Extracellular interaction between programmed death ligand-1 (PD-L1) and programmed cell death protein-1 (PD-1) leads to tumour-associated immune escape. Here we show that the immunosuppression activity of PD-L1 is stringently modulated by ubiquitination and N-glycosylation. We show that glycogen synthase kinase 3β (GSK3β) interacts with PD-L1 and induces phosphorylation-dependent proteasome degradation of PD-L1 by β-TrCP. In-depth analysis of PD-L1 N192, N200 and N219 glycosylation suggests that glycosylation antagonizes GSK3β binding. In this regard, only non-glycosylated PD-L1 forms a complex with GSK3β and β-TrCP. We also demonstrate that epidermal growth factor (EGF) stabilizes PD-L1 via GSK3β inactivation in basal-like breast cancer. Inhibition of EGF signalling by gefitinib destabilizes PD-L1, enhances antitumour T-cell immunity and therapeutic efficacy of PD-1 blockade in syngeneic mouse models. Together, our results link ubiquitination and glycosylation pathways to the stringent regulation of PD-L1, which could lead to potential therapeutic strategies to enhance cancer immune therapy efficacy.

SUMMARY Pro-inflammatory cytokines produced in the tumor microenvironment lead to eradication of anti-tumor immunity and enhanced tumor cell survival. In the current study, we identified tumor necrosis factor alpha (TNF-α) as a major factor triggering cancer cell immunosuppression against T cell surveillance via stabilization of programmed cell death-ligand 1 (PD-L1). We demonstrated that COP9 signalosome 5 (CSN5), induced by NF-κB p65, is required for TNF-α-mediated PD-L1 stabilization in cancer cells. CSN5 inhibits the ubiquitination and degradation of PD-L1. Inhibition of CSN5 by curcumin diminished cancer cell PD-L1 expression and sensitized cancer cells to anti-CTLA4 therapy.

Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformin's role in cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy.

The engagement of programmed cell death protein 1 (PD-1; encoded by the PDCD1 gene) receptor expressed on activated T cells and its ligand, programmed death-ligand 1 (PD-L1; encoded by the CD274 gene), is a major co-inhibitory checkpoint signaling that controls T cell activities. Various types of cancers express high levels of PD-L1 and exploit PD-L1/PD-1 signaling to evade T cell immunity. Blocking the PD-L1/PD-1 pathway has consistently shown remarkable anti-tumor effects in patients with advanced cancers and is recognized as the gold standard for developing new immune checkpoint blockade (ICB) and combination therapies. However, the response rates of anti-PD-L1 have been limited in several solid tumors. Therefore, furthering our understanding of the regulatory mechanisms of PD-L1 can bring substantial benefits to patients with cancer by improving the efficacy of current PD-L1/PD-1 blockade or other ICBs. In this review, we provide current knowledge of PD-L1 regulatory mechanisms at the transcriptional, posttranscriptional, post-translational, and extracellular levels, and discuss the implications of these findings in cancer diagnosis and immunotherapy.