Twist1 expression is induced in Th1, but not Th2 or Th17 cells. CD4⁺CD62L^hi OVA-specific T lymphocytes were stimulated in vitro under Th1 or Th2, or under Th17-polarizing conditions. Functional polarization of Th1, Th2, and Th17 cells, i.e., the cytokine expression profile, was confirmed by intracellular immunofluorescence (Fig. S1). (A) Twist1 mRNA in resting cells 6 d after the last stimulation (Th1; circles) or after 3 h of restimulation with anti-CD3/CD28 and IL-2 (Th1; squares) or PMA/ionomycin and IL-2 (Th1; triangles: Th2; diamonds) was determined by RT-PCR and normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT). (B) Kinetics of twist1 mRNA expression in 6-d-old (open bars) and 24-d-old (shaded bars) Th1 cells, after stimulation with anti-CD3/CD28 and IL-2 or IL-2 alone. (C) Twist1 transcript levels in 6-d-old Th1, Th2, and Th17 cells after reactivation with PMA/ionomycin. (D) Twist1 protein expression in resting (-) and 5 h PMA/ionomycin-restimulated (+) 6- and 24-d-old Th1 and Th2 cells. Control: α-Tubulin immunoblot (bottom). (E) Kinetics of Twist1 protein expression in 24-d-old Th1 cells, before and at the indicated time intervals of stimulation with anti-CD3/CD28 and IL-2. Data are representative of two experiments. Fig. S1 is available.