Analyses of in vitro polarized Th2 cells. Supporting data for Figs. 3, 4, and 5. (A) WT and Pik3cd^E1020K/+ naïve CD4 T cells were Th2-polarized in the presence or absence of an αIFNγ blocking antibody. Left: Percentages of IFNγ⁺ cells from the indicated groups. Right: Fold Tbet expression (MFI normalized to control WT) in live CD4⁺ cells from the indicated groups. n = 6–8 for each group, from six to eight independent experiments. (B) Gene expression (RPKM) of Il4ra, Il12rb1, and Il12rb2 in WT and Pik3cd^E1020K/+ Th2-polarized cells. n = 3, bulk RNAseq. (C) Naïve CD4 T cells from the indicated mice were Th2-polarized for 24 h in the absence of inhibitors. At 24 h of culture, PI3Kδ inhibitor Cal101 (10 nM) or mTOR inhibitor rapamycin (200 nM) was added to polarizing media and cells were cultured for an additional 48 h. Representative flow cytometry plots showing IFNγ and IL-4 staining in live CD4⁺ cells. Data are representative of three independent experiments, n = 3. (D) GSEA comparing WT and Pik3cd^E1020K/+ transcriptomes for expression of TFT gene sets. (E) Fold induction of Foxo1 expression in Th2-polarized live CD4⁺ cells, measured by flow cytometry and calculated by normalizing Foxo1 MFIs to the 0 time point of the corresponding genotype. n = 5 for each group, from five independent experiments. (F) Naïve CD4 T cells from the indicated mice were Th2-polarized in the presence or absence of αIFNγ blocking antibody, and pFoxo1(S256) was measured in live CD4⁺ cells from the indicated groups by flow cytometry. Fold induction was calculated by normalizing pFoxo1(S256) MFIs to WT control cells. n = 5 for each group, from five independent experiments. (G) Naïve CD4 T cells were nucleofected with Cas9-gRNA complexes containing NC or Foxo1-targeting gRNAs and underwent Th2 polarization. Representative flow cytometry histogram showing Foxo1 expression from the indicated cells, one example from nine independent experiments. (H) Naïve CD4 T cells from WT and Pik3cd^E1020K/+ mice were transduced with GFP only–, Foxo1-WT– or Foxo1-AAA–encoding retroviruses under Th2-polarizing conditions. Top: Representative flow cytometry plots showing IFNγ and IL-4 expression in live GFP⁺ CD4⁺ T cells cultured under Th2-polarizing conditions from the indicated groups. Bottom, percentages of IL-4⁺ (left), IL-13⁺ (middle), and IFNγ⁺ (right) Th2-polarized liveCD4 GFP⁺ T cells from the indicated groups. n = 6 for each group, from six independent experiments. (I) WT Naïve CD4 T cells were nucleofected with NC or Foxo1-targeting gRNA-Cas9 complexes and polarized under Th2 conditions in the presence or absence of an IL-2 blocking antibody. Representative flow cytometry plots showing IFNγ and IL-4 expression in live CD4⁺ cells. Data are representative of two independent experiments, n = 5. (J) Comparison of transcriptomes from Foxo1 KO Treg cells and Pik3cd^E1020K/+ Th2 cells. Genes upregulated (FC >1.5, P < 0.05) in Foxo1 KO Treg³⁶ (versus WT Treg) and Pik3cd^E1020K/+ Th2 (versus WT Th2) were compared (left), and pathway analysis (Enrichr; Xie et al., 2021) was performed on common DEGs (right). (K–M) Supplemental data related to Fig. 5, I and J, transfer of Foxo1 gRNA-treated cells. (K) Cell counts of lung CD4 T cells (liveCD45⁺TCRβ⁺CD4⁺CD8⁻) from the indicated groups. (L) Frequencies of IL-4⁺ lung CD4 T cells from the indicated groups. (M) Cell counts of lung eosinophils (liveCD45⁺CD3⁻NK1.1⁻CD19⁻CD11b⁺Ly6G⁻SiglecF⁺) from the indicated groups. n = 6–9 for each group, pooled from two independent experiments. Statistical comparisons were made using ratio paired t tests (A, B, E, F, and H) or unpaired t tests (K–M). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NC, negative control.