ISG15 targets glycosylated PD-L1 and promotes its degradation to enhance antitumor immune effects in lung adenocarcinoma

Immunocheckpoint inhibitors (ICIs) have been widely used in the clinical treatment of lung cancer. Although clinical studies and trials have shown that patients can benefit significantly after PD-1/PD-L1 blocking therapy, less than 20% of patients can benefit from ICIs therapy due to tumor heterogeneity and the complexity of immune microenvironment. Several recent studies have explored the immunosuppression of PD-L1 expression and activity by post-translational regulation. Our published articles demonstrate that ISG15 inhibits lung adenocarcinoma progression. Whether ISG15 can enhance the efficacy of ICIs by modulating PD-L1 remains unknown.

The ubiquitination modification of PD-L1 by ISG15 increases K48-linked ubiquitin chain modification, thereby increasing the degradation rate of glycosylated PD-L1-targeted proteasome pathway. More importantly, ISG15 enhanced the sensitivity to immunosuppressive therapy. Our study shows that ISG15, as a post-translational modifier of PD-L1, reduces the stability of PD-L1 and may be a potential therapeutic target for cancer immunotherapy.

The relationship between ISG15 and lymphocyte infiltration was identified by IHC. The effects of ISG15 on tumor cells and T lymphocytes were assessed using RT-qPCR and Western Blot and in vivo experiments. The underlying mechanism of PD-L1 post-translational modification by ISG15 was revealed by Western blot, RT-qPCR, flow cytometry, and Co-IP. Finally, we performed validation in C57 mice as well as in lung adenocarcinoma tissues.

ISG15 promotes the infiltration of CD4+ T lymphocytes. In vivo and in vitro experiments demonstrated that ISG15 induces CD4+ T cell proliferation and invalidity and immune responses against tumors. Mechanistically, we demonstrated that the ubiquitination-like modifying effect of ISG15 on PD-L1 increased the modification of K48-linked ubiquitin chains thus increasing the degradation rate of glycosylated PD-L1 targeting proteasomal pathway. The expression of ISG15 and PD-L1 was negatively correlated in NSCLC tissues. In addition, reduced accumulation of PD-L1 by ISG15 in mice also increased splenic lymphocyte infiltration as well as promoted cytotoxic T cell infiltration in the tumor microenvironment, thereby enhancing anti-tumor immunity. Conclusions: The ubiquitination modification of PD-L1 by ISG15 increases K48-linked ubiquitin chain modification, thereby increasing the degradation rate of glycosylated PD-L1-targeted proteasome pathway. More importantly, ISG15 enhanced the sensitivity to immunosuppressive therapy. Our study shows that ISG15, as a post-translational modifier of PD-L1, reduces the stability of PD-L1 and may be a potential therapeutic target for cancer immunotherapy.