In this study, we gained an in-depth understanding of the intrinsic innate and adaptive immunity, and their ICs expressions in CHB patients at different stages. The results showed that compared with HC, CHB patients retained their innate antiviral cytokines immunity, the ability of CD4 + T cells to secrete antiviral factors was enhanced, while that of CD8 + T cells was weakened. CHB patients had weakened innate immune NK cells cytotoxicity, while the cytotoxicity of NKT cells and adaptive immune cells was enhanced. Compared to IA patients, although adaptive immunity of GZ and IT was impaired, their innate antiviral cytokines immunity was decreased while their cytotoxicity function was increased. We further found that ICs were expressed differently on innate and adaptive immune cells in CHB patients at different clinical stages and related with their immune function. Our results implied the possibility of innate immune cells via ICs regulating adaptive immunity.
Our previous research has shown that almost half of patients who do not meet treatment guidelines had high levels of antiviral cytokines production by NK cells. NK cells immunity existed in different natural history stages of untreated chronic HBV infected patients, and CHB patients in the IT and GZ phases exhibited partially preserved NK cells immunity and antiviral activity [21, 24]. The preservation of NK cells lysis ability is an important aspect of NK cells antiviral function, which is necessary for eliminating HBV cccDNA and solving chronic infections [25, 26]. In this study, compared with IA, GZ and IT patients showed a decrease in their adaptive immune antiviral cytokines secretion and cytotoxic function (T cells). However, the cytotoxic function of innate immunity (NK and NKT cells) was preserved or even elevated, and the ability to secrete antiviral cytokines was preserved in GZ's NKT cells. The results indicate that the cellular functions of NK and NKT in the IT and GZ subgroups of CHB patients exhibit the functional dichotomy and they also retained some ability to clear HBV in their bodies in addition to IA patients, which were previously proposed by other scholars [21, 24, 27]. In four stages of CHB patients, in IA patients, NK cells transit towards more NKbright cells and NKbright cells specifically release cytokines [28], which is the main subtypes of NK cells found in inflammatory lesions [29–31]. Consistent with other researches, our results suggest that the definition of IT and GZ status of CHB patients based on clinical parameters may not accurately reflect their internal immune status and should be reconsidered. Moreover, some researches has reported evidence of histological activity or even fibrosis in liver biopsies of patients with sustained normal ALT levels and high HBV DNA levels, indicating the complexity of defining host immune responses to viruses and the limitations of disease classification based solely on serological or biochemical markers [21, 24, 32]. Since GZ and IT patients may have disease progression (hepatitis activity, liver fibrosis, cirrhosis, liver cancer), it is necessary to strengthen follow-up and even start antiviral treatment as soon as possible.
ICs are the important regulators of host immunity and contribute to the development of the pathogenesis in patients with chronic HBV infection [14, 33–36, 37–39]. The role of PD1 may be different in various viral entities [40–43], and it has functional duality during chronic viral infection [44]. Tim3 seems to have a dual role in tuberculosis infection [45], and it has obvious inhibitory effect on CD 4 + and CD 8 + T cells [46]. However, their roles of innate and adaptive immune cells in control HBV infection are not fully understood. In our study, the expressions of PD1 and Tim3 were upregulated in innate immune cells of patients with chronic hepatitis B, while the expressions of different ICs on adaptive immune cells were differential. Compared to IA patients, the ICs levels of NK cells in GZ and IT patients were similar, the expressions of Tim3 on NKT cells in GZ and IT patients were significantly increased, while the expressions of PD1 on T cells in these two groups were significantly increased, and the expressions of Tim3 were significantly reduced in the IT group. Our findings revealed that ICs were expressed differently on innate and adaptive immune cells in CHB patients at different clinical stages. Moreover, among those ICs, NK cells mainly expressed different levels of PD1 and Tim3, and T cells mainly expressed different levels of PD1 and LAG3. A system evaluation showed that the expression levels of ICs on CD8 + T cells in patients with chronic hepatitis B infection and some results of PD1 expressions are inconsistent with our study[47]. CD4 + T cells also showed some different results compared to other studies[48]. The reason for the above inconsistency may attribute to the number and status of patients in this study. Furthermore, our correlations analysis found that the expressions of PD1 on NK cells were positively correlated with their TNFα levels, while the expressions of Tim3 on NK cells and NKT cells were positively correlated with their cytotoxic molecules.In summary, our findings indicate that the different innate and acquired immune states in CHB patients at different stages are related to the expression levels of ICs, mainly determined by the expressions of PD1 and Tim3.
In addition, the innate immune cells can affect the infectious disease progression via regulating T cells immunity. However, the mechanism remains unclear. NK cells can actively influence the responses of CD4 + T and CD8 + T cells to various antigens through cells lysis, production of cytokines and chemokines and altering antigen presentation [49–53]. Our study showed a negative correlation between NK cytotoxic functional molecules(GranzymeB and Perforin) with CD4 + T cytotoxic functional molecules (Perforin, CD107a) and antiviral cytokine (TNFα), suggesting that NK cells may inhibit CD4 + T cells immune function through cytotoxic functional molecules.There is also research indicating that NKT cells play a crucial role in activating adaptive responses [54], and NKT cells may be an important checkpoint in HBV control [14]. In the absence of NKT cells activation, the adaptive immune and HBV inhibitory effects are greatly weakened [54]. In this study, the expressions of Tim3 in NKT cells were negatively correlated with the CD8 + T toxic function(Perforin), indicating that Tim3 expressions on NKT cells possibly have a negative regulatory effect on CD8 + T cells and simultaneously cause a decrease in toxic function, which is consistent with previous researches [14]. The expressions of PD1 in NK and NKT cells were positively correlated with TNFα levels on CD4 + T cells, while Tim3 levels of NKT cells were negatively correlated with TNFα levels on CD4 + T cells, and Tim3 levels on NK and NKT cells were negatively correlated with CD4 + T cells toxicity. The negative regulation of Tim3 is consistent with literature reports [46]. The positive correlations between PD1 expressions on innate immune cells and CD4 + T cells function may be related to the expressions of these co-inhibitory receptors in other immune cells and the complex interactions and regulation between immune cells. At present, it is difficult to cure hepatitis B with existing treatments, which mainly attributing to the dysfunction of host immunity. Therefore, there is an urgent need for new strategies to enhance the antiviral immune response to treat CHB infection. HBV immunotherapy aims to inhibit HBV replication in the long term by reactivating the host immune response [17]. It can target both innate and adaptive immunity, restore exhausted immune responses and significantly improve patient prognosis. The restoration of T cells immunity through immune regulation has become an active research field, and immunotherapy to enhance the immune response to chronic HBV infection is currently in the preclinical development stage [55]. This study provides clues for the potential targets of immunotherapy based antiviral therapy that NKT cells negative regulation of T cells immune function by Tim3.
Our research has some limitations. Firstly, the sample size and types of ICs studied are relatively small. We can collect more multi-center samples and detect more ICs in the future study to strengthen our findings' authority. Secondly, our study is a cross-sectional study, and all of our samples were obtained at a single time point. In the future, our longitudinal queue can be used to observe the dynamic changes of immunity, ICs expressions and their impact on disease progression or antiviral efficacy.