Figure 8.
Fas-induced T cell activation occurs in the absence of FADD. (A) Naïve CD4 T cells from WT and Pik3cdE1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fadd-targeting gRNAs and polarized under Th2 conditions in the presence or absence of FasL-LZ (25 ng/ml). Top: Representative flow cytometry histograms showing pS6(S240/44) staining in live CD4+ cells. Bottom: Fold induction of pS6(S240/44) in Th2-polarized (−/+ FasL-LZ) live CD4+ T cells. Fold induction was calculated by normalizing MFIs to NC WT cells. n = 4–5 for each group, from four to five independent experiments. (B and C) Naïve CD4 T cells from WT and Pik3cdE1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fadd-targeting gRNAs and polarized under Th2 conditions. n = 6 for each group, from six independent experiments. (B) Frequencies of IFNγ+ Th2-polarized cells. (C) Th2-polarized cells were gated as FasLlow, FasLmid, and FasLhigh. Frequencies of IFNγ+ cells were measured in the indicated groups. (D–G) Fas was tagged with BioID2 on its intracellular C terminus (Fas-BioID) and stably expressed in Fas-deficient Jurkat cells. Fas-BioID Jurkat cells were cultured in the presence or absence of recombinant multimeric FasL (FasL-LZ). Jurkat cells expressing BioID alone were used as a control. (D) Venn diagram showing proteins identified following mass spectrometry analysis of biotinylated proteins (streptavidin pull-down) that were common between or specific to BioID-alone Jurkat cells versus Fas-BioID + FasL-LZ (P < 0.05, n = 3 for all groups). (E) Heatmap (row z-score) showing % normalized spectral abundance of proteins specifically upregulated in Fas-BioID ± FasL-LZ Jurkat cells relative to BioID-alone Jurkat cells. (F) Pathway enrichment of Reactome gene sets was performed using Enrichr (Xie et al., 2021), with significantly enriched gene sets colored in blue; proteins specifically upregulated in Fas-BioID + FasL-LZ Jurkat cells relative to BioID-alone Jurkat cells were used as input for pathway enrichment. (G) Normalized spectral abundance (%) of the indicated proteins in BioID-alone, Fas-BioID, and Fas-BioID + FasL-LZ Jurkat cells, measured by mass spectrometry. Statistical comparisons were made using ratio paired t tests (G). *P < 0.05, **P < 0.01. NC, negative control.

Fas-induced T cell activation occurs in the absence of FADD. (A) Naïve CD4 T cells from WT and Pik3cdE1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fadd-targeting gRNAs and polarized under Th2 conditions in the presence or absence of FasL-LZ (25 ng/ml). Top: Representative flow cytometry histograms showing pS6(S240/44) staining in live CD4+ cells. Bottom: Fold induction of pS6(S240/44) in Th2-polarized (−/+ FasL-LZ) live CD4+ T cells. Fold induction was calculated by normalizing MFIs to NC WT cells. n = 4–5 for each group, from four to five independent experiments. (B and C) Naïve CD4 T cells from WT and Pik3cdE1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fadd-targeting gRNAs and polarized under Th2 conditions. n = 6 for each group, from six independent experiments. (B) Frequencies of IFNγ+ Th2-polarized cells. (C) Th2-polarized cells were gated as FasLlow, FasLmid, and FasLhigh. Frequencies of IFNγ+ cells were measured in the indicated groups. (D–G) Fas was tagged with BioID2 on its intracellular C terminus (Fas-BioID) and stably expressed in Fas-deficient Jurkat cells. Fas-BioID Jurkat cells were cultured in the presence or absence of recombinant multimeric FasL (FasL-LZ). Jurkat cells expressing BioID alone were used as a control. (D) Venn diagram showing proteins identified following mass spectrometry analysis of biotinylated proteins (streptavidin pull-down) that were common between or specific to BioID-alone Jurkat cells versus Fas-BioID + FasL-LZ (P < 0.05, n = 3 for all groups). (E) Heatmap (row z-score) showing % normalized spectral abundance of proteins specifically upregulated in Fas-BioID ± FasL-LZ Jurkat cells relative to BioID-alone Jurkat cells. (F) Pathway enrichment of Reactome gene sets was performed using Enrichr (Xie et al., 2021), with significantly enriched gene sets colored in blue; proteins specifically upregulated in Fas-BioID + FasL-LZ Jurkat cells relative to BioID-alone Jurkat cells were used as input for pathway enrichment. (G) Normalized spectral abundance (%) of the indicated proteins in BioID-alone, Fas-BioID, and Fas-BioID + FasL-LZ Jurkat cells, measured by mass spectrometry. Statistical comparisons were made using ratio paired t tests (G). *P < 0.05, **P < 0.01. NC, negative control.

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