Figure 4.
PD1 triggering affects PKCθ recruitment and vimentin phosphorylation at the IS. (A) Left: immunofluorescent labeling of PD1 and PKCθ upon 60 minutes of coculture of Jurkat-PD1 WT cells with Raji Ctrl cells (upper panel) or PDL2 expressing Raji cells (lower panel), preincubated with 6 ng/mL of SEE and mixed with Jurkat-PD1 WT cells at a 1:1 ratio. Right: quantification of the PKCθ signal intensity in Jurkat cells within proximity of the T and B cell interface from 1 experiment. Representative results of 4 independent experiments are shown. Each dot represents a value from 1 Jurkat cell, 46 and 56 cells were quantified, respectively. (B) ImageStream-based quantification of the colocalization between PD1 and PKCθ in Jurkat-PD1 WT cells cocultured for 60 minutes with Raji Ctrl or Raji PDL2 cells, preincubated with 6 ng/mL of SEE and mixed with Jurkat-PD1 WT cells at a 1:1 ratio. Upon fixation and permeabilization, samples were stained for PKCθ, PD1 and nuclei (DAPI) and analyzed by ImageStream 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 PKCθ upon 30 minutes of coculture of human primary CD4 T cells 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. Images shown are from a single Z plane, which likely accounts for the concentration of staining in the T cell in the absence of PDL2 ligation, whereas the signal is weaker in the Raji PDL2 condition (bottom panel). Right: quantification of the PKCθ signal intensity in T cells within proximity of the T cell–B cell interface from 1 experiment. Representative results of 3 independent experiments are shown. Each dot represents a value from 1 primary T cell, 15 and 12 cells were quantified, respectively. (D) Left: immunofluorescent labeling of PD1 and pS39 Vim upon 60 minutes of coculture of human primary CD4 T cells 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 a 1:2 (Raji:T cells) ratio. Right: quantification of the total pVim39 signal intensity in T cells from 1 experiment. Representative results of 3 independent experiments are shown. Each dot represents a value from 1 primary T cell, 19 and 10 cells were quantified, respectively. Scale bar: 5 μm (panels A,C-D). Graphs in panels A,C-D show mean value ± SEM. Statistical analysis was performed using a 2-tailed, unpaired t-test. ∗∗∗∗P < .0001.

PD1 triggering affects PKCθ recruitment and vimentin phosphorylation at the IS. (A) Left: immunofluorescent labeling of PD1 and PKCθ upon 60 minutes of coculture of Jurkat-PD1 WT cells with Raji Ctrl cells (upper panel) or PDL2 expressing Raji cells (lower panel), preincubated with 6 ng/mL of SEE and mixed with Jurkat-PD1 WT cells at a 1:1 ratio. Right: quantification of the PKCθ signal intensity in Jurkat cells within proximity of the T and B cell interface from 1 experiment. Representative results of 4 independent experiments are shown. Each dot represents a value from 1 Jurkat cell, 46 and 56 cells were quantified, respectively. (B) ImageStream-based quantification of the colocalization between PD1 and PKCθ in Jurkat-PD1 WT cells cocultured for 60 minutes with Raji Ctrl or Raji PDL2 cells, preincubated with 6 ng/mL of SEE and mixed with Jurkat-PD1 WT cells at a 1:1 ratio. Upon fixation and permeabilization, samples were stained for PKCθ, PD1 and nuclei (DAPI) and analyzed by ImageStream 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 PKCθ upon 30 minutes of coculture of human primary CD4 T cells 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. Images shown are from a single Z plane, which likely accounts for the concentration of staining in the T cell in the absence of PDL2 ligation, whereas the signal is weaker in the Raji PDL2 condition (bottom panel). Right: quantification of the PKCθ signal intensity in T cells within proximity of the T cell–B cell interface from 1 experiment. Representative results of 3 independent experiments are shown. Each dot represents a value from 1 primary T cell, 15 and 12 cells were quantified, respectively. (D) Left: immunofluorescent labeling of PD1 and pS39 Vim upon 60 minutes of coculture of human primary CD4 T cells 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 a 1:2 (Raji:T cells) ratio. Right: quantification of the total pVim39 signal intensity in T cells from 1 experiment. Representative results of 3 independent experiments are shown. Each dot represents a value from 1 primary T cell, 19 and 10 cells were quantified, respectively. Scale bar: 5 μm (panels A,C-D). Graphs in panels A,C-D show mean value ± SEM. Statistical analysis was performed using a 2-tailed, unpaired t-test. ∗∗∗∗P < .0001.

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