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

Abstract

Background: 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.

Methods: 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.

Results: 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.

Keywords: Anticancer immunity; Glycosylated PD-L1; ISG15/ISGylation; Lung adenocarcinoma.

Fig. 1

Expression of ISG15 and lymphcytes in lung adenocarcinoma. A Activation of memory CD4⁺ T cells, infiltration of CD8a⁺ T cells and expression of CD8⁺ T lymphocytotoxic factors in high/low ISG15 expressing tumour tissues obtained from the TCGA collection. B Representative IHC images showing the expression of ISG15, CD3, CD4, CD8. Scale bars 100 μm. Magnification, ×100, ×400. C The relationship between CD3⁺, CD4⁺, CD8⁺ T cells and ISG15 was analysed by IHC staining of LUAD

Fig. 2

ISG15 promotes the infiltration of immune cells in vivo. A Western blot analysis of ISG15 in LLC with over-expressed ISG15 lentiviruses. B The bar graph exhibited the tumor weight of wild-type (WT) and over-expressed ISG15 (Lv-ISG15) LLC cells in immunocompetent C57BL/6 mice. C General views of the lungs and HE staining remove from the mice in WT and LvSG15 groups. Black arrows represent images of metastasis in the lungs. Magnification, ×100, ×400. D Survival curves demonstrating the WT and Lv-ISG15 groups of C57BL/6 mice during one experimental cycle (30 days). E The curve graph exhibited the tumor volume formation of WT and Lv-ISG15 LLC cells in C57BL/6 mice. F Representative images of tumors from C57BL/6 mice with HE and immunohistochemical staining of CD31, KI67, CD3, CD4, and CD8. Scale bars 100 μm. Magnification, ×400. G Populations of tumor-infiltrating CD3⁺ T cells and relative ratio of CD4⁺IFN-γ⁺ and CD8⁺GzmB⁺ cells in CD3⁺ TILs in WT and Lv-ISG15 groups detected by flow cytometry. H Populations of CD4⁺ and CD8⁺ T cells in spleen from the mice in WT and Lv-ISG15 groups detected by flow cytometry. The data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

ISG15 enhances the anti-tumor immunity of CD4⁺ T lymphocytes, which is related to PD-L1. A Growth of control or overexpressed with ISG15 (Lv-ISG15) or shRNA-ISG15 (Sh-ISG15) A549/ H1299 cells before and after co-culture with CD4⁺T cells. The bar chart below indicates the number of cells. B Western blot analysis the expression of apoptosis-related proteins in tumor cells after co-culture. C The IFN-γ secreted by CD4⁺ T cells co-cultured with A549 and H1299 under the three treatment conditions were evaluated by ELISA. D The expansion of CD4⁺ T cells co-cultured with irradiated A549/ H1299 cells or A549/ H1299 cells overexpressed/shRNA of ISG15 were assessed by CCK-8. E The secretion of ISG15 in the supernatant of tumor cells under three treatment conditions detected by ELISA. The data are shown as the mean ± SD. F Typical images of IHC staining for ISG15, granzyme B, perforin and PD-L1 in two patients with LUAD. Scale bars 100 μm. Magnification, ×400. G Number of cells positive for Granzyme B or Perforin in each high magnification (×400) field of view. H, correlation analysis of PD-L1 and ISG15 expression in 40 lung adenocarcinoma tissues. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

ISG15 down-regulates the expression of glycosylated PD-L1. A Western blot analysis of the protein level of PD-L1 in control or Lv-ISG15 or Sh-ISG15 A549, H1299 and LLC cells. B After 5 ng/ml IFN-γ treatment of cells in the control group or Lv-ISG15 group for 12 h, mRNA levels of ISG15 were measured by quantitative qRT-PCR and protein expression levels of PD-L1 were measured by Western blot. C Cellular immunofluorescence staining was used to observe the expression of PD-L1 in Sh-ISG15 and Lv-ISG15 or the control A549/H1299 cells. D The plasma membrane PD-L1 in control or sh-ISG15 A549/H1299 cells was detected by flow cytometry. The data are shown as the mean ± SD. Ns means no significance. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Exogenous ISG15 also reduces the expression of glycosylated PD-L1. A Western blot and RT-qPCR were used to detect the expression of different molecular weights of PD-L1 at concentrations of 0 μg, 1 μg, 1.5 μg, 2 μg, and 4 μg of exogenous ISG15 in A549 and H1299. B H1299 or A549 treated with 4 μg or 0.8 μg of exogenous ISG15, the changes of PD-L1 with different molecular weights (left panel) and mRNA level (right panel) in 0–48 h.The data are shown as the mean ± SD. Ns means no significance. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

ISG15 and PD-L1 form ISGylation and promote its ubiquitination for proteasome-dependent degradation. A Quantitative qRT-PCR analysis of the mRNA level of PD-L1 in Sh-ISG15 and Lv-ISG15 or the control A549/H1299 cells. B Western blotting was performed to detect the expression level of glycosylated PD-L1 at different times in A549 and H1299, after CHX (25 μg/ml) treatment. The data were normalized for comparative purposes (bottom panel). C Ubiquitination level of PD-L1 detected by Immunoprecipitation (IP) assay in Lv-ISG15 or control cells after treated with MG132 (20 μmol) for 8 h. D Immunoprecipitation of ISG15 and PD-L1 at lysine48-linked ubiquitination sites after H1299 and Pc-9 treated with 20 μM MG132 6 h. E Western blot was used to examine the effect of bafilomycin or MG132 on PD-L1 levels in the presence of CHX in Lv-ISG15 cells. F IP experiment detects the interaction between ISG15 and PD-L1. G Co-IP assay detects whether the interaction between ISG15 and PD-L1 is specific to ISGylation and whether it is dissociated by USP18 in H1299 and Pc-9 cells co-transfected ISG15 and USP18 or control plasmids

Fig. 7

ISG15 elevation-induced destabilization of PD-L1 improve PD-L1-targeted immunotherapy