Figure S2.

Identification of Th2 Trm cells via flow cytometry. C57BL/6 mice were sensitized and challenged with i.n. HDM and then rested for 6–12 wk followed by lung tissue harvest. (A) Representative flow cytometry on lung cells in HDM-memory mice to identify Th2 cells (anti-CD45 i.v. unlabeled, FoxP3⁻GATA3⁺CD4⁺ T cells). (B) Quantitation of lung Th2 cells (anti-CD45 i.v. unlabeled, FoxP3⁻GATA3⁺CD4⁺ T cells) in naive and HDM-memory mice via flow cytometry. (C) Representative histogram demonstrating CD69 staining (and fluorescence minus one control staining) of lung Th2 cells from HDM-memory mice (left panel) with quantitation of percent CD69⁺ cells from lung Th2 cells and FoxP3⁻GATA3⁻CD4⁺ T cell populations (right panel). (D) C57BL/6 mice were sensitized and challenged with i.n. A. alternata, rested for 6–8 wk, and left untreated or rechallenged with a single dose of i.n. A. alternata. Quantitation of lung Th2 cells (anti-CD45 i.v. unlabeled, FoxP3⁻GATA3⁺CD4⁺ T cells) in indicated groups. Representative data show individual mice with mean ± SEM from one of two independent experiments with three or four mice per group. For statistical analysis, a two-tailed t test was performed for parametric data, and a two-tailed Mann-Whitney U test was performed for nonparametric data. *, P < 0.05; ****, P < 0.0001. FSC-A, forward scatter area; FSC-H, forward scatter height; SSC-A, side scatter area.