Baseline characteristics of the study population
To study viral and immune correlation in the different CHB disease phases, we carefully selected a homogeneous cohort of untreated chronic HBV infected patients, without any other comorbidities, attending our outpatient clinic. To rule out the impact of advanced liver fibrosis on any identified immune parameter, patients with advanced fibrosis (F2 fibrosis or higher) were excluded. Typical for natural history of CHB patients, IT patients were youngest among the patient cohort. Owing to the stringent definition criteria, differences in age, ALT and HBV DNA levels were observed between clinical phases. Unlike some recent reports, qHBsAg level in this study was higher in GZ patients than IT patients [22, 23] (Table 1).
Cytokines profiles in CHB patients with different stages
To investigate whether CHB patients beyond current treatment criteria are characterized by a state of defective antiviral response, we analysed the expression profiles of three major effector cytokines, IFN-γ+, TNF-α+ and IL-2+, produced by innate and adaptive immunity. The representative dot plots and gating strategies of flow cytometry analysis for T cells, NK and NKT cells derived cytokines were shown in Supplementary Figure 1. We first analysed their T cells derived cytokines profiles and compared them with those in healthy controls. As expected, both frequencies of IFN-γ+ and TNF-α+ by CD4+ and CD8+ T cells were statistically significant higher in IA patients than IT patients. Similar result was found in frequency of IL-2+ by CD4+ T cells in current CHB patient cohort. There were no significant different distributions of these cytokines profiles found among IA, IC and GZ patient (Figure 1A).
We also measured the frequencies of cytokines by NK and NKT cells in current CHB cohort. As expected, statistically significant differences were observed in frequencies of NK and NKT cells secreting IFN-γ+, with progressive decrease level from patients of IA, IC, GZ, and to IT. The differences between the frequencies of IFN-γ+ and TNF-α+ by NKT and NK cells, respectively, in the patients of IA and GZ were not statistically significant. However, the frequencies of IFN-γ+ produced by NKT, NK cells and TNF-α+ produced by NK cells of patients in IA phase were all higher than those in IT phase (P = 0.004 and 0.0008 for IFN-γ+ by NKT and NK cells in IA vs IT; P = 0.004 for TNF-α+ by NK cells in IA vs IT, respectively, Figure 1B).
Taken together, these results indicate that a certain number of CHB patients beyond the current treatment guidelines, particularly, patients in GZ phase, still produce antiviral cytokines.
Distribution of distinct cytokine profiles in CHB with different disease phases
Because clinical-virological features from patients with CHB had association with TNF-α+, IFN-γ+ and IL-2+ by T cells and NK cells, respectively, we then assessed whether their combined evaluation could be used to identify maturation of an efficient antiviral response to therapy in individual treatment-naïve CHB patient. We firstly investigated the correlation among current 3 pairs of T-cell subsets cytokines and 2 pairs of NK and NKT cell cytokines. The overall correlation among these 10 cytokines was shown in Figure 2A. After correlation analysis, expressions of 6 cytokines (CD4_IFN-γ+, CD4_IL-2+, CD8_TNF-α+, CD8_IL-2+, NK_IFN-γ+, NKT_TNF-α+) were selected to construct an IA-similar cytokines profiles. The assumption was that acquisition of an IA-similar cytokines profiles could reflect a vigorous response to antiviral therapy. A threshold was thus established as shown by the mean value found in IA patients plus one standard deviation for the above selected parameters, and calculation of their expressions in individual patient was conducted to compare with each matched threshold. Individual cytokines distribution profile was distinguished according to the altered number of applicable parameters beyond the threshold for all patients. A profile with all the applicable parameters altered was assumed to reflect immune active response to therapy whereas the one with no applicable parameters altered was predicted to be associated with an awakening response to therapy. IA and IC with active immune response to HBV showed a prevalent expression of more inflammatory patterns with 6 and 4 altered applicable parameters respectively. In contrast, IT patients showed an immune depletive pattern with only 2 altered application parameters. GZ patients instead showed an intermediate behavior with 5 altered applicable parameters, as a likely result of the transition from an immune depletive to an inflammatory pattern of typical IA patients (Figure 2B). Based on Spearman’s rank correlation analysis, CD4_TNF-α+, CD8_IL-2+ and NK_IFN-γ+ were selected to be the representative cytokines in current CHB cohort for principal component analysis, which further confirmed that most IT patients significantly differed from IA and IC patients (red, blue and black circles, respectively), both of who clustered homogeneously and an intermediate distribution was observed for GZ patients who were widely scattered (green circles, Figure 2C).
Association among T-cell secreting cytokines and correlation with clinical-virological characteristics
The linear regression analysis was used to examine the association between T cells producing cytokines and clinical-virological parameters. Univariate analysis revealed that positive HBeAg, higher levels of ALT and HBV DNA were associated with increased level of CD4+ T cells secreting TNF-α+. Older age and higher ALT level were associated with more proportion of IFN-γ+ by CD4+ T cells, while IL-2+ by CD4+T cells was linked to the increased HBV genotypes. After adjusting for other confounding factors, multivariate analysis revealed that both higher ALT level and older age were significantly associated with increased IFN-γ+ and TNF-α+ produced by CD4+T cells (Table 2)
Univariate analysis of relationship between CD8+ T cells derived cytokines and clinical-virological factors showed age and ALT were associated with both TNF-α+ and IFN-γ+ by CD8+ T cells, respectively. Multivariate analyse indicated older age and high ALT were still associated with increased IFN-γ+, while only ALT was significantly related to the higher TNF-α+ by CD8+ T cells. There was no statistically significant association between IL-2+ by CD8+ T cells and viral parameters (Table 2).
Therefore, IFN-γ+ by either CD4+ or CD8+ T cells had significantly associated with older age and higher ALT, while TNF-α+ from these T cells subsets was associated with ALT.
Association among NK and NKT-cell secreting cytokines and correlation with clinical-virological characteristics
Similarly, the linear regression analysis was used to examine the association between clinical-virological factors and NK or NKT-cell expressing cytokines in current CHB cohort. Univariate analysis on the NK-cell cytokines profiles showed more frequencies of IFN-γ+ and TNF-α+ were correlated with higher ALT level, while only TNF-α+ was also associated with HBV DNA. Multivariate analysis also showed similar results regarding the association between cytokines and ALT (Table 3). We also detected cytokines produced by a subset of T cells that express NK cell markers, NKT cells. HBeAg, ALT, and HBV genotype were found to be associated with NKT secreting TNF-α+ via univariate analysis (Table 4).
In summary, multivariate analysis from 10 clinical-virological parameters and 10 cytokines implied that ALT was statistically significant association with 8 cytokines from T cells and NK cells. Age and HBV DNA had association with 2 cytokines, while gender was correlated with only one cytokine frequency (supplementary figure 2).