Percentages of lymphocyte subsets after Tofa treatment.

<p>Cells were analysed before treatment (day 0), after 4 days of incubation with Tofa (day 4) and then four days after the withdrawal of the drug (day 4+4). Percentage of CD3, CD19 and CD16/56 cells were evaluated on total lymphocytes, while CD4 cells, CD8 cells and CD4/CD8 ratio was evaluated on CD3 positive T lymphocytes. Data are expressed as mean ± SD of four independent experiments.</p

Lymphocytes activation and proliferation after Tofa treatment.

<p>Cells were analysed after four days of incubation with Tofa (day 4) and four days after the withdrawal of the drug (day 4+4). The percentage of proliferating cells was assessed by measuring the level of CFSE fluorescence. Activation of cells is indicated as MFI of CD25. Data are expressed as mean ± SD of four independent experiments.</p

Representative data of cell proliferation and activation.

<p>Cells were incubated with PHA and the two concentrations of Tofa (grey histograms); after four days the medium was removed, and the incubation continued in drug-free medium for four additional days (black line histograms). The panel on the left represents the percentage of proliferating cells based on dilution of CFSE dye. On the right expression of CD25 is reported and evaluated as MFI.</p

Absolute cell counts and viability.

<p>The plots on the left represent the absolute number of cells. Histograms on the right report the percentage of dead cells, as 7AAD positive cells. Counts and viability are reported for each subpopulation of interest, before treatment (day 0) and after the first four days of incubation in presence or absence of PHA and Tofa (day 4). Data are expressed as mean ± SD of two independent experiments.</p

Cytokine determination in culture supernatants at different time frames.

<p>Cytokines were evaluated by magnetic bead-based multiplex immunoassays. The analysis was performed at day 4 and at day 4+4 on three series of culture supernatants. Data are expressed as mean concentration (ng/mL) ± SD. Only the histograms of the cytokines mainly involved in the effects evaluated in the study are reported in the figure. Grey bars: unstimulated cells; Black bars: PHA stimulated cells. Statistical significance was calculated using one-way analysis of variance (ANOVA) and Bonferroni post-test. *p<0.05.</p

Proliferation and cell activation at the different time frames.

<p>Cell activation at day 4 (A) and after four days after withdrawal of Tofa (B) is represented as mean fluorescence index (MFI) of CD25 ± SD of four independent experiments. Cell proliferation at day 4 (C) and after four days after withdrawal of Tofa (D) is represented as percentage of cells with reduced fluorescence of CFSE ± SD of four independent experiments. Statistical significance was calculated using one-way analysis of variance (ANOVA) and Bonferroni post-test. *** p<0.0001, **p<0.001.</p

Phosphorylation profile of STAT proteins.

<p>Phosphorylation profile of STAT1, STAT2, STAT3, STAT5A/B, STAT6 was evaluated by magnetic bead-based multiplex immunoassays. Data are expressed as Median Intensity Fluorescence (MFI) ± SD of three independent experiments. Grey bars: unstimulated cells; Black bars: PHA stimulated cells. Statistical significance was calculated using one-way analysis of variance (ANOVA) and Bonferroni post-test. *p<0.05.</p