transplantation or sham surgery was subsequently performed and mice were treated either with or without TIM-4 mAb. All mice underwent analysis to determine the RAI score on day 7 following surgery. (I) The expression of key phosphoproteins involved in the NF-κB and p38 MAPK signaling pathways, including p65 and p38, respectively, were analyzed using western blotting. Data are presented as the mean ± standard deviation. a P

    Article Snippet: The primary antibodies used included: TIM-4 (RMT4-53, cat. no. GTX14149; 1:100 dilution; GeneTex Inc.), tumor necrosis factor-α (TNF-α; 52B38, cat. no. ab1793; 1:100 dilution; Abcam), interferon-γ (IFN-γ; #37895R, cat. no. MAB485R; 1:100 dilution; R & D Systems Inc., Minneapolis, MN, USA), chemokine ligand 2 (CCL2; 2D8, cat. no. GTX60582; 1:100 dilution; GeneTex Inc.), C-X-C motif chemokine ligand 2 (CXCL2; cat. no. GTX74085; 1:200 dilution; GeneTex Inc.), TGF-β (cat. no. ab92486; 1:100 dilution), phosphorylated (p)-signal transducer and activator of transcription 6 (STAT6; phospho Y641, cat. no. ab28829; 1:100 dilution), p-p65 (phospho S536, cat. no. ab86299; 1:100 dilution), p-p38 (phospho Y182, cat. no. ab47363; 1:100 dilution), forkhead box P3 (Foxp3; 236A/E7, cat. no. ab20034; 1:100 dilution), transcription factor gata3 (Gata3; cat. no. ab106625, 1:100 dilution) and β-actin (cat. no. ab8226; 1:100 dilution) (all from Abcam).

    Techniques: Isolation, Mouse Assay, Staining, Labeling, Confocal Microscopy, Flow Cytometry, Cytometry, AST Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Transplantation Assay, Expressing, Standard Deviation

    Intradermal vaccination of C57BL/6 wild-type mice with peptide antigens and photochemical internalization (PCI) effectively induces antigen-specific CD8+ effector T cell responses. Groups of five C57BL/6 wild-type mice were intradermally immunized with TRP-2 180–188 or HPV 43–78 peptide ± PCI treatment. Mice received three identical vaccination treatments with 14-day intervals. (A) 10 days after the last vaccination, frequencies of activated, antigen-specific CD8+ CTLs were analyses from blood samples with HPV 49–57 or TRP-2 180–188 peptide-loaded MHC class I pentamers (CD8+ CD44+ pentamer+ cells). Plots show representative examples of flow cytometric analysis for HPV 43–78 (upper panels) or TRP-2 180–188 (lower panels) vaccinated mice without (left) or with PCI treatment (right). (B) Quantification of activated HPV 43–48 -specific (CD8+ CD44+ H-2Kb/HPV 49–57 -pentamer+ cells, left) and TRP-2 180–188 -specific (CD8+ CD44+ H-2Kb/TRP-2 180–188 -pentamer+ cells, right) activated CD8+ T cells from all five mice in each group of the experiment. (C) Frequencies of interferon (IFN)-γ effector cytokine producing CD8+ CTLs were analyzed from spleen cells after overnight stimulation with TRP-2 180–188 or HPV 43–78 peptide, respectively (CD8+ CD44+ IFN-γ+ cells). Plots show representative examples of flow cytometric analysis for HPV 43–78 (upper panels) or TRP-2 180–188 (lower panels) vaccinated mice without (left) or with PCI treatment (right). (D) Quantification of IFN-γ effector cytokine production from CD8+ T cells (CD8+ CD44+ IFN-γ+ T cells) from HPV 43–48 -vaccinated (left) and TRP-2 180–188 -vaccinated mice for all five mice in each group of the experiments. Horizontal lines in scatter graphs indicate median values; P values were calculated using an unpaired two-tailed t -test to compare pairs of datasets (* P

    Journal: Frontiers in Immunology

    Article Title: Photochemical Internalization of Peptide Antigens Provides a Novel Strategy to Realize Therapeutic Cancer Vaccination

    doi: 10.3389/fimmu.2018.00650

    Figure Lengend Snippet: Intradermal vaccination of C57BL/6 wild-type mice with peptide antigens and photochemical internalization (PCI) effectively induces antigen-specific CD8+ effector T cell responses. Groups of five C57BL/6 wild-type mice were intradermally immunized with TRP-2 180–188 or HPV 43–78 peptide ± PCI treatment. Mice received three identical vaccination treatments with 14-day intervals. (A) 10 days after the last vaccination, frequencies of activated, antigen-specific CD8+ CTLs were analyses from blood samples with HPV 49–57 or TRP-2 180–188 peptide-loaded MHC class I pentamers (CD8+ CD44+ pentamer+ cells). Plots show representative examples of flow cytometric analysis for HPV 43–78 (upper panels) or TRP-2 180–188 (lower panels) vaccinated mice without (left) or with PCI treatment (right). (B) Quantification of activated HPV 43–48 -specific (CD8+ CD44+ H-2Kb/HPV 49–57 -pentamer+ cells, left) and TRP-2 180–188 -specific (CD8+ CD44+ H-2Kb/TRP-2 180–188 -pentamer+ cells, right) activated CD8+ T cells from all five mice in each group of the experiment. (C) Frequencies of interferon (IFN)-γ effector cytokine producing CD8+ CTLs were analyzed from spleen cells after overnight stimulation with TRP-2 180–188 or HPV 43–78 peptide, respectively (CD8+ CD44+ IFN-γ+ cells). Plots show representative examples of flow cytometric analysis for HPV 43–78 (upper panels) or TRP-2 180–188 (lower panels) vaccinated mice without (left) or with PCI treatment (right). (D) Quantification of IFN-γ effector cytokine production from CD8+ T cells (CD8+ CD44+ IFN-γ+ T cells) from HPV 43–48 -vaccinated (left) and TRP-2 180–188 -vaccinated mice for all five mice in each group of the experiments. Horizontal lines in scatter graphs indicate median values; P values were calculated using an unpaired two-tailed t -test to compare pairs of datasets (* P

    Article Snippet: Intracellular cytokine staining was performed with antibodies to interferon (IFN)-γ (anti-IFN-γ APC, clone XMG1.2, eBioscience) and flow cytometric analysis performed as described above.

    Techniques: Mouse Assay, Flow Cytometry, Two Tailed Test

    Effects of cytokine-specific antibodies on LPS-induced IFN-γ production. Isolated splenocytes were incubated with LPS (1 μg/ml) or 10 8 CFU of heat-killed bacteria per ml for 24 h in the presence of nonspecific goat IgG (10 μg/ml), anti-IL-10 (10 μg/ml), anti-IL-12 (1 μg/ml), anti-IL-15 (10 μg/ml), or anti-IL-18 (10 μg/ml). The IFN-γ levels in the conditioned media were determined by ELISA ( n = 8 to 12 wells/group). †, significantly ( P

    Journal: Clinical and Diagnostic Laboratory Immunology

    Article Title: Endotoxin-Induced Gamma Interferon Production: Contributing Cell Types and Key Regulatory Factors

    doi: 10.1128/CDLI.9.3.530-543.2002

    Figure Lengend Snippet: Effects of cytokine-specific antibodies on LPS-induced IFN-γ production. Isolated splenocytes were incubated with LPS (1 μg/ml) or 10 8 CFU of heat-killed bacteria per ml for 24 h in the presence of nonspecific goat IgG (10 μg/ml), anti-IL-10 (10 μg/ml), anti-IL-12 (1 μg/ml), anti-IL-15 (10 μg/ml), or anti-IL-18 (10 μg/ml). The IFN-γ levels in the conditioned media were determined by ELISA ( n = 8 to 12 wells/group). †, significantly ( P

    Article Snippet: Isolated splenocytes (106 in 0.1 ml of PBS) were incubated on ice with marker-specific antibodies or isotype controls (0.5 μg of antibody/106 cells) in polystyrene tubes for 30 min. For intracellular staining with anti-IFN-γ, the cells were fixed with Cytofix/Cytoperm buffer and were incubated with anti-IFN-γ diluted in Permwash buffer (B-D Pharmingen).

    Techniques: Isolation, Incubation, Enzyme-linked Immunosorbent Assay

    LPS-induced IFN-γ production is class II MHC independent. Isolated splenocytes were cultured with LPS (1 μg/ml) or 10 8 CFU of heat-killed E. coli , P. auruginosa , or S. aureus per ml for 24 h in the presence of nonspecific goat IgG (10 μg/ml) or anti-MHC class II (10 ng/ml). The IFN-γ levels in the conditioned media were determined by ELISA ( n = 4 to 8 wells/group). ∗, significantly ( P

    Journal: Clinical and Diagnostic Laboratory Immunology

    Article Title: Endotoxin-Induced Gamma Interferon Production: Contributing Cell Types and Key Regulatory Factors

    doi: 10.1128/CDLI.9.3.530-543.2002

    Figure Lengend Snippet: LPS-induced IFN-γ production is class II MHC independent. Isolated splenocytes were cultured with LPS (1 μg/ml) or 10 8 CFU of heat-killed E. coli , P. auruginosa , or S. aureus per ml for 24 h in the presence of nonspecific goat IgG (10 μg/ml) or anti-MHC class II (10 ng/ml). The IFN-γ levels in the conditioned media were determined by ELISA ( n = 4 to 8 wells/group). ∗, significantly ( P

    Article Snippet: Isolated splenocytes (106 in 0.1 ml of PBS) were incubated on ice with marker-specific antibodies or isotype controls (0.5 μg of antibody/106 cells) in polystyrene tubes for 30 min. For intracellular staining with anti-IFN-γ, the cells were fixed with Cytofix/Cytoperm buffer and were incubated with anti-IFN-γ diluted in Permwash buffer (B-D Pharmingen).

    Techniques: Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay

    Characterization of IFN-γ-producing cells in the splenocytes after endotoxin (LPS) challenge. Mice were challenged with LPS (4 mg/kg of body weight, intraperitoneally), and their spleens were harvested at the indicated time points. The amounts of IFN-γ produced by DX5 + (NK) and CD3 + (T) cells were determined by flow cytometry. The DX5 + IFN-γ + and CD3 + IFN-γ + populations are circumscribed within the box at the 8-h time point, at which the level of IFN-γ production was maximal.

    Journal: Clinical and Diagnostic Laboratory Immunology

    Article Title: Endotoxin-Induced Gamma Interferon Production: Contributing Cell Types and Key Regulatory Factors

    doi: 10.1128/CDLI.9.3.530-543.2002

    Figure Lengend Snippet: Characterization of IFN-γ-producing cells in the splenocytes after endotoxin (LPS) challenge. Mice were challenged with LPS (4 mg/kg of body weight, intraperitoneally), and their spleens were harvested at the indicated time points. The amounts of IFN-γ produced by DX5 + (NK) and CD3 + (T) cells were determined by flow cytometry. The DX5 + IFN-γ + and CD3 + IFN-γ + populations are circumscribed within the box at the 8-h time point, at which the level of IFN-γ production was maximal.

    Article Snippet: Isolated splenocytes (106 in 0.1 ml of PBS) were incubated on ice with marker-specific antibodies or isotype controls (0.5 μg of antibody/106 cells) in polystyrene tubes for 30 min. For intracellular staining with anti-IFN-γ, the cells were fixed with Cytofix/Cytoperm buffer and were incubated with anti-IFN-γ diluted in Permwash buffer (B-D Pharmingen).

    Techniques: Mouse Assay, Produced, Flow Cytometry, Cytometry

    Additive effects of cytokine-specific antibodies and CTLA4-Ig on LPS-induced IFN-γ production. Isolated splenocytes were incubated with LPS (1 μg/ml) or 10 8 CFU of heat-killed bacteria per ml for 24 h in the presence of nonspecific goat IgG (10 μg/ml), anti-IL-12 (1 μg/ml), anti-IL-15 (10 μg/ml), or anti-IL-18 (10 μg/ml) in combination with CTLA4-Ig (1 μg/ml). The IFN-γ levels in the conditioned media were determined by ELISA ( n = 6 to 8 wells/group.). ∗, significantly ( P

    Journal: Clinical and Diagnostic Laboratory Immunology

    Article Title: Endotoxin-Induced Gamma Interferon Production: Contributing Cell Types and Key Regulatory Factors

    doi: 10.1128/CDLI.9.3.530-543.2002

    Figure Lengend Snippet: Additive effects of cytokine-specific antibodies and CTLA4-Ig on LPS-induced IFN-γ production. Isolated splenocytes were incubated with LPS (1 μg/ml) or 10 8 CFU of heat-killed bacteria per ml for 24 h in the presence of nonspecific goat IgG (10 μg/ml), anti-IL-12 (1 μg/ml), anti-IL-15 (10 μg/ml), or anti-IL-18 (10 μg/ml) in combination with CTLA4-Ig (1 μg/ml). The IFN-γ levels in the conditioned media were determined by ELISA ( n = 6 to 8 wells/group.). ∗, significantly ( P

    Article Snippet: Isolated splenocytes (106 in 0.1 ml of PBS) were incubated on ice with marker-specific antibodies or isotype controls (0.5 μg of antibody/106 cells) in polystyrene tubes for 30 min. For intracellular staining with anti-IFN-γ, the cells were fixed with Cytofix/Cytoperm buffer and were incubated with anti-IFN-γ diluted in Permwash buffer (B-D Pharmingen).

    Techniques: Isolation, Incubation, Enzyme-linked Immunosorbent Assay

    IFN-γ mRNA expression after endotoxin (LPS) challenge. Mice were challenged with LPS (4 mg/kg of body weight, intraperitoneally), spleens were harvested at the specified times, and total RNA was isolated. IFN-γ mRNA levels were determined by RPA. L32, a housekeeping gene, served both as a loading standard and as an internal control.

    Journal: Clinical and Diagnostic Laboratory Immunology

    Article Title: Endotoxin-Induced Gamma Interferon Production: Contributing Cell Types and Key Regulatory Factors

    doi: 10.1128/CDLI.9.3.530-543.2002

    Figure Lengend Snippet: IFN-γ mRNA expression after endotoxin (LPS) challenge. Mice were challenged with LPS (4 mg/kg of body weight, intraperitoneally), spleens were harvested at the specified times, and total RNA was isolated. IFN-γ mRNA levels were determined by RPA. L32, a housekeeping gene, served both as a loading standard and as an internal control.

    Article Snippet: Isolated splenocytes (106 in 0.1 ml of PBS) were incubated on ice with marker-specific antibodies or isotype controls (0.5 μg of antibody/106 cells) in polystyrene tubes for 30 min. For intracellular staining with anti-IFN-γ, the cells were fixed with Cytofix/Cytoperm buffer and were incubated with anti-IFN-γ diluted in Permwash buffer (B-D Pharmingen).

    Techniques: Expressing, Mouse Assay, Isolation, Recombinase Polymerase Amplification

    Roles of accessory cells in endotoxin (LPS)-induced IFN-γ production. (A) Isolated splenic T and NK cells were incubated with resident peritoneal macrophages at the indicated ratios for 24 h in the presence of LPS (1 μg/ml). The levels of IFN-γ secreted in conditioned medium were determined by ELISA ( n = 4 to 6 wells/group). ∗, significantly ( P

    Journal: Clinical and Diagnostic Laboratory Immunology

    Article Title: Endotoxin-Induced Gamma Interferon Production: Contributing Cell Types and Key Regulatory Factors

    doi: 10.1128/CDLI.9.3.530-543.2002

    Figure Lengend Snippet: Roles of accessory cells in endotoxin (LPS)-induced IFN-γ production. (A) Isolated splenic T and NK cells were incubated with resident peritoneal macrophages at the indicated ratios for 24 h in the presence of LPS (1 μg/ml). The levels of IFN-γ secreted in conditioned medium were determined by ELISA ( n = 4 to 6 wells/group). ∗, significantly ( P

    Article Snippet: Isolated splenocytes (106 in 0.1 ml of PBS) were incubated on ice with marker-specific antibodies or isotype controls (0.5 μg of antibody/106 cells) in polystyrene tubes for 30 min. For intracellular staining with anti-IFN-γ, the cells were fixed with Cytofix/Cytoperm buffer and were incubated with anti-IFN-γ diluted in Permwash buffer (B-D Pharmingen).

    Techniques: Isolation, Incubation, Enzyme-linked Immunosorbent Assay

    Effects of B7 inhibitors on LPS-induced IFN-γ production. Isolated splenocytes were incubated with LPS (1 μg/ml) or 10 8 CFU of heat-killed bacteria per ml in the presence of nonspecific goat IgG (10 μg/ml), CTLA4-Ig (1 μg/ml), anti-CD80 (10 μg/ml), or anti-CD86 (10 μg/ml) for 24 h. The IFN-γ levels in the conditioned media were determined by ELISA ( n = 4 to 8 wells/group). In some experiments, anti-CD80 and anti-CD86 were preabsorbed with CD80-Ig or CD86-Ig to assess specificity. ∗, significantly ( P

    Journal: Clinical and Diagnostic Laboratory Immunology

    Article Title: Endotoxin-Induced Gamma Interferon Production: Contributing Cell Types and Key Regulatory Factors

    doi: 10.1128/CDLI.9.3.530-543.2002

    Figure Lengend Snippet: Effects of B7 inhibitors on LPS-induced IFN-γ production. Isolated splenocytes were incubated with LPS (1 μg/ml) or 10 8 CFU of heat-killed bacteria per ml in the presence of nonspecific goat IgG (10 μg/ml), CTLA4-Ig (1 μg/ml), anti-CD80 (10 μg/ml), or anti-CD86 (10 μg/ml) for 24 h. The IFN-γ levels in the conditioned media were determined by ELISA ( n = 4 to 8 wells/group). In some experiments, anti-CD80 and anti-CD86 were preabsorbed with CD80-Ig or CD86-Ig to assess specificity. ∗, significantly ( P

    Article Snippet: Isolated splenocytes (106 in 0.1 ml of PBS) were incubated on ice with marker-specific antibodies or isotype controls (0.5 μg of antibody/106 cells) in polystyrene tubes for 30 min. For intracellular staining with anti-IFN-γ, the cells were fixed with Cytofix/Cytoperm buffer and were incubated with anti-IFN-γ diluted in Permwash buffer (B-D Pharmingen).

    Techniques: Isolation, Incubation, Enzyme-linked Immunosorbent Assay

    IFN-γ production by isolated splenic T and NK cells in response to IFN-γ-regulating factors. Isolated splenic T and NK cells were incubated with IL-12 (2 ng/ml) alone or with IL-12 plus IL-15 (20 ng/ml), IL-18 (20 ng/ml), agarose-coupled anti-CD28 (10 μg/ml), CD80-Ig (10 μg/ml), CD86-Ig (10 μg/ml), or anti-CD3 (10 μg/ml) in the indicated combinations for 24 h. The IFN-γ levels in the conditioned media were determined by ELISA ( n = 4 to 8 wells/group).

    Journal: Clinical and Diagnostic Laboratory Immunology

    Article Title: Endotoxin-Induced Gamma Interferon Production: Contributing Cell Types and Key Regulatory Factors

    doi: 10.1128/CDLI.9.3.530-543.2002

    Figure Lengend Snippet: IFN-γ production by isolated splenic T and NK cells in response to IFN-γ-regulating factors. Isolated splenic T and NK cells were incubated with IL-12 (2 ng/ml) alone or with IL-12 plus IL-15 (20 ng/ml), IL-18 (20 ng/ml), agarose-coupled anti-CD28 (10 μg/ml), CD80-Ig (10 μg/ml), CD86-Ig (10 μg/ml), or anti-CD3 (10 μg/ml) in the indicated combinations for 24 h. The IFN-γ levels in the conditioned media were determined by ELISA ( n = 4 to 8 wells/group).

    Article Snippet: Isolated splenocytes (106 in 0.1 ml of PBS) were incubated on ice with marker-specific antibodies or isotype controls (0.5 μg of antibody/106 cells) in polystyrene tubes for 30 min. For intracellular staining with anti-IFN-γ, the cells were fixed with Cytofix/Cytoperm buffer and were incubated with anti-IFN-γ diluted in Permwash buffer (B-D Pharmingen).

    Techniques: Isolation, Incubation, Enzyme-linked Immunosorbent Assay