Viral infection parameters not nucleoside analogue itself correlates with host immunity in nucleoside analogue therapy for chronic hepatitis B

Abstract

Aim: To determine the relationship between host immunity and the characteristics of viral infection or nucleoside analogues (NAs) themselves in patients with chronic hepatitis B (CHB) receiving NA therapy.

Methods: Fifty-two hepatitis B envelope antigen (HBeAg) positive CHB patients were enrolled and divided equally into two groups. One group received telbivudine (LDT, 600 mg/d), and the other group received lamivudine (LAM, 100 mg/d). Clinical, virological and immunological parameters were assessed at the baseline and at 4, 12, 24, 36 and 48 wk.

Results: Both groups achieved significant hepatitis B virus (HBV) replication inhibition and alanine aminotransferase normalization at 48 wk. At the baseline, compared to healthy controls, CHB patients had a lower circulating CD8 T cell frequency (29.44% ± 11.55% vs 37.17% ± 7.30%, P = 0.03) and higher frequencies of programmed death 1 positive CD8 T cells (PD-1+ CD8 T) (16.48% ± 10.82% vs 7.02% ± 3.62%, P = 0.0001) and CD4+ CD25+ FoxP3+ T regulatory cells (Tregs) (23.64% ± 9.38% vs 13.60% ± 6.06%, P = 0.001). On therapy, at the beginning 24 wk with the levels of hepatitis B virus deoxyribonucleic acid (HBV DNA) and HBeAg declining, the frequencies of PD-1+ CD8 T cells and Treg cells gradually and significantly declined at 12 and 24 wk in both therapy groups. At treatment week 4, patients treated with LDT had a lower frequency of PD-1+ CD8 T cells compared to patients treated with LAM (10.08% ± 6.83% vs 20.51% ± 20.96%, P = 0.02). The frequency of PD-1+ CD8 T cells in all of the CHB patients was significantly correlated with both the HBV DNA level (r = 0.45, P = 0.01) and HBeAg level (r = 0.47, P = 0.01) at treatment week 24, but the frequency of Treg cells was only significantly correlated with the HBeAg level (r = 0.44,P = 0.02). Furthermore, the ability of CD8 T cells to secrete pro-inflammatory cytokines was partially restored after 24 wk of therapy.

Conclusion: NA-mediated HBV suppression could down-regulate the production of negative regulators of host immunity during the first 24 wk of therapy and could partially restore the ability of CD8 T cells to secrete pro-inflammatory cytokines. This immune modulating response may be correlated with the levels of both HBV DNA and HBeAg.

Trial registration: ClinicalTrials.gov NCT01480492.

Keywords: CD4+ CD25+ FoxP3+ T regulatory cells; Chronic hepatitis B; Immune modulation; Nucleoside analogues; Programmed death-1.

Figures

Figure 1

Pretreatment cellular phenotype of patients with chronic hepatitis B. A: Representative dot plots of CD4, CD8, programmed death-1 (PD-1) and FoxP3 staining in chronic hepatitis B (CHB) patients; B: Representative dot plots of CD4, CD8, PD-1 and FoxP3 staining in healthy controls (HCs); C: Peripheral frequency of CD8+ T cells in the CHB patients and HCs; D: Peripheral frequency of CD4+ T cells in the CHB patients and HCs; E: Peripheral frequency of PD-1+ CD8 T cells of the total CD8 T cells in CHB patients and HCs (P = 0.0001); F: Peripheral frequency of PD-1+ CD4 T cells of the total CD4 T cells in CHB patients and HCs; G: Frequency of circulating CD4+CD25+FoxP3+ T cells (FoxP3+ Treg) in the CHB patients and HCs. PBMC: Peripheral blood mononuclear cell.

Figure 2

Suppression of hepatitis B virus deoxyribonucleic acid and hepatitis B envelope antigen levels and the expansion of programmed death 1 positive CD8 T cells and CD4+ CD25+ FoxP3+ T regulatory cells. A, B: In patients treated with either lamivudine (LAM) or telbivudine (LDT), the hepatitis B virus (HBV) DNA and hepatitis B envelope antigen (HBeAg) levels decreased significantly compared to the baseline; C: In the LAM group, a significant decrease in the peripheral frequency of programmed death 1 positive CD8 T (PD-1+ CD8 T) cells was observed at treatment week 12; D: In the LDT group, the frequency of PD-1+ CD8 T cells significantly decreased at both week 4 and week 12 compared to the baseline; E and F: In both the LAM group (E) and LDT group (F), a significant decrease in the frequency of T regulatory cells was observed at weeks 12 and 24 compared to the baseline. aP < 0.05; bP < 0.01. HBV DNA: Hepatitis B virus deoxyribonucleic acid.

Figure 3

The relationship between the peripheral frequency of CD4+CD25+FoxP3+ T regulatory cells or programmed death 1 positive CD8 T cells and hepatitis B virus DNA or hepatitis B envelope antigen at different time points during therapy. A: Peripheral frequency of programmed death 1 positive CD8 T (PD-1+ CD8 T) cells in all chronic hepatitis B (CHB) patients at different points during therapy; B: Peripheral frequency of T regulatory cell (Treg) in all CHB patients at different points during therapy; C: Relationship between the HBeAg level and the peripheral frequency of Treg cells in all CHB patients during therapy; D: Relationship between the HBeAg level and the peripheral frequency of PD-1+ CD8 T cells in all CHB patients during therapy; E: Relationship between the hepatitis B virus (HBV) DNA level and the peripheral frequency of PD-1+ CD8 T cells in all CHB patients during therapy; F: Relationship between the HBV DNA level and the peripheral frequency of Treg cells in all CHB patients during therapy. The X-axis shows the different time points during treatment, the left Y-axis shows the HBV DNA or HBeAg levels, and the right Y-axis shows the frequency of Treg cells or PD-1+ CD8 T cells. In the CHB patients, the HBeAg and HBV DNA levels start declining after treatment week 4, along with a decline in FoxP3+ Treg and PD-1+ CD8 T cells. aP < 0.05; bP < 0.01.

Figure 4

Correlations between viral infection parameters and the frequency of pro-inflammatory cytokine-secreting T cells in chronic hepatitis B patients on nucleoside analogue therapy. The frequency of pro-inflammatory cytokine-secreting T cells increased significantly at treatment weeks 12 and 24. A: Frequency of interleukin-2 positive CD8 T cells (IL-2+ CD8 T); B: Frequency of interferon-gamma positive CD8 T cells (IFN-γ+ CD8 T); C: Frequency of interleukin-17A positive CD4 T cells (IL-17A+ CD4 T); D: Correlation between the hepatitis B envelope antigen (HBeAg) level and the frequency of IL-2+ CD8 T cells; E: Correlation between the HBeAg level and the frequency of IFN-γ+ CD8 T cells; F: Correlation between the HBeAg level and the frequency of IL-17A+ CD4 T cells; G: Correlation between the HBV DNA level and the frequency of IL-2+ CD8 T cells; H: Correlation between the HBV DNA level and the frequency of IFN-γ+CD8 T cells; I: Correlation between the HBV DNA level and the frequency of IL-17A+ CD4 T cells. aP < 0.05; bP < 0.01.