Figure 3.
Hyperactivated PI3Kδ disrupts Th2 lineage restriction. (A–E) Naïve CD4 T cells were activated with αCD3 + αCD28 in the presence of WT T-depleted APCs under Th2-polarizing conditions (IL-4 + αIL-12) for 72 h. (A) Left: Representative flow cytometry plots showing IL-4 and IL-13 staining in Th2-polarized live CD4+ cells. Right: Percentages of IL-4+ and IL-13+ cells from the indicated mice. n = 15 for each group, from 15 independent experiments. (B) Left: Representative flow cytometry histograms showing GATA3 expression in Th2-polarized live CD4+ cells from the indicated mice. Right: Fold GATA3 expression (MFI normalized to WT) in Th2-polarized live CD4+ cells from the indicated mice. n = 14 for each group, from 14 independent experiments. (C) Left: Representative flow cytometry plots showing IFNγ and IL-4 staining in Th2-polarized live CD4+ cells. Right: Percentages of IFNγ+ cells. (D) Left: Representative flow cytometry histograms showing Tbet expression in Th2 and WT control Th1-polarized live CD4+ cells. Right: Fold Tbet expression (MFI normalized to WT) in Th2-polarized live CD4+ cells from the indicated mice. (C and D)n = 14 for each group, from 14 independent experiments. (E) Percentages of IFNγ+ (left) and IL-13+ (right) cells over a time course of Th2 differentiation (0, 24, 48, and 72 h). n = 10 for each group, from 10 independent experiments. (F) Bulk RNAseq of WT and Pik3cdE1020K/+ naïve CD4 T cells and in vitro polarized Th2 cells. n = 3 biological replicates for each group. Volcano plots showing DEGs in red (WT upregulated) and blue (Pik3cdE1020K/+ upregulated); DEGs defined using fold change >1.5, P < 0.05. (G) Enrichment of hallmark pathways among DEGs comparing WT and Pik3cdE1020K/+ Th2 cells. (H) Time course of fold induction of pAKT(T308), pS6(S240/44), and pAKT(S473) in WT and Pik3cdE1020K/+ live CD4+ cells during Th2 differentiation (0, 24, 48, and 72 h), measured by flow cytometry. Fold induction calculated using MFIs of the indicated readouts normalized to the 0 time point of the corresponding genotype. n = 5–9 for each group, from five to nine independent experiments. Statistical comparisons were made using ratio paired t tests. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Hyperactivated PI3Kδ disrupts Th2 lineage restriction. (A–E) Naïve CD4 T cells were activated with αCD3 + αCD28 in the presence of WT T-depleted APCs under Th2-polarizing conditions (IL-4 + αIL-12) for 72 h. (A) Left: Representative flow cytometry plots showing IL-4 and IL-13 staining in Th2-polarized live CD4+ cells. Right: Percentages of IL-4+ and IL-13+ cells from the indicated mice. n = 15 for each group, from 15 independent experiments. (B) Left: Representative flow cytometry histograms showing GATA3 expression in Th2-polarized live CD4+ cells from the indicated mice. Right: Fold GATA3 expression (MFI normalized to WT) in Th2-polarized live CD4+ cells from the indicated mice. n = 14 for each group, from 14 independent experiments. (C) Left: Representative flow cytometry plots showing IFNγ and IL-4 staining in Th2-polarized live CD4+ cells. Right: Percentages of IFNγ+ cells. (D) Left: Representative flow cytometry histograms showing Tbet expression in Th2 and WT control Th1-polarized live CD4+ cells. Right: Fold Tbet expression (MFI normalized to WT) in Th2-polarized live CD4+ cells from the indicated mice. (C and D)n = 14 for each group, from 14 independent experiments. (E) Percentages of IFNγ+ (left) and IL-13+ (right) cells over a time course of Th2 differentiation (0, 24, 48, and 72 h). n = 10 for each group, from 10 independent experiments. (F) Bulk RNAseq of WT and Pik3cdE1020K/+ naïve CD4 T cells and in vitro polarized Th2 cells. n = 3 biological replicates for each group. Volcano plots showing DEGs in red (WT upregulated) and blue (Pik3cdE1020K/+ upregulated); DEGs defined using fold change >1.5, P < 0.05. (G) Enrichment of hallmark pathways among DEGs comparing WT and Pik3cdE1020K/+ Th2 cells. (H) Time course of fold induction of pAKT(T308), pS6(S240/44), and pAKT(S473) in WT and Pik3cdE1020K/+ live CD4+ cells during Th2 differentiation (0, 24, 48, and 72 h), measured by flow cytometry. Fold induction calculated using MFIs of the indicated readouts normalized to the 0 time point of the corresponding genotype. n = 5–9 for each group, from five to nine independent experiments. Statistical comparisons were made using ratio paired t tests. **P < 0.01, ***P < 0.001, ****P < 0.0001.

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