Figure 2.

Induced Foxp3⁺ Tregs were not necessary for ET to abrogate colitis. (A) Schematic demonstrating the design of the experiments: ET, Epicutaneous immunotherapy. RAG1^−/− mice were injected with colitogenic T cells (CD4⁺CD45RB^hi) from wild-type mice. Once mice exhibited symptoms (weight loss, loose stool, or blood in the stool) of colitis at week 3 or 4, mice were injected with OVA TCR enriched T cells from Foxp3-DTR mice that were immunized with IP OVA with alum followed by gavage feeding with OVA with cholera toxin (CT). After this injection, mice were exposed on the skin (+ET) with Viaskin containing OVA or vehicle alone (–ET) weekly for 3 weeks. Mice were then injected or not with DT to deplete any induced Foxp3⁺ T cells. All mice then received an oral dose of OVA given by gavage. (B) Depletion of Foxp3⁺ T cells after administration of DT. (C) The percentage of initial body weight of mice with colitis induced via the CD4⁺CD45RB^hi transfer with the addition of OVA TCR enriched T cells from Foxp3-DTR mice without or with DT (+DT) and exposed to OVA-Viaskin (+ET) or not (control). (D) The final percentage of initial body weight as measured when sacrificing them. (E) Colon length of the mice after sacrificing them. (F) Histological score of colonic tissue as determined by a pathologist blinded to the treatment group. Representative H&E sections of colon at 40× magnification that demonstrate both control groups (control and control+DT) with areas of complete loss of mucosa (ulceration) with replacement by abundant inflammatory cells that also infiltrate the submucosa and muscularis and both treated groups (+ET and +ET+DT) with less inflammation and preservation of normal architecture including an intact mucsoca. The total histological score of the representative section of control, control+DT, +ET, and +ET+DT were 17, 17, 10, and 11, respectively. (G) Cytokine production by cultured colon samples (3 pooled experiments of 4–5 mice/group; *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant).