Fig. 6.

Domain-deleted forms of Hhex lose their effects in Treg cells. (A) Schematic diagram of Hhex domains showing the NT1 (N-terminal 1), NT2 (N-terminal 2), HD (homeodomain), and CT (C-terminal) domains. (B–E) Retroviral vectors encoding WT Hhex or deletion mutants of Hhex without each domain (ΔNT1, ΔNT2, ΔHD, or ΔCT) were transduced into iTreg cells. Foxp3⁺ cells were measured by flow cytometry (B), and the ratio of Foxp3⁺ cells among GFP⁺ cells is shown (C). Error bars represent the SD, and P values were calculated using Student’s t tests. **P < 0.01, ****P < 0.0001. GFP⁺ cells were sorted, and the relative amount of Foxp3 protein was measured by immunoblot analysis (D). mRNA levels of Foxp3 and other Treg signature genes were measured by qRT-PCR (E). (F) Binding of Hhex and Foxp3 was determined by co-IP. Hhex-FLAG and Foxp3 were overexpressed in 293T cells. Cell lysates were immunoprecipitated with anti-Foxp3, anti-FLAG (for Hhex), or normal IgG (as a negative control). Then, the proteins were immunoblotted with anti-Hhex or anti-Foxp3 antibodies. (G) WT Hhex-FLAG, ΔNT1-FLAG, ΔNT2-FLAG, ΔHD-FLAG, or ΔCT-FLAG was cotransfected along with Foxp3 into 293T cells. Cell lysates were immunoprecipitated with an anti-FLAG antibody. The immunoprecipitated proteins were detected with an anti-Foxp3 antibody. Input cell lysates were immunoblotted with anti-Foxp3 and anti-FLAG antibodies. (H and I) Transactivation of the promoters of Il2ra and Ctla4 by Foxp3 and Hhex was measured by transient reporter assay. EL4 cells were transfected with CD25P- or Ctla4P–LUC reporter constructs together with a Foxp3 expression vector and a vector expressing full-length or domain-deleted forms of Hhex (dNT1, dNT2, dHD, or dCT). Promoter activities are shown as the fold change (FC) relative to ctrl vector-transduced cells. Error bars represent the SD, and P values were calculated using Student’s t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant.