Figure S5.
Fas-FasL signaling potentiates T cell activation. Supporting data for Figs. 7, 8, and 9. (A) Naïve CD4+ T cells from the indicated mice were Th2-polarized in the presence or absence of αIL-2 blocking antibody, and surface FasL expression was measured by flow cytometry. Left: Representative flow cytometry histograms of surface FasL. Right: Fold FasL expression (MFI normalized to WT control) on Th2-polarized cells from the indicated groups. n = 5 for each group, from five independent experiments. (B and C) Supplemental data for Fig. 7 C. (B) Frequencies of FasLlow, FasLmid, and FasLhigh live CD4 T cells from the indicated groups. n = 7 for each group, from seven independent experiments. (C) Frequencies of IFNγ+ cells in the indicated FasL expression category from the indicated mice. n = 7 for each group, from seven independent experiments. (D) Frequencies of dead Th2-polarized cells (gated as total CD4+) from the indicated groups. Left: Frequencies of dead cells within FasLlow, FasLmid, and FasLhigh. Right: Frequencies of dead Th2 cells among total CD4+ cells. n = 7 for each group, from seven independent experiments. (E) Linear regression analyses comparing FasL MFIs with percentages of dead cells from the indicated groups. Correlation R2 and P values are indicated. (F and G) Naïve CD4 T cells from WT and Pik3cdE1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC, or Fas- or Fasl-targeting gRNAs and underwent Th2 polarization. (F) Representative flow cytometry histograms showing surface Fas expression in the indicated groups. (G) Representative flow cytometry histograms showing surface FasL expression in the indicated groups. (H) Representative flow cytometry histograms showing pAKT(T308), pFoxo1(S256), and pS6(S240/44) staining in NC, Fas, and Fasl gRNA-targeted Th2-polarized live CD4+ T cells from the indicated mice. Supporting data for Fig. 7 F. n = 8–10 for each group, from 8 to 10 independent experiments. (I) Fold induction of p-p65(S529) in Th2-polarized live CD4 T cells from the indicated groups, measured by flow cytometry. For all readouts, fold induction was calculated by normalizing MFIs to NC WT cells. n = 7 for each group, from seven independent experiments. (J) WT (CD45.1+CD45.2+) and Pik3cdE1020K/+ (CD45.1−CD45.2+) naïve CD4 T cells were Th2-polarized either alone (top) or cocultured (bottom) at a 1:1 ratio. Representative flow cytometry plots showing IFNγ and IL-4 expression in live CD4+ cells. Data are representative of six independent experiments. (K) Supporting data for Fig. 8, A–C. Representative flow cytometry histograms showing FADD staining in NC and Fadd gRNA-Cas9–treated cells from the indicated groups. (L and M) Naïve CD4+ T cells from WT and Pik3cdE1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fas-targeting gRNAs and underwent αCD3/CD28 stimulation in the presence of hIL-2 for 72 h. Cells were subsequently rested in serum-free media, underwent αCD3 (1 μg/ml) crosslinking over a time course (0, 1, 2, 5, 15, 60 min), and were analyzed by flow cytometry. n = 6–7 for each group, from six to seven independent experiments. (L) Fold induction of pAKT(T308) and (M) pERK(T202/04) over time for the indicated groups. Fold induction was calculated using MFIs normalized to the 0 time point of the corresponding sample. Right: AUC quantification of pAKT(T308) and pERK(T202/04) time courses for the indicated groups. (N and O) Naïve CD4+ T cells from WT and Pik3cdE1020K/+ mice underwent αCD3/CD28 stimulation in the presence of hIL-2 for 72 h. Cells were subsequently rested in serum-free media, underwent αCD3 (1 μg/ml) crosslinking in the presence or absence of FasL-LZ over a time course (0, 1, 2, 5, 15, 60 min), and were analyzed by flow cytometry. n = 6–7 for each group, from six to seven independent experiments. (N) Fold induction of pAKT(T308) and (O) pERK(T202/04) over time for the indicated groups. Fold induction was calculated using MFIs normalized to the 0 time point of the corresponding sample. Right: AUC quantification of pAKT(T308) and pERK(T202/04) time courses for the indicated groups. Statistical comparisons were made using ratio paired t tests, unless otherwise indicated. *P < 0.05, **P < 0.01, ***P < 0.001. AUC, area under the curve; NC, negative control.

Fas-FasL signaling potentiates T cell activation. Supporting data for Figs. 7, 8, and 9. (A) Naïve CD4+ T cells from the indicated mice were Th2-polarized in the presence or absence of αIL-2 blocking antibody, and surface FasL expression was measured by flow cytometry. Left: Representative flow cytometry histograms of surface FasL. Right: Fold FasL expression (MFI normalized to WT control) on Th2-polarized cells from the indicated groups. n = 5 for each group, from five independent experiments. (B and C) Supplemental data for Fig. 7 C. (B) Frequencies of FasLlow, FasLmid, and FasLhigh live CD4 T cells from the indicated groups. n = 7 for each group, from seven independent experiments. (C) Frequencies of IFNγ+ cells in the indicated FasL expression category from the indicated mice. n = 7 for each group, from seven independent experiments. (D) Frequencies of dead Th2-polarized cells (gated as total CD4+) from the indicated groups. Left: Frequencies of dead cells within FasLlow, FasLmid, and FasLhigh. Right: Frequencies of dead Th2 cells among total CD4+ cells. n = 7 for each group, from seven independent experiments. (E) Linear regression analyses comparing FasL MFIs with percentages of dead cells from the indicated groups. Correlation R2 and P values are indicated. (F and G) Naïve CD4 T cells from WT and Pik3cdE1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC, or Fas- or Fasl-targeting gRNAs and underwent Th2 polarization. (F) Representative flow cytometry histograms showing surface Fas expression in the indicated groups. (G) Representative flow cytometry histograms showing surface FasL expression in the indicated groups. (H) Representative flow cytometry histograms showing pAKT(T308), pFoxo1(S256), and pS6(S240/44) staining in NC, Fas, and Fasl gRNA-targeted Th2-polarized live CD4+ T cells from the indicated mice. Supporting data for Fig. 7 F. n = 8–10 for each group, from 8 to 10 independent experiments. (I) Fold induction of p-p65(S529) in Th2-polarized live CD4 T cells from the indicated groups, measured by flow cytometry. For all readouts, fold induction was calculated by normalizing MFIs to NC WT cells. n = 7 for each group, from seven independent experiments. (J) WT (CD45.1+CD45.2+) and Pik3cdE1020K/+ (CD45.1−CD45.2+) naïve CD4 T cells were Th2-polarized either alone (top) or cocultured (bottom) at a 1:1 ratio. Representative flow cytometry plots showing IFNγ and IL-4 expression in live CD4+ cells. Data are representative of six independent experiments. (K) Supporting data for Fig. 8, A–C. Representative flow cytometry histograms showing FADD staining in NC and Fadd gRNA-Cas9–treated cells from the indicated groups. (L and M) Naïve CD4+ T cells from WT and Pik3cdE1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fas-targeting gRNAs and underwent αCD3/CD28 stimulation in the presence of hIL-2 for 72 h. Cells were subsequently rested in serum-free media, underwent αCD3 (1 μg/ml) crosslinking over a time course (0, 1, 2, 5, 15, 60 min), and were analyzed by flow cytometry. n = 6–7 for each group, from six to seven independent experiments. (L) Fold induction of pAKT(T308) and (M) pERK(T202/04) over time for the indicated groups. Fold induction was calculated using MFIs normalized to the 0 time point of the corresponding sample. Right: AUC quantification of pAKT(T308) and pERK(T202/04) time courses for the indicated groups. (N and O) Naïve CD4+ T cells from WT and Pik3cdE1020K/+ mice underwent αCD3/CD28 stimulation in the presence of hIL-2 for 72 h. Cells were subsequently rested in serum-free media, underwent αCD3 (1 μg/ml) crosslinking in the presence or absence of FasL-LZ over a time course (0, 1, 2, 5, 15, 60 min), and were analyzed by flow cytometry. n = 6–7 for each group, from six to seven independent experiments. (N) Fold induction of pAKT(T308) and (O) pERK(T202/04) over time for the indicated groups. Fold induction was calculated using MFIs normalized to the 0 time point of the corresponding sample. Right: AUC quantification of pAKT(T308) and pERK(T202/04) time courses for the indicated groups. Statistical comparisons were made using ratio paired t tests, unless otherwise indicated. *P < 0.05, **P < 0.01, ***P < 0.001. AUC, area under the curve; NC, negative control.

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