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Chronic HBV infection afflicts 350 million people worldwide and causes 1 million deaths annually26. Effective control of chronic HBV infection likely requires the generation of effector T cells in the secondary lymphoid organs and a robust CTL response in the liver. Due to practical reasons, previous studies mainly focused on studying HBV-specific CD8+ T cells from the peripheral blood and livers of CHB patients. These studies suggested several possible causes for the paucity of HBV-specific CD8+ T cells in the peripheral blood and livers of CHB patients, including impaired proliferation and enhanced apoptosis9, 11, 17, 18. However, these studies did not address the ongoing anti-HBV T cell response in the lymphoid organ and the signature of these effector T cells. Addressing this question is critical to understand the failure of the host anti-viral response in chronic HBV infection.
By studying CD8+ T cells from the spleens, peripheral blood, and livers of the same group of CHB patients, we have made several findings. First, we found that HBV-specific CD8+ T cells are generated in the spleens of CHB patients. However, they have impaired capacity to expand after stimulation. Second, we have identified miR-720 as a key regulator of CD8+ T cell proliferation by targeting the expression of the cell cycle regulators FOSB and c-Myc. Third, we have identified TCR signaling and TGFβ as stimuli of miR-720 expression in T lymphocytes and demonstrated that the expression of miR-720 in CD8+ T cells is strongly correlated with the treatment outcome of CHB patients. Taken together, our results suggest that upregulation of miR-720 in CD8+ T cells may play an important role in the development of chronic HBV infection: it inhibits antigen-specific CD8+ T cell expansion in secondary lymphoid organs, leading to insufficient antigen-specific CD8+ T cells migrating to the liver. The effects of miR-720 on the proliferation of HBV-specific and total CD8+ T cells during HBV infection may result in the transition from acute hepatitis B to persistent infection and hepatitis.
Our results suggest that upregulation of miR-720 in HBV-specific T lymphocytes plays a critical role in host immunity during chronic HBV infection. Our data are consistent with a model in which HBV-specific CD8+ T cells are activated in the spleen by HBV antigens, resulting in miR-720 upregulation. The elevated miR-720 expression is further sustained by high levels of TGFβ, thus preventing the generation of sufficient antigen-specific effector T cells.
Our results indicate that miR-720 is an important regulator of T cell proliferation. Primary T cells from healthy donors contain ~200 copies of miR-720 per cell, whereas total CD8+ T cells and HBV-specific CD8+ T cells from CHB patients contain ~500 and ~1500 copies of miR-720, respectively. When we overexpressed miR-720 in normal T cells at a level of ~500 copies per cell, T cell proliferation was impaired. Furthermore, treatment of primary T cells with miR-720 antagomir promotes their entry into the cell cycle. miR-720 regulates T cell proliferation by targeting two known cell cycle regulators, AP-1 and Myc. A previous study suggests that cooperation of NFAT and AP-1 is important for T cell activation and proliferation27. Our microarray data show that NFAT expression is intact; however, AP-1 expression is reduced. Moreover, FOSB silencing inhibits the proliferation of human primary T cells. These data support the idea that miR-720 regulates T cell exhaustion partially by altering the NFAT:AP-1 balance. Importantly, c-Myc expression is also suppressed by miR-720 during T cell proliferation. In T cells, c-Myc is induced upon T cell activation and drives T cells into the cell cycle28. Thus, miR-720 regulates the cell cycle at both early and later stages during T cell activation.
Our data suggest that the elevated expression of miR-720 in HBV-specific CD8+ T cells may be caused by a combined signal from TCR engagement and TGFβ signaling. Both viral antigens and TGFβ have been implicated in CD8+ T cell functional exhaustion in chronic viral infections19, 29, 30. In mouse LCMV chronic infection, TGFβ signaling mediates virus-specific CD8+ T cell deletion and viral persistence. In chronic HBV infection in humans, TGFβ1 signaling downregulates activating NK receptor expression and may contribute to HBV persistence31. Our results provide important insights into the roles of viral antigen and TGFβ in causing T cell exhaustion: these signals also upregulate miR-720 expression, leading to repressed expression of FOSB, and c-Myc and impaired proliferation of effector CD8+ T cells.
Clinically, nucleotide analogues and IFN-α are used to treat HBV infection25. Nucleotide analogues can efficiently inhibit viral replication; however, they also induce HBV DNA mutation, drug resistance, and poor HBV e antigen (HBeAg) seroconversion26. IFN-α exerts its anti-HBV effect through specific and non-specific antiviral immune responses32. Despite the comprehensive application of IFN-α treatment to chronic HBV infection, the outcome of this treatment is unpredictable. Our data indicate that miR-720 expression and plasma TGFβ levels are associated with the outcome of IFN-α treatment and suggest that together, miR-720 expression in CD8+ T cells and plasma TGFβ levels could be used as biomarkers for IFN-α treatment outcome prediction in CHB patients. In summary, our findings provide evidence that miR-720 plays a key role in HBV-specific T cell exhaustion during chronic HBV infection. Hence, targeting miR-720 may be a novel strategy to reverse T cell exhaustion and alleviate liver damage. |
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发表于 2015-7-29 22:49
