Supplementary Material for: Induction of Regulatory T Cells as a Novel Mechanism Underlying the Therapeutic Action of Kakkonto, a Traditional Japanese Herbal Medicine, in a Murine Food Allergy Model

<b><i>Background:</i></b> The number of patients with food allergy (FA) has dramatically increased. Although satisfactory drug therapies for FA are not available, we have found that kakkonto, a traditional Japanese herbal medicine, suppressed the occurrence of allergic symptoms in an FA mouse model. Thus, we investigated whether kakkonto could regulate the activation and differentiation of T cells in the colon. <b><i>Methods:</i></b> BALB/c mice were systemically sensitized and then orally challenged with ovalbumin. FA mice were orally treated with kakkonto. Lamina propria (LP) cells from their colons were isolated and analyzed. <b><i>Results:</i></b> Kakkonto significantly reduced the proportion of CD69<sup>+</sup> cells and the elevated helper T cell type 2-specific transcription factor GATA-3 mRNA expression in the LP CD4<sup>+</sup> T cells, showing that kakkonto has a suppressive effect on the activation and Th2 differentiation of LP effector CD4<sup>+</sup> T cells of the FA mouse colon. Furthermore, kakkonto significantly increased the proportion of Foxp3<sup>+</sup>CD4<sup>+</sup> regulatory T cells in the LP CD4<sup>+</sup> T cells of the FA mouse colon. Similarly, the number of Foxp3-positive cells was dramatically increased in the colonic mucosa of kakkonto-administered FA mice. However, the pharmacological effect and Foxp3<sup>+</sup>CD4<sup>+</sup> regulatory T cell-inducing ability of kakkonto were not attenuated by the administration of an anti-CD25 monoclonal antibody in the FA model. <b><i>Conclusions:</i></b> The induction of Foxp3<sup>+</sup>CD4<sup>+</sup>CD25<sup>-</sup> regulatory T cells in the colon as a novel mechanism underlying the therapeutic action of kakkonto could be utilized for the development of a novel anti-FA drug.