*3.4. Th1, Th2 and Treg Cell Di*ff*erentiation*

For Th1 polarization, purified CD4<sup>+</sup> Th0 cells were cultured in expansion media supplemented with IL-12 (10 ng/mL) (Peprotech) and anti-IL-4 neutralizing antibodies (5 μg/mL) (BD Bioscience, San Jose, CA, USA) for 5 days. For Th2 induction, purified CD4<sup>+</sup> Th0 cells were cultured in expansion media with IL-4 (20 ng/mL) (Peprotech) and anti-IFN-γ neutralizing antibodies (5 μg/mL) (BioxCell, West Lebanon, NH, USA) for 5 days. To evaluate the spontaneous induction ratio of Treg cells, purified CD4<sup>+</sup> Th0 cells were cultured in expansion media for 5 days without any lineage-specific stimulants. To assess the Ech A impact on Th cell polarization, Ech A or vehicle was also treated during the culture period.
