Journal: Scientific Reports

Article Title: Multivalent Fc γ -receptor engagement by a hexameric Fc-fusion protein triggers Fc γ -receptor internalisation and modulation of Fc γ -receptor functions

doi: 10.1038/s41598-017-17255-8

Figure Lengend Snippet: Characterisation of hexameric-Fc binding. ( A ) The binding of fluorescently tagged γ1 hexameric-Fc and IVIg to PBMC subsets was analysed by flow cytometry. Shown is 1 representative donor out of 3 independent donors. ( B ) Flow cytometric analysis of FcγR expression on PBMCs. Cells from the same donor as in ( A ) were stained with FcγR antibodies to analyse FcγRI, FcγRII and FcγRIII expression on PBMC subsets. ( C ) Binding of hexameric-Fc to HEK293 cells transfected with indicated FcγR constructs. Data shows the mean of 3 independent experiments + /- SEM. ( D ) Representative SPR traces showing the binding of γ4-hexameric-Fc and IgG4 to FcγRIIa. Hexameric-Fc was titrated in a two-fold dilution series from 1μM to 7.8 nM. IgG4 was titrated in a two-fold dilution series between 50μM and 0.39 μM.

Article Snippet: Other assay conditions included TT only (1.0 μg/ml), or TT-ICs with hexameric-Fc (50 μg/ml) or FcγR blocking antibodies (R&D Systems, AF1330 anti-CD16 and AF1597anti-CD16, 20 μg/ml per antibody).

Techniques: Binding Assay, Flow Cytometry, Expressing, Staining, Transfection, Construct

Journal: Scientific Reports

Article Title: Multivalent Fc γ -receptor engagement by a hexameric Fc-fusion protein triggers Fc γ -receptor internalisation and modulation of Fc γ -receptor functions

doi: 10.1038/s41598-017-17255-8

Figure Lengend Snippet: In vitro disruption of FcγR function. ( A ) Macrophages were incubated with hexameric-Fc s or IvIg at 75 µg/ml for 1 hour. Cells were then washed and incubated for the indicated period. Cells were then labelled with fluorescently-conjugated hexameric-Fc. Data shows means from three donors ± SEM. ( B ) Pre-incubation with hexameric-Fc inhibits phagocytosis for 72hrs. Human monocyte-derived macrophages were incubated with the indicated hexameric-Fcs or IVIg at 75 µg/ml or 300 µg/ml for 1 hour, washed and cultured for 1 to 72 hours before co-culture with autologous CFSE-labelled B cell targets in the presence of 0.1 μg/ml anti-CD20. The disappearance of target cells was measured by flow cytometry after 18hrs and plotted as % inhibition of total antibody-dependent phagocytosis. Data are the mean ± SEM for three donors.

Article Snippet: Other assay conditions included TT only (1.0 μg/ml), or TT-ICs with hexameric-Fc (50 μg/ml) or FcγR blocking antibodies (R&D Systems, AF1330 anti-CD16 and AF1597anti-CD16, 20 μg/ml per antibody).

Techniques: In Vitro, Disruption, Incubation, Derivative Assay, Cell Culture, Co-Culture Assay, Flow Cytometry, Inhibition

Journal: Scientific Reports

Article Title: Multivalent Fc γ -receptor engagement by a hexameric Fc-fusion protein triggers Fc γ -receptor internalisation and modulation of Fc γ -receptor functions

doi: 10.1038/s41598-017-17255-8

Figure Lengend Snippet: In vivo effects of hexameric-Fc. ( A ) 125 I γ1 Hexameric-Fc was administered to mice at 0.5 mg/kg, 2 mg/kg or 10 mg/kg. At indicated timepoints, plasma was collected from three mice per timepoint and concentration of protein bound radioactivity in plasma determined by direct measurement in a gamma counter. Values were corrected to calculate μg-equivalents of hexameric-Fc per mL of plasma. ( B ) γ4eng F234L F296Y hexameric-Fc administered to cynomolgus monkeys by IV bolus at 1 dose of 2 mg/kg. Concentrations of hexameric-Fc and smaller and larger human Fc-containing moieties were detected in plasma by mass spectroscopy. n of 3 animals, ± SEM. ( C ) Binding of γ1-hexameric-Fc to immobilised recombinant FcRn was investigated by SPR. Hexameric-Fc was titrated in a two-fold dilution series from 2.5μM to 39 nM. ( D ) To assess hexameric-Fc mediated FcγR blockade in cynomolgus monkeys, whole blood samples were collected after a 30 mg/kg IV dose of γ4eng F234L F296Y hexameric-Fc. Surface labelling of samples was carried out to identify monocytes (CD14 + ) and occupancy of FcγRs assessed using a AF647-conjugated γ4eng F234L F296Y prior to analysis by flow-cytometry. 3 animals, ± SEM. ( E ) To assess the effect of hexameric-Fc in an ITP model, 10 mg/kg hexameric-Fc was administered to mice IV, at the timepoints indicated, prior to addition of anti-CD41 (MWReg30) to induce platelet depletion. Whole blood samples were taken immediately prior to and 24 hour post anti-CD41 in order to determine platelet numbers. n = 6 mice per group, graph shows mean ± SEM, *=p < 0.05, ***=p < 0.01, by ANOVA and Dunnetts multiple comparison test.

Article Snippet: Other assay conditions included TT only (1.0 μg/ml), or TT-ICs with hexameric-Fc (50 μg/ml) or FcγR blocking antibodies (R&D Systems, AF1330 anti-CD16 and AF1597anti-CD16, 20 μg/ml per antibody).

Techniques: In Vivo, Clinical Proteomics, Concentration Assay, Radioactivity, Mass Spectrometry, Binding Assay, Recombinant, Flow Cytometry, Comparison

Journal: International Journal of Molecular Sciences

Article Title: TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2

doi: 10.3390/ijms23169306

Figure Lengend Snippet: TNF-α does not impact the levels of TNFR1 and TNFR2 on Th1 and Th17 cells. Purified CD4 + T cell subsets were stimulated with 1 µg/mL of TNF-α for 24 h and stained, for flow cytometry, with anti-human TNFR1 or TNFR2 mAbs. ( A ) Representative histograms showing the expression levels of TNFR1 and TNFR2 on Th1 and Th17 cells at baseline conditions and upon TNF-α stimulus. Solid black line histograms, isotype control; black histograms, Th1 cells; grey histograms, Th17 cells; dashed line histograms, TNF-α-treated Th1 cells; long dashed line histograms, TNF-α-treated Th17 cells. The levels of TNFR1 ( B ) and TNFR2 ( C ) were measured on purified CD4 + T cell subpopulations obtained from two to four healthy controls. Bars represent the mean values ± SD. Statistical analyses were carried out with Kruskal–Wallis followed by Dunn’s multiple comparison tests. ** p < 0.01.

Article Snippet: Cells were then stained with a mouse anti-human TNFR1-APC mAb (clone 16803) (R&D Systems, Minneapolis, MN, USA) and a rat anti-human TNFR2-PE mAb (clone hTNFR-M1) (BD Biosciences) for 30 min at 4 °C in the dark.

Techniques: Purification, Staining, Flow Cytometry, Expressing, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2

doi: 10.3390/ijms23169306

Figure Lengend Snippet: Effect of TNFR1 or TNFR2 blockade on the production of IFN-γ and IL-17 by Th1 and Th17 cells. Purified CD4 + T lymphocyte subpopulations were incubated with neutralizing antibodies to TNFR1 or TNFR2 for 1 h prior to a 4-day stimulus with TNF-α (1 µg/mL). Intracellular staining of IFN-γ and IL-17 was assessed by flow cytometry. ( A ) Representative dot plots of Th1 and Th17 cells treated with anti-TNFR1 or anti-TNFR2 in the presence or absence of TNF-α. An isotype control was used to discard non-specific effects of the neutralizing antibodies. The fold increase in the percentages of IFN-γ ( B , D ) or IL-17 ( C , E ) producers was measured on Th1 and Th17 cells obtained from two to six healthy donors. Bars represent the mean values ± SD. Data were normalized against cells that did not receive treatment with TNF-α or TNF-α plus TNFRs blocking mAbs. For statistical analysis, Kruskal–Wallis and Dunn’s multiple comparison tests were performed. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cells were then stained with a mouse anti-human TNFR1-APC mAb (clone 16803) (R&D Systems, Minneapolis, MN, USA) and a rat anti-human TNFR2-PE mAb (clone hTNFR-M1) (BD Biosciences) for 30 min at 4 °C in the dark.

Techniques: Purification, Incubation, Staining, Flow Cytometry, Control, Blocking Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2

doi: 10.3390/ijms23169306

Figure Lengend Snippet: Expression of TNFR1 and TNFR2 on Th1 and Th17 cells present in the peripheral blood of rheumatoid arthritis (RA) patients treated with adalimumab. Cell staining for flow cytometry analysis was performed on PBMC samples from healthy controls ( n = 9) and RA patients ( n = 10) before (PRE) and after (POST) treatment with adalimumab. The levels of TNFR1 ( A ) and TNFR2 ( C ) are expressed in MFI values. The frequency of TNFR1 ( B ) and TNFR2 ( D )-expressing lymphocytes are also shown. Each symbol represents data for one individual. Mean values ± SD are indicated. Significance was assessed with non-parametric Kruskal–Wallis test followed by Dunn’s multiple comparison test (for MFI data) or parametric one-way ANOVA plus Tukey’s post-test (for lymphocyte frequencies data). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cells were then stained with a mouse anti-human TNFR1-APC mAb (clone 16803) (R&D Systems, Minneapolis, MN, USA) and a rat anti-human TNFR2-PE mAb (clone hTNFR-M1) (BD Biosciences) for 30 min at 4 °C in the dark.

Techniques: Expressing, Staining, Flow Cytometry, Comparison