Hybrid Th1/2 cells develop <i>in vivo</i> in response to parasite infections.

<p>(A) WT C57BL/6 or Balb/c mice were infected or immunized/challenged with the indicated pathogens. T-bet and GATA-3 expression by CD4⁺ T cells from indicated organs was analyzed <i>ex vivo</i> without restimulation. Numbers indicate frequencies. Data are representative of three <i>H. polygyrus</i> and three <i>S. mansoni</i> experiments with <i>n</i> = 3–4 mice each. (B) WT C57BL/6 mice were infected with <i>H. polygyrus</i>. T-bet and GATA-3 expression in CD4⁺ splenocytes was analyzed in a kinetic fashion. Mean frequencies ± SD of T-bet⁻GATA-3⁺ (Th2) and T-bet⁺GATA-3⁺ (Th1/2) cells are shown (<i>n</i> = 3 mice/time point). Inserted dot plot shows a typical staining on d 6. Data are representative of two independent experiments. (C) Spleen and mLN cells from WT C57BL/6 mice infected with <i>H. polygyrus</i> were restimulated with adult worm lysate at the peak of infection on d 21. Antigen-specific CD4⁺ T cells as identified by CD40L expression were analyzed for transcription factor and cytokine expression. Numbers indicate frequencies. Data are representative of three independent experiments. (D–F) WT C57BL/6 mice were infected with <i>H. polygyrus</i>. CD4⁺ splenocytes were analyzed at the peak of infection on d 21. Th2 cells were gated as CD4⁺T-bet⁻GATA-3⁺ and Th1/2 cells as CD4⁺T-bet⁺GATA-3⁺ as shown in (B). (D) Cytokine expression was analyzed upon PMA/ionomycin restimulation (<i>n</i> = 6 mice). Numbers indicate frequencies. Data are representative of two independent experiments. (E) Geometric mean indices ± SD of GATA-3 protein staining are shown (<i>n</i> = 3 mice). Data are representative of three independent experiments. (F) Spleen cells were restimulated as in (D). Geometric mean indices ± SD of cytokine expression calculated as the geometric mean of cytokine⁺ cells divided by the geometric mean of cytokine⁻ cells are shown (<i>n</i> = 6 mice). Data are pooled from two independent experiments. (G) Expression of CXCR3 and T1/ST2 by CD4⁺ T cells was analyzed on day 14 of infection. Th2 cells were gated as CD4⁺T-bet⁻GATA-3⁺ and Th1/2 cells as CD4⁺T-bet⁺GATA-3⁺ as shown in (B). As a Th1 reference, the T-bet⁺GATA-3⁻ population was gated as in the lower right quadrant of the inset in (B). Numbers indicate geometric means. Data are representative of two independent experiments.