Figure 5.

Effect of different therapeutics on the induction of regulatory T cells in vitro and in vivo. Studies on the effect of different drugs on Treg response. (A) In vitro study on Tregs. Human Tregs were isolated from human PBMCs of healthy volunteers, sorted from pre-enriched CD4⁺ cells and in vitro stimulated for 36 h with anti-CD3/CD28 coated MACSiBead in the presence or absence of class one MEK-inhibitors, followed by Foxp3 staining and flow cytometry analysis. The data are shown as %Tregs (of gated lymphocytes) normalized to untreated control and presented as mean ± SD (n = 5–8). One-way ANOVA with Sidak correction was used for statistical analysis. p = 0.0001 is represented with *** and p< 0.0001 with ****. (B-D) Mice were infected with 3 x 10⁵ pfu influenza A virus and treated twice daily with either zapnometinib, baloxavir, oseltamivir or solvent control for 3 days and sacrificed 6 days p.i. Lungs were harvested on day 6 p.i. and analyzed for the presence of Tregs. (B) Schematic of the in vivo efficacy study. Clinical signs were assessed once daily and included body weight and disease symptom score. This figure was created with BioRender.com. (C) Gating strategy for the quantification of Tregs in the lung of IAV infected and treated mice. CD4⁺ cells were gated for CD4⁺CD25⁺ cells and further for FoxP3⁺. (D) In vivo effect of zapnometinib, baloxavir and oseltamivir on the induction of Tregs after infection with IAV compared to control. Data presented as mean ± SD (n = 5). One-way ANOVA with Sidak correction was used for statistical analysis (*p< 0.05, **p< 0.005, ***p< 0.001, ns, not significant).