Figure 1. SCFA produced by commensal bacteria stimulate in vitro generation of Treg cells.

a) Effect of fecal extracts from SPF, antibiotic-treated (AVNM), or germ-free (GF) mice on in vitro induction of Foxp3 expression in naïve CD4⁺ T cells stimulated with CD3 antibody in the presence of Flt3L-elicited DC and TGF-β. Foxp3 expression was assessed by flow cytometric analysis on day 4 of culture. Naïve CD25⁺CD62L^hiCD44^loCD4⁺ T cells were FACS-purified from B6 mice. Fecal extracts were prepared in 70% ethanol. Data are shown as fold induction over corresponding dilution of vehicle and are representative of 2 independent experiments.

b) HPLC fractionation of 2-nitrophenylhydrazine-HCl derivatized SCFA present in indicated fecal extracts. Red and yellow arrows indicate peaks corresponding to propionate and butyrate, respectively. Internal standard peak is indicated with a star. The HPLC fractionation profile of fecal extracts pooled from three animals each is representative of two independent experiments.

c) Effect of indicated purified SCFA on in vitro induction of Foxp3 expression in naïve CD4⁺ T cells isolated from B6 or Foxp3^GFP mice as described in (a). Data are representative of 3 independent experiments.

d) Effect of butyrate on Foxp3 induction in CNS1-sufficient and -deficient naïve CD4⁺ T cells from Foxp3^GFP and Foxp3^ΔCNS1 as described in (a). Data are representative of at least two independent experiments.