Stable T-bet+GATA-3+ Th1/Th2 Hybrid Cells Arise In Vivo, Can Develop Directly from Naive Precursors, and Limit Immunopathologic Inflammation
Figure 1
Hybrid Th1/2 cells develop in vivo in response to parasite infections.
(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 ex vivo without restimulation. Numbers indicate frequencies. Data are representative of three H. polygyrus and three S. mansoni experiments with n = 3–4 mice each. (B) WT C57BL/6 mice were infected with H. polygyrus. 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 (n = 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 H. polygyrus 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 H. polygyrus. 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 (n = 6 mice). Numbers indicate frequencies. Data are representative of two independent experiments. (E) Geometric mean indices ± SD of GATA-3 protein staining are shown (n = 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 (n = 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.