Figure 1.
PD1 triggering induced a reduction in S/T phosphorylation of cytoskeleton-related proteins. (A) Scheme of the SILAC labeling and mass spectrometry approach used to identify PD1-mediated changes in the phosphoproteome of human primary CD4 T cells. Cells were labeled with heavy or light SILAC media for 2 weeks and treated for 10 or 30 minutes with beads coated with anti-CD3 and anti-CD28 antibodies in combination with TACI-Fc (stimulatory beads) or PDL1-Fc (inhibitory beads). After mixing, lysates were digested with trypsin and phosphorylated peptides were identified by LC-MS/MS analysis. (B) Graphs summarizing phosphorylation events that differed between stimulatory (S) and inhibitory (I) conditions. Left graph: all identified sites. Dots in the upper right quarter represent sites for which phosphorylation decreased upon PD1 triggering at both, the 10- and 30-minute time points. Right graph: only cytoskeleton-related proteins, including vimentin and LCP1. (C) Western blot analysis monitoring phosphorylation of S39 (pS39) of vimentin (Vim) in human primary CD4 T cells from 2 independent donors, left untreated (NT) or triggered for the indicated time points with S or I beads. Black vertical line indicates removal of an irrelevant lane. (D) Quantification of the experiments performed as in panel C: pS39 Vim levels were normalized to total Vim levels for the 30-minute condition for 3 independent experiments. (E) Western blot analysis monitoring phosphorylation of S5 of L-plastin (pLCP1) in human primary CD4 and CD8 T cells from 2 independent donors. Cells were left untreated (NT) or triggered for the indicated time points with S or I beads. Black vertical line indicates the cut of the image performed to remove 5-minute time point, which was not relevant for this figure. (F) Quantification of experiments performed as in panel E: pS5 LCP1 levels in CD4 (upper panel) and CD8 (lower panel) were normalized to total LCP1 levels for the 30-minute condition, for 6 and 11 independent experiments, respectively. (G) Western blot analysis monitoring phosphorylation of S5 of L-plastin (pLCP1) in Jurkat Ctrl cells or in Jurkat cells expressing WT PD1 (PD1) triggered for the indicated time points with Raji Ctrl cells or Raji PDL2 in presence or absence of SEE at 6 ng/mL. (H) Quantification of experiments performed as in panel G: pS5 LCP1 level in Jurkat cells was normalized to total LCP1 level for the 30-minute condition for 3 independent experiments. Graphs in panels D,F,H show mean value ± SEM. Statistical analysis for graphs in panels D,F was performed using a 2-tailed, unpaired t test. Statistical analysis for graph in panel H was performed using 1-way ANOVA with Šídák's multiple comparisons test. ∗∗∗∗P < .0001; ∗∗∗P < .001; ∗∗P < .01. ANOVA, analysis of variance; SEM, standard error of the mean.

PD1 triggering induced a reduction in S/T phosphorylation of cytoskeleton-related proteins. (A) Scheme of the SILAC labeling and mass spectrometry approach used to identify PD1-mediated changes in the phosphoproteome of human primary CD4 T cells. Cells were labeled with heavy or light SILAC media for 2 weeks and treated for 10 or 30 minutes with beads coated with anti-CD3 and anti-CD28 antibodies in combination with TACI-Fc (stimulatory beads) or PDL1-Fc (inhibitory beads). After mixing, lysates were digested with trypsin and phosphorylated peptides were identified by LC-MS/MS analysis. (B) Graphs summarizing phosphorylation events that differed between stimulatory (S) and inhibitory (I) conditions. Left graph: all identified sites. Dots in the upper right quarter represent sites for which phosphorylation decreased upon PD1 triggering at both, the 10- and 30-minute time points. Right graph: only cytoskeleton-related proteins, including vimentin and LCP1. (C) Western blot analysis monitoring phosphorylation of S39 (pS39) of vimentin (Vim) in human primary CD4 T cells from 2 independent donors, left untreated (NT) or triggered for the indicated time points with S or I beads. Black vertical line indicates removal of an irrelevant lane. (D) Quantification of the experiments performed as in panel C: pS39 Vim levels were normalized to total Vim levels for the 30-minute condition for 3 independent experiments. (E) Western blot analysis monitoring phosphorylation of S5 of L-plastin (pLCP1) in human primary CD4 and CD8 T cells from 2 independent donors. Cells were left untreated (NT) or triggered for the indicated time points with S or I beads. Black vertical line indicates the cut of the image performed to remove 5-minute time point, which was not relevant for this figure. (F) Quantification of experiments performed as in panel E: pS5 LCP1 levels in CD4 (upper panel) and CD8 (lower panel) were normalized to total LCP1 levels for the 30-minute condition, for 6 and 11 independent experiments, respectively. (G) Western blot analysis monitoring phosphorylation of S5 of L-plastin (pLCP1) in Jurkat Ctrl cells or in Jurkat cells expressing WT PD1 (PD1) triggered for the indicated time points with Raji Ctrl cells or Raji PDL2 in presence or absence of SEE at 6 ng/mL. (H) Quantification of experiments performed as in panel G: pS5 LCP1 level in Jurkat cells was normalized to total LCP1 level for the 30-minute condition for 3 independent experiments. Graphs in panels D,F,H show mean value ± SEM. Statistical analysis for graphs in panels D,F was performed using a 2-tailed, unpaired t test. Statistical analysis for graph in panel H was performed using 1-way ANOVA with Šídák's multiple comparisons test. ∗∗∗∗P < .0001; ∗∗∗P < .001; ∗∗P < .01. ANOVA, analysis of variance; SEM, standard error of the mean.

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