Figure 4.

PD-1 interferes with immune synapse maturation and T cell adhesion.A, heatmap of phosphosites within proteins important for cytoskeletal organization targeted by PD-1 showing Log2FC values relative to the unstimulated control. B, histogram plots showing surface expression levels of PD-1 ligands in Raji B cells and (C) diagram of Raji-Jurkat conjugate assay. D, microscopic evaluation of IS formation following SEE-coated Raji cell co-incubation with either WT Jurkat cells, which express low levels of surface PD-1, or PD-1–transduced Jurkat clone. Schematic representation of conjugate formation (D, left), confocal images of Raji-Jurkat conjugates in parental Jurkat cells, and PD-1–transduced Jurkat clone (D, center), transfected with LifeAct, and bar graph of mean ± S.E. (error bars) of the quantitated proportion of conjugate formation in each group (D, right) from three independent experiments. E, Western blotting analysis and quantitation of PAK2 Ser-197 phosphorylation from n = 3 independent experiments in Jurkat T cells after 5-min stimulation with Dynabeads coated with either αCD3 alone or αCD3+PD-L2. F, a representative blot of active GTP-bound Rap1 pulldown assay following 5-min crosslinking stimulation in WT Jurkat T cells from three independent experiments (bar graph shows mean ± S.D. (error bars)). Statistical analysis was performed using unpaired (D) or paired (F) Student's t test, where *p ≤ 0.05 and **p ≤ 0.01.