*3.2. Mixed Leukocyte Reaction*

For MLR, human MNCs were labeled using the CellTrace CFSE cell labeling kit (Invitrogen, Grand Island, NY, USA) following the manufacturer's instructions. Cells were then seeded in a 6-well plate at a density of 5 <sup>×</sup> <sup>10</sup><sup>4</sup> /well and cultured in RPMI 1640 (Gibco, Grand Island, NY, USA) with supplement of 10% FBS (Gibco) and 100 U/mL penicillin/streptomycin (Gibco) in the presence of Con A (5 μg/mL for MNC proliferation) (Sigma, St. Lois, MO, USA) or anti-CD3/anti-CD28 (5 μg/mL and 2 μg/mL, respectively, for T cell proliferation) (eBioscience, San Diego, CA, USA) for 5 days. To assess the Ech A impact on cell proliferation, Ech A or vehicle was also treated during the culture period. After 5 days, cells were harvested and prepared for flow cytometry analysis.
