USP18 is crucial for IFN-γ-mediated inhibition of B16 melanoma tumorigenesis and antitumor immunity

Abstract

Background: Interferon (IFN)-γ-mediated immune response plays an important role in tumor immunosurveillance. However, the regulation of IFN-γ-mediated tumorigenesis and immune response remains elusive. USP18, an interferon stimulating response element, regulates IFN-α-mediated signaling in anti-viral immune response, but its role in IFN-γ-mediated tumorigenesis and anti-tumor immune response is unknown.

Method: In this study, USP18 in tumorigenesis and anti-tumor immune response was comprehensively appraised in vivo by overexpression or downregulation its expression in murine B16 melanoma tumor model in immunocompetent and immunodeficient mice.

Results: Ectopic expression or downregulation of USP18 in B16 melanoma tumor cells inhibited or promoted tumorigenesis, respectively, in immunocompetent mice. USP18 expression in B16 melanoma tumor cells regulated IFN-γ-mediated immunoediting, including upregulating MHC class-I expression, reducing tumor cell-mediated inhibition of T cell proliferation and activation, and suppressing PD-1 expression in CD4+ and CD8+ T cells in tumor-bearing mice. USP18 expression in B16 melanoma tumor cells also enhanced CTL activity during adoptive immunotherapy by prolonging the persistence and enhancing the activity of adoptively transferred CTLs and by reducing CTL exhaustion in the tumor microenvironment. Mechanistic studies demonstrated that USP18 suppressed tumor cell-mediated immune inhibition by activating T cells, inhibiting T-cell exhaustion, and reducing dendritic cell tolerance, thus sensitizing tumor cells to immunosurveillance and immunotherapy.

Conclusion: These findings suggest that stimulating USP18 is a feasible approach to induce B16 melanoma specific immune response.

Figure 1

USP18 expression in tumor cells with IFN-γ signaling. B16 tumor cells were stimulated in vitro with IFN-γ (10 ng/ml) (R&D systems), IFN-α (10 ng/ml) (R&D systems), or LPS (1 μg/ml) (Invivogen) for 24 hr, then harvested for qRT-PCR assay of USP18 mRNA (A) and Western blot assay (B) of USP18 and pStat1 protein expression. EMT6 and 4 T1 tumor cells were untreated or treated with IFN-γ (5 ng/ml) for 24 hr, USP18 expression was analyzed by Western blot analysis (C). B16-GFP cells were subcutaneously inoculated into C57BL/6 mice until tumor sizes reached about 5 mm. Some mice received adoptive transfer of 5 × 10⁶ activated OT-1 cells for 48 hr. B16-GFP tumor cells were collected and qRT-PCR analysis of USP18 expression (D). B16 tumor cells were cocultured with activated OT-1 cells for 24 hr and analyzed for USP18 and pStat1 expression (E).

Figure 2

USP18 expression in tumor cells suppresses tumor growth in vivo. 3 × 10⁵ B16-OVA-shUSP18 or B16-OVA-shCon tumor cells were intravenously inoculated into C57BL/6 mice. Tumor growth was monitored by counting the lung tumor foci (A-B) and the survival rate (C). 2 × 10⁵ B16-OVA-USP18 or B16-OVA-GFP tumor cells were intravenously inoculated into C57BL/6 mice. Tumor growth was monitored by counting the lung tumor foci (D-E), and mouse survival rate over time (F). 1 × 10⁶ B16-OVA-USP18 or B16-OVA-GFP, or B16-OVA-shUSP18 tumor cells were subcutaneously inoculated into C57BL/6 mice. The tumor growth was monitored as tumor volume and by H&E staining (G-I).

Figure 3

USP18 function in tumor growth depends on the immune system. 3 × 10⁵ B16-OVA-USP18 or B16-OVA-GFP tumor cells were intravenously inoculated into NSG mouse. The tumor burden (A-B) and mice survival rate (C) were analyzed. 3 × 10⁵ B16-OVA-USP18 or B16-OVA-GFP tumor cells were intravenously inoculated into C57BL/6 mice with or without depletion of CD8⁺ or CD4⁺ T cells, or NK1.1⁺ NK cells, and the tumor burden in the lung was monitored (D-E). 3 × 10⁵ B16-OVA-GFP, or B16-OVA-USP18 tumor cells were intravenously inoculated into C57BL/6, or Ifng^-/- B6 mice. Tumor growth was monitored (F-G).

Figure 4

USP18 expression in tumor modulates immune cell population and phenotype. C57/BL6 mice received intravenous inoculation of 3 × 10⁵ B16-OVA-USP18 or B16-OVA-GFP tumor cells. 2 weeks later, T cell, CD11b⁺ myeloid cell and NK cell infiltration in the lung was analyzed by flow cytometry (A). Antigen-specific OT-1 tetramer-positive CD8⁺ T cells in the lung draining lymph nodes (DLN), lung, and spleen were analyzed (B). PD-1 expression on CD8⁺ T cells (C) was analyzed. NKG2D expression on NK cells in tumor microenvironment was monitored by flow cytometry (D). ISG15 levels in B16-OVA-GFP, B16-OVA-USP18 or B16-OVA-shUSP18 tumor lysate was analyzed by ELISA (E). CD11c⁺ dendritic cells were isolated from B16-OVA-GFP, or B16-OVA-USP18, and B16-OVA-shUSP18 lung melanoma and cultured in vitro with or without LPS. Dendritic cell maturation and cytokine were assayed by flow cytometry and ELISA, respectively (F-G).

Figure 5

Immune suppression by tumor cells is regulated by USP18 expression. B16-OVA-GFP or B16-OVA-USP18 tumor cells without (A-B) or with IFN-γ sensitization (5 ng/ml) (C) were irradiated and cocultured with naïve OT-1 T cells. Cell proliferation (A) and IL-2 and IFN-γ secreteion (B-C) of the T cells were analyzed. PD-1 expression in cocultured OT-1 cells was monitored by flow cytometry (D). The CTL activity of activated OT-1 cells against B16-OVA-GFP and B16-OVA-USP18 was analyzed (E). B16-OVA-USP18 cells with or without IFN-γ (10 ng/ml) sensitization were irradiated and cocultured with naive OT-2 T cells for 72 hrs, and OT-2 T-cell proliferation (F) and cytokines IL-2 and IFN-γ secreteion (G) were analyzed.

Figure 6

Inhibition of USP18 activity in tumor cells compromises antigen-specific CTL activity. CD45.1⁺ C57BL/6 mice received intravenous inoculation of 1 × 10⁶ B16-OVA-shCon and B16-OVA-shUSP18 tumor cells. At day 8, 5 × 10⁶ activated CD45.2⁺ OT-1 CTLs were intravenously injected into tumor-bearing mice. On day 10, the mice were sacrificed; CD45.2⁺ CTLs in lung, lung draining lymph nodes, and spleen were analyzed for OT-1 tetramer expression on CD8⁺ T cells (gating on CD8⁺ cell population) (A-B), and IFN-γ and TNF-α (C), and PD-1 and KLRG1 (D).

Figure 7

Combination of Ad-USP18 and antigen-specific CTLs in subcutaneous B16 melanoma therapy. 1 × 10⁶ B16-OVA-GFP or B16-OVA-USP18 cells were subcutaneously inoculated into C57BL/6 mice (n = 5). Tumor MHC class-I and PD-L1 expression was analyzed by flow cytometry (A). Antigen-specific CTLs in the spleen and tumor were analyzed as OT-1 tetramer-positive CD8⁺ T cells (B). 1 × 10⁶ B16-OVA cells were subcutaneously inoculated into CD45.1⁺ C57BL/6 mice. At day 5, tumor-bearing mice were treated with 5 × 10⁶ activated CD45.2⁺ OT-1 cells (intravenous) and intra-tumor injection of Ad-GFP or Ad-USP18 (10¹⁰ pfu in 50 μl). B16-OVA tumor growth was monitored. (C). Effector CD45.2⁺ CD8⁺ T-cell persistence was measured 15 days after tumor inoculation (D). 1 × 10⁶ B16 cells were subcutaneously inoculated into C57BL/6 mice. The tumor-bearing mice were treated with 5 × 10⁶ activated pmel-1⁺ CTLs (intravenous) along with intra-tumor injection of Ad-GFP or Ad-USP18 (10¹⁰ pfu in 50 μl) with or without dendritic cell vaccination. B16 tumor growth was monitored after CTL and Ad-USP18 treatment (E).