Simultaneous commitment of naive T cells to the Th1 and Th2 differentiation programs.

<p>(A–E) FACS-sorted naive CD4⁺CD62L^hiCD44^loCD25⁻CXCR3⁻Gr-1⁻ LCMV-TCR^tg Th cells were activated with APCs and GP_61–80 peptide in the presence of the indicated cytokines and cytokine-blocking antibodies. (A) T-bet and GATA-3 expression is shown in naive cells (top row) or on d 5 of culture (middle row). Geometric mean indices are indicated. Cytokine expression upon PMA/ionomycin restimulation is shown on d 5 of culture (lower row). Numbers indicate frequencies. Data are representative of five independent experiments. (B) Geometric mean indices ± SD of T-bet and GATA-3 stainings versus isotype control stainings on d 5 of culture are depicted as normalization to Th1 cells for T-bet or to Th2 cells for GATA-3. Data are pooled from five independent experiments. (C) Cytokine expression was analyzed after PMA/ionomycin restimulation on d 5 of culture. Mean frequencies ± SD of cytokine⁺ cells are shown. Data are pooled from five independent experiments. (D) Expression of the indicated cytokine and chemokine receptors (upper row) and transcription factors (lower row) was analyzed on d 5 of culture. Tfh cells were obtained from spleens of WT C57BL/6 mice and identified as CD4⁺CXCR5⁺PD-1⁺ cells. Numbers indicate geometric means. Data are representative of two independent experiments. (E) On d 2 of culture, levels of phosphorylated (upper row) and total (lower row) STAT proteins were determined. Numbers indicate geometric mean indices. Data are representative of two independent experiments. (F) Naive Th cells from WT Balb/c mice were cultured with anti-CD3/anti-CD28 in the presence of polarizing cytokines and cyokine-blocking antibodies to induce Th1, Th2, and hybrid Th1/2 cells. T-bet and GATA-3 expression was analyzed in a kinetic fashion and plotted as geometric mean indices. Data are representative of three independent experiments.