Phosphorylation of PD-1-Y248 is a marker of PD-1-mediated inhibitory function in human T cells

PD-1 is a target of cancer immunotherapy but responses are limited to a fraction of patients. Identifying patients with T cells subjected to PD-1-mediated inhibition will allow selection of suitable candidates for PD-1-blocking therapy and will improve the therapeutic success. We sought to develop an approach to detect PD-1-mediated inhibitory signaling. The cytoplasmic tail of PD-1 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) encompassing Y223 and an immunoreceptor tyrosine-based switch motif (ITSM) encompassing Y248, which is indispensable for interaction of SHP-2 and delivery of PD-1 inhibitory function. We generated an antibody specific for phosphorylated PD-1-Y248 and examined PD-1pY248(+) (pPD-1) expression in human T cells. pPD-1 was upregulated by TCR/CD3 + CD28 stimulation and simultaneous PD-1 ligation. pPD-1(+)CD8(+) T cells were identified in human peripheral blood and had impaired effector function. pPD-1(+) T cells were also detected in tumor-draining lymph nodes of tumor bearing mice and in biopsies of patients with glioblastoma multiform. Detection of pPD-1(+) T cells might serve as a biomarker for identification of T cells subjected to PD-1-mediated immunosuppression.