EmTh-1 supernatant impairs TGF-β–induced iTreg generation and promotes Tbet⁺ T-lymphocyte differentiation. CD4⁺CD25⁻CD62L⁺ naive T cells were cultured for 72 hours with T-cell expander beads (cell:bead ratio 1:1) with or without TGF-β1 (5 ng/mL) in the presence or absence of the emTh-1 supernatant (emTh-1 sup). Cells were then analyzed by flow cytometry. (A-B) Representative dot plots or histogram plots from 10 independent experiments. (C) Percentage of CD4⁺CD25⁺FoxP3⁻- activated T cells in total CD4⁺ T lymphocytes. *P < .01, a significant difference compared with cells cultured without emTh-1 supernatant. (D) The expression of the transcription factors FoxP3, Tbet, and GATA-3 was determined in CD4⁺CD25⁻CD62L⁺ T cells cultured for 72 hours with T-cell expander beads with or without TGF-β1 treated or not with emTh-1 supernatant. Results are representative of 3 independent experiments.