Figure 3

NAD⁺ promotes Treg conversion into Th17 cells through the transcription factors STAT3 and RORγt.

CD4⁺ CD25⁺ Tregs were cultured in presence of α-CD3, α-CD28 and IL-2 with increasing concentrations of NAD⁺; by (a) 24 hrs or (b) 96 hrs of culture mRNA levels of Tbx21 (Tbet), GATA3, STAT3, STAT5, RORγt and Foxp3 were measured by real-time PCR (n = 6 per group), NAD⁺ was added at increasingly concentreations of 0, 5 and 50 μM. (c) CD4⁺ CD25⁺ Tregs isolated from STAT3^−/− mice were cultured for 96 hrs in the presence of α-CD3, α-CD28 and IL-2 with or without NAD⁺ 250 μM ; frequencies of CD4⁺ IL-17A⁺ Foxp3⁺ cells and IL-17A protein levels were determined by flow cytometry and ELISA (n = 6 per group). Data derived from two independent experiments. Data represent mean ± s.d. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant. ANOVA tests were used to compare groups.