PD1 inhibits PKCθ-dependent phosphorylation of cytoskeleton-related proteins and immune synapse formation.

The inhibitory cell surface receptor programmed death 1 (PD1) is a major target for antibody-based cancer immunotherapies. Nevertheless, a substantial number of patients fail to respond to the treatment or experience adverse effects. An improved understanding of intracellular pathways targeted by PD1 is thus needed to develop better predictive and prognostic biomarkers. Here, via unbiased phosphoproteome analysis of primary human T cells, we demonstrate that PD1 triggering inhibited the phosphorylation and physical association with PKC theta (PKCof a variety of cytoskeleton-related proteins. PD1 blocked activation and recruitment of PKCθ to the forming immune synapse (IS) in a SHP1/SHP2 tyrosine phosphatase-dependent manner. Consequently, PD1 engagement led to impaired synaptic phosphorylation of cytoskeleton-related proteins and formation of smaller IS. TCR-induced phosphorylation of the PKCθ substrate and binding partner Vimentin was long-lasting and it could be durably inhibited by PD1 triggering. Vimentin phosphorylation in intratumoral T cells also inversely correlated with PDL1 levels in human lung carcinoma. Thus, PKCθ and its substrate Vimentin represent important targets of PD1-mediated T-cell inhibition, and low levels of Vimentin phosphorylation may serve as a biomarker for the activation of the PD1 pathway.