Colons were harvested, rinsed with PBS, and colonic patches were removed. Isolation of splenic and LP cellular populations was performed as previously described (28). Colonic Treg subpopulations were identified as 7AAD-, CD45+, CD3+, CD4+, Foxp3+, and RORγt+. Foxp3 was identified using GFP signal. To detect intracellular antigens (RORγt, cMAF, GATA3, and Tbet) and cytokines (IL13), cells were fixed and permeabilized overnight and stained per the manufacturer’s recommendations (eBioscience). Flow cytometry was performed with a FACScan cytometer (BD Biosciences, San Jose, CA) retrofitted with additional lasers, or an Attune NXT four-laser flow cytometer (Invitrogen). Data acquisition and analysis were performed using Attune NXT software and FlowJo software (Tree Star, Ashland, OR). viSNE analysis was performed with FlowJo software.

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