Figure 5.
Inactivation of Foxo1 in Pik3cdE1020K/+CD4+T cells impairs Th2 lineage restriction. (A) Pathway enrichment of TF perturbations followed by expression gene sets performed using Enrichr (Xie et al., 2021): significantly enriched gene sets colored in blue. Genes upregulated in HDM-treated Pik3cdE1020K/+ CD4 T cells relative to WT counterparts (Fig. 2 A) were used as input for pathway enrichment. (B) Time course (0, 24, 48, 72 h) of pFoxo1(S256) in Th2-polarized live CD4+ cells from indicated mice. Left: Representative flow cytometry plots. Right: Fold induction of pFoxo1(S256) over time. Fold induction was calculated by normalizing pFoxo1(S256) MFIs to the 0 time point of the corresponding genotype. n = 8 for each group, from eight independent experiments. (C) GSEA comparing Th2-polarized WT and Pik3cdE1020K/+ transcriptomes for the expression of Foxo1-activated (left) and Foxo1-repressed (right) gene sets. (D) Gene expression heatmap (row z-score) showing normalized RPKM values of leading edge genes from Foxo1-activated and Foxo1-repressed GSEA described in Fig. 4 C. (E) Naïve CD4+ T cells from the indicated mice were Th2-polarized in the presence or absence of αIL-2 blocking antibody. Left: Representative flow cytometry plots showing pFoxo1(S256). Right: Fold induction of pFoxo1(S256). Fold induction was calculated by normalizing pFoxo1(S256) MFIs to WT control cells. n = 10 for each group, from 10 independent experiments. (F) GSEA comparing control and αIL-2–treated Th2-polarized CD4 T cell transcriptomes for the expression of Foxo1-activated (top) and Foxo1-repressed (bottom) gene sets in the indicated groups. (G and H) Naïve CD4 T cells were nucleofected with gRNA-Cas9 complexes containing NC or Foxo1-targeting gRNAs and differentiated under Th2 conditions. n = 9 for each group, from nine independent experiments. (G) Left: Representative flow cytometry plots showing IFNγ and IL-4 expression in live CD4+ T cells. Right: Percentages of IFNγ+ and IL-4+ cells in Th2-polarized cells. (H) Left: Representative flow cytometry plots showing IL-2 and CD4 expression in live CD4+ T cells. Right: Percentages of IL-2+ cells in Th2-polarized cells from the indicated groups. (I and J) TCRα-deficient recipient mice were injected with 1 × 106 NC or Foxo1 gRNA-Cas9–nucleofected naïve CD4 T cells 14 days prior to HDM sensitization. n = 6–9 for each group, pooled from two independent experiments. (I) Experimental design. (J) Frequencies of IFNγ+ (left) and IL-2+ (right) lung CD4 T cells. Statistical comparisons used ratio paired t tests (B and E–G) or unpaired t tests (I). **P < 0.01, ***P < 0.001, ****P < 0.0001. NC, negative control.

Inactivation of Foxo1 in Pik3cd E1020K/+ CD4 + T cells impairs Th2 lineage restriction. (A) Pathway enrichment of TF perturbations followed by expression gene sets performed using Enrichr (Xie et al., 2021): significantly enriched gene sets colored in blue. Genes upregulated in HDM-treated Pik3cdE1020K/+ CD4 T cells relative to WT counterparts (Fig. 2 A) were used as input for pathway enrichment. (B) Time course (0, 24, 48, 72 h) of pFoxo1(S256) in Th2-polarized live CD4+ cells from indicated mice. Left: Representative flow cytometry plots. Right: Fold induction of pFoxo1(S256) over time. Fold induction was calculated by normalizing pFoxo1(S256) MFIs to the 0 time point of the corresponding genotype. n = 8 for each group, from eight independent experiments. (C) GSEA comparing Th2-polarized WT and Pik3cdE1020K/+ transcriptomes for the expression of Foxo1-activated (left) and Foxo1-repressed (right) gene sets. (D) Gene expression heatmap (row z-score) showing normalized RPKM values of leading edge genes from Foxo1-activated and Foxo1-repressed GSEA described in Fig. 4 C. (E) Naïve CD4+ T cells from the indicated mice were Th2-polarized in the presence or absence of αIL-2 blocking antibody. Left: Representative flow cytometry plots showing pFoxo1(S256). Right: Fold induction of pFoxo1(S256). Fold induction was calculated by normalizing pFoxo1(S256) MFIs to WT control cells. n = 10 for each group, from 10 independent experiments. (F) GSEA comparing control and αIL-2–treated Th2-polarized CD4 T cell transcriptomes for the expression of Foxo1-activated (top) and Foxo1-repressed (bottom) gene sets in the indicated groups. (G and H) Naïve CD4 T cells were nucleofected with gRNA-Cas9 complexes containing NC or Foxo1-targeting gRNAs and differentiated under Th2 conditions. n = 9 for each group, from nine independent experiments. (G) Left: Representative flow cytometry plots showing IFNγ and IL-4 expression in live CD4+ T cells. Right: Percentages of IFNγ+ and IL-4+ cells in Th2-polarized cells. (H) Left: Representative flow cytometry plots showing IL-2 and CD4 expression in live CD4+ T cells. Right: Percentages of IL-2+ cells in Th2-polarized cells from the indicated groups. (I and J) TCRα-deficient recipient mice were injected with 1 × 106 NC or Foxo1 gRNA-Cas9–nucleofected naïve CD4 T cells 14 days prior to HDM sensitization. n = 6–9 for each group, pooled from two independent experiments. (I) Experimental design. (J) Frequencies of IFNγ+ (left) and IL-2+ (right) lung CD4 T cells. Statistical comparisons used ratio paired t tests (B and E–G) or unpaired t tests (I). **P < 0.01, ***P < 0.001, ****P < 0.0001. NC, negative control.

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