Inhibition of Ikaros activity results in defective anergy induction and il2 promoter deacetylation in Th1 cells. (A) Th1 cells were transfected with the pSUPER-IK-19i vector (Ik-siRNA) or empty pSUPER (Control) and pGFP. After 36 hours, GFP⁺ sorted cells were treated with or without ionomycin (Iono) and stimulated with anti-CD3 and anti-CD28. IL-2 production was determined by EILSA. Bars show mean + SEM of 3 different experiments. Following ionomycin treatment, Ikaros levels from both cell populations were measure by Western blot. Blots were also probed with antiactin antibody to control loading. (B) ChIP assays were performed on Th1 cells transduced with virus expressing GFP or Ikaros-6 and GFP (Ik6) and sorted for GFP expression. Cells were rested or treated with ionomycin (Io). Samples precipitated with an antiacetylated H4 antibody were amplified with primers for the il2 promoter. Products are shown for precipitated complexes and inputs. One experiment of 2 is shown. Numbers below bands represent relative intensity (to RV-GFP–infected cells). Precipitated complexes were also amplified using primers for the CD3ϵ promoter. Anergy induction in sorted cells was also analyzed by ELISA following stimulation with anti-CD3 and anti-CD28. Bars show mean + SEM of the anergy index value (ratio of IL-2 produced by control cells and anergized cells) of 2 different experiments performed in triplicate.