Figure 1. TGFβ1 regulates the expression of SMYD3 in inducible Treg cells.

(A) Naïve T cells were cultured under TH0 or skewed towards iTregs. After 48 hours the levels of methyltransferases were each assessed by PCR arrays. Data are representative of 3 independent experiments. (B) SMYD3 expression was assessed by q-RT-PCR in naïve T cells skewed under iTreg conditions over 120 hours. At 48 hours some wells had TGFβ1 withdrawn from the culture, mRNA extracted, and gene expression of SMYD3 assessed. (C) Bar graph shows expression of H3K4me3 in iTreg cells normalized with TH0 and histogram show H3K4me3 expression in gated CD4+CD25+T cells in iTreg and TH0 cells at 3 days of differentiation. (D) Naïve CD4⁺T cells from Foxp3^egfp mice were skewed towards iTreg cells. Converted CD4⁺CD25⁺Foxp3^egfp+ iTregs and CD4⁺CD25⁺Foxp3^egfp− effector cells were FACS sorted from 5-day cultures and mRNA was extracted with Foxp3 and SMYD3 expression was then analyzed by q-RT-PCR. (E) Naïve CD4⁺T cells and iTreg cells were stained with anti-CD4 and anti-SMYD3 and evaluated by confocal microscopy. (F) Naïve CD4⁺T cells were isolated from mouse spleens and human PBMCs, activated under TH0 or iTreg skewing condition for 5 days, mRNA extracted, and gene expression of Foxp3 and SMYD3 assessed. Data are means ± SD of triplicate samples and are representative of at least two independent experiments. *P<0.05, **P<0.01, ***P<0.001.