Monitoring PD-1 Phosphorylation to Evaluate PD-1 Signaling during Antitumor Immune Responses

PD-1 expression marks activated T cells susceptible to PD-1-mediated inhibition but not whether a PD-1-mediated signal is being delivered. Molecular predictors of response to PD-1 immune checkpoint blockade (ICB) are needed. We describe a monoclonal antibody (mAb) that detects PD-1 signaling through the detection of phosphorylation of the immunotyrosine switch motif (ITSM) in the intracellular tail of mouse and human PD-1 (phospho-PD-1). We showed PD-1⁺ tumor-infiltrating lymphocytes (TILs) in MC38 murine tumors had high phosphorylated PD-1, particularly in PD-1⁺TIM-3⁺ TILs. Upon PD-1 blockade, PD-1 phosphorylation was decreased in CD8⁺ TILs. Phospho-PD-1 increased in T cells from healthy human donors after PD-1 engagement and decreased in patients with Hodgkin lymphoma following ICB. These data demonstrate that phosphorylation of the ITSM motif of PD-1 marks dysfunctional T cells that may be rescued with PD-1 blockade. Detection of phospho-PD-1 in TILs is a potential biomarker for PD-1 immunotherapy responses.