(A) Murine naive CD4⁺ T cells were cultured under Th17-polarizing conditions at indicated concentrations of IL-6 and infected with retroviruses of an LMP-IRES-NGFR vector of mTAp63KD or a nonsilencing vector. Representative flow cytometry profiles and the frequency of Foxp3- or RORγt-expressing cells in infected cells are shown. Data are reported as mean ± SEM. n = 3–4 experiments. *P < 0.05 by unpaired t test. (B) Murine naive CD4⁺ T cells were cultured under iTreg-polarizing conditions and infected with retroviruses of LMP-nonsilencing-IRES-NGFR or LMP-mTAp63KD-IRES-NGFR. Representative flow cytometry profiles of annexin V versus DAPI and the frequency of annexin V⁺ cells among DAPI⁻ cells at day 3 are shown (left panels). Data are compiled from 3 independent experiments (n = 3 each) and analyzed by unpaired t test. (C) Naive CD45.2⁺CD4⁺ T cells from Foxp3^YFP-Cre mice were cultured under iTreg-polarizing conditions and infected with retroviruses of LMP-mTAp63KD-IRES-NGFR (TAp63KD) or LMP-nonsilencing-IRES-NGFR (NS). Infected NGFR⁺ Foxp3^YFP+ cells (iTreg) were sorted and co-cultured with CellTrace Violet–labeled naive CD45.1⁺CD4⁺ T cells (responder) at the indicated ratio. Representative histograms of CellTrace Violet and mean ± SEM of division index of responder cells are shown (left panels). Representative flow cytometry profiles of Foxp3 versus CD45.2 and the frequency of Foxp3⁺ cells among CD45.2⁺ cells at day 2 are shown (right panels). Data are compiled from 3 independent experiments (n = 3 each). *P < 0.05, unpaired t test. (D) iTreg cells were prepared as described in C, and DNA methylation status for the Foxp3 CNS2 region was determined by the bisulfite sequencing analysis. Data are shown as a lollipop methylation diagram. The black circle indicates a 100% ratio of methylation of CpGs.