(A) Schematic depicting genetic targeting of the endogenous Areg locus via homologous recombination with the P2A-Thy1.1-STOP-Neomycin knockin construct, with subsequent crossing to the FLPeR mouse to remove the neomycin cassette and create the final Areg^Thy1.1 allele. Inset: Depiction of Areg transcription/translation in the Areg^Thy1.1 mouse; for each molecule of Areg mRNA translated, a single Thy1.1 mRNA is also translated (as a separate protein, due to the P2A site) and traffics separately to the surface of the cell, where it can be targeted by fluorescently conjugated antibodies. (B) Mouse splenocytes from Areg^Thy1.1 mice underwent a short-term stimulation protocol (PMA/ionomycin, 3 hours), then were stained for endogenous AREG (see Methods) and Thy1.1 (live staining). Representative plots for stimulated Tregs from AREG^– and AREG⁺ populations shown (including IgG control for the AREG⁺ population); plots for unstimulated Tregs not shown. Percentage staining shown on plots. Gating strategy shown in Supplemental Figure 2. n = 5 per group, all values included from 2 experiments. (C) Schematics depicting the models of lung damage used in this study, including general time course and disease characteristics, and Treg increases/Areg production status. dpi, days post-instillation. (D) Thy1.1 staining by flow cytometry on live lung Tregs from Foxp3^GFP Areg^Thy1.1 mice during the IAV or bleomycin (bleo.) models of lung damage or from control (saline-treated) lungs. Dpi for each model indicated in figure. Representative plots shown, including fluorescence-minus-one (FMO) control (from IAV 8 dpi staining). Gating strategy shown in Supplemental Figure 3. n = 3–5 per group, all values included from 2 experiments. Mean ± SEM displayed on graphs. Statistical analysis was done using Bonferroni’s multiple comparisons test. **: 0.001 < P < 0.01, ***: 0.0001 < P < 0.001, ****: P < 0.0001.