Eradication of immune destruction is a hallmark of cancer, which is primarily due to the abnormal activation of immune checkpoints and the termination of antitumor immune response. Programmed cell death (PD-1)/programmed death ligand 1 (PD-L1) have been demonstrated as a pair of major immune checkpoint molecules and valuable therapeutic targets for melanoma treatment.1 For instance, the binding of membrane PD-L1 on tumor cells to PD-1 on T cells evokes an immunosuppressive signal that results in the dysfunction and even the apoptosis of cytotoxic T cells, thereby impairing antitumor immunity.2 Therefore, PD-1/PD-L1 blockade is in a position to disrupt the interaction between tumor cells and T cells, which restores tumor-specific immune response.3 For melanoma patients receiving anti-PD-1 antibody monotherapy, the median overall survival could be up to 32.7 months after almost 5 years of follow-up.4 However, a proportion of 40%–60% of patients does not achieve any significant therapeutic response, and some patients even show complete resistance to PD-1/PD-L1 blockade.5 Accumulative evidence has revealed multiple primary and acquired alterations leading to treatment resistance of immunotherapy, including low mutational burden, defective antigenicity and antigen presentation of tumor cell and genomic dysregulation of interferon-γ (IFN-γ) signaling pathway.6–9 These reports have prompted potential molecular profile for predicting the treatment response and effective synergized therapeutic strategy for cancer immunotherapy. Notably, tumor cells can adaptively increase the expression of PD-L1 or other immune checkpoints under the control of IFN-γ after the treatment with anti-PD-1 antibody, rendering melanomas refractory to immunotherapy.10–12 Hence, the molecular mechanism underlying aberrant tumorous PD-L1 expression associated with the resistance to immunotherapy in melanoma is in urgent need to be clarified to improve the therapeutic efficacy.

Inflammation is closely related to the development of various cancers including melanoma, with its pivotal role in the regulation of cancer immune response demonstrated recently.13–15 To be specific, tumor necrosis factor-α (TNF-α) that is a common proinflammatory cytokine and its downstream pathway could induce tumor immune escape by elevating the expressions of immune checkpoint molecules.13 16 On the other, TNF-α is also one of the major cytokines secreted by activated CD8⁺ T cells in tumor microenvironment that potentiates antitumor immune response.17 18 These reports highlight the vital impact of inflammatory signaling on antitumor immunity, though the actual function remains controversial. A20 is a primary responsive gene of TNF-α and is documented as a crucial negative mediator of inflammation as well as immune response in multiple immune cells.19 As a ubiquitin-editing enzyme, it is composed of an N-terminal OTU domain with deubiquitinase activity and seven Cys2-Cys2 zinc finger C-terminal domains functioning as a ubiquitin ligase.20 The cooperative activity of these two ubiquitin-editing domains mediates the negative regulatory role of A20 in NF-κB signaling,20 so that the genetic deficiency of TNFAIP3 that encodes A20 protein can result in the onset and progression of multiple autoimmune diseases by amplifying the pro-inflammatory NF-κB signaling.21 For cancer pathogenesis, previous investigations emphasized on A20 expressed in tumor-infiltrating immune cells to clarify its effect on antitumor immunity. In B16 mouse melanoma tumor model, silencing of A20 in dendritic cells (DCs) enhanced NF-κB activity followed by elevated expression of interleukin 6 (IL-6), TNF-α and IL-12, leading to potentiated antitumor immune responses.22 In addition, tumor-infiltrating CD8⁺ T with the deletion of A20 had stronger antitumor capacity by relieving the brake on NF-κB signaling pathway.23 Of note, A20 is also abundantly expressed in tumor cells and can directly influence their biological behaviors.24 25 Therefore, A20 expressed in immune cells and tumor cells in tumor microenvironment are both greatly implicated in the pathogenesis of cancer. Previous studies regarding antitumor immunity mainly focused on the role of A20 that is expressed in immune cells. However, whether tumorous A20 can affect the function of tumor-infiltrating immune cells and the therapeutic effect of immunotherapy remains unknown.

In the present study, A20 was initially identified to be highly related with the effect of anti-PD-1 immunotherapy among melanoma patients. Preclinical melanoma mouse model and in vitro coculture system were then established to evaluate the therapeutic effect of anti-PD-1 immunotherapy with synergized suppression of tumorous A20 expression. Subsequently, the potential mechanism of A20-mediated resistance to immunotherapy was further investigated by bioinformatics, mass spectrum analysis and a set of biochemical analyzes, with a particular emphasis on the regulation of PD-L1 expression. Furthermore, the relationship between tumorous A20 expression and the invigoration of circulating exhausted-phenotype CD8⁺ T cells after anti-PD-1 antibody treatment was analyzed in melanoma patients.

A detailed description of the methods used in this study is available in online supplemental methods.