Licorice extract and its fractions promote Treg cell induction in vitro.
(a) Naive CD4+ T cells were stimulated with immobilized anti-CD3, soluble anti-CD28 monoclonal antibodies and TGFβ (1 μg/ml) under the indicated concentrations of licorice extract and analyzed by FACS. (b) Naive CD4+ T cell were stimulated as in (a) under the indicated licorice fractions and analyzed by FACS. (c) Analysis of Foxp3 protein expression of a per-cell basis in Treg cells generated in the presence of licorice extract or Gly1 fraction treatment. Data are shown as mean fluorescence intensity (MFI). (d,e) CD4+CD25+ Treg cells untreated or treated with Gly1 were incubated with CFSE labelled CD4+CD25− conventional T cells in an in vitro suppression assay. The suppression was assayed by FACS analysis for dilution of CFSE in gated conventional T cells. Results are expressed as means ± SEM and are representative of more than three experiments. *P < 0.05, **P < 0.01 and ***P < 0.001, as determined by one-way ANOVA followed by Bonferroni’s test.