PD-1 marks dysfunctional regulatory T cells in malignant gliomas

Immunotherapies targeting the immune checkpoint receptor programmed cell death protein 1 (PD-1) have shown remarkable efficacy in treating cancer. CD4(+)CD25(hi)FoxP3(+) Tregs are critical regulators of immune responses in autoimmunity and malignancies, but the functional status of human Tregs expressing PD-1 remains unclear. We examined functional and molecular features of PD-1(hi) Tregs in healthy subjects and patients with glioblastoma multiforme (GBM), combining functional assays, RNA sequencing, and cytometry by time of flight (CyTOF). In both patients with GBM and healthy wsubjects, circulating PD-1(hi) Tregs displayed reduced suppression of CD4(+) effector T cells, production of IFN-gamma, and molecular signatures of exhaustion. Transcriptional profiling of tumor-resident Tregs revealed that several genes coexpressed with PD-1 and associated with IFN-gamma production and exhaustion as well as enrichment in exhaustion signatures compared with circulating PD-1(hi) Tregs. CyTOF analysis of circulating and tumor-infiltrating Tregs from patients with GBM treated with PD-1-blocking antibodies revealed that treatment shifts the profile of circulating Tregs toward a more exhausted phenotype reminiscent of that of tumor-infiltrating Tregs, further increasing IFN-gamma production. Thus, high PD-1 expression on human Tregs identifies dysfunctional, exhausted Tregs secreting IFN-gamma that exist in healthy individuals and are enriched in tumor infiltrates, possibly losing function as they attempt to modulate the antitumoral immune responses.