Fig. 5.

Reduced inhibitory function and elevated apoptosis of iTreg cells differentiated from Met-treated naïve CD4⁺ CD25⁻ cells. (A) CD4⁺ CD25⁺ cells induced as in Fig. 4C (day 5) were enriched in the iTreg cell population using CD25⁺ magnet beads. iTreg cells were incubated with conventional naïve CD4⁺ CD25⁻ T cells (Tconv) labeled with CFSE at the indicated ratios in the presence of anti-CD3 mAb and rhIL-2 for 3 days for the Treg suppression assay. iTreg cells that differentiated from naïve CD4 T cells pretreated with Met exhibited less of an inhibitory effect on Tconv cell division. (B) iTreg cells (day 3 and day 5 cultures) were induced from CTV-labeled naïve CD4⁺ CD25⁻ cells of Foxp3^Cre-GFP mice. The GFP (Foxp3)-positive population was characterized as iTreg cells undergoing cell division (gate a) and those without division (gate b). Only the Met-pretreated iTreg population expanded by IL-2 treatment (day 5, gate a, bottom panel) was positive for annexin V binding, i.e., undergoing apoptosis.