Figure 7. In vitro–generated Tregs using periweaning colonic APCs rescues Th2 skewing and oral tolerance in desynchronized pups.

(A) Naive splenic T cells (RFP^–GFP^–) isolated from Foxp3^RFPRORγt^GFP mice were cultured for 7 days with MHCII⁺CD11c⁺ cells isolated from colon, SI, spleen, or MLN of DOL21 mice and analyzed by flow cytometry for Foxp3 and RORγt expression. (B–D) A total of 5 × 10⁴ Foxp3⁺ cells generated by culture with DOL21 colon LP-APC or by culture with DOL21 splenic APCs was transferred into DOL56 cross-fostered mice. (B) Serum IL-13 concentrations measured weekly after transfer. (C) Increase in footpad thickness following dietary Ova, Ova immunization, and footpad challenge in mice treated as in B. (D) Absolute number of Foxp3⁺ or IL13⁺ Ova-specific OTII T cells 7 days following transfer into mice treated as in B and given dietary Ova. Data are presented as the mean ± SEM, *P < 0.05. A is representative of 1 of 3 independent experiments with n = 3 replicate culture wells, B–D is representative of 2 independent experiments with 4 or more mice in each group, and offspring from 3 litters per condition were used in A–D. Significance was calculated using 1-way ANOVA with a Dunnett’s post hoc test in A, C, and D and 2-way ANOVA with a Dunnett’s post hoc test in B.