Figure 1.

Tregs inducing assay. (A) Treatment of canine PBMCs with TcES at 150 µg/mL caused an increase in Foxp3+ and Foxp3^high. Although single individuals show increased frequencies of Foxp3+ lymphocytes after treatment with AcES at 150 µg/mL (illustrated by the flow cytometric plots) this effect did not achieve statistical significance. In contrast, AcES at 15 µg/mL was associated with a slight decrease in Foxp3 expression in lymphocytes. (B) Three-colour flow cytometry revealed the TcES-associated increase in Foxp3^high lymphocytes to be associated with CD4+, CD4+ CD8+ double-positive and CD4- CD8- double-negative subsets, while this effect was lower in CD8+ T cells. Compared to TcES, treatment with AcES at 150 µg/mL was associated with a much lower elevation in Foxp3^high expression by lymphocytes and CD4+ CD8+ T cells. (C) Cultivating the cells in the presence of 150 µg/mL AcES or TcES induced marked increase in Helios expression by Foxp3^high lymphocytes. (D) Treatment of canine PBMCs with ES products was associated with decreased frequencies of CD25+ and CD25++ cells within Foxp3^high lymphocytes and this effect was most obvious at 150 µg/mL. P-values were calculated according to paired t-test. Error bars represent standard error of mean (SEM). FSC = forward scatter, SSC = side scatter. Experiments were repeated six times at two different time points.