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