TGF-β1Down-regulation Of NKG2D/DAP10and2B4/SAP Expression On Human NK Cells Contributes To HBV Persistence

The mechanism underlying persistent HBV infection remains unclear. The course of HBV infection is complicated. There are three phases of chronic HBV (CHB) infection that are now widely accepted:"Immune Tolerant"(IT) phase;"Immune Active"(IA) phase and inactive (IN) phase. During the IT phase of CHB infection, the virus evolves strategies to evade immune clearance in the majority of patients. Previous investigations have demonstrated that NK cells may be particularly important in patients with CHB. However, the tolerance mechanism of the IT phase and the contribution of NK cells to the control of HBV persistence infection are unclear.NK cells have been viewed as the most important effectors of the initial antiviral innate immune response. Their activation depends on the integration of signals from "co-activation" receptors, and the cytotoxic effects of NK cells on target cells are tempered by a need for combined signals from multiple activating receptors, such as NKG2D and2B4. We investigated the role of innate immune responses to persistent HBV infection in154HBV-infected patients and95healthy controls. The expression of NKG2D-and2B4-activating receptors on NK cells was significantly decreased, and moreover, the expression of DAP10and SAP, the intracellular adaptor proteins of NKG2D and2B4(respectively), were lower, which then impaired NK cell-mediated cytotoxic capacity and interferon-γ production. Higher concentrations of transforming growth factor-beta1(TGF-β1) were found in sera from persistently infected HBV patients. TGF-β1down-regulated the expression of NKG2D and2B4on NK cells in our in vitro study, leading to an impairment of their effector functions. Anti-TGF-β1antibodies could restore the expression of NKG2D and2B4on NK cells in vitro. Furthermore, TGF-β1induced cell-cycle arrest in NK cells by up-regulating the expression of p15and p21in NK cells from immunotolerant (IT) patients. We conclude that TGF-β1may reduce the expression of NKG2D/DAP10and2B4/SAP, and those IT patients who are deficient in these double-activating signals have impaired NK cell function, which is correlated with persistent HBV infection. Collectively, these findings may contribute to our understanding of the immune tolerance mechanism and aid in the development of novel therapeutic methods to clear HBV infection during the initial phase.Here, we provide evidence demonstrating that high levels of TGF-β1may reduce the expression of NKG2D/DAP10and2B4/SAP, these IT patients deficient in double-activating signals have impaired the function of NK cell, which was correlated to persistent infection of HBV. There are several key points in this study.(1) We provide evidence of down-regulation of NKG2D/DAP10and2B4/SAP in NK cells in IT phase patients. We observed that NKG2D and2B4expression were decreased on circulating NK cells during IT phase HBV infection, but not in other-phase patients. We further demonstrated that lower levels of intracellular adaptor proteins were associated with lower surface expressions of NKG2D and2B4, which implied that the NK cell activation signalling pathway might be impeded.(2) We provide evidence that the synergistically co-activated signalling pathway initiated by NKG2D plus2B4does not operate properly in IT phase patients. We further demonstrated down-regulation of the Ca2+mobilisation triggered by NKG2D and2B4receptor synergy in primary NK cells from the patients in the phase we designated IT. Moreover, there was a significant reduction in the production of IFN-γ by NK cells from patients in the IT phase compared to healthy controls and patients in the IA phase. These observations indicate that the incomplete function of NK cells in the IT phase may contribute to persistent HBV infection.(3) We provide evidence that anti-TGF(31antibodies partially restores the expression of NKG2D and2B4on NK cells and the potential function of NK cells in patients with persistent HBV infection. Using a cytometric bead array (CBA) inflammation kit and ELISA technology, we found that high levels of soluble TGF-β1were associated with the reduction of NKG2D and2B4in patients with IT phase. Furthermore, anti-TGF-β1partially restored the expression of NKG2D and2B4and also restored the Ca2+flux on NK cells when co-cultured with sera from IT Phase patients.(4) We provide evidence that TGF-(31causes cell-cycle arrest of NK cells by up-regulating the levels of p15and p21in NK cells from IT patients. We demonstrated that high levels of TGF-β1in patients with IT phase could facilitate cell-cycle arrest in NK cells. In addition, we observed relatively high levels of Smad-2P, Smad2and p15on NK cells from IT phase patients. Altogether, these data demonstrate that TGF-(31signalling is responsible for the insufficiency in NK cell phenotypes and performance in IT phase patients, and NK cells found in patients facilitate persistent HBV infection. Our study provides a basis for improving current therapies for IT patients by blockade of TGF-β1inhibitory pathways, which could result in an additive efficacy in eliminating virus at the initial phase of CHB infection.