BACKGROUND: The programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) are validated cancer targets; however, emerging mechanisms and impact of PD-L1 intracellular signaling on cancer behavior are poorly understood. METHODS: We investigated the cancer cell intrinsic role of PD-L1 in multiple patient-derived models in vitro and in vivo. PD-L1 overexpression, knockdown, and PD-L1 intracellular domain (PD-L1-ICD) deletion (Δ260-290PD-L1) models were assessed for key cancer properties: clonogenicity, motility, invasion, and immune evasion. To determine how PD-L1 transduces signals intracellularly, we used the BioID2 platform to identify the PD-L1 intracellular interactome. Both human papillomavirus-positive and negative patient-derived xenografts were implanted in NOD-scid-gamma and humanized mouse models to investigate the effects of recombinant PD-1, anti-PD-L1, and anti-signal transducer and activator of transcription 3 (STAT3) in vivo. RESULTS: PD-L1 intracellular signaling increased clonogenicity, motility, and invasiveness in multiple head and neck squamous cell carcinoma (HNSCC) models, and PD-1 binding enhanced these effects. Protein proximity labeling revealed the PD-L1 interactome, distinct for unbound and bound PD-1, which initiated cancer cell-intrinsic signaling. PD-L1 binding partners interleukin enhancer binding factors 2 and 3 (ILF2-ILF3) transduced their effect through STAT3. Δ260-290PD-L1 disrupted signaling and reversed pro-growth properties. In humanized HNSCC in vivo models bearing T-cells, PD-1 binding triggered PD-L1 signaling, and dual PD-L1 and STAT3 inhibition were required to achieve tumor control. CONCLUSIONS: Upon PD-1 binding, the PD-L1 extracellular and intracellular domains exert a synchronized effect to promote immune evasion by inhibiting T-cell function while simultaneously enhancing cancer cell-invasive properties.

In the setting of conventional radiation therapy, even when combined with immunotherapy, head and neck cancer often recurs locally and regionally. Elective nodal irradiation (ENI) is commonly employed to decrease regional recurrence. Given our developing understanding that immune cells are radio-sensitive, and that T cell priming occurs in the draining lymph nodes (DLNs), we hypothesize that radiation therapy directed at the primary tumor only will increase the effectiveness of immunotherapies. We find that ENI increases local, distant, and metastatic tumor growth. Multi-compartmental analysis of the primary/distant tumor, the DLNs, and the blood shows that ENI decreases the immune response systemically. Additionally, we find that ENI decreases antigen-specific T cells and epitope spreading. Treating the primary tumor with radiation and immunotherapy, however, fails to reduce regional recurrence, but this is reversed by either concurrent sentinel lymph node resection or irradiation. Our data support using lymphatic sparing radiation therapy for head and neck cancer.