E2A promotes IL-10 expression in activated CD8⁺ T cells. (A) Activated Il10^gfp/+ CD8⁺ T cells were transduced with control-Cherry, E12-Cherry, or E47-Cherry retroviruses. Two days later, Cherry⁺ cells were purified by fluorescence-activated cell sorting and cells were analyzed. IL-10-GFP expression was analyzed 3 days later. Data are representative of 2 independent experiments. (B-C) Wild-type and Id2^fl/flLck^Cre+ OT-I cells were transduced with shRNA-tcfe2a-GFP and shRNA-control-GFP retroviruses. GFP⁺ cells (95% positive) were restimulated for 4 to 5 hours with SIINFEKL peptide and then analyzed for intracellular IFN-γ and IL-10 expression. (C) Frequency of IL-10⁺ cells among OT-I CD8⁺ T cells transduced with Tcfe2a shRNA normalized to the frequency of IL-10⁺ cells among OT-I CD8⁺ T cells transduced with control shRNA. Data are pooled from 3 independent experiments. Statistically significant differences of the test group to 1 were determined using an unpaired 2-tailed Student t test; *P < .05, **P < .01. (D) E2A and IRF4 binding sites within the CNS-9 are boxed; * indicates nucleotides conserved between humans and mice. (E) NIH-3T3 were transfected with firefly luciferase (Luc) reporter construct containing the CNS-9 enhancer with the Il10 minimal promoter together with empty vector, vectors encoding either IRF4 or E47, or with both vectors. Transfection efficiency was controlled by cotransfection of a plasmid constitutively expressing the Renilla luciferase. Results show the ratio of the firefly luciferase to the Renilla luciferase luminescence normalized to the ratio in cells transfected with CNS-9/minimal promoter construct alone. Data show the mean ± SEM pooled from 3 independent experiments. Statistically significant differences of the test group to 1 were determined using an unpaired 2-tailed Student t test; ***P < .001. (F) Ratio of Il10 mRNA expression evaluated by quantitative real-time PCR in wild-type or Irf4^−/− OT-I T cells transduced with E12-Cherry vs control-Cherry retroviruses cultured for 2 days in the presence of IL-2 plus IL-21. Data are pooled from 3 independent experiments. (G) Il10 mRNA expression was measured by quantitative real-time PCR and normalized to Hprt expression in wild-type or Irf4^−/− OT-I CD8⁺ T cells activated with OVA peptide and cultured for 2 days in the presence of IL-2 or IL-2 plus IL-21. Data are pooled from 3 independent experiments. (F-G) Statistical differences were determined using an unpaired 2-tailed Student t test; *P < .05, **P < .001.