Mouse il4

T_reg progenitors were isolated as previously described ^{2 (link)}. Briefly, Foxp3-GFP thymi were dissected, dissociated, and pooled CD4SP cells were enriched by magnetically depleting with biotinylated anti-CD8 and Ter119 antibodies (eBioscience) followed by secondary labeling with streptavidin-conjugated microbeads (Miltenyi Biotec). Enriched CD4SP cells were stained with fluorchrome-conjugated anti-CD4, CD25, CD73 and streptavidin prior to sorting CD4⁺CD73⁻CD25⁺GFP⁻, CD4⁺CD73⁻CD25⁻GFP⁺, or CD4⁺CD73⁻CD25⁺GFP⁺ cells using a BD Facs Aria sorter (BD Biosciences). Purified T_reg cell progenitors or T_reg cells were incubated in complete RPMI and supplemented with human IL2 from R&D Systems (Minneapolis, MN) or mouse IL4 from Tonbo Biosciences (San Diego, CA). After 72 hours, cells were harvested, stained with anti-CD4, CD8 and CD25 antibodies and analyzed by flow cytometry for the percentage or number of cells that express CD25 and GFP after incubation.