Id2 deficiency does not alter the expression of key IL-10 transcriptional activators or the epigenetic status of the Il10 locus. (A) Quantitative analysis of mRNA expression by real-time PCR within wild-type D^bNP₃₆₆⁺ (KLRG1⁻) or Id2^fl/flLck^Cre+ CD8⁺ T cells from PR8-primed HKx31-infected mice (day 9) and naïve splenic CD44⁻CD62L⁺CD8⁺ T cells. Data show the mean ± SEM of gene expression relative to Hprt from 3 independent samples. (B) In vitro activation of wild-type and Id2^fl/flLck^Cre+ CD8⁺ T cells (day 2 cultures) with IL-21 (50 ng/mL) or IL-12 (5 ng/mL) prior to intracellular staining for pSTAT3 and pSTAT4. Histograms show representative staining after 15 min (solid blue line) or 30 min (solid red line; unstimulated cells, gray shading). Bar graphs show the mean ± SEM of pSTAT3 or pSTAT4 mean fluorescence intensity (MFI) pooled from 3 independent experiments. (C) Quantitative analysis of Il10 mRNA expression by real-time PCR within in vitro–activated wild-type or Id2^fl/flLck^Cre+ OT-I CD8⁺ T cells cultured 4 days in presence of IL-2 alone or IL-2 and IL-21. Data show the mean ± SEM of gene expression relative to Hprt from 3 independent samples. (D) CNSs in the Il10 loci of mouse and human are shown. The mouse genomic sequence is used as the base sequence on the x-axis. Red represents intergenic regions with a minimal length of 100 bp and at least 70% of conservation between human and mouse as shown in the ECR Browser. The CNS-9 and CNS-3 regions are indicated. (E) ChIP analysis of the Il10 locus from in vitro–activated wild-type or Id2^fl/flLck^Cre+ OT-I CD8⁺ T cells cultured as in (C) with antiserum specific for acetylation of histone H3 at Lys27 (H3K27ac) or trimethylation of histone H3 at Lys27 (H3K27me3). Data show 1 of 2 independent experiments with similar results.