PD1 induces long-lasting dephosphorylation of cytoskeleton proteins in vitro and in human lung adenocarcinoma tissues. (A) Western blot analysis monitoring phosphorylation of S5 of L-plastin (pS5 LCP1), S39 of vimentin (pS39 Vim), and S72 of vimentin (pS72 Vim) in human primary CD4 T cells. Cells were left untreated (NT) or triggered for the indicated time points with stimulatory (S) or inhibitory (I) beads. Quantification of the signal at 24 hours was performed by normalization of the phosphorylation signal to the signal of nonphosphorylated total proteins and calculated for 4 (pS39 Vim) and 5 independent experiments (pS5 LCP1 and pS72 Vim). Mean value ± SEM. Statistical analysis was performed using a 2-tailed, unpaired t test. ∗P < .05; ∗∗∗P < .001; ∗∗∗∗P < .0001. (B) Immunohistochemistry staining of human lung adenocarcinoma tissue samples characterized by low or high PDL1 expression. Samples where fluorescently labeled for PanCK, PDL1, PD1, CD3 and pS39 Vim. Nuclei were visualized with DAPI. Insets show intratumoral CD3⁺ lymphocytes, positive for PD1, coexpressing pS39 Vim in the case of PDL1^low adenocarcinoma, and negative for pS39 Vim in PDL1^high adenocarcinoma. Images were acquired using Vectra Polaris imaging system. Images were segmented into “tumor” or “stroma” sections using the inForm software and based on PanCK and DAPI signals. (C) Quantification of the ratio of pS39Vim⁺ cells amongst CD3⁺PD1⁺ cells (left panel) or CD3⁺ cells (right panel) in tumor and stroma regions performed in 5 cases of PDL1^low and in 5 cases of PDL1^high human lung adenocarcinomas. Number of imaging fields collected per each case for PDL1^low: 1-21; 2-16; 3-19; case 4-15; case 5-18. Number of imaging fields collected per each case for PDL1^high: 1-19; 2-22; 3-13; case 4-20; case 5-11. Number of tumor cells analysed per each case for PDL1^low: 1-30 853; 2-81 983; 3-60 988; 4-14 298; 5- 35 166. Number of tumor cells analysed per each case for PDL1^high: 1-5 900; 2-89 931; 3-27 363; 4-89 448; 5-20 981. Number of stroma cells analysed per each case for PDL1^low: 1-39 006; 2-31 193; 3-19 132; 4-32 378; 5-36 296. Number of stroma cells analysed per each case for PDL1^high: 1-112 832; 2-15 749; 3-10 887; 4-2; 5-51 525. Statistical analysis was performed using a 2-tailed, unpaired t test. ∗P < .05.