*2.3. Ech A Induced the Generation of Regulatory T Cells In Vitro*

It is well known that naive Th (Th0) cells can differentiate into various subtypes of active Th cells such as Th1, Th2 and Treg cells upon specific cues and each type of mature Th cell plays a distinct and pivotal role in various disease developments including IBD [17,18]. Therefore, next we isolated CD4<sup>+</sup> naive Th0 cells from human cord blood then performed a differentiation experiment following the typical procedure with or without Ech A to evaluate the impact of Ech A on Th cell polarization. As shown in Figure 3, flow cytometry analysis on cell surface markers revealed that Ech A provided no significant impact on Th1/2 polarization in vitro (Figure 3A,B). On the other hand, Ech A treatment could stimulate the generation of Treg cells; indeed, approximately 10% of total Ech A-treated cells expressed the marker of the Treg cell marker Foxp3, while the spontaneous induction ratio of Treg cells were less than 1% on average (Figure 3C).

**Figure 2.** Ech A impact on mixed lymphocyte reaction (MLR) in vitro. CFSE-labeled human mononuclear cells (MNCs) were cultured for five days in the presence of mitogen or antibodies for CD3/28 with or without Ech A treatment, then the percentage of proliferated cells was evaluated using flow cytometry. The proliferation of both MNCs (**A**,**B**) and T cells (**C**,**D**) was reduced by Ech A treatment in a dose-dependent manner. VC, vehicle-treated control. \*\* *P* < 0.01. Results are shown as mean ± SD.

**Figure 3.** Ech A impact on Th cell polarization in vitro. (**A**,**B**) the percentages of IFN-γ <sup>+</sup> and IL-4<sup>+</sup> cells among CD4<sup>+</sup> Th cells were evaluated using flow cytometry to determine the induction ratio of Th1 and Th2 cells, respectively. No significant change was observed upon Ech A treatment. (**C**) Spontaneous generation of Foxp3<sup>+</sup> Treg cells among CD4<sup>+</sup> Th cells upon vehicle and Ech A treatment was assessed using flow cytometry. Ech A treatment led to an increase in Treg population compared to the vehicle-treated group. VC, vehicle-treated control. \* *P* < 0.05, \*\* *P* < 0.001. Results are shown as mean ± SD.
