Monitoring PD-1 Signaling in Tumor Infiltrating Lymphocytes

Abstract Programmed death (PD)-1 pathway blockade is a successful strategy for cancer immunotherapy. However, only a limited number of patients respond. There is great need to identify predictors of therapeutic response to PD-1 immunotherapy. One challenge is that PD-1 expression can mark activated T cells, which are susceptible to PD-1-mediated inhibition, but does not indicate whether a PD-1-mediated immunoinhibitory signal is being delivered. Currently there are no methods to detect active PD-1 signaling and, in turn, successful PD-1 inhibition. Here, we describe a novel antibody that detects PD-1 signaling. This antibody detects phosphorylation of the immunotyrosine switch motif (ITSM) in the intracellular tail of both human and mouse PD-1 (phospho-PD-1). Using this anti-phospho-PD-1 mAb, we show that PD-1+ tumor infiltrating lymphocytes (TILs) in MC38 murine colorectal tumors have high levels of phosphorylated PD-1, particularly in PD-1+TIM-3+TILs. Upon PD-1 blockade, PD-1 phosphorylation is markedly decreased in TIM-3+CD8+ TILs prior to tumor clearance. We also observed decreased phospho PD-1 levels in T cells from peripheral blood of human lymphoma patients following treatment with PD-1 and LAG-3 mAb. These data demonstrate that phosphorylation of the ITSM of PD-1 marks dysfunctional T cells that may be rescued with PD-1 blockade. Analysis of phospho-PD-1 may serve as a potential biomarker for effective PD-1 immunotherapy.