Nat Commun. 2019 Jan 15;10(1):221. doi: 10.1038/s41467-018-08096-8.
Breakdown of adaptive immunotolerance induces hepatocellular carcinoma in HBsAg-tg mice.
Zong L1,2, Peng H1,2, Sun C1,2, Li F1,2, Zheng M3, Chen Y1,2, Wei H1,2, Sun R1,2, Tian Z4,5.
Author information
1
Division of Molecular Medicine, Hefei National Laboratory for Physical Sciences at Microscale, the CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences, University of Science and Technology of China, 230027, Hefei, Anhui, China.
2
Institute of Immunology, University of Science and Technology of China, 230027, Hefei, Anhui, China.
3
Clinical Laboratory, The First Affiliated Hospital of Anhui Medical University, 230022, Hefei, Anhui, China.
4
Division of Molecular Medicine, Hefei National Laboratory for Physical Sciences at Microscale, the CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences, University of Science and Technology of China, 230027, Hefei, Anhui, China. [email protected].
5
Institute of Immunology, University of Science and Technology of China, 230027, Hefei, Anhui, China. [email protected].
Abstract
Hepatitis B virus (HBV) can induce chronic inflammation, cirrhosis, and eventually hepatocellular carcinoma (HCC). Despite evidence suggesting a link between adaptive immunity and HBV-related diseases in humans, the immunopathogenic mechanisms involved are seldom described. Here we show that expression of TIGIT, a promising immune checkpoint in tumor immunotherapy, increases with age on hepatic CD8+ T cells in HBsAg-transgenic (HBs-tg) mice whose adaptive immune system is tolerant to HBsAg. TIGIT blockade or deficiency leads to chronic hepatitis and fibrosis, along with the emergence of functional HBsAg-specific cytotoxic T lymphocytes (CTLs), suggesting adaptive immune tolerance could be broken by TIGIT blockade or deficiency. Importantly, HBsAg vaccination further induces nonresolving inflammation and HCC in a CD8+ T cell-dependent manner in TIGIT-blocked or -deficient HBs-tg mice. Therefore, CD8+ T cells play an important role in adaptive immunity-mediated tumor progression and TIGIT is critical in maintenance of liver tolerance by keeping CTLs in homeostatic balance.
Breakdown of adaptive immunotolerance induces hepatocellular carcinoma in HBsAg-tg mice
Lu Zong, Hui Peng, Cheng Sun, Fenglei Li, Meijuan Zheng, Yongyan Chen, Haiming Wei, Rui Sun & Zhigang Tian
Nature Communicationsvolume 10, Article number: 221 (2019) | Download Citation
Abstract
Hepatitis B virus (HBV) can induce chronic inflammation, cirrhosis, and eventually hepatocellular carcinoma (HCC). Despite evidence suggesting a link between adaptive immunity and HBV-related diseases in humans, the immunopathogenic mechanisms involved are seldom described. Here we show that expression of TIGIT, a promising immune checkpoint in tumor immunotherapy, increases with age on hepatic CD8+ T cells in HBsAg-transgenic (HBs-tg) mice whose adaptive immune system is tolerant to HBsAg. TIGIT blockade or deficiency leads to chronic hepatitis and fibrosis, along with the emergence of functional HBsAg-specific cytotoxic T lymphocytes (CTLs), suggesting adaptive immune tolerance could be broken by TIGIT blockade or deficiency. Importantly, HBsAg vaccination further induces nonresolving inflammation and HCC in a CD8+ T cell-dependent manner in TIGIT-blocked or -deficient HBs-tg mice. Therefore, CD8+ T cells play an important role in adaptive immunity-mediated tumor progression and TIGIT is critical in maintenance of liver tolerance by keeping CTLs in homeostatic balance.
Introduction
Chronic hepatitis B virus (HBV) infection affects more than 350 million people worldwide, despite the effective HBV vaccination among the young generation. Current antiviral treatment in the clinic is hardly effective to clear the virus1. Accumulating evidence has shown that chronic HBV (CHB) infection is an important risk factor for hepatocellular carcinoma (HCC)2,3,4. Virologists attribute HBV-mediated hepatocarcinogenesis to the integration of the viral DNA into the host DNA and oncoprotein regulatory X protein (HBx)5,6. However, it has been increasingly accepted that HBV is a non-cytopathic virus and HBV pathogenesis lies mostly in immune-mediated liver injury7,8,9,10, which triggers the development of HCC without viral transactivation, insertional mutagenesis, and genotoxic chemicals11. Despite such progress, the lack of appropriate animal models that mimic HBV-related HCC has impeded studies of immune mechanisms underlying HBV-induced HCC development.
The liver is a unique immune organ that favors the induction of immune tolerance rather than immune activation12. During CHB infection, virus-specific CD8+ T cells gradually acquire expression of numerous co-inhibitory receptors13,14,15,16, such as PD-1, CTLA-4, and Tim-317,18. Considering the contribution of immune-mediated injury in HBV pathogenesis, co-inhibitory receptors expressed by hepatic CD8+ T cells are important for preventing immune-driven pathology, but also result in CTL exhaustion and thereby limit viral clearance19,20. Blockade of co-inhibitory receptors, such as PD-1, CTLA-4, 2B4, and Tim-317,21,22,23,24, and/or activation of costimulatory signals from CD137 or OX4025,26,27, could rescue CD8+ T cell function during HBV infection, as evidenced by improved production of interferon (IFN)-γ and cytotoxic capacity of effector CD8+ T cells. On the other hand, CD8+ T cell response could also promote hepatic inflammatory development during acute or chronic virus infection7, as implied by clinical and animal studies28,29,30.
The co-inhibitory receptor T cell immunoglobulin and immune receptor tyrosine-based inhibitory motif domain (TIGIT), highly expressed on activated T cells, could inhibit T cell functions after engagement with its ligand CD155 on antigen-presenting cells31. Moreover, it has been demonstrated that TIGIT is a characteristic marker of exhausted CD4+ T32 and CD8+ T cells33 in tumor tissue, and enforces CD8+ T cell exhaustion during chronic lymphocytic choriomeningitis virus (LCMV) infection33. In the clinic, downregulated expression of TIGIT on both CD8+ T and CD4+ T cells were observed in hepatitis C virus (HCV) patients who were cured by direct-acting antivirals, suggesting a role for TIGIT in T cell dysfunction during HCV infection34. In addition, TIGIT expression on T cells correlated with disease progression induced by human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) infection35,36. Nevertheless, whether TIGIT contributes to HBV-mediated immune tolerance and HBV-related HCC has not been explored.
Here, a high expression of TIGIT was found on hepatic CD8+ T cells of HBsAg transgenic (HBs-tg) mice, which are immunologically tolerant to HBV. TIGIT blockade or TIGIT deficiency could break CD8+ T cell tolerance to the viral antigen in HBs-tg mice, leading to chronic hepatitis and fibrosis. Importantly, HBsAg vaccination in combination with TIGIT blockade or TIGIT deficiency in HBs-tg mice triggered HCC development in a CD8+ T cell-dependent manner. Thus, this study has developed a mouse model of HBV-related HCC, providing experimental evidence supporting chronic inflammation in promoting cancer and revealing unfavorable consequences of the immune checkpoint blockade.
Results
TIGIT blockade or deficiency leads to chronic hepatitis
It has been demonstrated that HBs-tg mice, whose hepatocytes continuously express HBV surface antigens and adaptive immune system is tolerant to HBV, can be used as a model for HBV carriers37,38. Given that previous studies have shown that blockade of co-inhibitory receptors could restore CD8+ T cell functions in HBV-carrier people23, we explored the consequences of blocking the TIGIT pathway in the HBs-tg mouse model. HBs-tg mice were injected weekly with anti-TIGIT monoclonal antibodies (mAb) or control rat IgG (Fig. 1a). Consistent with published reports39, young HBs-tg mice had normal serum alanine aminotransferase (ALT) levels compared to wild-type (WT) mice (below 50 U/L), while elder HBs-tg mice displayed slightly elevated ALT levels (Fig. 1b). We verified the blocking efficiency by flow cytometric analysis of TIGIT staining on NK cells and CD8+ T cells from HBs-tg mice treated with anti-TIGIT mAbs (Supplementary Fig. 1a). To further confirm that the anti-TIGIT mAb is a blocking antibody rather than depleting antibody, the absolute number of peripheral blood CD8+ T cells and NK cells were counted. It was found that there was no change after antibody treatment in vivo (Supplementary Fig. 1b). In addition, flow cytometric analysis with a different clone of TIGIT antibody showed that TIGIT+ NK cells and TIGIT+CD8+ T cells were not reduced after TIGIT blockade (Supplementary Fig. 1a), confirming that the anti-TIGIT mAb injected to mice did not induce depletion of immune cells that express TIGIT. It has been reported that, when some inhibitory pathways are ablated, there will be compensatory responses induced by increased expression of other immunosuppressive receptors40. To test this possibility, we examined expression of other important inhibitory receptors, such as PD-1, CTLA-4, Tim-3, KLRG-1, and Lag-3 on CD8+ T cells and NK cells. No changes were detected after TIGIT blockade, thus excluding the potential compensatory effects of these inhibitory receptors (Supplementary Fig. 1d). Expression of CD96, a co-inhibitory receptor sharing the same ligand CD155 with TIGIT41 also remained low on CD8+ T cells after TIGIT blockade. However, it was expressed at a lower level on NK cells in TIGIT-blocked mice compared to control mice (Supplementary Fig. 1c). The co-stimulatory molecule CD226, which competes with CD96 and TIGIT for binding to CD15541, maintained a high expression on CD8+ T cells and NK cells after TIGIT blockade (Supplementary Fig. 1c).