RIG-I is essential for radiation-induced IFN-β production and PD-L1/PD-L2 upregulation in HPV-negative head and neck squamous cell carcinoma cells

The RIG-I pathway, a key component of the innate immune system that classically detects cytosolic viral RNA, has also been implicated in regulating tumor cell viability, radiosensitivity, and patient survival across multiple cancer types. However, these effects appear to be context-dependent and partly contradictory. Whether radiotherapy modulates tumor-intrinsic RIG-I signaling in head and neck squamous cell carcinoma (HNSCC) depending on HPV status, and whether such modulation is associated with changes in the tumor immune phenotype, remains unknown. Four human HNSCC cell lines (two HPV-negative and two HPV-positive), along with siRNA-mediated RIG-I knockdown and a CRISPR/Cas9-generated RIG-I knockout derivative, were treated with radiotherapy. Tumor cells were analyzed for their immune phenotype, including RIG-I expression via Western blot and flow cytometry, IFN-β secretion via ELISA, and PD-L1 and PD-L2 expression via flow cytometry. Radiotherapy, dose- and fraction-dependent, induced the upregulation of RIG-I expression and IFN-β secretion from HPV-negative HNSCC cells. The knockdown and knockout of the RIG-I-encoding gene DDX58 in the HPV-negative cell line HSC4 minimized both IFN-β secretion and PD-L1 and PD-L2 expression. These effects could be replicated by pre-exposing HPV-negative cell lines to the RIG-I inhibitor RIG012 before irradiation. Interestingly, HPV-positive cells showed less radiotherapy-mediated modulation of RIG-I and of the tumor cell phenotype of immune-related surface markers and showed no significant reaction to the inhibitor treatment. The previously unrecognized RIG-I-IFN-β axis is a central regulator of RT-induced immune checkpoint modulation for HPV-negative HNSCC. These results provide new opportunities to reshape the immune response following RT in HNSCC.