Figure 6. IFN-γ/STAT1-induced IRF1 suppresses IL-9 production in human Th9 cells.

Naive CD4⁺CD25⁻CD45RA⁺CD45RO⁻ T cells were isolated from human PBMCs and cultured under Th0 or Th9 conditions in the presence or absence of human IFN-γ as indicated. (a) Intracellular staining for IL-9 and IFN-γ after 3 days of culture (bars to the right give mean±s.d. of three independent experiments). (b) Expression of IL9 and IFNG mRNA on day 2 in Th0 and Th9 cells; results were normalized to 18S and are presented relative to Th0 cells. (c) ELISA of IL-9 in supernatants of Th0 and Th9 cultures after 3 days. (d) Phospho-flow for p-STAT1 in Th9 cells with or without IFN-γ treatment, day 2 of culture (values show mean fluoresence intensity). (e) Intracellular staining for IRF1 and IRF4, day 2 of culture (bars to the right give mean±s.d. of three independent experiments). (f–h) Human naive CD4⁺CD25⁻CD45RA⁺CD45RO⁻ T cells were activated under Th0 condition for 16 h, then transfected with scrambled (scr) or IRF1-specific siRNA (siIRF1) and then cultured for further 72 h under Th9 conditions with/without IFN-γ. (f) Intracellular staining of IRF1 in scr- or siIRF1-transfected cells 24 h post transfection upon culture under Th9 conditions with IFN-γ. (g) Expression of IL9 mRNA in transfected cells at 48 h post transfection; results were normalized to 18S and are presented relative to Th9 cells treated with scr siRNA and IFN-γ. (h) ELISA of IL-9 in supernatants of transfected cells 72 h post transfection. (a–h) Data are representative of three independent experiments with different donors; *P<0.05, **P<0.005, ***P<0.001 (two-tailed Student's t-test).