Figure 1. NAD⁺ promotes conversion of CD4⁺ CD25⁺ Foxp3⁺ Tregs into IL-17A producing cells.

(a) CD4⁺ CD25⁺ Tregs were FACS-sorted from EGFP-Foxp3 mice and cultured in presence of α-CD3, α-CD28, and IL-2 with increasing concentrations of NAD⁺ for 96h. (b) Frequencies of CD4⁺ CD25^HighFoxp3^EGFP and CD4⁺ CD25^LowFoxp3^EGFP T cells were assessed in a dose-dependent manner. (c) CD4⁺ CD25⁺ Tregs from EGFP-Foxp3 mice were isolated and frequencies of CD4⁺ Foxp3⁺ IL-17A⁺ cells were assessed in a dose-dependent manner for 96h (n = 12 per group, representative plots shown). (d) CD4⁺ CD25⁺ Tregs from wild type mice were isolated and frequencies of CD4⁺ Foxp3⁺ IL-17A⁺ cells were assessed in a dose and time-dependent manner (n = 12 per group, representative plots shown). Data derived from three independent experiments. Data represent mean ± s.d. *P < 0.05; **P < 0.01. Student’s t-test and ANOVA tests were used accordingly to compare groups.