In vitro polarization of human Th naive precursors. CD4⁺CD45RA⁺ T cells were isolated from peripheral blood by negative selection as specified in “Materials and methods.” Cells were stimulated with anti-CD3/anti-CD8 beads (0.5 beads per cell) and cultured for 7 to 21 days under Th1 or Th2 polarizing conditions (see “Materials and methods”). (A) Shown is a typical experiment in which 7-day Th1 and Th2 cultures were restimulated for 5 hours with PMA (10 ng/mL) and ionomycin (1 μg/mL) in the presence of monensin and then fixed and permeabilized for single-cell detection of IL-4 and IFN-γ. (B) Percentages of IL-4– and IFN-γ–producing cells in similar experiments performed with 7-day Th-cell preparations from 15 donors. Differences were highly significant (P < .001; paired t test). (C) IL-4 and IFN-γ secretion from human in vitro–polarized cultures. IL-4 and IFN-γ were measured by ELISA in the supernatants of cells stimulated (20 hours) with PMA and ionomycin as specified. Means ± SEM of 6 donors. *P < .05 relative to Th1.