Figure 9.
Fas interacts with the TCR complex and costimulates TCR signaling. (A) Confocal imaging of CD3ε and Fas in CD4 T cells. Naïve and stimulated (αCD3+αCD28 or αCD3+αCD28+FasL-LZ) CD4 T cells from the indicated mice were stained with αCD3ε-AF488 (green) and αFas-AF555 (red), and colocalization (yellow) was measured. Data are representative of three individual experiments, n = 19–24 unique images for each group. Left: Representative images of CD3ε and Fas costaining from the indicated groups. Right: Quantification of % colocalization between CD3ε and Fas in the indicated groups. (B and C) FLIM-FRET microscopy of CD3ε-Fas interactions in CD4 T cells. Naïve and stimulated (αCD3+αCD28 or αCD3+αCD28+FasL-LZ) CD4 T cells from the indicated mice were stained with αCD3ε-AF488 alone (donor control) or costained with αCD3ε-AF488 (donor) and αFas-AF555 (acceptor), and FL was measured. Results are representative of three independent experiments. (B) Representative images from the indicated groups. Scale bars in images indicate 4 μm. (C) Left: FL (ns) measurements of individual pixels from ROIs on cells from indicated groups. Right: FRET efficiency (%) within ROI from individual cells from the indicated groups. n = 18 for each group. (D) Naïve CD4 T cells from WT and Pik3cdE1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fas-targeting gRNAs and stimulated with αCD3/CD28 in the presence of hIL-2 for 72 h. Cells were rested in serum-free media, treated with αCD3 (1 μg/ml) (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. Top: Fold induction of pS6(S240/44) over time. Fold induction was calculated using pS6(S240/44) MFIs normalized to the 0 time point of the corresponding sample. Bottom: AUC quantification of pS6(S240/44) time courses for the indicated groups. (E) 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, treated with αCD3 (1 μg/ml) in the presence or absence of FasL-LZ over a time course (0, 1, 2, 5, 15, 60 min), and analyzed by flow cytometry. n = 6–7 for each group, from six to seven independent experiments. Top: Fold induction of pS6(S240/44) over time for the indicated groups. Fold induction was calculated using pS6(S240/44) MFIs normalized to the 0 time point of the corresponding sample. Bottom: AUC quantification of pS6(S240/44) time courses. Statistical comparisons were made using ratio paired t tests (D and E) and unpaired t tests (A and C). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. AUC, area under the curve; NC, negative control; ROIs, regions of interest.

Fas interacts with the TCR complex and costimulates TCR signaling. (A) Confocal imaging of CD3ε and Fas in CD4 T cells. Naïve and stimulated (αCD3+αCD28 or αCD3+αCD28+FasL-LZ) CD4 T cells from the indicated mice were stained with αCD3ε-AF488 (green) and αFas-AF555 (red), and colocalization (yellow) was measured. Data are representative of three individual experiments, n = 19–24 unique images for each group. Left: Representative images of CD3ε and Fas costaining from the indicated groups. Right: Quantification of % colocalization between CD3ε and Fas in the indicated groups. (B and C) FLIM-FRET microscopy of CD3ε-Fas interactions in CD4 T cells. Naïve and stimulated (αCD3+αCD28 or αCD3+αCD28+FasL-LZ) CD4 T cells from the indicated mice were stained with αCD3ε-AF488 alone (donor control) or costained with αCD3ε-AF488 (donor) and αFas-AF555 (acceptor), and FL was measured. Results are representative of three independent experiments. (B) Representative images from the indicated groups. Scale bars in images indicate 4 μm. (C) Left: FL (ns) measurements of individual pixels from ROIs on cells from indicated groups. Right: FRET efficiency (%) within ROI from individual cells from the indicated groups. n = 18 for each group. (D) Naïve CD4 T cells from WT and Pik3cdE1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fas-targeting gRNAs and stimulated with αCD3/CD28 in the presence of hIL-2 for 72 h. Cells were rested in serum-free media, treated with αCD3 (1 μg/ml) (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. Top: Fold induction of pS6(S240/44) over time. Fold induction was calculated using pS6(S240/44) MFIs normalized to the 0 time point of the corresponding sample. Bottom: AUC quantification of pS6(S240/44) time courses for the indicated groups. (E) 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, treated with αCD3 (1 μg/ml) in the presence or absence of FasL-LZ over a time course (0, 1, 2, 5, 15, 60 min), and analyzed by flow cytometry. n = 6–7 for each group, from six to seven independent experiments. Top: Fold induction of pS6(S240/44) over time for the indicated groups. Fold induction was calculated using pS6(S240/44) MFIs normalized to the 0 time point of the corresponding sample. Bottom: AUC quantification of pS6(S240/44) time courses. Statistical comparisons were made using ratio paired t tests (D and E) and unpaired t tests (A and C). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. AUC, area under the curve; NC, negative control; ROIs, regions of interest.

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