Figure 2: Type I IFNs directly activate Eomes gene expression in CD8⁺ T cells.

(a) Purified CD8⁺ T cells were incubated in medium alone or stimulated with rIFNβ (100 U ml⁻¹) and/or rIL-4 (20 ng ml⁻¹). After 6 h of stimulation, Eomes mRNA levels were quantified and normalized against β-actin mRNA levels. Histograms represent mean±s.e.m. of triplicates (representative of three independent experiments). Eomes expression (mean fluorescence intensity (MFI)) in total or the indicated subpopulation of CD8⁺ T cells was assessed by flow cytometry after 16 h of stimulation. (b) Splenocytes from WT, IRF9^−/− or STAT1^−/− mice were cultured for 16 h with or without rIFNβ (100 U ml⁻¹) in presence of rIL-2 (100 U ml⁻¹). Eomes expression in total CD8⁺ T cells was assessed by flow cytometry. (c) Naive CD44⁻CD8⁺ T cells and CD44⁺CD62L⁺ memory CD8⁺ T cells were sorted and cultured for the indicated time with or without rIFNβ (100 U ml⁻¹). Eomes mRNA levels were normalized against β-actin mRNA levels (experiment performed in triplicates). (d) WT or IRF9^−/− Thy1.2⁺ CD8⁺ T cells were cultured with WT Thy1.1⁺ splenocytes for 16 h with or without rIFNβ (100 U ml⁻¹). Eomes expression in WT or IRF9^−/− Thy1.2⁺ CD8⁺ T cells is shown. Mean±s.e.m. of two independent experiments is shown. (e) WT or IRF9^−/− Thy1.2⁺ CD8⁺ T cells were adoptively transferred i.v. into WT Thy1.1⁺ mice 24 h before PBS or polyI:C (200 μg per mouse) injection (intraperitoneally). Mice were killed 16 h post injection. Eomes expression within the Thy1.2⁺ CD8⁺ T-cell population was analysed by flow cytometry. Histograms represent mean±s.e.m. (five mice per group). (f) Total CD8⁺ T cells from WT and IRF9^−/− mice were stimulated for 4 h with IFNβ or medium alone. H3K4me3 and H3K9Ac modifications in the proximal promoter region of the Eomes or Gapdh loci were quantified by quantitative (q)PCR after chromatin immunoprecipitation (ChIP) experiments (normalization against input controls). Values are expressed as mean±s.e.m. of duplicates and are representative of two independent experiments. (g) Total CD8⁺ T cells from WT mice were incubated with rIFNβ or medium alone for the indicated time. STAT1 recruitment to the proximal region or to a control region located +14.6 kb downstream of the transcriptional start site of the Eomes gene was quantified by qPCR after ChIP experiments (normalization against input controls). Values represent mean±s.e.m. of three independent experiments. *P<0.05 (non-parametric Mann–Whitney).