Discussion
IL-35 has been reported to have a role in liver disease, including viral hepatitis, hepatic fibrosis, liver cirrhosis, chemical-induced liver injury, as well as HCC [10, 14-18]. Additionally, a large amount of HCC is associated with impairment of the proliferation, cytokine production, and cytotoxic effector functions accompanied by a chronic hepatitis virus-specific T cell immune reaction [19]. In this study we found large amounts of IL-35 in HCC patient serum and tumor tissues, which indicated that IL-35 might be involved in HCC initiation and progression. Thus, a better understanding of the relationship between the level of IL-35 expression and the anti-tumor immune changes seemed necessary.
The immune cells and secreted cytokines present in the tumor microenvironment contribute to tumor progression or active repression of tumor development. A total of 1270 DEGs were detected in the tumor and non-tumor tissues. Through online IPA pathway analysis we found that most of the overlapped DEGs were involved in T cell immune activities. Based on our previous work in which over-expressed IL-35 was shown to exert strong inhibition on CD4+T cells through Tregs-derived IL-10, we determined the levels of Foxp3 and IL-10 expression to explore the changes in Tregs [20]. We found that there were higher Foxp3 and IL-10 expression in IL-35 high group cases, which suggested that IL-35 was positively correlated with Tregs frequency and function.
Peripheral and tissue resident CD8+T cells have roles in the anti-tumor effect and IL-35 suppressed cytotoxicity and pro-inflammatory cytokine secretion [21]. Hacohen et al. (17) developed cytolytic activity as a quantitative measure of the activity of tumor-infiltrating lymphocytes, especially CD8+T cells, based on two key effector genes (GZMA and PRF1[22]. In this study we calculated the index “CYT” and detected the level of IFN-γ expression to reflect the cytotoxic status and anti-tumor cytokine secretion capability between the two compared groups. Our results showed that both the cytolytic activity and IFN-γ secretion were significantly decreased when there was a large amount of IL-35 present in the liver microenvironment.
In the past few years, immune inhibitory checkpoints have been increasingly recognized as important elements in the immunosuppression of chronic inflammation and tumor immunity. There are studies that have shown Tregs-derived IL-35 induce CD8+T cell exhaustion that limits effective anti-tumor immunity [13]. We checked the expression of T cell inhibitory receptor to reflect the intratumoral T cell exhaustion status. Our results revealed that both PD1 and LAG3 are highly expressed in the IL-35 high group samples, which indicated that IL-35 might promote inhibitory receptor induction and exhaustion.