PD1-triggered inhibition of LCP1 phosphorylation is SHP1/SHP2-dependent. (A) Overview of the Jurkat cell lines used to assess the relevance of SHP1/2 phosphatases in the PD1-induced dephosphorylation of cytoskeleton proteins. Upper section: Ctrl cells expressing WT PD1. Middle section: Jurkat cells expressing PD1 DM, Y223F/Y248F, unable to bind SHP1/SHP2. Lower section: CRISPR/Cas9-mediated silencing of SHP1 and SHP2 in Jurkat-PD1-NFAT cells. (B) Upper panel: western blot monitoring phosphorylation of S5 of L-plastin (pLCP1) in the indicated cell lines. Jurkat-PD1 WT and Jurkat-PD1 DM cells were cocultured for the indicated times with PDL2–expressing Raji cells (Raji PDL2) in presence or absence of SEE at 6 ng/mL. Lower panel: quantification of experiments, pS5 LCP1 was normalized to total LCP1 for the 60-minute condition for 6 independent experiments. (C) Western blot monitoring phosphorylation of S5 of L-plastin (pLCP1) in 2 independent Jurkat-PD1-NFAT cell lines stably transduced with SHP1- and SHP2–specific CRISPR constructs and cocultured for 30 minutes with Raji Ctrl cells or Raji PDL2 in presence of SEE at 6 ng/mL. Representative data from 4 independent experiments.