Generation of a mouse model that recapitulates key aspects of human AD-HIES. Depiction of mouse Stat3 gene within RP24-236G5 BAC. Exons 15 and 16, which encode part of STAT3 DNA binding domain, are highlighted. Based on mutations observed in HIES patients, the BAC was modified to delete valine 463 from the binding domain (from PVVVI in WT to PVVI in mutant) (A). Throughout the text, the Stat3-ΔV463 allele is referred to as mut-Stat3. The intron separating exons 15 and 16 was also deleted to permit estimation of transgene copy number (relative to WT) by Southern blotting after tail DNA digestion with PstI, as shown in panel (B). Transgenic line L1, which contains 2 transgene copies, was used in all subsequent experiments. Western blot analysis of STAT3 and Y705 phosphorylated STAT3 levels from CD4⁺ T cells unstimulated or stimulated with 100 ng/mL IL-21 for 5 to 15 minutes. Cells were isolated from 6- to 8-week-old WT and mut-Stat3 mice, expanded in IL-2 and rested for 2 hours prior to stimulation. Data are representative of 3 independent experiments (C). Flow cytometry analysis of Y705 phosphorylated STAT3 levels in freshly isolated splenic B cells left unstimulated (shaded) or stimulated with 50 ng/mL IL-6, IL-10, or IL-21 for 30 minutes (unshaded). Cells were isolated from 6- to 12-week-old WT (solid line) or mut-Stat3 (dotted-line) mice. Data are representative of 6 mice in 2 independent experiments (D). CD4⁺ T cells were prepared as in (C) and stimulated for 15 minutes with 100 ng/mL IL-21. STAT3 binding to a candidate oligonucleotide was assayed using the TransAm STAT3 Assay Kit (Active Motif) according to the manufacturer’s instructions (E). Flow cytometry analysis of IL-17A expression in CD4⁺ T cells stimulated with αCD3/αCD28, TGF-β-1, and IL-6 for 70 hours. Cells were isolated from 6-week-old WT and mut-Stat3 mice. Data are representative of 8 independent experiments (F).