HBV is a double-stranded DNA virus that can cause acute or chronic liver infection in humans. The antigen cellular immune response plays an important role in the pathogenesis of HBV infection and the clearance of HBV, as has been emphasized in the literature [7
CTLs can kill the hepatitis B virus via cytolytic and non-cytolytic pathways, but they are also the main effector cells for liver injury. In acute HBV infection, HLA class I-restricted CTLs respond specifically to antigen epitopes of the viral membrane, capsid and polymerase proteins; thus, multi-epitope- and multi-clone-specific CD8+
CTLs can be detected. On the other hand, the number of specific CD8+
CTLs in the periphery in patients with chronic hepatitis B is very small. Nakamoto et al. [8
] found that in AHB patients with normal immune function, a substantial number of HBV particles are cleared before the liver cells are injured, but a certain amount of the virus still replicates in liver cells, leading to the induction of cytolytic mechanisms and liver cell injury. Thus, HBV is eventually completely cleared, although short-term liver injury can be seen, which is clinically manifested as self-limited AHB. Guidotti et al. [9
] studied two chimpanzee models with HBV infection and found that HBV DNA in the serum and liver disappears before the peak in serum transaminases. In the current study, we found that HBcAg-specific CTLs can be detected in the peripheral blood of AHB patients even in the early stages, and their frequency changes dynamically. During hospitalization, HBV CTLs frequencies in AHB patients were significantly higher than those in CHB patients and healthy controls. When serum ALT levels were high, HBV DNA levels decreased significantly. Among AHB patients, 26.4% had negative serum HBV DNA before admission; 50.9% had negative serum HBeAg; and 17.0% had negative serum HBsAg. These findings suggest that serum HBV levels were suppressed or partially cleared before liver transaminase levels became elevated. Meanwhile, peak levels of peripheral blood epitope-specific CTLs in AHB patients occurred in the second week after admission, when the ALT levels had significantly decreased. Peak levels of HBV epitope-specific CTLs appeared later than serum ALT peak levels and lagged behind serum HBsAg and HBV DNA peak levels; furthermore, the higher the frequency of peripheral blood HBV-specific CTLs, the earlier of the antigenic diversion of HBeAg, suggesting that the high frequency of specific CTLs in AHB patients may have played an important role in clearance of the virus. However, the frequency of specific CTLs in AHB patients was not synchronous with liver cell injury or serum HBV clearance, in other words, clearance of the virus occurred earlier than liver cell injury, suggesting that not only cytotoxic effects but also non-cytotoxic effects might be involved in the virus clearance of AHB patients.
Our study indicates that not only was the frequency of specific CTLs significantly higher in AHB patients than in CHB patients, but also the response ability of HBV antigen epitope-specific CD8+ was higher in AHB patients (p < 0.05). The ability of different antigens to stimulate specific CTL to produce INF-γ was different; the pattern observed was as follows: Core18-27> Env348-357> Pol575-583, which may be related to the different immunogenicity of the different regions of HBV antigens [10
]. The level of HBV Core18-27-specific response was significantly higher in the acute phase in AHB patients compared with those in convalescence (p < 0.05), which proved that this antigen epitope might be the main target site for host immune clearance of virus [11
In addition to the characteristics of the specific cell-mediated immune response observed in this study, the non-specific cellular immune response had its own features. The total numbers of peripheral blood cytotoxic T lymphocytes (CD3+CD8+) in AHB patients in the early stage of infection were higher than in the healthy control group and among CHB patients. The peak of CD3+CD8+ cell numbers, meanwhile, appeared at the second week after admission, and the dynamic changes of this cell number were basically the same as that of HBcAg-specific CTL. A correlation was observed between the growth and decline of CD3+CD8+ cell numbers and HBcAg-specific CTL (r = 0.492, p = 0.01). These results suggest that the number of peripheral blood CD8 + lymphocytes might reflect CTLs frequency indirectly in AHB patients and was helpful to predict the prognosis of an HBV infection.
NK cells are the first line of host defense against the foreign invasion of pathogenic microorganisms [12
]. They are usually activated in the early stage of a viral infection and are particularly abundant in the liver [13
]. Activated NK cells play an important role in the recirculation of virus-specific T cells and the induction of anti-viral immunity in the liver [15
]. Previously, we compared lymphocytes in the liver and peripheral blood of patients with different types of hepatitis and found that NK cells in the liver were significantly more abundant than in the peripheral blood [16
]. This tendency shows that NK cells play an important role in local immunity in the liver. Our study analyzed peripheral blood NK cells in AHB patients and found that dynamic changes in peripheral blood NK cells were different from the trend of the frequency of HBcAg-specific CTLs. The NK cells in these patients gradually increased to the normal range as the virus was cleared in the peripheral blood and liver function recovered and the dynamic changes of peripheral blood NK cells negatively correlated with the frequency of HBV-specific CTL (r = -0.266, p = 0.05). These results suggest that NK cells play an extremely important role in the effective clearance of HBV in the early stage of infection.
NKT cells are highly heterogeneous effector cells with immune and regulatory functions [17
]. They possess the characteristics of both NK cells (CD56+
) and T lymphocytes (CD3+
]. NKT cells are very rich in the mouse liver and account for 50% of intrahepatic lymphocytes, mainly located at the hepatic sinusoids [19
]. They play an important role in the prevention of tumor metastasis and the removal of viruses [20
]. It was shown, by a mouse hepatitis model induced by Con A or LPS plus IL-12, that NKT cells could induce liver damage, and the activated NKT cells disappeared from liver [21
]. The activation of NKT cells is often accompanied by the activation of NK cells, which then release cytokines that prevent viral replication [23
]. Therefore, it is thought that NK cells might be effector cells for NKT cells [24
]. In the healthy control group, 2% of the CD3+
cells in the peripheral blood expressed CD56, whereas in the liver, this value was 30% [25
]. In our study, the number of NKT cells in the peripheral blood of AHB patients in the early stage of infection was significantly lower than in the control group, and the dynamic changes in NKT cells were basically the same as those in NK cells. Based on these findings, we suggest that NK and NKT cells accumulate in the liver and play an important role in local immunity, resulting in a lower frequency of NKT cells in the peripheral blood.