Figure 6: Aiolos binds to the IL10 genomic region in TNFi-exposed IL-17+ CD4+ T cells and drives IL10 expression in primary CD4+ effector T cells.

(a) Analysis of sequence conservation in genomic IL10. Pair-wise sequence alignment to the human genome is depicted (50% lower cut-off), with evolutionarily conserved regions identified where identity above 70% over 100 bp was scored. These conserved genomic regions were searched for conserved consensus Aiolos DNA-binding motifs, shown at the top of the graph and numbered from 1–5. (b) Multiple sequence alignment of putative Aiolos motifs 3, 4 and 5. The consensus binding site is shown in bold and highlighted in blue. (c) ChIP-PCR of sorted TNFi-exposed IL-17+ CD4+ T cells shows Aiolos enrichment at motifs 3, 4 and 5. Analysed DNA motifs are demonstrated on the horizontal axes and numbered according to Fig. 6a. PROM indicates the previously found Ikaros-binding sites in the promoter of mouse IL10. The values were normalized to two negative control regions (titin exons) to find enrichment of Aiolos binding at a region of interest over background signal (fold enrichment is shown±s.e.m.). (d) Memory CD4+ T cells were co-transfected with a CMV-eGFP reporter vector and either CMV-IKZF3 or CMV-empty vector (EV). At 48 h eGFP+ cells were sorted and IKZF3 and IL10 expression determined by real-time PCR (data normalized to SDHA, mean±s.e.m., n=3).