Figure 7.
Fas-FasL signaling potentiates T cell activation, exacerbating CD4+T cell dysregulation in the presence of activated PI3Kδ. (A) Fold surface FasL expression (MFI normalized to WT) on Th2-polarized live CD4+ T cells, measured by flow cytometry (gated on live CD4+). n = 8 for each group, from eight independent experiments. (B) Fold surface FasL expression (MFI normalized to NC) on NC and Foxo1 gRNA-targeted naïve CD4 T cells cultured under Th2-polarizing conditions. n = 4 for each group, from four independent experiments. (C) Left: Th2 polarized cells (live CD4+) from WT and Pik3cdE1020K/+ animals were gated as FasLlow, FasLmid, and FasLhigh. Right: Linear regressions comparing FasL MFIs in each gate with percentages of IFNγ+ cells from the indicated groups. Correlation R2 and P values are indicated. n = 7 for each group, from seven independent experiments. (D–F) Naïve CD4 T cells from WT and Pik3cdE1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC, or Fas- or Fasl-targeting gRNAs and polarized under Th2 conditions. n = 8–10 for each group, from 8 to 10 independent experiments. (D) Representative flow cytometry plots showing IFNγ and IL-4 expression in live CD4+ T cells. (E) Percentages of IFNγ+ (left), IL-4+ (middle), and IL-13+ (right) in Th2-polarized live CD4+ T cells. (F) Fold induction of pAKT(T308) (left), pFoxo1(S256) (middle), and pS6(S240/44) (right) in Th2-polarized live CD4 T cells, measured by flow cytometry. For all readouts, fold induction was calculated by normalizing MFIs to NC WT cells. (G and H) NC, Fas, and Fasl gRNA-treated naïve CD4 T cells underwent Th2 polarization in the presence or absence of recombinant multimeric FasL (FasL-LZ), and phosphorylation of AKT(T308), Foxo1(S256), and S6(S240/44) was analyzed by flow cytometry. n = 6 for each group, from six independent experiments. (G) Representative flow cytometry histograms showing pS6(S240/44) staining in Th2-polarized live CD4+ T cells. Dashed lines represent cells cultured without FasL-LZ; solid lines show cells cultured with FasL-LZ. (H) Fold induction of pAKT(T308), pFoxo1(S256), and pS6(S240/44) in Th2-polarized (–/+ FasL-LZ) live CD4+ T cells, measured by flow cytometry. For all readouts, fold induction was calculated by normalizing MFIs to NC WT cells. Statistical comparisons were made using ratio paired t tests, unless otherwise indicated. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NC, negative control.

Fas-FasL signaling potentiates T cell activation, exacerbating CD4 + T cell dysregulation in the presence of activated PI3Kδ. (A) Fold surface FasL expression (MFI normalized to WT) on Th2-polarized live CD4+ T cells, measured by flow cytometry (gated on live CD4+). n = 8 for each group, from eight independent experiments. (B) Fold surface FasL expression (MFI normalized to NC) on NC and Foxo1 gRNA-targeted naïve CD4 T cells cultured under Th2-polarizing conditions. n = 4 for each group, from four independent experiments. (C) Left: Th2 polarized cells (live CD4+) from WT and Pik3cdE1020K/+ animals were gated as FasLlow, FasLmid, and FasLhigh. Right: Linear regressions comparing FasL MFIs in each gate with percentages of IFNγ+ cells from the indicated groups. Correlation R2 and P values are indicated. n = 7 for each group, from seven independent experiments. (D–F) Naïve CD4 T cells from WT and Pik3cdE1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC, or Fas- or Fasl-targeting gRNAs and polarized under Th2 conditions. n = 8–10 for each group, from 8 to 10 independent experiments. (D) Representative flow cytometry plots showing IFNγ and IL-4 expression in live CD4+ T cells. (E) Percentages of IFNγ+ (left), IL-4+ (middle), and IL-13+ (right) in Th2-polarized live CD4+ T cells. (F) Fold induction of pAKT(T308) (left), pFoxo1(S256) (middle), and pS6(S240/44) (right) in Th2-polarized live CD4 T cells, measured by flow cytometry. For all readouts, fold induction was calculated by normalizing MFIs to NC WT cells. (G and H) NC, Fas, and Fasl gRNA-treated naïve CD4 T cells underwent Th2 polarization in the presence or absence of recombinant multimeric FasL (FasL-LZ), and phosphorylation of AKT(T308), Foxo1(S256), and S6(S240/44) was analyzed by flow cytometry. n = 6 for each group, from six independent experiments. (G) Representative flow cytometry histograms showing pS6(S240/44) staining in Th2-polarized live CD4+ T cells. Dashed lines represent cells cultured without FasL-LZ; solid lines show cells cultured with FasL-LZ. (H) Fold induction of pAKT(T308), pFoxo1(S256), and pS6(S240/44) in Th2-polarized (–/+ FasL-LZ) live CD4+ T cells, measured by flow cytometry. For all readouts, fold induction was calculated by normalizing MFIs to NC WT cells. Statistical comparisons were made using ratio paired t tests, unless otherwise indicated. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NC, negative control.

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