HBZ induced Foxp3 expression in naive T cells through Smad3. (A) HBZ modulated the expression of selected TGF-β target genes. (Left panel) Mouse naive T cells were transduced with pMXs-IG vector encoding sHBZ or empty vector. Forty-eight hours after viral infection, total RNA was extracted from sorted green fluorescent protein-positive cells. The level of Pdgfb, Sox4, Cdkn1a, Cdkn2b, Ctgf, Foxp3, Runx1, Myc, Tsc22d1, Id2, β-actin, and HBZ mRNA was analyzed by semiquantitative RT-PCR. (Right panel) Schema of the effect of TGF-β and sHBZ on TGF-β target gene transcription. ↑ indicates up-regulation; ↓, down-regulation; and -, no effect. (B) CTLL-2/pME18Sneo and CTLL-2/sHBZ, cells were plated in 96-well plates. Cells were treated with increasing concentrations of TGF-β for 72 hours. Proliferation of each cell was examined by methyl thiazolyl tetrazolium assay. Expression of HBZ was detected by RT-PCR (bottom panel). (C) SB431542, an inhibitor of the TGF-β receptor, could not inhibit the induction of Foxp3 by HBZ. Mouse CD4⁺CD25⁻ T cells were transduced with pGCDNsamI/N vector encoding sHBZ, or with empty vector. Three days after TGF-β (0.2 ng/mL) and SB431542 (5μM) treatment, cells were stained with anti-Foxp3 in addition to anti-NGFR and then analyzed by flow cytometry. Numbers indicate the percentage of Foxp3-positive cells among NGFR-positive cells. (D) HBZ induced FoxP3 in human naive T cells. Human CD4⁺CD25⁻ T cells were transfected with lentiviral vectors expressing sHBZ, or with empty vector. Two days after stimulating with TGF-β (0.1 ng/mL), cells were stained with antibodies for CD4, NGFR, and Foxp3 and then analyzed by flow cytometry. (E) SIS3 inhibited the HBZ-induced Foxp3 induction. Mouse CD4⁺CD25⁻ T cells were transduced with pGCDNsamI/N vector encoding sHBZ or empty vector. Fifteen hours after viral infection, SIS3 (5μM) and TGF-β (1 ng/mL) were added. Thirty-six hours after treatment, Foxp3 expression was detected by flow cytometry. Numbers indicate the percentage of Foxp3-positive cells among NGFR-positive cells. Representative data from 3 independent experiments are shown. (F) HBZ activated transcription of the Foxp3 promoter through its Smad site of enhancer. EL4 cells were transfected with the Foxp3 reporter plasmid or its mutants with or without the sHBZ-expressing plasmid (pcDNA3.1-mycHis-sHBZ). Luciferase activity was measured 48 hours after stimulation by TGF-β. Expression of sHBZ was detected by Western blot. CBB staining was shown as the loading control. (G) HBZ formed complex with Smad3/p300 in FoxP3 enhancer. MT-2 cells treated with 5 ng/mL of TGF-β for 2 hours, and chromatin immunoprecipitated by each indicated antibody. The precipitated DNAs and 1% of the input cell lysates were amplified by the specific primers for FoxP3 enhancer.