Figure 5.
PD1-induced inhibition of PKCθ localization to the IS is SHP1/SHP2-dependent. (A) Left: immunofluorescent labeling of PD1 and PKCθ of Jurkat-PD1 WT cells (upper panel) and Jurkat-PD1 DM cells (lower panel) upon 60 minute of coculture with Raji PDL2 cells, preincubated with 6 ng/mL of SEE and mixed with Jurkat cells at a 1:1 ratio. Right: representative histogram of the colocalization profile prepared with RGB profiler plugin of ImageJ. Representative images of 3 independent experiments are shown. (B) ImageStream-based quantification of the colocalization between PD1 and PKCθ in Jurkat-PD1 WT cells and Jurkat-PD1 DM cells cocultured for 60 minutes with Raji PDL2 cells. Raji PDL2 cells were preincubated with 6 ng/mL of SEE and mixed with Jurkat cells at 1:1 ratio. Upon fixation and permeabilization, samples were stained for PKCθ, PD1, and nuclei (DAPI) and analyzed by Amnis Image Stream imaging flow cytometry. Left: histogram measuring the extent of signal overlap between PKCθ and PD1 with PD1 signal in the IS used as a mask; right: examples of images acquired. Representative results of 2 independent experiments are shown. (C) Left: immunofluorescent labeling of PD1 and pY90 PKCθ of human primary CD4 T cells upon 60 minute of coculture with Raji Ctrl cells (upper panel) or Raji PDL2 cells (lower panel). Raji cells were preincubated with 1 ng/mL of SEA/SEB/SEE and mixed with CD4 cells at a 1:2 (Raji:T cells) ratio. Right panel: quantification of total pY90 PKCθ signal intensity in T cells from 1 experiment. Representative results of 3 independent experiments are shown. Each dot represents the value from 1 primary T cell, 14 and 16 cells were quantified, respectively. (D) Left: immunofluorescent labeling of PD1 and pY90 PKCθ of Jurkat-PD1 WT cells upon 30 minutes of coculture with Raji Ctrl cells (upper panel) or Raji PDL2 cells (lower panel). Raji cells were preincubated with 6 ng/mL of SEE and mixed with Jurkat-PD1 WT cells at 1:1 ratio. Right panel: quantification of total pY90 PKCθ signal intensity in Jurkat-PD1 WT cells. Representative results of 3 independent experiments are shown. Each dot represents a value from 1 Jurkat cell, 19 and 19 cells were quantified, respectively. (E) Left: immunofluorescent labeling of PD1 and pY90 PKCθ of Jurkat-PD1 DM cells upon 60 minute of coculture with Raji Ctrl cells (upper panel) or Raji PDL2 cells (lower panel). Raji cells were preincubated with 6 ng/mL of SEE and mixed with Jurkat-PD1 DM cells at 1:1 ratio. Right panel: quantification of total pY90 PKCθ signal intensity in Jurkat-PD1 DM cells. Representative results of 3 independent experiments are shown. Each dot represents a value from 1 Jurkat cell, 19 and 24 cells were quantified, respectively. Scale bar, 5 μm. Graphs in panels C-E show mean value ± SEM. Statistical analysis was performed using a 2-tailed, unpaired t test. ∗∗∗∗P < .001; ∗∗∗P < .001; ns, not significant.

PD1-induced inhibition of PKCθ localization to the IS is SHP1/SHP2-dependent. (A) Left: immunofluorescent labeling of PD1 and PKCθ of Jurkat-PD1 WT cells (upper panel) and Jurkat-PD1 DM cells (lower panel) upon 60 minute of coculture with Raji PDL2 cells, preincubated with 6 ng/mL of SEE and mixed with Jurkat cells at a 1:1 ratio. Right: representative histogram of the colocalization profile prepared with RGB profiler plugin of ImageJ. Representative images of 3 independent experiments are shown. (B) ImageStream-based quantification of the colocalization between PD1 and PKCθ in Jurkat-PD1 WT cells and Jurkat-PD1 DM cells cocultured for 60 minutes with Raji PDL2 cells. Raji PDL2 cells were preincubated with 6 ng/mL of SEE and mixed with Jurkat cells at 1:1 ratio. Upon fixation and permeabilization, samples were stained for PKCθ, PD1, and nuclei (DAPI) and analyzed by Amnis Image Stream imaging flow cytometry. Left: histogram measuring the extent of signal overlap between PKCθ and PD1 with PD1 signal in the IS used as a mask; right: examples of images acquired. Representative results of 2 independent experiments are shown. (C) Left: immunofluorescent labeling of PD1 and pY90 PKCθ of human primary CD4 T cells upon 60 minute of coculture with Raji Ctrl cells (upper panel) or Raji PDL2 cells (lower panel). Raji cells were preincubated with 1 ng/mL of SEA/SEB/SEE and mixed with CD4 cells at a 1:2 (Raji:T cells) ratio. Right panel: quantification of total pY90 PKCθ signal intensity in T cells from 1 experiment. Representative results of 3 independent experiments are shown. Each dot represents the value from 1 primary T cell, 14 and 16 cells were quantified, respectively. (D) Left: immunofluorescent labeling of PD1 and pY90 PKCθ of Jurkat-PD1 WT cells upon 30 minutes of coculture with Raji Ctrl cells (upper panel) or Raji PDL2 cells (lower panel). Raji cells were preincubated with 6 ng/mL of SEE and mixed with Jurkat-PD1 WT cells at 1:1 ratio. Right panel: quantification of total pY90 PKCθ signal intensity in Jurkat-PD1 WT cells. Representative results of 3 independent experiments are shown. Each dot represents a value from 1 Jurkat cell, 19 and 19 cells were quantified, respectively. (E) Left: immunofluorescent labeling of PD1 and pY90 PKCθ of Jurkat-PD1 DM cells upon 60 minute of coculture with Raji Ctrl cells (upper panel) or Raji PDL2 cells (lower panel). Raji cells were preincubated with 6 ng/mL of SEE and mixed with Jurkat-PD1 DM cells at 1:1 ratio. Right panel: quantification of total pY90 PKCθ signal intensity in Jurkat-PD1 DM cells. Representative results of 3 independent experiments are shown. Each dot represents a value from 1 Jurkat cell, 19 and 24 cells were quantified, respectively. Scale bar, 5 μm. Graphs in panels C-E show mean value ± SEM. Statistical analysis was performed using a 2-tailed, unpaired t test. ∗∗∗∗P < .001; ∗∗∗P < .001; ns, not significant.

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