Figure 7. Dll4/Notch activated iT_reg cells retain Foxp3 expression and are resistant to Th17 skewing in vitro.

A. Splenic naïve CD4 T cells from DNMAML or wild type (wt) B6 mice were skewed toward iT_reg for 48 hours. Il17a expression in iT_reg culture were measured.

B. Foxp3 expression in iT_reg culture were measured by realtime PCR after 48 hours of skewing.

C. IL-17A secretion from iT_reg were measured by luminax system after 48 hours of skewing

D. Naïve CD4 T cells from wild type B6 mice were skewed toward iT_reg with or without Dll4 stimulation for 72 hours, then rested in 10 ng/mL of IL-2 for another 72 hours. At day 6, 5 × 10⁵ of viable cells were re-stimulated in Th17 conditions. After 3 days, IL-17A in the supernatant was accessed

E. Percentage of RORγt⁺ in viable CD4 T cells after Th17 skewing

F. Percentage of Foxp3⁺ in viable iT_reg or Dll4-exposed iT_reg after Th17 skewing

G. Expression level of Foxp3 in viable Foxp3⁺CD4 T cells in iT_reg or Dll4-exposed iT_reg culture after Th17 skewing

H. Naïve CD4 T cells from wild type B6 mice were skewed toward iT_reg with or without Dll4 stimulation for 72 hours, then rested in 10 ng/mL of IL-2 for another 72 hours. 5 × 10⁵ of viable Foxp3-eGFP⁺ cells were sorted out and re-stimulated in Th17 conditions at day 6. After 3 days, the percentage of RORγt⁺Foxp3⁺ was determined

I. Percentage of RORγt⁻Foxp3⁺ after Th17 re-stimulation on Foxp3-eGFP⁺ cells