Figure 4.
Dysregulated IL-2 signaling rewires Th2 differentiation of Pik3cdE1020K/+CD4 T cells. (A) Left: Representative flow cytometry plots showing IL-2 and CD4 expression in Th2-polarized cells from the indicated mice. Right: Percentages of IL-2+ Th2 cells. n = 9 for each group, from nine independent experiments. (B, C, and E) Time course analysis (0, 24, 48, 72 h) of IL-2 production, and pSTAT5(Y694) and CD25 during Th2 polarization, measured by flow cytometry. n = 7–10 for each group, from 7–10 independent experiments. (B) Percentages of IL-2+ cells over time from the indicated mice. (C) Fold induction of pSTAT5(Y694) over time from the indicated mice. Fold induction was calculated using pSTAT5(Y694) MFIs normalized to the 0 time point of the corresponding genotype. (D) Schematic describing experiments shown in F and G. (E) Fold induction of CD25 expression from the indicated mice. Fold induction was calculated using CD25 MFIs normalized to the 0 time point of the corresponding genotype. (F and G) Th2-polarized CD4 T cells from WT and Pik3cdE1020K/+ were rested in serum-free media for 4 h and subsequently stimulated with hIL-2 over the indicated time course (0, 15, 60, 120 min). n = 4–5 for each group, from four to five independent experiments. (F) Fold induction of pSTAT5(Y694) over time from the indicated mice, measured by flow cytometry. (G) Fold induction of pS6(S240/44) over time from the indicated mice, measured by flow cytometry. Fold induction was calculated using MFIs (pSTAT5(Y694) or pS6(S240/44)) normalized to the 0 time point of the corresponding genotype. (H–J) Naïve CD4 T cells were Th2-polarized in the presence or absence of αIL-2 blocking antibody (20 μg/ml). (H) Representative flow cytometry plots showing IFNγ and IL-4 staining in Th2-polarized live CD4+ cells. (I) Percentages of IL-4+ (left), IL-13+ (middle), and IFNγ+ (right) cells from the indicated mice, in the presence or absence of αIL-2. n = 11 for each group, from 11 independent experiments. (J) Left: Representative flow cytometry plots showing pAKT(T308) in Th2-polarized live CD4+ cells in the presence or absence of αIL-2. Right: Fold induction of pAKT(T308) in Th2-polarized live CD4+ cells from the indicated groups. Fold induction was calculated by normalizing pAKT(T308) MFIs to WT control cells. n = 5 for each group, from five independent experiments. Statistical comparisons were made using ratio paired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Dysregulated IL-2 signaling rewires Th2 differentiation of Pik3cd E1020K/+ CD4 T cells. (A) Left: Representative flow cytometry plots showing IL-2 and CD4 expression in Th2-polarized cells from the indicated mice. Right: Percentages of IL-2+ Th2 cells. n = 9 for each group, from nine independent experiments. (B, C, and E) Time course analysis (0, 24, 48, 72 h) of IL-2 production, and pSTAT5(Y694) and CD25 during Th2 polarization, measured by flow cytometry. n = 7–10 for each group, from 7–10 independent experiments. (B) Percentages of IL-2+ cells over time from the indicated mice. (C) Fold induction of pSTAT5(Y694) over time from the indicated mice. Fold induction was calculated using pSTAT5(Y694) MFIs normalized to the 0 time point of the corresponding genotype. (D) Schematic describing experiments shown in F and G. (E) Fold induction of CD25 expression from the indicated mice. Fold induction was calculated using CD25 MFIs normalized to the 0 time point of the corresponding genotype. (F and G) Th2-polarized CD4 T cells from WT and Pik3cdE1020K/+ were rested in serum-free media for 4 h and subsequently stimulated with hIL-2 over the indicated time course (0, 15, 60, 120 min). n = 4–5 for each group, from four to five independent experiments. (F) Fold induction of pSTAT5(Y694) over time from the indicated mice, measured by flow cytometry. (G) Fold induction of pS6(S240/44) over time from the indicated mice, measured by flow cytometry. Fold induction was calculated using MFIs (pSTAT5(Y694) or pS6(S240/44)) normalized to the 0 time point of the corresponding genotype. (H–J) Naïve CD4 T cells were Th2-polarized in the presence or absence of αIL-2 blocking antibody (20 μg/ml). (H) Representative flow cytometry plots showing IFNγ and IL-4 staining in Th2-polarized live CD4+ cells. (I) Percentages of IL-4+ (left), IL-13+ (middle), and IFNγ+ (right) cells from the indicated mice, in the presence or absence of αIL-2. n = 11 for each group, from 11 independent experiments. (J) Left: Representative flow cytometry plots showing pAKT(T308) in Th2-polarized live CD4+ cells in the presence or absence of αIL-2. Right: Fold induction of pAKT(T308) in Th2-polarized live CD4+ cells from the indicated groups. Fold induction was calculated by normalizing pAKT(T308) MFIs to WT control cells. n = 5 for each group, from five independent experiments. Statistical comparisons were made using ratio paired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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