Regulatory T cell population in LTAC mice.

<p>(A) Flow cytometry analysis with thymocytes from 6-week-old (w.o.) or 11 w.o. WT and LTAC mice. Staining for CD25 and Foxp3 determined Treg in CD4 single positive cells. The plots are representative of three independent experiments. (B) Foxp3 expression in TCRβ^<b>+</b>CD4^<b>+</b> cells in tissues indicated. The histograms are representative of at least five independent experiments. (C and D) Frequencies (left) and absolute numbers (right) of Foxp3^<b>+</b> cells of TCRβ^<b>+</b>CD4^<b>+</b> cells from the mLNs (C) and cLP (D) of WT (n = 5–8, filled circle) and LTAC mice (n = 5–8, open circle), determined by flow cytometry analysis from (B). Horizontal bars represent mean. (E) Genomic DNAs from the sorted cells indicated, followed by PCR amplification to detect flox or Δ allele in TAK1 genomic locus like <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128761#pone.0128761.g002" target="_blank">Fig 2C</a>. (F) CFSE-labeled CD45.1^<b>+</b>CD4^<b>+</b>CD25^<b>–</b> T cells (Tresp) were cultured with soluble anti-CD3 antibody and T cell-depleted splenocytes in the absence (Tresp alone) or presence of CD4^<b>+</b>CD25^<b>+</b> regulatory T cells (Treg) purified from wild-type or LTAC mice for 3 days (The ratio of Tresp to Treg is 2:1.). The histograms are representative of three independent experiments. In (C) and (D), unpaired <i>t</i> tests were performed. Statistical significance was indicated by **<i>P</i> < 0.01, ***<i>P</i> < 0.001; ns, not significant.